这项工作得到了国家自然科学基金（82004031）和四川省科技计划（2022NSFSC1303）的支持。我们非常感谢成都中医药大学中药创新研究所的孙佳怡在蛋白质印迹方面的帮助。

1. 下载 xanthatin 和 Keap1 的结构
<ol><li>打开 PubChem 数据库 （https://pubchem.ncbi.nlm.nih.gov/），输入 xanthatin（小分子），然后按 搜索并单击 第一个结果。单击" 下载 "，然后单击"2D结构"下的" 保存 "，以.sdf格式保存化合物。</li>
	<li>下载蛋白质的晶体结构。<ol><li>打开 UniProt 数据库 （https://www.uniprot.org/），输入 Keap1 并点击搜索。单击左列中"流行生物"下的" 人类 "，然后单击右列中的" 名为 Q14145 的第一个条目 "。</li>
		<li>单击左列函数下的 Structure ，找到 PDB ID 为 2FLU 的结构，然后单击 RCSB-PDB。单击" 下载文件 "并选择 "PDB 格式 "以下载 Keap1 蛋白的晶体结构。</li>
	</ol></li>
</ol>2. 分子对接
<ol><li>在桌面上新建一个名为 molecular docking 的文件夹，并将下载的 xanthatin（小分子）和 Keap1（蛋白质）结构保存在此文件夹中。 注意:文件夹名称和路径必须为英文。</li>
	<li>打开 Maestro 软件，点击文件，选择更改工作目录，点击桌面，双击选择新创建的分子对接文件夹，点击选择选项。</li>
	<li>小分子加工<ol><li>单击 "文件 并导入结构选项"，单击 "桌面"，双击"分子对接"文件夹，单击" xanthatin结构文件"，然后单击" 打开 "以导入小分子文件。</li>
		<li>单击软件右上角 的任务 ，然后选择 LigPrep 选项。使用工作区中的结构;其余参数为默认参数，单击" 运行 "以执行小分子处理。</li>
	</ol></li>
	<li>蛋白质结构加工<ol><li>单击" 文件"和"导入结构 "选项，单击 "桌面"，然后双击" 分子对接 "文件夹。单击" Keap1 蛋白质结构 "文件，然后单击" 打开 "以导入蛋白质结构文件。</li>
		<li>单击软件右上角 的"任务 "，然后选择" 蛋白质制备向导 "选项。选中 "从基团中删除超过 5 Å 的水"框 ，并输入 pH 值为 7.4+/-0.0。单击" 预处理"，单击" 优化" ，然后单击"优化">"删除 Waters >最小化"。</li>
		<li>上一个任务完成后，单击下一个任务。单击 "任务" ，然后选择" 站点地图 "选项，对所有参数使用默认设置，然后单击" 运行"。</li>
		<li>单击" 文件"和"导入结构 "选项，单击 "桌面"，双击分子对接文件夹，然后打开名为"sitemap_1"的文件夹。单击名为 Sitemap_1_out.maegz 的第一个文件，然后单击" 打开"。</li>
		<li>双击圆点以在工作区中选择sitemap_1_protein，然后单击 Sitemap_1_site_1 以选择它。单击 "任务 "，然后选择" 受体网格生成 "选项。 注意:站点地图预测的蛋白质位点根据评分进行排名，得分最高的位点排在第一位。</li>
		<li>在 receptor 下选择 Entry 选项，其余参数不变。点击蛋白质结构中的 小白球 ，生成默认大小的对接盒，并将作业名称更改为 glide-grid_2FLU。单击 "运行"。</li>
	</ol></li>
	<li>分子对接<ol><li>单击 "任务" ，然后选择" 配体对接 "选项。从文件中选择 Receptor Grid As ，然后单击 Browse。单击 "桌面"，双击"分子对接"文件夹，双击滑动grid_2FLU文件，单击"Glide-grid_2FLU.zip文件"，然后单击" 打开"。</li>
		<li>单击" Use Ligands from files">浏览>桌面。双击分子对接文件夹，双击ligprep_1文件，单击Ligprep_1-out.maegz文件，然后单击 打开。</li>
		<li>单击 "设置 "，然后选择 "精度为XP（额外精度） "选项，将作业名称更改为"glide-dock_XP_2FLU"，然后单击" 运行"。</li>
	</ol></li>
	<li>查看对接结果<ol><li>单击" 文件"和"导入结构 "选项，单击" 桌面"，然后双击" 分子对接 "文件夹。双击 glide-dock_XP_2FLU 文件，单击 glide-dock_XP_1_pv.maegz 文件，然后单击" 打开"。</li>
		<li>双击圆点以在工作区中选择配体，然后单击以选择sitemap_1_protein。单击 "表"，滑向最右侧，然后在"停靠分数"下查看分数。</li>
	</ol></li>
	<li>2D 结果的可视化<ol><li>双击圆点以在工作区中选择配体，然后单击以同时选择配体sitemap_1_protein。单击 Ligand Interaction > View ，然后选中 LID 图例 框。</li>
		<li>单击 "文件"，选择" 保存屏幕截图"，在宽度处输入 6000，取消选中 "透明背景"框，然后单击" 确定"。单击 "桌面"，将文件命名为 2D-xanthatin-2FLU，然后保存图像。</li>
	</ol></li>
	<li>3D结果的可视化<ol><li>双击圆点以在工作区中选择配体，然后单击以同时选择配体sitemap_1_protein。</li>
		<li>在"快速选择"中单击" L "以选择小分子，然后单击" 样式"。单击样式中 的第二行 以更改小分子键的大小，然后单击颜色原子以更改小分子颜色。</li>
		<li>选择图中的小分子，单击鼠标右键，选择 扩展选择，然后选择 4 Å。单击"在样式中 应用标签 "以显示标签。</li>
		<li>单击样式中的第二行以更改氨基酸键的大小，然后单击 "颜色原子 "以更改氨基酸颜色。</li>
		<li>单击 "下一步">"反转"，按住控制键并单击"样式"下的第一个眼睛位置，以仅显示带有标签的氨基酸。</li>
		<li>单击" 设置色带>样式 "以将蛋白质显示为色带样式，然后单击"色带"下的" 编辑色带 "以更改色带颜色。</li>
		<li>单击屏幕右下角的 交互切换 ，单击鼠标右键并取消选中联系人/冲突。</li>
		<li>在任务中搜索"测量"，选择 "距离"，然后单击屏幕中由黄色虚线（氢键）连接的两个原子，以测量氢键的长度。</li>
		<li>按住鼠标滚轮旋转蛋白质，将蛋白质调整到屏幕的中心，并确保每个氨基酸标签都可见。</li>
		<li>单击 "工作区">"将图像另存为 "，然后单击桌面上的。默认情况下将图像保存为600dpi，将文件命名为3D-xanthatin-2FLU，选择文件类型为tiff格式，然后保存图像。</li>
	</ol></li>
</ol>3. 细胞培养和CETSA样品制备
<ol><li>细胞培养<ol><li>将 5 x 106 RAW264.7 细胞/mL 加入 100 mm 培养皿中，并与 6 mL 含有 10% FBS 和 1% 抗生素 - 抗真菌溶液的 DMEM 培养基在 37 °C、95% 湿度和 5% CO2 下孵育。准备三个培养皿，当细胞达到80%汇合度时进行实验。</li>
	</ol></li>
	<li>CETSA样品制备<ol><li>吸出旧培养基，向每个培养皿中加入 2 mL 加热至室温的磷酸盐缓冲盐水 （PBS），洗涤 2 次。</li>
		<li>向每个细胞培养皿中加入 2 mL PBS，混合细胞，并将细胞收集到两个 1.5 mL 微量离心管中。在室温下以377× g 离心3分钟。</li>
		<li>弃去上清液，用含有 1% 广谱蛋白酶抑制剂混合物的 2 mL PBS 重悬细胞。分装到单独的微量离心管中，每个离心管中含有 1 mL 细胞悬液。</li>
		<li>将微量离心管在液氮中冷冻至白色固体，然后立即在37°C水浴中解冻并重复3次。在4°C下以12,000× g 离心10分钟。</li>
		<li>取两个微量离心管，一个含有100μMxanthatin，另一个含有等体积的DMSO，并向每个管中加入450μL离心上清液，在37°C孵育30分钟。</li>
		<li>将来自两个微量离心管的上清液加入体积为 60 μL/管的 14 个 PCR 管中，并在不同温度（45 °C、48 °C、51 °C、54 °C、57 °C、60 °C 和 63 °C）下同时加热管 3 分钟，每个温度下有两个 PCR 管，用于 DMSO 和 xanthatin 组， 分别。</li>
		<li>对于未加热的组，从两个微量离心管中的每一个中取出上清液，并加入14个PCR管中，7个用于DMSO组，7个用于黄素组，每个体积为60μL。不要在这里加热样品。</li>
		<li>在室温下冷却加热组的管子。将所有管（加热和未加热）在4°C下以12,000× g 离心10分钟，并从每个管中取出48μL上清液到新的1.5mL微量离心管中。向每个试管中加入12μL SDS-PAGE蛋白上样缓冲液，涡旋混合充分，并在100°C的水浴中加热5分钟。 注意:制备的CETSA样品必须储存在-80°C，并在1周内通过蛋白质印迹进行测试。</li>
	</ol></li>
</ol>4. 蛋白质印迹
<ol><li>SDS-PAGE凝胶的制备<ol><li>用超纯水冲洗一块长玻璃板和一块短玻璃板。将板放入夹紧槽中，然后放在透明板上。加入超纯水并进行泄漏检查 10 分钟。</li>
		<li>将 8 mL 下凝胶溶液和 8 mL 下凝胶缓冲液加入锥形瓶中，轻轻摇动混合。向凝胶溶液中加入 160 μL 凝血促进剂并充分混合。 注意: 下层粘合剂的体积不应超过夹紧槽上的绿线。</li>
		<li>从板中排出超纯水，并用滤纸吸干。加入准备好的下凝胶溶液。加入 2 mL 异丙醇，等待 20 分钟凝固。</li>
		<li>取 2 mL 上凝胶溶液和 2 mL 上凝胶缓冲液加入锥形瓶中，轻轻摇动瓶进行混合。</li>
		<li>从凝胶层中取出异丙醇，并用滤纸吸干多余的异丙醇。</li>
		<li>将 40 μL 凝血促进剂加入混合的上凝胶溶液中，充分混合，然后在长板和短玻璃板之间加入并插入 1.5 mm 15 孔梳子，等待 20 分钟。 注意: 梳子应垂直于上层插入，梳子和溶液之间没有气泡。</li>
	</ol></li>
	<li>电泳<ol><li>向烧杯中加入1 L超纯水，加入一袋SDS-PAGE电泳缓冲液粉末，用玻璃棒充分搅拌，并将制备的凝胶加入电泳单元中。将设备放入盒子中，然后拉出梳子。</li>
		<li>在室温下解冻标记物和 CETSA 样品，涡旋并下降。根据预定顺序，向第一个孔中加入 2.5 μL 标记物，向剩余的每个孔中加入 20 μL CETSA 样品。 注:根据步骤3.2.6中描述的温度处理，从45°C到63°C依次加入CETSA样品;首先加入DMSO组，然后加入xanthatin组（图1）。</li>
		<li>将剩余的电泳溶液加入电泳单元中，盖上盖子，插入电极，正极对正极，负极对负极。</li>
		<li>打开电源开关并将电压开关调整到 80 V 以开始电泳。</li>
	</ol></li>
	<li>转移到膜上<ol><li>将 850 mL 超纯水、100 mL 无水乙醇和 50 mL 快速转印缓冲液加入烧杯中，充分搅拌。准备泡沫板、塑料刀、镊子、托盘和膜转移装置。</li>
		<li>根据凝胶的大小切开PVDF膜，做标记，然后将其浸泡在甲醇中30秒以使其活化。</li>
		<li>将转移溶液倒入托盘中，放置并打开转移夹，并放置海绵垫和三层转移滤纸。</li>
		<li>停止电泳，然后打开电泳盒的盖子。取电泳装置，倒出电泳液，取出板。将板放在泡沫板上，用塑料刀轻轻撬开，然后切开凝胶，以清楚地看到蛋白质标记物和靶蛋白。</li>
		<li>用镊子握住标记的一侧，在装有跨膜溶液的托盘中洗涤，然后将其平放在滤纸上。将活化的 PVDF 膜浸入转印溶液中，用镊子握住标记面，然后面朝下覆盖凝胶。 注意: PVDF 膜标记的一侧是正面，请注意在放置过程中不会产生气泡。</li>
		<li>将两层转移滤纸和一层海绵垫放在PVDF膜上，盖住并夹紧转移夹，然后放入转移罐中。 注意: 传输夹的黑色到传输插槽的黑色。</li>
		<li>将剩余的跨膜溶液加入跨膜盒中，盖上盖子，阳性转阳性，阴性转阴性。</li>
		<li>将电流调节为 400 mA，时间调整为 30 分钟，然后按 Run 开始在室温下转移膜。</li>
	</ol></li>
	<li>阻塞<ol><li>向玻璃瓶中加入 2 L 超纯水，加入一袋 TBS 粉末，然后加入 2 mL 吐温制成 TBST 溶液。称量BSA粉末，用TBST制备5%BSA溶液，并向培养箱中加入6mL BSA溶液。</li>
		<li>停止膜转移，然后打开转移夹。夹住 PVDF 膜的标记侧并将其放入培养箱中。将培养箱放在振荡器上，并在室温下块1.5小时。</li>
	</ol></li>
	<li>一抗的孵育<ol><li>用 5% BSA 溶液以 1:1000 制备一抗（详见 材料表 ），在培养箱中回收 BSA 溶液，并向培养箱中加入 6 mL 一抗。将培养箱放入装有冰袋的泡沫盒中，并在摇摇杯上放置过夜。</li>
	</ol></li>
	<li>二抗的孵育<ol><li>第二天在培养箱中收集一抗，然后加入 6 mL TBST 溶液，并将其放在振荡器上洗涤 5 分钟。</li>
		<li>倒出洗净的TBST溶液，加入新鲜的洗涤;重复 5 次，并用 5% BSA 溶液以 1:5000 制备二抗。</li>
		<li>倒出 TBST 溶液，向孵育箱中加入 6 mL 二抗。将培养箱放在振荡器上，在室温下孵育1.5小时。</li>
		<li>收集二抗（详见 材料表 ），然后加入 6 mL TBST 溶液，并将其置于振荡器上洗涤 5 分钟。倒出洗净的TBST溶液，加入新鲜的洗涤;重复 5 次。</li>
	</ol></li>
	<li>露出条带<ol><li>在最后一次洗涤结束时保留 TBST 溶液。取等体积的化学发光试剂 A 和 B，摇匀，充分混合。将溶液避光。</li>
		<li>打开凝胶成像系统，点击" 样本"，检查校准，等待校准完成，然后点击" 标记"，检查校准。</li>
		<li>用镊子拾取条带的标记侧，并完全浸入化学发光试剂溶液中。夹住标记的一侧，将条带正面朝下放入成像仪中，合上成像仪的盖子，单击 曝光，并将曝光时间设置为 1 秒。 注意:开发商必须完全覆盖条带，并且开发应在黑暗的环境中进行。</li>
		<li>单击 "保存"，为图像命名，然后将其另存为.tif文件。分析蛋白质印迹的光密度。将DMSO组各温度的灰度值与45 °C的灰度值进行比较，将xanthatin组各温度的灰度值与xanthatin在45 °C的灰度值进行比较。</li>
	</ol></li>
</ol>

验证黄素-Keap1 与 Docking 和 CETSA 的相互作用

<ol>
	<li>Soleymani, F., Paquet, E., Viktor, H., Michalowski, W., Spinello, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-protein+interaction+prediction+with+deep+learning:+A+comprehensive+review.">Protein-protein interaction prediction with deep learning: A comprehensive review.</a> Computat Struct Biotechnol J. 20, 5316-5341 (2022).</li><li>Du, X., 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=Insights+into+protein-ligand+interactions:+mechanisms,+models,+and+methods.">Insights into protein-ligand interactions: mechanisms, models, and methods.</a> Int J Mol Sci. 17 (2), 144 (2016).</li><li>Wu, Q., Peng, Z., Zhang, Y., Yang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=COACH-D:+improved+protein-ligand+binding+sites+prediction+with+refined+ligand-binding+poses+through+molecular+docking.">COACH-D: improved protein-ligand binding sites prediction with refined ligand-binding poses through molecular docking.</a> Nucleic Acids Res. 46, W438-W442 (2018).</li><li>Zhang, F., 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=PROBselect:+accurate+prediction+of+protein-binding+residues+from+proteins+sequences+via+dynamic+predictor+selection.">PROBselect: accurate prediction of protein-binding residues from proteins sequences via dynamic predictor selection.</a> Bioinfo. 36, i735-i744 (2020).</li><li>Kandel, J., Tayara, H., Chong, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PUResNet:+prediction+of+protein-ligand+binding+sites+using+deep+residual+neural+network.">PUResNet: prediction of protein-ligand binding sites using deep residual neural network.</a> J Cheminfo. 13 (1), 65 (2021).</li><li>Dhakal, A., Mckay, C., Tanner, J. J., Cheng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+intelligence+in+the+prediction+of+protein-ligand+interactions:+recent+advances+and+future+directions.">Artificial intelligence in the prediction of protein-ligand interactions: recent advances and future directions.</a> Brief Bioinform. 23 (1), 476 (2022).</li><li>Hatty, C. R., Banati, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-ligand+and+membrane-ligand+interactions+in+pharmacology:+the+case+of+the+translocator+protein+(TSPO).">Protein-ligand and membrane-ligand interactions in pharmacology: the case of the translocator protein (TSPO).</a> Pharmacol Res. 100, 58-63 (2015).</li><li>Chaires, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calorimetry+and+thermodynamics+in+drug+design.">Calorimetry and thermodynamics in drug design.</a> Ann Rev Biophys. 37, 135-151 (2008).</li><li>Huang, S. Y., Zou, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+and+challenges+in+protein-ligand+docking.">Advances and challenges in protein-ligand docking.</a> Int J Mo Sci. 11 (8), 3016-3034 (2010).</li><li>Li, J., Fu, A., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+scoring+functions+used+for+protein-ligand+interactions+in+molecular+docking.">An overview of scoring functions used for protein-ligand interactions in molecular docking.</a> Interdiscip Sci. 11 (2), 320-328 (2019).</li><li>Crampon, K., Giorkallos, A., Deldossi, M., Baud, S., Steffenel, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Machine-learning+methods+for+ligand-protein+molecular+docking.">Machine-learning methods for ligand-protein molecular docking.</a> Drug Discov Today. 27 (1), 151-164 (2022).</li><li>Pinzi, L., Rastelli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+docking:+Shifting+paradigms+in+drug+discovery.">Molecular docking: Shifting paradigms in drug discovery.</a> Int J Mol Sci. 20 (18), 4331 (2019).</li><li>Abdolmaleki, A., Ghasemi, F., Ghasemi, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer-aided+drug+design+to+explore+cyclodextrin+therapeutics+and+biomedical+applications.">Computer-aided drug design to explore cyclodextrin therapeutics and biomedical applications.</a> Chem Biol Drug Des. 89 (2), 257-268 (2017).</li><li>Chen, G., Seukep, A. J., Guo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+molecular+docking+for+the+research+and+discovery+of+potential+marine+drugs.">Recent advances in molecular docking for the research and discovery of potential marine drugs.</a> Mar Drugs. 18 (11), 545 (2020).</li><li>Li, T., Guo, R., Zong, Q., Ling, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+molecular+docking+in+elaborating+molecular+mechanisms+and+interactions+of+supramolecular+cyclodextrin.">Application of molecular docking in elaborating molecular mechanisms and interactions of supramolecular cyclodextrin.</a> Carbohydr Polym. 276, 118644 (2022).</li><li>Martinez Molina, D., 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=Monitoring+drug+target+engagement+in+cells+and+tissues+using+the+cellular+thermal+shift+assay.">Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay.</a> Science. 341 (6141), 84-87 (2013).</li><li>Tu, Y., Tan, L., Tao, H., Li, Y., Liu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CETSA+and+thermal+proteome+profiling+strategies+for+target+identification+and+drug+discovery+of+natural+products.">CETSA and thermal proteome profiling strategies for target identification and drug discovery of natural products.</a> Phytomedicine. 116, 154862 (2023).</li><li>Dai, L., 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=Horizontal+cell+biology:+Monitoring+global+changes+of+protein+interaction+states+with+the+proteome-wide+cellular+thermal+shift+assay+(CETSA).">Horizontal cell biology: Monitoring global changes of protein interaction states with the proteome-wide cellular thermal shift assay (CETSA).</a> Annu Rev Biochem. 88, 383-408 (2019).</li><li>Lade, D. M., Nicoletti, R., Mersch, J., Agazie, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+synthesis+of+improved+active-site+SHP2+inhibitors+with+anti-breast+cancer+cell+effects.">Design and synthesis of improved active-site SHP2 inhibitors with anti-breast cancer cell effects.</a> Eur J Med Chem. 247, 115017 (2023).</li><li>Jiang, X., 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=Novel+chemical-structure+TPOR+agonist,+TMEA,+promotes+megakaryocytes+differentiation+and+thrombopoiesis+via+mTOR+and+ERK+signalings.">Novel chemical-structure TPOR agonist, TMEA, promotes megakaryocytes differentiation and thrombopoiesis via mTOR and ERK signalings.</a> Phytomedicine. 110, 154637 (2023).</li><li>Mateus, A., 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=Thermal+proteome+profiling+for+interrogating+protein+interactions.">Thermal proteome profiling for interrogating protein interactions.</a> Mol Syst Biol. 16 (3), 9232 (2020).</li><li>Sanchez, T. W., 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=Real-time+cellular+thermal+shift+assay+to+monitor+target+engagement.">Real-time cellular thermal shift assay to monitor target engagement.</a> ACS Chem Biol. 17 (9), 2471-2482 (2022).</li><li>Tolvanen, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+advances+in+CETSA.">Current advances in CETSA.</a> Front Mol Biosci. 9, 866764 (2022).</li><li>Liu, M., 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=Xanthatin+inhibits+STAT3+and+NF-&#954;B+signalling+by+covalently+binding+to+JAK+and+IKK+kinases.">Xanthatin inhibits STAT3 and NF-&#954;B signalling by covalently binding to JAK and IKK kinases.</a> J Cell Mol Med. 23 (6), 4301-4312 (2019).</li><li>Liu, Y., Chen, W., Zheng, F., Yu, H., Wei, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xanthatin+alleviates+LPS-Induced+inflammatory+response+in+RAW264.7+macrophages+by+Inhibiting+NF-&#954;B,+MAPK+and+STATs+activation.">Xanthatin alleviates LPS-Induced inflammatory response in RAW264.7 macrophages by Inhibiting NF-&#954;B, MAPK and STATs activation.</a> Molecules. 27 (14), 4603 (2022).</li><li>Dinkova-Kostova, A. T., Kostov, R. V., Canning, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keap1,+the+cysteine-based+mammalian+intracellular+sensor+for+electrophiles+and+oxidants.">Keap1, the cysteine-based mammalian intracellular sensor for electrophiles and oxidants.</a> Arch Biochem Biophys. 617, 84-93 (2017).</li><li>Tabana, Y., Babu, D., Fahlman, R., Siraki, A. G., Barakat, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+identification+of+small+molecules:+An+overview+of+the+current+applications+in+drug+discovery.">Target identification of small molecules: An overview of the current applications in drug discovery.</a> BMC Biotechnol. 23 (1), 44 (2023).</li><li>Schenone, M., Dan&#269;&#237;k, V., Wagner, B. K., Clemons, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+identification+and+mechanism+of+action+in+chemical+biology+and+drug+discovery.">Target identification and mechanism of action in chemical biology and drug discovery.</a> Nat Chem Biol. 9 (4), 232-240 (2013).</li><li>Hughes, J. P., Rees, S., Kalindjian, S. B., Philpott, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+early+drug+discovery.">Principles of early drug discovery.</a> Br J Pharmacol. 162 (6), 1239-1249 (2011).</li><li>Chakraborty, C., 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=SARS-CoV-2+protein+drug+targets+landscape:+A+potential+pharmacological+insight+view+for+the+new+drug+development.">SARS-CoV-2 protein drug targets landscape: A potential pharmacological insight view for the new drug development.</a> Expert Rev Clin Pharmacol. 14 (2), 225-238 (2021).</li><li>Ziegler, S., Pries, V., Hedberg, C., Waldmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+identification+for+small+bioactive+molecules:+Finding+the+needle+in+the+haystack.">Target identification for small bioactive molecules: Finding the needle in the haystack.</a> Angew Chem Int Ed Engl. 52 (10), 2744-2792 (2013).</li><li>Zhang, X., 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=Highly+effective+identification+of+drug+targets+at+the+proteome+level+by+pH-dependent+protein+precipitation.">Highly effective identification of drug targets at the proteome level by pH-dependent protein precipitation.</a> Chem Sci. 13 (42), 12403-12418 (2022).</li><li>Zhang, X., 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=A+simplified+thermal+proteome+profiling+approach+to+screen+protein+targets+of+a+ligand.">A simplified thermal proteome profiling approach to screen protein targets of a ligand.</a> Proteomics. 20, 1900372 (2020).</li><li>Hou, Y., 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=Salidroside+intensifies+mitochondrial+function+of+CoCl2-damaged+HT22+cells+by+stimulating+PI3K-AKT-MAPK+signaling+pathway.">Salidroside intensifies mitochondrial function of CoCl2-damaged HT22 cells by stimulating PI3K-AKT-MAPK signaling pathway.</a> Phytomedicine. 109, 154568 (2022).</li><li>Wang, X., 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=Salidroside,+a+phenyl+ethanol+glycoside+from+Rhodiolacrenulata,+orchestrates+hypoxic+mitochondrial+dynamics+homeostasis+by+stimulating+Sirt1/p53/Drp1+signaling.">Salidroside, a phenyl ethanol glycoside from Rhodiolacrenulata, orchestrates hypoxic mitochondrial dynamics homeostasis by stimulating Sirt1/p53/Drp1 signaling.</a> J Ethnopharmacol. 293, 115278 (2022).</li></ol>

作者没有什么可透露的。

疾病靶点的确定与药物的发现和开发密切相关27.通过精确靶向特定靶点，可以开发候选药物以更有效地治疗特定疾病，同时最大限度地减少与药物相关的副作用28,29。最常用的靶标是蛋白质靶标30。然而，由于细胞内蛋白质的广泛多样性，特殊蛋白质靶标的鉴定带来了巨大的挑战30,31<sup class="...

蛋白质是生物体中非常重要的大分子，在细胞内具有多种独特的功能，例如膜组成、细胞骨架形成、酶活性、运输、细胞信号传导以及参与细胞内和细胞外机制 1,2,3。蛋白质主要通过与多种分子的特异性相互作用来表现出其生物学功能，包括其他蛋白质、核酸、小分子配体和金属离子 1,4。配体是与生物体中的蛋白质特异性结合的小分子化合物。蛋白质和配体之间的相互作用发生在蛋白质上的特定位点，称为结合位点，也称为结合口袋5。在药物化学研究中，重点在于识别与疾病明显相关的关键蛋白质，这些蛋白质是药物的靶标6。因此，深入了解蛋白质和配体之间的结合位点对于促进药物发现、设计和研究至关重要7,8。
分子对接是一种广泛使用的用于研究蛋白质-配体结合的计算工具，它利用蛋白质和配体的三维结构来探索它们在形成稳定复合物时的主要结合模式和亲和力9,10,11。分子对接技术的应用起源于1970年代。基于锁键配对原理，利用分子对接软件的算法，通过分析对接结果，可以确定化合物与分子靶标之间的相互作用。这种方法可以预测化合物和靶分子的活性结合位点。因此，它有助于识别配体-受体相互作用的最佳结合构象（此处称为结合模型），这对于理解这些分子结合的机制至关重要 12,13,14,15。虽然分子对接提供了有价值的基于计算机的配体-受体相互作用预测，但重要的是要注意这些是初步发现。因此，进一步的实验验证对于确认这些相互作用至关重要。
细胞热位移测定 （CETSA） 最初由 Pär Nordlund 的研究团队于 2013 年提出，是一种验证药物-靶蛋白相互作用的方法。该技术专门测试药物结合诱导的靶蛋白的热稳定性，为确认分子相互作用提供了一种实用的方法16,17,18。这种方法基于配体结合在靶蛋白内启动热位移的基本原理，适用于各种生物样品，包括细胞裂解物、完整活细胞和组织19,20。CETSA 通过检测由于配体结合导致的蛋白质热力学稳定性并将观察到的表型反应与目标化合物联系起来，支持完整细胞中小分子的直接靶向参与21,22。在源自 CETSA 的各种方法中，Western Blot-CETSA （WB-CETSA） 被认为是一种经典方法。在使用 CETSA 方法制备样品后，使用蛋白质印迹分析来检测靶蛋白热稳定性的变化。这使得可以精确测定细胞系统内的药物-蛋白质相互作用17,23。
Xanthatin 是从植物 Xanthium L. 中分离出的一种生物活性化合物，具有抗炎等特性，在中医中已用于治疗鼻窦炎和关节炎等疾病24,25。Kelch 样 ECH 相关蛋白 1 （Keap1） 是基于 Cullin3 的 Cullin-RING E3 泛素连接酶多亚基蛋白复合物的组成部分，也是细胞内氧化还原稳态的重要调节因子，它通过调节细胞内氧化还原状态来影响炎症反应的强度和持续时间26。在这项研究中，我们首先利用分子对接来研究xanthatin（小分子）与Keap1蛋白之间的相互作用，旨在预测它们的结合模式。随后，我们采用 CETSA 方法通过评估 xanthatin 对 Keap1 蛋白热稳定性的影响来验证这种相互作用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.45 &#956;m Polyvinylidene fluoride membrane</td>
			<td>Millipore</td>
			<td>PR05509</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous ethanol</td>
			<td>Chron chemicals</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>BioFroxx</td>
			<td>4240GR100</td>
			<td></td>
		</tr>
		<tr>
			<td>Broad-spectrum protease inhibitor mixtures</td>
			<td>Boster Biological Technology Co., Ltd</td>
			<td>AR1193</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Boster Biological Technology Co., Ltd</td>
			<td>PYG0040</td>
			<td></td>
		</tr>
		<tr>
			<td>Enhanced chemiluminescence reagent</td>
			<td>Beyotime Biotechnology Co., Ltd</td>
			<td>P0018S</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH antibody</td>
			<td>ProteinTech Group Co., Ltd</td>
			<td>10494-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Imaging Instrument</td>
			<td>E-BLOT</td>
			<td>Touch Imager Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>Gradient PCR instrument</td>
			<td>Biometra TADVANCED</td>
			<td>Biometra Tadvanced 96SG</td>
			<td></td>
		</tr>
		<tr>
			<td>High-speed freezing centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Allegra X-30R</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxidase-conjugated affiniPure goat antibody</td>
			<td>ProteinTech Group Co., Ltd</td>
			<td>SA00001-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol&#160;</td>
			<td>Chron chemicals</td>
			<td>67-63-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Keap1 antibody</td>
			<td>Zen BioScience Co., Ltd</td>
			<td>R26935</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal bath</td>
			<td>Analytik Jena</td>
			<td>TSC</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Chron chemicals</td>
			<td>67-56-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ncmblot rapid transfer buffer (20&#215;)</td>
			<td>NCM Biotech Co., Ltd</td>
			<td>WB4600</td>
			<td></td>
		</tr>
		<tr>
			<td>Omni-Easy OneStep PAGE gel fast preparation kie</td>
			<td>Epizyme Biotech Co., Ltd</td>
			<td>PG212</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer saline</td>
			<td>Boster Biological Technology Co., Ltd</td>
			<td>PYG0021</td>
			<td></td>
		</tr>
		<tr>
			<td>Prestained Color Protein Marker</td>
			<td>Biosharp&#160;</td>
			<td>BL741A</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein Blotting Electrophoresis System</td>
			<td>Bio-Rad</td>
			<td>MiniPROTEAN&#210;Tetra Cell</td>
			<td></td>
		</tr>
		<tr>
			<td>RAW264.7 cell</td>
			<td>Beyotime Biotechnology Co., Ltd</td>
			<td>C7505</td>
			<td></td>
		</tr>
		<tr>
			<td>RAW264.7 cell-specific medium</td>
			<td>Procell Life Science&#38;Technology Co., Ltd</td>
			<td>CM-0597</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE protein loading buffer</td>
			<td>Boster Biological Technology Co., Ltd</td>
			<td>AR1112-10</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE running buffer powder</td>
			<td>Servicebio</td>
			<td>G2018</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris buffered saline powder</td>
			<td>Servicebio</td>
			<td>G0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>BioFroxx</td>
			<td>1247ML100</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Memmert</td>
			<td>WNE10</td>
			<td></td>
		</tr>
		<tr>
			<td>Water purifier</td>
			<td>Millipore</td>
			<td>Milli- IQ 7005</td>
			<td></td>
		</tr>
		<tr>
			<td>Xanthatin</td>
			<td>ChemConst Biotechnology Co., Ltd</td>
			<td>CONST210706</td>
			<td></td>
		</tr>
	</tbody>
</table>

protein target prediction and validation of small molecule compound

蛋白质是人体生理学的基础，其靶点在研究和药物开发中至关重要。关键蛋白质靶标的识别和验证已成为药物开发不可或缺的一部分。分子对接是一种广泛用于研究蛋白质-配体结合的计算工具，特别是在药物和蛋白质靶标相互作用的背景下。为了进行结合的实验验证并直接获得药物与其靶标的结合，使用细胞热位移测定 （CETSA） 方法。本研究旨在将分子对接与CETSA相结合，以预测和验证药物与重要蛋白质靶点之间的相互作用。具体来说，我们通过分子对接分析预测了xanthatin和Keap1蛋白之间的相互作用及其结合模式，然后使用CETSA法验证了该相互作用。结果表明，黄素可以与Keap1蛋白的特定氨基酸残基建立氢键，降低Keap1蛋白的热稳定性，表明黄素可以直接与Keap1蛋白相互作用。

分子对接分析预测了黄素与Keap1蛋白之间的相互作用。 图 2 显示了 xanthatin 与 Keap1 蛋白的氨基酸残基 Gly-367 和 Val-606 之间形成氢键，Gly-367 的氢键长度为 2.17 Å，Val-606 的氢键长度为 2.13 Å。此外，计算出的对接分数为 -5.69 kcal/mol 表明 xanthatin 和 Keap1 蛋白之间具有良好的结合亲和力。
CETSA方法显示，黄素结合改变了Keap1蛋白的热稳定性，证实了黄素与Keap1?...

Watch this Scientific Journal Video about Author Spotlight: Streamlining Protein Target Prediction and Validation via Molecular Docking and CETSA at JoVE.com

作者聚焦:通过分子对接和 CETSA 简化蛋白质靶标预测和验证

这里使用的实验展示了一种分子对接结合细胞热位移测定的方法，以预测和验证小分子和蛋白质靶标之间的相互作用。

小分子化合物的蛋白质靶点预测与验证

Proteins are fundamental to human physiology, with their targets being crucial in research and drug development. The identification and validation of ...

我们的研究重点是蛋白质靶点、小分子化合物的预测和验证。这里使用的实验展示了一种分子对接方法与类似的热位移测定相结合，以预测和验证小分子和蛋白质靶标之间的相互作用。CETSA的这些步骤很复杂，操作过程中的任何步骤错误都会对结果产生很大影响。我们的协议将预测与验证相结合，有效降低了时间消耗和经济成本。此外，实验仪器和材料很容易选择使用。最后，CETSA样品易于在测试中制备，保证了样品的可重复性。

protein-target-prediction-and-validation-of-small-molecule-compound

Research

JoVE Journal

Medicine

Protein Target Prediction and Validation of Small Molecule Compound

2.3K 次观看.  Chengdu University of Traditional Chinese Medicine. 我们的研究重点是蛋白质靶点、小分子化合物的预测和验证。这里使用的实验展示了一种分子对接方法与类似的热位移测定相结合，以预测和验证小分子和蛋白质靶标之间的相互作用。CETSA的这些步骤很复杂，操作过程中的任何步骤错误都会对结果产生很大影响。我们的协议将预测与验证相结合，有效降低了时间消耗和经济成本。此外，实验仪器和材料很容易选择使用?...

视频: 作者聚焦:通过分子对接和 CETSA 简化蛋白质靶标预测和验证

An Experimental Paradigm for the Prediction of Post-Operative Pain (PPOP)

Many women undergo cesarean delivery without problems, however some experience significant pain after cesarean section. Pain is associated with negative short-term and long-term effects on the mother. Prior to women undergoing surgery, can we predict who is at risk for developing significant postoperative pain and potentially prevent or minimize its negative consequences? These are the fundamental questions that a team from the University of Washington, Stanford University, the Catholic University in Brussels, Belgium, Santa Joana Women's Hospital in S&atilde;o Paulo, Brazil, and Rambam Medical Center in Israel is currently evaluating in an international research collaboration. The ultimate goal of this project is to provide optimal pain relief during and after cesarean section by offering individualized anesthetic care to women who appear to be more 'susceptible' to pain after surgery.
A significant number of women experience moderate or severe acute post-partum pain after vaginal and cesarean deliveries. 1 Furthermore, 10-15% of women suffer chronic persistent pain after cesarean section. 2 With constant increase in cesarean rates in the US 3 and the already high rate in Brazil, this is bound to create a significant public health problem. When questioning women's fears and expectations from cesarean section, pain during and after it is their greatest concern. 4 Individual variability in severity of pain after vaginal or operative delivery is influenced by multiple factors including sensitivity to pain, psychological factors, age, and genetics. The unique birth experience leads to unpredictable requirements for analgesics, from 'none at all' to 'very high' doses of pain medication. Pain after cesarean section is an excellent model to study post-operative pain because it is performed on otherwise young and healthy women. Therefore, it is recommended to attenuate the pain during the acute phase because this may lead to chronic pain disorders. The impact of developing persistent pain is immense, since it may impair not only the ability of women to care for their child in the immediate postpartum period, but also their own well being for a long period of time.
In a series of projects, an international research network is currently investigating the effect of pregnancy on pain modulation and ways to predict who will suffer acute severe pain and potentially chronic pain, by using simple pain tests and questionnaires in combination with genetic analysis. A relatively recent approach to investigate pain modulation is via the psychophysical measure of Diffuse Noxious Inhibitory Control (DNIC). This pain-modulating process is the neurophysiological basis for the well-known phenomenon of 'pain inhibits pain' from remote areas of the body. The DNIC paradigm has evolved recently into a clinical tool and simple test and has been shown to be a predictor of post-operative pain.5 Since pregnancy is associated with decreased pain sensitivity and/or enhanced processes of pain modulation, using tests that investigate pain modulation should provide a better understanding of the pathways involved with pregnancy-induced analgesia and may help predict pain outcomes during labor and delivery. For those women delivering by cesarean section, a DNIC test performed prior to surgery along with psychosocial questionnaires and genetic tests should enable one to identify women prone to suffer severe post-cesarean pain and persistent pain. These clinical tests should allow anesthesiologists to offer not only personalized medicine to women with the promise to improve well-being and satisfaction, but also a reduction in the overall cost of perioperative and long term care due to pain and suffering. On a larger scale, these tests that explore pain modulation may become bedside screening tests to predict the development of pain disorders following surgery.

An Experimental Paradigm for the Prediction of Post-Operative Pain PPOP

Diffuse noxious inhibitory control, temporal summation and wound hyperalgesia testing are demonstrated in the obstetric patient. These tests evaluate inhibitory and excitatory mechanisms of pain processing and are here utilized to evaluate endogenous analgesia at different time-points during pregnancy and the peripartum period to help reveal individual s risk for persistent pain.

用于预测手术后的疼痛的实验范式（PPOP）

Many women undergo cesarean delivery without problems, however some experience significant pain after cesarean section. Pain is associated with negative ...

科研

医学

Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

持续时间较长的小鼠全身热疗协议

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease

Unbiased discovery proteomics strategies have the potential to identify large numbers of novel biomarkers that can improve diagnostic and prognostic testing in a clinical setting and may help guide therapeutic interventions. When large numbers of candidate proteins are identified, it may be difficult to validate candidate biomarkers in a timely and efficient fashion from patient plasma samples that are event-driven, of finite volume and irreplaceable, such as at the onset of acute graft-versus-host disease (GVHD), a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT).
Here we describe the process of performing commercially available ELISAs for six validated GVHD proteins: IL-2R&alpha;5, TNFR16, HGF7, IL-88, elafin2, and REG3&alpha;3 (also known as PAP1) in a sequential fashion to minimize freeze-thaw cycles, thawed plasma time and plasma usage. For this procedure we perform the ELISAs in sequential order as determined by sample dilution factor as established in our laboratory using manufacturer ELISA kits and protocols with minor adjustments to facilitate optimal sequential ELISA performance. The resulting plasma biomarker concentrations can then be compiled and analyzed for significant findings within a patient cohort. While these biomarkers are currently for research purposes only, their incorporation into clinical care is currently being investigated in clinical trials.
This technique can be applied to perform ELISAs for multiple proteins/cytokines of interest on the same sample(s) provided the samples do not need to be mixed with other reagents. If ELISA kits do not come with pre-coated plates, 96-well half-well plates or 384-well plates can be used to further minimize use of samples/reagents.

High throughput validation of multiple candidate biomarkers can be performed by sequential ELISA in order to minimize freeze/thaw cycles and use of precious plasma samples. Here, we demonstrate how to sequentially perform ELISAs for six different validated plasma biomarkers1-3 of graft-versus-host disease (GVHD)4 on the same plasma sample.

高通量顺序酶联免疫吸附试验的急性移植物抗宿主病的生物标志物的验证

Unbiased discovery proteomics strategies have  the potential to identify large numbers of novel biomarkers that can improve  diagnostic and prognostic ...

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

目标移植的肌肉祖细胞的分子成像

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the prognosis of idiopathic scoliosis in asymptomatic and affected children. The assay consists of the evaluation of the functional status of Gi and Gs proteins in peripheral blood mononuclear cells (PBMCs) by cellular dielectric spectroscopy (CDS), using an automated CDS-based instrument, and the classification of children into three functional groups (FG1, FG2, FG3) with respect to the profile of imbalance between the degree of response to Gi and Gs proteins stimulation. The classification is further confirmed by the differential effect of osteopontin (OPN) on response to Gi stimulation among groups and the severe progression of disease is referenced by FG2. Approximately, a volume of 10 ml of blood is required to extract PBMCs by Ficoll-gradient and cells are then stored in liquid nitrogen. The adequate number of PBMCs to perform the assay is obtained after two days of cell culture. Essentially, cells are first incubated with phytohemmaglutinin (PHA). After 24 hr incubation, medium is replaced by a PHA-free culture medium for an additional 24 hr prior to cell seeding and OPN treatment. Cells are then spectroscopically screened for their responses to somatostatin and isoproterenol, which respectively activate Gi and Gs proteins through their cognate receptors. Both somatostatin and isoproterenol are simultaneously injected with an integrated fluidics system and the cells&#39; responses are monitored for 15 min. The assay can be performed with fresh or frozen PBMCs and the procedure is completed within 4 days.

A structured protocol is presented for a cell-based assay as a functional test to predict the prognosis of idiopathic scoliosis using cellular dielectric spectroscopy (CDS). The assay can be performed with fresh or frozen peripheral blood mononuclear cells (PBMCs) and the procedure is completed within 4 days.

特发性脊柱侧弯的使用蜂窝介电谱的预后预测细胞为基础的检测协议

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

在体内MRI 对小鼠股骨骨愈合过程中骨折愈伤组织发育的评价

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

Fat-Water Phantoms for Magnetic Resonance Imaging Validation: A Flexible and Scalable Protocol

As new techniques are developed to image adipose tissue, methods to validate such protocols are becoming increasingly important. Phantoms, experimental replicas of a tissue or organ of interest, provide a low cost, flexible solution. However, without access to expensive and specialized equipment, constructing stable phantoms with high fat fractions (e.g., &#62;50% fat fraction levels such as those seen in brown adipose tissue) can be difficult due to the hydrophobic nature of lipids. This work presents a detailed, low cost protocol for creating 5x 100 mL&#160;phantoms with fat fractions of 0%, 25%, 50%, 75%, and 100% using basic lab supplies (hotplate, beakers, etc.) and easily accessible components (distilled water, agar, water-soluble surfactant, sodium benzoate, gadolinium-diethylenetriaminepentacetate (DTPA) contrast agent, peanut oil, and oil-soluble surfactant). The protocol was designed to be flexible; it can be used to create phantoms with different fat fractions and a wide range of volumes. Phantoms created with this technique were evaluated in the feasibility study that compared the fat fraction values from fat-water magnetic resonance imaging to the target values in the constructed phantoms. This study yielded a concordance correlation coefficient of 0.998 (95% confidence interval: 0.972-1.00). In summary, these studies demonstrate the utility of&#160;fat phantoms for validating adipose tissue imaging techniques across a range of clinically relevant tissues and organs.

The purpose of this work is to describe a protocol for creating a practical fat-water phantom that can be customized to produce phantoms with varying fat percentages and volumes.

磁共振成像验证中的脂肪水幽灵: 一种灵活且可伸缩的协议

As new techniques are developed to image adipose tissue, methods to validate such protocols are becoming increasingly important. Phantoms, experimental ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

一种从成年大鼠肾脏中分离完整型肾小球的有效筛分方法

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

Virtual Prism Adaptation Therapy: Protocol for Validation in Healthy Adults

Hemispatial neglect is a common impairment after stroke. It is associated with poor functional and social outcomes. Therefore, an adequate intervention is imperative for the successful management of hemispatial neglect. However, the clinical use of various interventions is limited in real clinical practice. Prism adaptation therapy is one of the most evidence-based rehabilitation modalities to treat hemispatial neglect. To overcome any possible shortcoming that may occur with prism therapy, we developed a new system using immersive virtual reality and depth-sensing camera to create a virtual prism adaptation therapy (VPAT). To validate the VPAT system, we designed an experimental protocol investigating the behavioral errors and changes in cortical activation via the VPAT system. Cortical activation was measured by functional near infrared spectroscopy (fNIRS). The experiment consisted of four phases. All four included clicking, pointing or rest applied to right-handed healthy people. Clicking versus pointing was used for investigating the cortical region related with the gross motor task, and pointing with VPAT versus pointing without VPAT was used for investigating the cortical region associated with visuospatial perception. The preliminary results from four healthy participants showed that pointing errors by the VPAT system was similar to the conventional prism adaptation therapy. Further analysis with more participants and fNIRS data, as well as a study in patients with stroke may be required.

This experimental protocol demonstrates the use of virtual prism adaptation therapy (VPAT) in healthy adults and the association between VPAT and functional near infrared spectroscopy to determine the effect of VPAT on cortical activation. Results suggest that VPAT may be feasible and could induce similar behavioral adaptation as conventional prism adaptation therapy.

虚拟棱镜适应疗法:健康成人验证协议

Hemispatial neglect is a common impairment after stroke. It is associated with poor functional and social outcomes. Therefore, an adequate intervention is ...

Network Pharmacology Prediction and Experimental Validation of Trichosanthes-Fritillaria thunbergii Action Mechanism Against Lung Adenocarcinoma

We aimed to study the mechanism of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma (LUAD) based on network pharmacology and experimental verification. The effective components and potential targets of Trichosanthis and Fritillaria thunbergii were collected by high-throughput experiment and reference-guided (HERB) database of traditional Chinese medicine and a similarity ensemble approach (SEA) database, and the LUAD-related targets were queried by the GeneCards and Online Mendelian Inheritance in Man (OMIM) databases. A drug-component-disease-target network was constructed by Cytoscape software. Protein-protein interaction (PPI) network, gene ontology (GO) function, and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses were conducted to obtain core targets and key pathways. An aqueous extract of Trichosanthes-Fritillaria thunbergii and A549 cells were used for the subsequent experimental validation. Through the HERB database and literature search, 31 effective compounds and 157 potential target genes of Trichosanthes-Fritillaria thunbergii were screened, of which 144 were regulatory targets of Trichosanthes-Fritillaria thunbergii in the treatment of lung adenocarcinoma. The GO functional enrichment analysis showed that the mechanism of action of Trichosanthes-Fritillaria thunbergii against lung adenocarcinoma is mainly protein phosphorylation. The KEGG pathway enrichment analysis suggested that the treatment of lung adenocarcinoma by Trichosanthes-Fritillaria thunbergii mainly involves the PI3K/AKT signaling pathway. The experimental validation showed that an aqueous extract of Trichosanthes-Fritillaria thunbergii could inhibit the proliferation of A549 cells and the phosphorylation of AKT. Through network pharmacology and experimental validation, it was verified that the PI3K/AKT signaling pathway plays a vital role in the action of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma.

Network Pharmacology Prediction and Experimental Validation of Trichosanthes-Fritillaria thunbergii Action Mechanism Against Lung Adenocarcinoma

This study reveals the mechanism of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma based on network pharmacology and experimental verification. The study also demonstrates that the PI3K/AKT signaling pathway plays a vital role in the action of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma.