这项工作由 NIH 主任的新创新奖 (奖号) 资助。DP2HD091793)。Q.D.M. 得到 NSF 研究生研究奖学金计划的支持 (批准号:DGE-1650044)。H 获得美国国立卫生研究院 GT BioMAT 训练补助金的支持, 5T32EB006343 和格鲁吉亚技术总裁的奖学金。G.A.K. 在 "宝来欢迎基金" 的科学界面上担任职业奖。内容完全是作者的责任, 不一定代表国家卫生研究院的官方意见。

在研究机构的机构动物护理和使用委员会 (IACUC) 的机构批准是进行以下动物实验的必要。此外, 标准的动物保育设施 (例如,住房室、无菌的动物罩、麻醉的 isofluorane 室和为道德端点 euthanization 的 CO2室) 是必要的, 以便适当地执行这些实验。对这些设施的特殊培训和协助可以由一个机构的生理学研究实验室提供。所有的动物工作都是通过 IACUC 在佐治亚理工大学 (礼宾: A14100) 批准的。
1. 氧化铁纳米颗粒 (IONP) 合成
注: 安全: 整个氧化铁纳米颗粒的合成应使用个人防护设备和在化学油烟机下面。
<ol><li>在一个15毫升圆锥管, 冷却1毫升30% 氢氧化铵在一个冰桶30分钟。</li>
	<li>用去离子水洗250毫升锥形瓶, 加入10毫升双蒸馏水 (ddH2O)。将瓶子的外面浸入一个放在搅拌/热板上的冰浴中。使用塑料吸管将 N2气体流动到瓶中的 ddH2O。气泡15分钟, N2到 deoxygenate。</li>
	<li>称224µmol (4.5 克) 葡聚糖 (分子量 (兆瓦) = 20 kDa) 为50毫升圆锥管。添加 H2O, 使混合物的最终体积, 包括葡聚糖, 是20毫升。漩涡大力溶解葡聚糖。</li>
	<li>将290µmol (78.5 克) 的氯化铁 (III) 的重量加到六水合物中, 加入葡聚糖溶液和涡旋溶解。</li>
	<li>通过0.2 µm 筛选器筛选结果解决方案。</li>
	<li>将1毫升冷却氢氧化铵转化为9毫升的脱氧水。返回到4°c。</li>
	<li>重367µmol (73.0 克) 的氯化铁 (II), 溶解在1毫升无氧 ddH2O, 并过滤通过0.2 µm 过滤器。</li>
	<li>将葡聚糖铁 (III) 溶液转化成250毫升锥形瓶和 deoxygenate, 在冰上15分钟。与 1600 rpm 旋转的磁力搅拌杆混合。</li>
	<li>要创造一个均匀的氮气气氛, 用橡胶隔膜盖上烧瓶。用18口径的针刺穿隔膜, 将2到烧瓶中流动。插入一个单独的18测量针作为流量出口。</li>
	<li>添加467µL 的铁 (II) 溶液的葡聚糖铁 (III) 溶液与1毫升注射器, 搅拌与磁搅拌杆在 1600 rpm。铁 (II) 与铁 (III) 的比值导致了平衡反应, 将产生磁铁矿, 或 Fe3O4。</li>
	<li>加入冷稀释氢氧化铵滴入铁 (II)-铁 (III)-葡聚糖溶液, 以启动成核过程21。确保每个液滴在掉入另一粒之前混合良好。在加入1-2 毫升氢氧化铵后完成反应。</li>
	<li>停止流动的 N2和消除橡胶隔膜。除去冰浴, 用温水浴代替, 同时继续搅拌溶液。确保溶液的温度达到75摄氏度, 孵育75分钟。</li>
	<li>从搅拌/热板中取出烧瓶, 然后通过0.2 µm 0.1 µm 过滤器将溶液加倍过滤, 去除粗颗粒。</li>
	<li>缓冲液将微粒转化成 ddH2O, 使用 100 kDa 分子量切断 (m.w.c.) 选矿厂, 从溶液中去除多余的葡聚糖, 这应该是非常粘性的。如果在 2-3 旋转之后, 即使流通过仍然是暗的, 则替换筛选器, 这表明筛选器可能已损坏。<ol>
<li>要使用自旋过滤器进行缓冲交换, 离心机在4°c 4800 X g 处15分钟, 丢弃流程, 并向 IONP 解决方案中添加新缓冲区。重复3-5 次。</li>
	</ol>
</li>
	<li>用分光光度计/平板阅读器测定纳米粒子的浓度。吸光度测量在400毫微米和使用 IONP 的摩尔吸收率在这个波长 (ε = 2.07 x 106 cm-1 M-1) 确定 IO 微粒的浓度。</li>
	<li>并用重悬 IONPs 到10毫克/毫升在 ddH2O (通常总体积是 ~ 3 毫升), 并确保使用聚丙烯管这一步骤的化学相容性。添加1.6 卷5米氢氧化钠, 然后添加0.65 容量的环氧氯丙烷。严格混合在室温下的平板振动筛12小时, 以交联葡聚糖。</li>
	<li>使用一个20毫升注射器与一个18口径的针将 IONP 溶液转移到 50 kDa m.w.c. 透析膜。将透析膜放入4升 ddH2o, 并更换 H2o 数次 (每 2-3 小时)。孵化过夜。</li>
	<li>测量浓度, 使 IONPs 5 毫克/毫升 (见步骤 1.15)。加入氢氧化铵达到 20% (v/v), 在室温下摇动 > 12 小时, aminate IONPs 表面。使用 30 kDa m.w.c. 过滤器进行缓冲交换 (见 1.14)。</li>
	<li>用快速蛋白液相色谱法 (FPLC) 在最终纯化前, 将音量调低至 2.5-3.5 毫升; 见凝胶过滤柱材料表)。</li>
	<li>使用动态光散射 (dl) 确定 IONPs 的水动力半径 (预期范围 10-100 nm, 平均大小 40-50 nm)。这样做, 通过 aliquoting 1 毫升100X 稀释 IONP 样品在试管, 在机器的地方, 并使用制造商的软件采取的测量。 注: IONPs 的总人口将在10和100毫微米之间由 dl 测量, 但多数人口是在平均直径附近, 范围从 40-50 毫微米。如果你想要限制大小范围, 你可以进一步使用大小排除色谱分离分数以不同的直径。IONPs 应存放在4摄氏度。</li>
</ol>2. 肽的设计, 共轭到 IONP, 并在体外验证
<ol><li>合成一种多肽基质 (例如,由核心设施或商业) 的目标蛋白酶与 n-终端荧光记者, 如荧光素异硫氰酸酯 (FITC) 和 C 末端半胱氨酸残渣, 以允许硫醇介导的耦合。 注: 在本研究的情况下 MMP9, 使用的肽是 FITC-PLGVRGK C, 与 k猫/km ~ 2.0 x 105 m-1s-1。</li>
	<li>整除0.5 毫克的 IONP (1 毫克/毫升) 和缓冲交换成耦合缓冲 (50 毫米硼酸钠与1毫米 EDTA, pH = 8.5) 使用 10 kDa m.w.c. o. 旋转过滤器 (见步骤 1.14)。</li>
	<li>溶解琥珀酰亚胺碘乙酸 (新加坡) 在甲基酰胺 (DMF) 达到浓度为30毫克/毫升, 并增加新加坡航空 IONP 摩尔比500新加坡航空: IONP。 注意: 新加坡航空是一种 heterobifunctional 交叉链接器, 它介导胺类在 IONPs 上的偶联与肽基板上的巯基团。</li>
	<li>在黑暗的室温下孵育 1-2 小时。如果一夜之间离开, 孵育4摄氏度。</li>
	<li>使用 10 kDa m.w.c. 旋转过滤器将缓冲区交换到耦合缓冲区中以删除反应 (参见步骤 1.14)。</li>
	<li>将最终产品解决方案带到1毫克/毫升 (0.5 毫升溶液)。将感兴趣的肽与 90:1 (肽: IONP) 的摩尔比与 20 kDa 硫醇终止的聚乙二醇 (peg) 混合, 摩尔比为 20:1 (peg: IONP)。将这种肽 PEG 溶液与 IONP 混合。</li>
	<li>在室温下以最高速度在平板振动筛上孵化, 以防止荧光分子漂白。</li>
	<li>添加 l-半胱氨酸的摩尔比为 500:1 (C: IONP), 以中和任何反应的新加坡航空分子。在室温下以最高速度在25摄氏度上孵育1小时。</li>
	<li>通过 FPLC 净化 (见 1.1.19)。使用分光光度计测量样品溶液的吸光度, 并计算肽: IONP 比根据以下。提纯后, 在 PBS 的4摄氏度贮存1毫克/毫升的产品。<ol>
<li>使用分光光度计测量样品溶液在 400 nm (sample,400) 的吸光度, 以及荧光所用的最大吸光度波长, 为 FITC (sample,488) 的 488 nm。测量同一两个波长 (400 nm 和 488 nm) 的股票胺 IONP 溶液的吸光度, 分别表示为NP,400和NP,488。</li>
		<li>使用等式1和2计算规范化的400和488值。 <img alt="Equation 1" src="/files/ftp_upload/57937/57937eq1.jpg">等式1 <img alt="Equation 2" src="/files/ftp_upload/57937/57937eq2.jpg">等式2</li>
		<li>400和488表示样本的归一化值。使用这些值计算肽的比率: IONP 与等式3。 <img alt="Equation 3" src="/files/ftp_upload/57937/57937eq3.jpg">等式3</li>
		<li>IONP <img alt="Equation 4" src="/files/ftp_upload/57937/57937eq4.jpg">和<img alt="Equation 5" src="/files/ftp_upload/57937/57937eq5.jpg">荧光的摩尔 absorptivities 分别在哪里和哪里。为这些微粒<img alt="Equation 4" src="/files/ftp_upload/57937/57937eq4.jpg"> = 2.06 x 106 M-1cm-1和为 FITC <img alt="Equation 5" src="/files/ftp_upload/57937/57937eq5.jpg"> = 7.2万 M-1cm-1, 因此价值<img alt="Equation 6" src="/files/ftp_upload/57937/57937eq6.jpg">应该是28.75。典型肽: IONP 比应在20:1 到50:1 的范围内。</li>
	</ol>
</li>
	<li>通过进行体外裂解试验验证探针的功能。<ol>
<li>与 PBS (1% BSA), 使一个18µL 纳米粒子溶液与 200 nM 浓度的肽。混合2µL 的蛋白酶的兴趣 (MMP9; 0.1-1 毫克/毫升)。</li>
		<li>在37摄氏度的平板阅读器中孵育1小时, 进行荧光测量 (使用适当的激发和发射波长, 分别为 FITC 485 nm 和 528 nm), 每 1-2 分钟来监测裂解。</li>
	</ol>
</li>
</ol>3. 癌症的纳米传感器和尿检管理
注: 有关示例模型的详细信息, 请参阅我们以前的报告13。
<ol><li>建立一个新陈代谢的笼子, 通过确保一个圆柱套到96井板顶部的尿收集。在收集尿液时, 将一只老鼠放在袖子内, 用培养皿盖盖住, 以防动物逃跑。</li>
	<li>在无菌盐水中, 用肽在3000毫克/千克 (~ 50 µmol) 的浓度下, 准备注射液 (200 µL 最大容积)。</li>
	<li>在雌性, 8 周大的裸鼠, 携带异种 LS174T 大肠肿瘤的负担约100-300 毫米3, 这应该发生在大约10天, 通过尾静脉注射管理 nanosensor 溶液。注射后立即将老鼠放入新陈代谢的笼子里, 注意每只老鼠注射的时间。</li>
	<li>注射后 60-90 分钟, 从96井板中取出圆柱套筒。抑制小鼠, 对膀胱施加轻微的压力, 诱导小鼠将剩余的尿液放到盘子里。收集所有尿液 (200-500 µL)。</li>
	<li>通过免疫沉淀分析尿样, 从尿液中纯化 FITC, 提高灵敏度。使用磁性珠子加上抗 FITC 抗体。<ol>
<li>首先, 用涂层缓冲器清洗25毫克的磁珠3次, 并将最终体积带到225µL。 注意: 要使涂层缓冲 (0.1 米硼酸钠) 是很重要的, ph 值 9.5), 阻断缓冲 (pbs, 0.5% BSA, 0.05% Tween-20, pH 7.4) 和洗涤缓冲 (PBS, 0.1% BSA, 0.05% Tween-20, pH 7.4) 每次新鲜, 每一个大约20毫升是必要的。</li>
		<li>添加200µL 抗 FITC (5 毫克/毫升) 和200µL 硫酸铵 (3 米)。在旋转的 37 C 上孵育16-24 小时。</li>
		<li>将解决方案添加到磁选机上, 取出上清, 替换<img alt="Equation 7" src="/files/ftp_upload/57937/57937eq7.jpg">为625Λ阻塞缓冲器, 并在一夜之间孵化 37 C。然后用洗涤缓冲器清洗3次, 然后在1.25 毫升的洗涤缓冲器中储存。</li>
		<li>孵化2µL 尿与5µL 磁性珠 (20 毫克/毫升) 和带来到总容量到50µL 与 PBS (0.01% 吐温 20)。孵育60分钟。</li>
		<li>每洗一次后, 用磁选机将50µL PBS (0.01% 吐温 20) 洗两次, 收集磁性珠子。</li>
		<li>洗脱两次, 32.5 µL 5% 冰乙酸。中和池洗脱 (70 µL) 与35µL 2 米三, 以达到最终的 pH 值为7。</li>
		<li>阅读在一个板块阅读器 (见材料表) 在适当的激发和发射波长, 以量化尿荧光。计算荧光记者对已知的自由荧光浓度阶梯的浓度。</li>
	</ol>
</li>
</ol>

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S., 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=Public+health+impact+of+achieving+80%+colorectal+cancer+screening+rates+in+the+United+States+by+2018.">Public health impact of achieving 80% colorectal cancer screening rates in the United States by 2018.</a> Cancer. 121 (13), 2281-2285 (2015).</li><li>Mehner, 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=Tumor+cell-produced+matrix+metalloproteinase+9+(MMP-9)+drives+malignant+progression+and+metastasis+of+basal-like+triple+negative+breast+cancer.">Tumor cell-produced matrix metalloproteinase 9 (MMP-9) drives malignant progression and metastasis of basal-like triple negative breast cancer.</a> Oncotarget. 5 (9), 2736-2749 (2014).</li><li>Bremer, C., Tung, C. H., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+molecular+target+assessment+of+matrix+metalloproteinase+inhibition.">In vivo molecular target assessment of matrix metalloproteinase inhibition.</a> Nature Medicine. 7 (6), 743-748 (2001).</li><li>Warren, A. 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=Disease+detection+by+ultrasensitive+quantification+of+microdosed+synthetic+urinary+biomarkers.">Disease detection by ultrasensitive quantification of microdosed synthetic urinary biomarkers.</a> Journal of the American Chemical Society. 136 (39), 13709-13714 (2014).</li><li>Warren, A. D., Kwong, G. A., Wood, D. K., Lin, K. Y., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Point-of-care+diagnostics+for+noncommunicable+diseases+using+synthetic+urinary+biomarkers+and+paper+microfluidics.">Point-of-care diagnostics for noncommunicable diseases using synthetic urinary biomarkers and paper microfluidics.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (10), 3671-3676 (2014).</li><li>Kwong, G. 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=Mathematical+framework+for+activity-based+cancer+biomarkers.">Mathematical framework for activity-based cancer biomarkers.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (41), 12627-12632 (2015).</li><li>Dudani, J. S., Jain, P. K., Kwong, G. A., Stevens, K. R., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivated+spatiotemporally-responsive+nanosensors+of+in+vivo+protease+activity.">Photoactivated spatiotemporally-responsive nanosensors of in vivo protease activity.</a> ACS Nano. 9 (12), 11708-11717 (2015).</li><li>Palmacci, S., Josephson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+polysaccharide+covered+superparamagnetic+oxide+colloids.">Synthesis of polysaccharide covered superparamagnetic oxide colloids.</a> US patent. , (1991).</li><li>Choi, H. S., 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=Renal+clearance+of+nanoparticles.">Renal clearance of nanoparticles.</a> Nature Biotechnology. 25 (10), 1165-1170 (2007).</li><li>Harris, T. J., von Maltzahn, G., Derfus, A. M., Ruoslahti, E., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolytic+actuation+of+nanoparticle+self-assembly.">Proteolytic actuation of nanoparticle self-assembly.</a> Angewandte Chemie (International edition in English). 45 (19), 3161-3165 (2006).</li><li>Kwon, E. J., Dudani, J. S., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasensitive+tumour-penetrating+nanosensors+of+protease+activity.">Ultrasensitive tumour-penetrating nanosensors of protease activity.</a> Nature Biomedical Engineering. 1, (2017).</li><li>Villanueva, J., Nazarian, A., Lawlor, K., Tempst, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+Peptidase+activities+in+complex+proteomes+by+MALDI-TOF+mass+spectrometry.">Monitoring Peptidase activities in complex proteomes by MALDI-TOF mass spectrometry.</a> Nature Protocols. 4 (8), 1167-1183 (2009).</li><li>Villanueva, J., 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=Differential+exoprotease+activities+confer+tumor-specific+serum+peptidome+patterns.">Differential exoprotease activities confer tumor-specific serum peptidome patterns.</a> The Journal of Clinical Investigation. 116 (1), 271-284 (2006).</li></ol>

邝其志是联合创始人, 并担任 Glympse 的顾问, 正在开发与本文所述研究相关的产品。这项研究可能影响他的个人财务状况。格鲁吉亚技术委员会根据其利益冲突政策审查并批准了这一安排的条款。

该方法描述了由共轭纳米粒核的蛋白酶基质组成的活性纳米传感器的发展。蛋白水解的事件被称为 "药代动力学开关", 因为裂解肽产品小于肾脏大小的过滤限制5毫微米23和过滤成尿产生无创信号。因此, 重要的是使用纳米粒子或载体的水动力半径大于 5 nm, 因为任何更小的将被肾脏迅速清除和混淆尿信号。我们使用 pegylated IONPs, 因为他们是 FDA 批准, 耐受性好, 并有循环半衰期 3-5 小...

蛋白酶是多功能酶, 专门研究肽键的水解, 对许多生物过程有显著的控制作用, 包括稳态、allostasis 和疾病1。蛋白酶活性的改变状态与各种疾病相关, 包括癌症和心血管疾病, 使蛋白酶有吸引力的候选者发展成为临床生物标志物2,3。此外, 蛋白酶活性在功能上与不同的梗死再发, 患者的结局和疾病的预后4。广泛地, 生物传感器被开发用于检测各种生物学现象和疾病, 如癌症, 神经退行性疾病, 电子转移过程5,6,7,8,9. 更具体地说, 以基板为基础的蛋白酶传感器用于检测蛋白酶活性, 包括荧光探针, 用于诊断成像10和 isotopically 标记的肽基片体外质谱检测11。此外, 还开发了基于活动的探针, 其中含有与基片类似的区域, 可以约束或修改目标蛋白酶12。采用这种方法, 当活性部位改变时, 目标蛋白酶不可逆转地受到抑制, 分析需要组织的收获, 这限制了体内的应用。然而,在体内对蛋白酶活性的感知是很重要的, 因为对蛋白酶活性的调节严重依赖于其他生物活动的上下文, 如内源性抑制剂的存在。
这项工作的目的是描述以活动为基础的纳米传感器的配方, 通过产生一个可测量的尿液来检测体内蛋白酶的活性。这个平台被用来作为一种无创诊断, 以鉴别复杂的疾病, 如癌症, 使用失调蛋白酶活动作为功能生物标志物。我们的 nanosensor 平台由氧化铁纳米粒子 (IONP) 与蛋白酶基体结合而成。这些基底被一个荧光记者终止, 当蛋白酶切割基体时释放。这些 IONPs在体内循环, 定位到疾病部位, 并暴露基质到活性疾病相关的蛋白酶。乳沟后, 荧光记者被释放, 由于其体积小, 被过滤成尿液, 而 uncleaved 基质上的 IONP 留在体内。因此,体内蛋白酶活性的增加会导致尿液中的记者浓度增高 (图 1)。由于我们的平台是尿液测试, 不需要成像平台和诊断信号丰富的尿液。
这个平台可以被设计来检测各种疾病, 包括癌症, 纤维化, 血栓形成13,14。在这里, 我们描述了纳米传感器的设计, 以检测基质 metallopeptidase 9 (MMP9) 活性作为结直肠癌的生物标志物的海拔。大肠癌是美国癌症死亡的第二大原因, 估计仅在2014年就有136800例新病例和50300人死亡.结直肠肿瘤细胞产生 MMP9, 这已经显示出推动恶性进展, 基质退化, 以及转移16。此外, 我们确定了一个合适的肽基质 (PLGVRGK) 为 MMP9 从文献17。该平台可用于早期癌症检测和低成本的护理点诊断13,14,18,19,20,21。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57937/57937fig1.jpg"> 图 1:体内Nanosensor 活动示意图.纳米传感器循环通过身体和本地化到疾病的地点。然后, IONPs 提出了与疾病相关的蛋白酶劈裂肽基。切割碎片的大小允许肾脏清除, 导致他们在尿中定位。在动物小便后, 这些肽片段可以通过他们的记者分子来分析。<a href="https://www.jove.com/files/ftp_upload/57937/57937fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

<table border="0" cellpadding="0" cellspacing="0" width="944">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">0.2 &#181;m syringe filters</td>
			<td style="width:171px;">VWR</td>
			<td style="width:233px;">4652</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">18G needle</td>
			<td style="width:171px;">VWR</td>
			<td style="width:233px;">89134-024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">15 mL conicals</td>
			<td style="width:171px;">VWR</td>
			<td style="width:233px;">89039-670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">250 mL Erlenmeyer flask</td>
			<td style="width:171px;">VWR</td>
			<td style="width:233px;">89000-362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Stir bar</td>
			<td style="width:171px;">VWR</td>
			<td style="width:233px;">58949-006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Hot Plate/Magnetic Stirrer</td>
			<td style="width:171px;">VWR</td>
			<td style="width:233px;">97042-634</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Glacial acetic acid</td>
			<td style="width:171px;">VWR</td>
			<td style="width:233px;">97064-482</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Albumin from Bovine Serum (BSA)</td>
			<td style="width:171px;">Thermo Fisher</td>
			<td style="width:233px;">A13100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Iron (III) chloride hexahydrate</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:233px;">236489</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Iron (II) chloride tetrahydrate</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:233px;">44939</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Epichlorohydrin</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:233px;">45340-500ML-F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">DMF</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:233px;">D4551</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Ammonium Hydroxide</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:233px;">320145-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sodium Hydroxide pellets</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:233px;">221465-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">EDTA</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:233px;">E9884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sodium Borate</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:233px;">B9876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">L-Cysteine</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:233px;">168149-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Tris-HCl</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:233px;">T5941</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Tris base</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:233px;">T6066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">PBS tablets</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:233px;">P4417</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Dextran</td>
			<td style="width:171px;">Pharmacosmos</td>
			<td>5510 0020 9006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Amicon 15 mL 10k filters, 24 pk</td>
			<td style="width:171px;">Millipore</td>
			<td style="width:233px;">UFC901024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Amicon 15 mL 30k filters, 24 pk</td>
			<td style="width:171px;">Millipore</td>
			<td style="width:233px;">UFC903024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Amicon 15 mL 100k filters, 24 pk</td>
			<td style="width:171px;">Millipore</td>
			<td style="width:233px;">UFC910024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Zetasizer Nano ZS</td>
			<td style="width:171px;">Malvern Panalytical</td>
			<td style="width:233px;">NanoZS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Slide-A-Lyzer Dialysis Cassette</td>
			<td style="width:171px;">LifeTech</td>
			<td style="width:233px;">66130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Dynabeads MyOne Tosylactivated</td>
			<td style="width:171px;">LifeTech</td>
			<td style="width:233px;">65501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">SIA</td>
			<td style="width:171px;">Life Tech</td>
			<td style="width:233px;">22349</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">PEG 20k</td>
			<td style="width:171px;">Laysan Bio</td>
			<td style="width:233px;">MPEG-SH-20K-1g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Fluorescein Antibody [2A3]</td>
			<td style="width:171px;">GeneTex</td>
			<td style="width:233px;">GTX10257</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Hiload 16/600 superdex 200</td>
			<td style="width:171px;">GE Healthcare</td>
			<td style="width:233px;">45-002-490</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Plate Reader</td>
			<td style="width:171px;">Fisher</td>
			<td style="width:233px;">BTCYT5M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">BD Insulin Syringes</td>
			<td style="width:171px;">Fisher</td>
			<td style="width:233px;">NC0872854</td>
			<td></td>
		</tr>
	</tbody>
</table>

nanosensors to detect protease activity in vivo for noninvasive diagnostics

蛋白酶是多功能酶, 专门研究肽键的水解和控制广泛的生物过程, 包括稳态和 allostasis。此外, 失调蛋白酶活性驱动发病机制, 是疾病的功能性生物标志物, 如癌症;因此, 检测体内蛋白酶活性的能力可以为生物医学诊断提供临床相关信息。该协议的目的是创造纳米传感器, 通过产生一个可量化的尿液信号, 探索体内蛋白酶的活性。这些蛋白酶纳米传感器由两个成分组成: 纳米粒子和基体。纳米微粒的作用是增加循环半衰期和基质输送到目标疾病地点。基板是一个短肽序列 (6-8 AA), 这是专门针对目标蛋白酶或一组蛋白酶。基体与纳米微粒的表面共轭, 并由记者 (如荧光标记) 终止, 用于检测。当失调蛋白酶切割肽基板时, 记者被过滤成尿液, 作为蛋白酶活性的生物标志物。本文描述了基质金属蛋白酶 9 (MMP9) nanosensor 的构建, 它与肿瘤的进展和转移有关, 用于在小鼠模型中检测大肠癌。

IONPs 的人口的多数是在平均直径附近, 范围从 40-50 毫微米。在聚乙二醇化之后, 这个大小范围有循环半衰期大约6小时13 在体内(参见图 2a)。如果你想选择一个特定的大小范围, 你可以使用大小排除色谱分离 IONP 分数与不同的直径。纳米粒子的透射电镜将显示为单独的球形氧化铁纳米粒子交联在一起的外层葡聚糖。...

Watch this Scientific Journal Video about Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics at JoVE.com

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

蛋白酶是严格调控的酶参与基本的生物学过程, 失调蛋白酶活动推动复杂疾病, 如癌症的进展。该方法的目的是创造纳米传感器, 以测量体内蛋白酶的活动, 产生一个可从宿主尿和歧视性疾病检测到的裂解信号。

纳米传感器检测体内蛋白酶活性的无创诊断

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

nanosensors-to-detect-protease-activity-vivo-for-noninvasive

Research

JoVE Journal

Bioengineering

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

16.2K 次观看.  Georgia Tech College of Engineering and Emory School of Medicine. 蛋白酶是严格调控的酶参与基本的生物学过程, 失调蛋白酶活动推动复杂疾病, 如癌症的进展。该方法的目的是创造纳米传感器, 以测量体内蛋白酶的活动, 产生一个可从宿主尿和歧视性疾病检测到的裂解信号。

视频: Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

科研

生物工程

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

病毒粒子为<em在体内</em&gt;肿瘤显像

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Quantitative FRET (F&#246;rster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein activity and have diverse roles in many biological processes. For example, SUMO covalently modifies a large number or proteins with important roles in many cellular processes, including cell-cycle regulation, cell survival and death, DNA damage response, and stress response 1-5. SENP, as SUMO-specific protease, functions as an endopeptidase in the maturation of SUMO precursors or as an isopeptidase to remove SUMO from its target proteins and refresh the SUMOylation cycle 1,3,6,7.
The catalytic efficiency or specificity of an enzyme is best characterized by the ratio of the kinetic constants, kcat/KM. In several studies, the kinetic parameters of SUMO-SENP pairs have been determined by various methods, including polyacrylamide gel-based western-blot, radioactive-labeled substrate, fluorescent compound or protein labeled substrate 8-13. However, the polyacrylamide-gel-based techniques, which used the "native" proteins but are laborious and technically demanding, that do not readily lend themselves to detailed quantitative analysis. The obtained kcat/KM from studies using tetrapeptides or proteins with an ACC (7-amino-4-carbamoylmetylcoumarin) or AMC (7-amino-4-methylcoumarin) fluorophore were either up to two orders of magnitude lower than the natural substrates or cannot clearly differentiate the iso- and endopeptidase activities of SENPs.
Recently, FRET-based protease assays were used to study the deubiquitinating enzymes (DUBs) or SENPs with the FRET pair of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) 9,10,14,15. The ratio of acceptor emission to donor emission was used as the quantitative parameter for FRET signal monitor for protease activity determination. However, this method ignored signal cross-contaminations at the acceptor and donor emission wavelengths by acceptor and donor self-fluorescence and thus was not accurate.
We developed a novel highly sensitive and quantitative FRET-based protease assay for determining the kinetic parameters of pre-SUMO1 maturation by SENP1. An engineered FRET pair CyPet and YPet with significantly improved FRET efficiency and fluorescence quantum yield, were used to generate the CyPet-(pre-SUMO1)-YPet substrate 16. We differentiated and quantified absolute fluorescence signals contributed by the donor and acceptor and FRET at the acceptor and emission wavelengths, respectively. The value of kcat/KM was obtained as (3.2 &plusmn; 0.55) x107 M-1s-1 of SENP1 toward pre-SUMO1, which is in agreement with general enzymatic kinetic parameters. Therefore, this methodology is valid and can be used as a general approach to characterize other proteases as well.

Quantitative FRET F&#246;rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

A novel method involving quantitative analysis of FRET (F&ouml;rster Resonance Energy Transfer) signals is described for studying enzyme kinetics. KM and kcat were obtained for the hydrolysis of the catalytic domain of SENP1 (SUMO/Sentrin specific protease 1) to pre-SUMO1 (Small Ubiquitin-like MOdifier). The general principles of this quantitative-FRET-based protease kinetic study can be applied to other proteases.

定量荧光共振能量转移（福斯特共振能量转移）SENP1蛋白酶的动力学测定分析

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein ...

FIBS-enabled Noninvasive Metabolic Profiling

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be validated for predictive purposes. Ideally such systems would be low volume, which precludes sampling and destructive analyses when time course data are to be obtained. What is needed is an in situ monitoring tool which can report the necessary information in real-time and noninvasively. An interesting option is the use of fluorescent, protein-based in vivo biological sensors as reporters of intracellular concentrations. One particular class of in vivo biosensors that has found applications in metabolite quantification is based on F&#246;rster Resonance Energy Transfer (FRET) between two fluorescent proteins connected by a ligand binding domain. FRET integrated biological sensors (FIBS) are constitutively produced within the cell line, they have fast response times and their spectral characteristics change based on the concentration of metabolite within the cell. In this paper, the method for constructing Chinese hamster ovary (CHO) cell lines that constitutively express a FIBS for glucose and glutamine and calibrating the FIBS in vivo in batch cell culture in order to enable future quantification of intracellular metabolite concentration is described. Data from fed-batch CHO cell cultures demonstrates that the FIBS was able in each case to detect the resulting change in the intracellular concentration. Using the fluorescent signal from the FIBS and the previously constructed calibration curve, the intracellular concentration was accurately determined as confirmed by an independent enzymatic assay.

A description of how to calibrate F&#246;rster Resonance Energy Transfer integrated biological sensors (FIBS) for in situ metabolic profiling is presented.&#160;The FIBS can be used to measure intracellular levels of metabolites noninvasively aiding in the development of metabolic models and high throughput screening of bioprocess conditions.

FIBS功能的无创性代谢轮廓

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

一个完整的皮肤缺损模型，以评估生物材料的血管化<em&gt;体内</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Magnetic Levitation Coupled with Portable Imaging and Analysis for Disease Diagnostics

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for specialized equipment and trained personnel require that many diagnostic tests be performed at remote, centralized clinical laboratories. Magnetic levitation is a simple yet powerful technique and can be applied to levitate cells, which are suspended in a paramagnetic solution and placed in a magnetic field, at a position determined by equilibrium between a magnetic force and a buoyancy force. Here, we present a versatile platform technology designed for point-of-care diagnostics which uses magnetic levitation coupled to microscopic imaging and automated analysis to determine the density distribution of a patient's cells as a useful diagnostic indicator. We present two platforms operating on this principle: (i) a smartphone-compatible version of the technology, where the built-in smartphone camera is used to image cells in the magnetic field and a smartphone application processes the images and to measures the density distribution of the cells and (ii) a self-contained version where a camera board is used to capture images and an embedded processing unit with attached thin-film-transistor (TFT) screen measures and displays the results. Demonstrated applications include: (i) measuring the altered distribution of a cell population with a disease phenotype compared to a healthy phenotype, which is applied to sickle cell disease diagnosis, and (ii) separation of different cell types based on their characteristic densities, which is applied to separate white blood cells from red blood cells for white blood cell cytometry. These applications, as well as future extensions of the essential density-based measurements enabled by this portable, user-friendly platform technology, will significantly enhance disease diagnostic capabilities at the point of care.

We present a magnetic levitation technique coupled with automated imaging and analysis in both a smartphone-compatible device and a device with embedded imaging and processing. This is applied to measure the density distribution of cells with two demonstrated biomedical applications: sickle cell disease diagnosis and separating white and red blood cells.

磁悬浮加上便携式成像与分析诊断疾病

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for ...

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow.

Calorespirometry: 一种功能强大、无创的研究细胞能量代谢的方法

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

用微计算机断层扫描法对无节段3D 组织学进行固定染色、全尺寸、毫米度试样的刚性嵌入

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Using Nanoplasmon-Enhanced Scattering and Low-Magnification Microscope Imaging to Quantify Tumor-Derived Exosomes

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes have the potential to serve as biomarkers for disease diagnosis and to monitor disease progression and treatment response. However, most exosome analysis procedures require exosome isolation and purification steps, which are usually time-consuming and labor-intensive, and thus of limited utility in clinical settings. This report describes a rapid procedure to analyze specific biomarkers on the outer membrane of exosomes without requiring separate isolation and purification steps. In this method, exosomes are captured on the surface of a slide by exosome-specific antibodies and then hybridized with nanoparticle-conjugated antibody probes specific to a disease. After hybridization, the abundance of the target exosome population is determined by analyzing low-magnification dark-field microscope (LMDFM) images of the bound nanoparticles. This approach can be easily adopted for research and clinical use to analyze membrane-associated exosome biomarkers linked to disease.

Clinical translation of exosome-derived biomarkers for diseased and malignant cells is hindered by the lack of rapid and accurate quantification methods. This report describes the use of low-magnification dark-field microscope images to quantify specific exosome subtypes in small volume serum or plasma samples.

纳米等离子体增强散射和低放大显微镜成像对肿瘤衍生外质体的定量

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes ...

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

研究脑癌和神经退行性疾病的人类神经器官

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...

纳米传感器检测体内蛋白酶活性的无创诊断

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