Name: bio-energetics investigation of candida albicans using real-time extracellular flux analysis
Uploaded: 2019-03-19T13:30:00
Description: Watch this Scientific Journal Video about Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis at JoVE.com

NC 实验室的研究得到了国家卫生研究院 R01AI24499 赠款和新泽西卫生基金会赠款的支持, #PC40-18日。

注: 分析的详细逐步协议如下所述, 原理图协议如图 1所示。
1.白色念珠菌菌株和生长条件
<ol><li>在30°c的孵化器振动台中, 在液体酵母提取物-苯并酮-右旋 (ypd) 中培养白色念珠菌菌株。 注: 保持念珠菌菌株作为冷冻库存和生长在 ypd 琼脂 (1% 酵母提取物, 2% 肽, 2% 葡萄糖, 和2% 琼脂)。</li>
</ol>2. 试剂的制备
<o...

<ol>
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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=Hidden+killers:+human+fungal+infections.">Hidden killers: human fungal infections.</a> Science Translational Medicine. 4 (165), (2012).</li><li>Wisplinghoff, H., 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=Nosocomial+bloodstream+infections+in+US+hospitals:+analysis+of+24,179+cases+from+a+prospective+nationwide+surveillance+study.">Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study.</a> Clinical Infectious Diseases. 39 (3), 309-317 (2004).</li><li>Ascioglu, 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=Defining+opportunistic+invasive+fungal+infections+in+immunocompromised+patients+with+cancer+and+hematopoietic+stem+cell+transplants:+an+international+consensus.">Defining opportunistic invasive fungal infections in immunocompromised patients with cancer and hematopoietic stem cell transplants: an international consensus.</a> Clinical Infectious Diseases. 34 (1), 7-14 (2002).</li><li>Stover, B. 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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=Niche-specific+regulation+of+central+metabolic+pathways+in+a+fungal+pathogen.">Niche-specific regulation of central metabolic pathways in a fungal pathogen.</a> Cellular Microbiology. 8 (6), 961-971 (2006).</li><li>Carman, A. J., Vylkova, S., Lorenz, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+acetyl+coenzyme+A+synthesis+and+breakdown+in+alternative+carbon+source+utilization+in+Candida+albicans.">Role of acetyl coenzyme A synthesis and breakdown in alternative carbon source utilization in Candida albicans.</a> Eukaryotic Cell. 7 (10), 1733-1741 (2008).</li><li>Fradin, 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=Granulocytes+govern+the+transcriptional+response,+morphology+and+proliferation+of+Candida+albicans+in+human+blood.">Granulocytes govern the transcriptional response, morphology and proliferation of Candida albicans in human blood.</a> Molecular Microbiology. 56 (2), 397-415 (2005).</li><li>Lorenz, M. C., Bender, J. A., Fink, G. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+alternative+carbon+utilization+pathways+in+Candida+albicans+attenuate+virulence+and+confer+pleiotropic+phenotypes.">Mutations in alternative carbon utilization pathways in Candida albicans attenuate virulence and confer pleiotropic phenotypes.</a> Eukaryotic Cell. 6 (2), 280-290 (2007).</li><li>Bambach, 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=Goa1p+of+Candida+albicans+localizes+to+the+mitochondria+during+stress+and+is+required+for+mitochondrial+function+and+virulence.">Goa1p of Candida albicans localizes to the mitochondria during stress and is required for mitochondrial function and virulence.</a> Eukaryotic Cell. 8 (11), 1706-1720 (2009).</li><li>Li, 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=Enzymatic+dysfunction+of+mitochondrial+complex+I+of+the+Candida+albicans+goa1+mutant+is+associated+with+increased+reactive+oxidants+and+cell+death.">Enzymatic dysfunction of mitochondrial complex I of the Candida albicans goa1 mutant is associated with increased reactive oxidants and cell death.</a> Eukaryotic Cell. 10 (5), 672-682 (2011).</li><li>Desai, C., Mavrianos, J., Chauhan, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+albicans+SRR1,+a+putative+two-component+response+regulator+gene,+is+required+for+stress+adaptation,+morphogenesis,+and+virulence.">Candida albicans SRR1, a putative two-component response regulator gene, is required for stress adaptation, morphogenesis, and virulence.</a> Eukaryotic Cell. 10 (10), 1370-1374 (2011).</li><li>Mavrianos, 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=Mitochondrial+two-component+signaling+systems+in+Candida+albicans.">Mitochondrial two-component signaling systems in Candida albicans.</a> Eukaryotic Cell. 12 (6), 913-922 (2013).</li><li>Koch, B., 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=The+Mitochondrial+GTPase+Gem1+Contributes+to+the+Cell+Wall+Stress+Response+and+Invasive+Growth+of+Candida+albicans.">The Mitochondrial GTPase Gem1 Contributes to the Cell Wall Stress Response and Invasive Growth of Candida albicans.</a> Frontiers in Microbiology. 8, 2555 (2017).</li><li>Qu, 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=Mitochondrial+sorting+and+assembly+machinery+subunit+Sam37+in+Candida+albicans:+insight+into+the+roles+of+mitochondria+in+fitness,+cell+wall+integrity,+and+virulence.">Mitochondrial sorting and assembly machinery subunit Sam37 in Candida albicans: insight into the roles of mitochondria in fitness, cell wall integrity, and virulence.</a> Eukaryotic Cell. 11 (4), 532-544 (2012).</li><li>Brandt, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Candida and Candidiasis. , (2002).</li><li>Huh, W. K., Kang, S. 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生物能学的额外助焊剂分析是通过实时测量氧化磷酸化 (OXPHOS) 依赖氧消耗来读出线粒体功能的一个很好的工具。此外, 还可以同时在实时分析中研究以细胞外酸化率 (细胞外 pH 值的变化) 来测量的糖酵解功能。
在检测板中成功电镀白色念珠菌是检测过程中的关键步骤之一, 因为 PDL 涂层板中细胞的培养使细胞能够粘附在检测板上。粘附白色念珠菌细胞的可视化是?...

侵袭性真菌感染每年在全球造成150多万人死亡。这一数字正在上升, 原因是免疫受损的人数增加, 包括老年人、早产儿、移植接受者和癌症患者1。白色念珠菌是一种机会性的人类真菌病原体, 是人类微生物群的一部分。它也居住黏膜表面和胃肠道作为一个共同的有机体。白色念珠菌在有免疫缺陷、接受过手术或接受过长期抗生素治疗的人身上会产生严重的全身性疾病。在人类2、3、4、5、6、7的医院传染病 (nid) 中,念珠菌物种名列前三到四个。每年全球念珠菌血液感染的数量估计约为 400, 000 例, 相关死亡率为 46-751。仅在美国, 因念珠菌病而导致的年死亡率就约为 10, 000 人。真菌引起的 NID 的范围也反映在天文数字的病人费用5中。在美国, 治疗侵袭性真菌感染的年度费用超过20亿美元, 给已经不堪重负的医疗系统增加了巨大的压力。目前, 由于毒性、耐药性日益普遍和药物相互作用, 现有的标准抗真菌疗法有限。因此, 迫切需要确定新的抗真菌药物靶点, 从而为高危患者提供更好的治疗选择。然而, 发现新的药物作用于真菌目标是复杂的, 因为真菌是真核生物。这极大地限制了特定于真菌的药物靶标的数量。
最近的研究表明, 线粒体是真菌毒力和抗真菌药物耐受性的关键因素, 因为线粒体对细胞呼吸、氧化代谢、信号转导和凋亡都很重要。 , 9,10,11。糖酵解代谢和非糖酵解代谢对哺乳动物宿主 12、13、14、15、16的生存至关重要。此外, 一些缺乏线粒体蛋白的白色念珠菌突变体, 如 goa1、Srr1、Gem1、sam37 等, 在丝状化方面也有缺陷, 这是白色念珠菌17的一个重要毒力因子,18,19,20,21,22. 此外, 在17、18、19、20、21的小鼠模型中, 这些突变体也因毒力而减弱 ,22。因此, 真菌线粒体是药物发现的一个有吸引力的靶点。然而,白色念珠菌线粒体功能的研究具有挑战性, 因为白色念珠菌是小阴性 23, 这意味着它不能生存没有线粒体基因组。
在这里, 我们描述了一个协议, 可以用来研究线粒体和糖酵解功能在c.白色, 而无需净化线粒体。该方法也可以进行优化, 以研究遗传操作或化学调节剂对白色念珠菌线粒体和糖酵解途径的影响。

<table border="0" cellpadding="0" cellspacing="0" style="width:664px;" width="664">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">RPMI 1640</td>
			<td style="width:84px;">Corning</td>
			<td style="width:119px;">MT50020PB</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Antimycin A</td>
			<td style="width:84px;">Sigma</td>
			<td>A8674</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCN</td>
			<td style="width:84px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Mito stress kit</td>
			<td style="width:84px;">Agilent</td>
			<td>103015-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Oligomycin</td>
			<td style="width:84px;">Calbiochem</td>
			<td>495455</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">pH meter</td>
			<td style="width:84px;">Accumet</td>
			<td>AR20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Phenol red</td>
			<td style="width:84px;">Sigma</td>
			<td>P5530</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Poly-D lysine</td>
			<td style="width:84px;">Sigma</td>
			<td>P6407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Rotenone</td>
			<td style="width:84px;">Santa cruz</td>
			<td>203242</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Seahorse XF24 FluxPak</td>
			<td style="width:84px;">Agilent</td>
			<td>100850-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SHAM</td>
			<td style="width:84px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Sodium Chloride</td>
			<td style="width:84px;">Amresco&#160;</td>
			<td>241</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Sodium hydroxie pellets</td>
			<td style="width:84px;">J.T Baker</td>
			<td>3722</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Tissue culture grade water</td>
			<td style="width:84px;">Gibco</td>
			<td>1523-0147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">XF assay calibrant solution</td>
			<td style="width:84px;">Agilent</td>
			<td>100840-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Yeast extract Peptone Dextrose</td>
			<td>Fisher scientific,</td>
			<td>BP2469</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Yeast extract Peptone Dextrose Agar</td>
			<td style="width:84px;">Sigma</td>
			<td>A1296</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:295px;">Yeast extract Peptone Glycerol</td>
			<td style="width:84px;">Sigma</td>
			<td>G2025</td>
			<td></td>
		</tr>
	</tbody>
</table>

bio-energetics investigation of candida albicans using real-time extracellular flux analysis

线粒体是细胞代谢和生存所必需的细胞器。线粒体中发生了多种关键事件, 如细胞呼吸、氧化代谢、信号转导和凋亡。因此, 线粒体功能障碍被报道在病原真菌的抗真菌药物耐受性和毒力中起着重要作用。最近的数据也使人们认识到线粒体作为真菌发病机制的重要因素的重要性。尽管线粒体在真菌生物学中的重要性, 但了解其功能的标准化方法还是很不发达。在这里, 我们提出了一个程序来研究基础耗氧率 (OCR), 线粒体呼吸的测量, 和细胞外酸化率 (ECAR), 一个测量糖酵解功能的 c. 白色念珠菌菌株。本文描述的方法可应用于任何念珠菌菌株, 而无需纯化完整的真菌细胞中的线粒体。此外, 该协议还可以定制, 以筛选线粒体功能抑制剂在白色念珠菌菌株。

本协议的重点是确定由额外通量分析仪评估的白色念珠菌的生物能量功能。缺乏线粒体蛋白 Mam33 的白色念珠菌突变体也随补体株一起被包括在内 , mam33 : mam33研究线粒体蛋白的缺失对 OCR 和 ecar 的影响。Sam33编码了假定的线粒体酸性基质蛋白及其在念珠菌中的功能尚不清楚。
用于额外通量...

Watch this Scientific Journal Video about Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis at JoVE.com

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

在这里, 我们提出了一个逐步的协议, 以研究线粒体呼吸和糖酵解功能在白色念珠菌使用一个额外的通量分析仪。

白念珠菌实时细胞外通量实时分析的生物能量学研究

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular ...

该协议对于确定念珠菌的糖解功能和线粒体呼吸非常重要。这项技术的主要优点是，它允许测量线粒体功能，而无需从念珠菌中纯化线粒体。这种方法可以优化，以研究基因操作或化学调制剂对线粒体和糖解通路在坎迪卡白细胞或其他真菌病原体的影响。这种技术相对简单。或者根据生物体的不同，它们的使用可能有助于优化细胞数量和线粒体抑制剂在测定中的浓度。在层流罩中，将聚-D-Lysine溶解在组织网格水中，最终浓度为每毫升50微克。将溶液混合好，并放入1.5毫升微离心管中，确保每管至少1.3毫升。将这种混合物的50微升加入检测板的每个井中，盖上盖子，在室温下孵育一到两个小时。然后吸进解决方案。用500微升无菌组织培养分级水冲洗一次检测板的井，打开盖子，让水井风干。如果不在准备当天使用盘子，请将其存放在四摄氏度，最多存放两到三天。在实验前一天，打开额外的通量检测套件并取出内容物。将传感器盒倒置放在公用用板旁边。用一毫升的卡兰特填充公用设施板的每个井，然后将传感器盒放回原处。确保含有荧光团的传感器被淹没在卡钳中。在非二氧化碳培养箱中孵化传感器盒在37摄氏度下过夜。在测定前一天，在YPD汤中接种准备好的C阿尔比肯，并在200转/小时在摇床中以30摄氏度过夜。在测定当天，稀释测定介质中适当数量的细胞，最终浓度为每100微升10万个细胞。除孔 A1、B4、C3 和 D6 外，将稀释的细胞的 100 微升添加到检测板的每个孔中。在这些井中，仅添加 100 微升的检测介质进行背景校正。在37摄氏度的温度下在非二氧化碳培养箱中孵育60分钟，让细胞粘附在板表面。在文本协议中概述的相应测定介质中，以10倍浓度为线粒体功能测定准备化合物。将50微升SHAM加入港口，将50微升的Oligomycin加入B端口，将62微升氰化钾加入C端口，将68微升的SHAM加入到D端口中。将50微升葡萄糖加入端口A，将55微升的Oligomycin加入端口B，将62微升的2-DG添加到端口C中，将68微升的抗霉素A添加到端口D中。按照分步说明操作，并填写设置过程中弹出的所有信息。生成组布局并设置文本协议中概述的协议。在检测之前保存这些布局。在检测时，通过打开检测向导选项卡中的打开文件选项中打开相应的文件来还原保存的协议。然后将 10X 化合物加载到包含校准器的水合传感器盒的各端口中。将传感器盒加载到额外通量分析仪的托架托盘中。单击屏幕上的"开始"按钮启动校准。接下来，沿着壁侧的细胞板轻轻添加 350 微升的检测介质，以最大限度地减少细胞干扰，使最终体积达到 450 微升。用检测板更换含有卡钳的实用板，然后继续检测。检测完成后，拆下传感器盒和板。将文件保存在相应的目标文件夹中。在这项研究中，通过特高量分析仪评估C albicans的生物能量功能。缺乏线粒体蛋白mam33的突变体也包括其补充应变，以研究线粒体蛋白的删除对耗氧率和细胞外酸化率的影响。C albicans 野生类型显示每分钟 145 个皮莫的耗氧率，这非常在任何细胞外通量测定的最佳范围内。野生型、mam33突变体和mam33互补菌株的基底耗氧率无差异。同样，菌株之间的基底细胞外酸化率没有显著差异。然而，通过抑制氰化钾的复杂IV，野生型和mam33突变体，mam33补充菌株显示向糖解的重大转变。然而，mam33突变菌株未能显示补偿性糖解移表明复合IV依赖通路在此菌株中受损。在糖解压力测试中，细胞缺葡萄糖一小时，测量基础氧消耗率和细胞外酸化率。饥饿时，与其他菌株相比，mam33突变菌株的耗氧量和细胞外酸化率显著降低。这表明当细胞被迫进入饥饿状态时，葡萄糖在呼吸和糖解的利用能力下降。注射葡萄糖后，所有菌株的耗氧率和细胞外酸化率均表现出刺激作用。检测板的涂层允许念珠菌细胞粘附在板上。念珠菌细胞的不当粘附会影响测定结果。在这项技术发展之后，它已被应用于广泛的细胞类型和疾病，解决线粒体在生理和病理条件下的功能。例如，在此分析中采用患者衍生细胞和控制细胞，可以解决线粒体功能如何改变的具体问题。在制备线粒体抑制剂（如氰化钾和抗霉素 A）时应小心，因为这些化合物具有剧毒性。

Research

JoVE Journal

Medicine

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

使用实时活体成像在肿瘤纳米粒子吸收的评价

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

科研

医学

Real-time fMRI Biofeedback Targeting the Orbitofrontal Cortex for Contamination Anxiety

实时功能磁共振成像生物反馈，针对污染焦虑的眶额皮层

We present a method for training subjects to control activity in a region of their orbitofrontal cortex associated with contamination anxiety using ...

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

实时定量PCR（qPCR的microRNA检测在前列腺肿瘤）

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Movement Retraining using Real-time Feedback of Performance

的运动再培训采用实时反馈的性能

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

采用实时定量PCR检测供体细胞植入一个具有竞争力的小鼠骨髓移植模型

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

脑脊液的microRNA分析采用实时定量PCR技术

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

化疗引起的血管毒性 - 实时<em&gt;在体内</em&gt;船舶减值成像

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

在实时的间皮细胞卵巢癌球体附件和入侵评估

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Real-time Cytotoxicity Assays in Human Whole Blood

实时的细胞毒性分析在人全血

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...