作者想感谢所有成员的博士巴列塔，博士的实验室权力的有益的意见而开发的协议。我们温迪奥斯汀的有益讨论和校对的稿子。这个手稿中描述的工作是由种子试点补助金每个上面列出大学内布拉斯加 - 林肯氧化还原生物学研究中心（母公司授予＃的NCRR 2P20RR 017675，D.贝克尔，PI）的研究者。此外，我们感谢奥费利娅查孔博士的研究用品和先生Halouska的部分工资支持，以规范本出版物中包含的NMR技术从她的R21授予（1R21AI087561-01A1）提供资金。

1。协议文本该协议强调的NMR方法，以适应M.肺结核 （III级代理）。因此，必须遵循生物安全3级（BSL3）的做法时，M.肺结核在每年认证的实验室研究。暴露于实验室产生的气溶胶是最重要的工作人员与这些微生物所遇到的危险。下面的过程是在我们的机构中​​进行的变化可能存在​​生物安全委员会的建议的基础上。常见的个人防护设备包括特卫强服，​​圆帽，鞋，N95呼吸器，保护眼睛，袖子，和双对丁腈手套。有关的工作，M.肺结核文化和/或打开操作与M.肺结核容器的类型A2或B2生物安全柜。被放置在塑料覆盖的吸收纸的w工作会有的表面上。将予出售或从设备中移除所有材料/耗材必须放置在两个生物危害袋和消毒的高压灭菌。与的1％Amphyl（结核菌，杀菌，杀菌和杀病毒剂），每个工作结束后，工作台面及机柜内的设备使用（FastPrep-24溶解均质机，分光光度计，桶与冰等）必须进行消毒。M.肺结核文化必须放置在双遏制运输的生物安全柜，冷冻箱，孵化器，离心机，冰箱等以外的更大的设备。离心进行封闭安全杯和O形圈螺旋盖的管。外BSL3实验室作进一步的分析，无细胞提取物被过滤通过0.2微米的过滤器或微生物在95℃下15分钟的加热杀死的。2样品均镀以验证的菌落形成单位的情况下除去遏制之前。 <ol><li>转移米德尔7H9完整的媒体，含有聚山梨酯80（吐温80，以防止结块）的110毫升肉汤（MADC-TW）或一个合适的培养基中的250毫升的锥形烧瓶中。一式三份的文化常规种植每条件时，使用13个 C-代谢物，10个相同的文化生长的一维1 H-NMR分析。两个条件（ 例如 ，不加药物），双量均的文化，每重复可以生长并分裂成两个相同的文化。所有的试剂和溶液的配方中所提供结束时的协议。 </li><li>接种到肉汤0.150毫升M.结核病 50％的甘油（允许在冰上解冻）。请参阅下面的注意事项1。 </li><li>让文化成长在37°C，100rpm振荡约6天（OD 600 0.6-0.8）。请参阅下文附注2。 </li><li>如果没有抗生素，替代增加或其他治疗是必要的，文化是随时可以收获。如果用于治疗，继续直接执行步骤5。从每个烧瓶中取出0.5毫升样品，转移到离心管中，在4°C文化滴定和质量控制污染检测，表型分析，PCR检测。请参阅下文附注3。在这一点上，继续进行第6步。 </li><li>将瓶中删除振动筛和执行所需的处理（ 如添加药物或代谢物）。地点烧瓶，振动筛，并培育一个额外的周期（ 如 6-18小时）。在这段时间结束后，另取1.0毫升，并确定OD 600。从每个烧瓶中取出0.5毫升样品，转移到离心管中，在4°C的文化滴定和质量控制。 </li><li>地点文化在冰上5分钟。在此步骤之后，留下细胞在冰上在整个的其它协议。通过离心收获培养在2,000 xg和4℃下15分钟在50毫升的试管，使用台式离心机。每个文化将需要用25毫升每个（共1的四根管00毫升是必需的，以获得足够的信号与噪声的用于2D 1 H-13 C NMR实验，其中只有50毫升每培养所需的1D 1 H NMR的实验）。 </li><li>每个细胞沉淀洗净两次与冰冷的DDH 2 O（大约15毫升的第一次和10毫升的第二次），如上所述通过离心参数。对于第二次洗涤，结合到相同的管中的10毫升等分试样纺丝前。在终体积为1.0ml，DDH 2 O（需要调整为细胞沉淀体积）中重悬细胞沉淀。如果在此步骤中，所需的单细胞悬浮液，无结块细胞可以简要地超声处理和/或通过一个27号针头的三倍。另外，将细胞沉淀可以在-80℃下冷冻直至进一步处理和存储。在后者的情况下，冷冻粒料之前，再悬浮于冰上解冻。重悬细胞沉淀，在最终体积为1.0毫升DDH 2 O，如上所述。 </li><li>内部转移r为1.0毫升细胞悬浮液至2.0ml含有溶胞矩阵B（0.1毫米的二氧化硅微球）的螺旋盖管。将FastPrep-24裂解匀浆内的生物安全柜。然后将材料放入的样本保持器，固定保持辐板在管的顶部。设定为6米/秒的速度，在均化器中处理样品60秒。 </li><li>在微型旋转样品在15,000 xg和4℃下进行10分钟以沉淀细胞碎片和完整细胞。 </li><li>到无菌管中取出上清液和通样品通过注射器过滤器（0.2μm）。板0.1毫升MADC琼脂上的样品（或有代表性的部分，例如10％）的样品，以验证是否存在没有存活的细胞。侦查人员也可以进行一个以上的过滤步骤，和/或验证的存在下存活的细胞提取物中使用活死的染色程序，以便防止或识别的任何潜在的生物安全顾虑。冷冻样品中的乙醇 - 干冰浴存储在-80℃下，直到他们准备要冷冻干燥和处理中的NMR设施。 </li><li> 2个月后，检查板来验证的情况下的菌落形成单位。如果没有CFU被发现，样本可能采取的BSL3实验室。进行进一步的分析，在标准的NMR的设施，这通常提供多用户和下运作没有具体的遏制要求。 </li><li>样品冷冻干燥至干，然后重新悬浮在0.7毫升的NMR缓冲区，并转移到一个离心管中。在13000×g下离心3分钟。取出0.6毫升和转移到一个5毫米的NMR管中。另外，冻干样品邮寄给非的生物危害性定期样本的外部设备。 </li><li> NMR数据立即被收集。尽管所有的预防措施，活性酶可能仍存在，样品通常并不稳定，很长一段时间。的NMR谱中的变化是引人注目的，当样品被排除为m，在室温或在4℃下矿石超过1周。此外，NMR数据收集未经治疗和药物治疗的文化，从每个类别中随机抽取的样品之间交替。这可以防止不必要的偏见，因为收集的NMR谱是之前，一个特定的类别有一个较长的延迟。如果所有未处理的样品的NMR光谱首先收集，然后依次由药物处理过的样品中的数据会发生甲偏置。如果样本不能被立即通过NMR分析，样品应在-80℃下储存在1毫升Eppendorf管中</li><li> BACS-120样品更换，仪器校准，这涉及到几个常规的措施，如锁，垫片，调整和优化的90°脉冲长度，插入样品后，需要最高质量的结果。单一样本是用来校准的90°脉冲的长度和对剩余的样品调整仪器。锁定，匀场，对每个样品的NMR数据采集的自动化全光照克ICONNMR和gradshim。 </li><li>甲1D 1 H NMR谱是使用布鲁克zgesgp脉冲序列收集与水抑制使用激发雕刻。一个32k的数据点用扫描宽度5482.5Hz，128扫描，和16个虚拟扫描。 A 2D 1 H-13 C HSQC谱的收集，使用的布鲁克hsqcetgp的脉冲序列。共2048个数据点与扫描宽度5000.0赫兹一起被采集的直接1 H尺寸，和64个数据点，沿间接13 C尺寸的扫描宽度18864.9赫兹。收集具有128扫描和16个虚拟扫描的频谱，以获得良好的信号噪声。 </li></ol><img alt="图1" src="/files/ftp_upload/3673/3673fig1.jpg" /> 图1，一种实验程序的流程图被描述。 NMR缓冲 原液的50mM磷酸钾的buff呃50μM第三次会议: <ul><li> 2.17克K 2 HPO 4（磷酸氢二钾） </li><li> 1.70克KH 2 PO 4（磷酸二氢钾） </li><li> 7.86毫克钠3 -三甲基甲硅烷基丙（TMSP-2 ,2,3,3-D 4（D，98％）） </li><li> "100％"D 2 O溶解于500毫升</li><li>最终的解决方案应该是在pH 7.2的（未校正的） </li></ul> MADC-TW或MOADC的TW（1L） <ul><li> 4.7克米德尔7H9肉汤基础</li><li> 900 ML DDH 2 O </li><li> 2 mL甘油</li><li>混合组分和25分钟的高压釜。清凉的液体接触，并添加以下解决方案: </li><li>百毫升ADC在准备MADC或100毫升OADC的在准备MOADC时</li><li> 2.5毫升20％的吐温80（可选地，1毫升的20％的非代谢泰洛沙泊） </li><li> 10毫升1％的放线菌酮</li></ul> ADC（1L）中 <ul><li> 20克D（+） - 葡萄糖</li><li> BSA分数50克V </li><li> 8.5克氯化钠</li><li> 800 ML DDH 2 O </li><li>溶解在一起，调整音量至1 L与0.2微米的过滤器和消毒。存放于4℃ </li></ul>另外，准备OADC（1L），添加1％的油酸（配方下面，使250毫升的批次）加50毫升以上列出的所有组件。溶解在一起，调整音量至1 L与0.2微米的过滤器和消毒。瓶需要被包装在铝箔和商店在4°C。 <ul><li> 2.5克油酸</li><li> 250毫升0.2M的NaOH </li><li>解冻油酸（固化制冷时）通过加​​热在55℃下10分钟，并在搅拌下进行60分钟，加至NaOH溶液中和热。储存，包裹在铝箔中的无菌玻璃瓶。密封瓶的封口膜和存储在4°C。 </li></ul>如果愿意，您可以购买商业BD BBL米德尔的ADC或OADC与过氧化氢酶的富集。 1％的放线菌酮（100ml）中 </STRONG&gt; <ul><li> 1克放线菌酮</li><li> 100 ML DDH 2 O </li></ul>溶解和消毒用0.2微米的过滤器。存放于4℃注意:放线菌酮是有毒的，所以格外小心处理。 20％（体积/体积）吐温80（100毫升） <ul><li> 20毫升吐温80 </li><li> 80 ML DDH 2 O </li></ul>另外，称量20克吐温80（约18.9毫升，密度1.06克/毫升），以制备20％w / v溶液。热到55°C，30分钟至完全溶解，混合。消毒液体，用0.2微米的过滤器。存储最终的溶液在室温下。 泰洛沙泊（100毫升）20％（体积/体积）的 <ul><li> 20毫升四丁酚醛</li><li> 80 ML DDH 2 O </li></ul>或者，称重20克泰洛沙泊（约18.2毫升，密度1.1克/毫升），以制备20％w / v溶液。 30分钟，到二55°C的热解决方案ssolve，完全混合。消毒液体，用0.2微米的过滤器。存储最终的溶液在室温下。 注1:甘油股票M.结核制备使用下面的协议。 放养M.肺结核 <ul><li>成长M.在50毫升MADC 肺结核饱和度（OD 600 = 1.5到2.0），在37℃下的振荡条件下（100转）。根据上的应变，这将花费7-14天。 </li><li>附带样品在4℃以2,000 xg离心15分钟以沉淀文化。除去上清液。 </li><li>重悬在无菌的50％甘油的6毫升。 </li><li>分装1.5毫升到4康宁的低温瓶和标签适当。 </li><li>立即闪光冻结于乙醇/干冰浴和存储的瓶在-80°C。 </li></ul>注2:接种菌株H37Rv（-80°C股票上涨1.5岁）为70毫升的MADC媒体产生的OD 600 &lt;/ sub&gt;的约0.6后5天的生长在37℃下在振荡条件下（100转），在一个伊诺40摇床。 注3:为了测试文化的污染，研究人员可以到标准营养丰富的培养基接种文化等分，并确认没有过夜培养后的增长。按常规，文化上都镀MADC琼脂相衬显微镜观察菌落形态和检查。如果需要，文化可以验证6110引物通过PCR方法。 2。代表性的成果一个样品已做好充分准备，将产生一个如图2所示的NMR光谱相似。这个频谱是表示在M.肺结核野生型代谢池。所识别的代谢产物被直接或间接相关联的D-丙氨酸的通路。另外，峰强度的大小是代谢物的浓度成比例呈现的细胞提取物中。因此，在处理培养物，药物治疗，和突变株之间的峰强度的变化可以指示的代谢产物和代谢途径中的扰动。该M.结核 1D 1 H NMR谱是在Bruker Avance 500兆赫光谱仪配备三重谐振，Z轴梯度冷冻探针收集。的频谱包含约。 400峰，它是可能的，以确定和量化40-50代谢产物，包括氨基酸，核苷酸前体，以及糖酵解和柠檬酸中间体的。一个BACS-120自动进样器与布鲁克图标软件用于自动NMR数据采集。 1D 1 H NMR谱，收集使用激发雕刻4有效地除去溶剂，并保持平坦的基线，消除了任何需要收集基线可能引起工件在随后的主成分分析（PCA）或正交偏最小二乘判别分析（OPLS- DA）。 A 1D 1 H NMR谱是在25℃下收集与5482.5 Hz和32K数据点的频谱宽度。总共有16个虚拟扫描和128扫描被用来以获得频谱。 ACD实验室1D NMR处理软件，用于半会自动处理所有1D 1 H-NMR谱。光谱傅立叶变换，分阶段实施，并参照第三次会议的峰值（0.0 PPM）。 NMRpipe软件包用于单独处理2D的1 H-13 C NMR光谱，并分析NMRDraw。布鲁克FID数据文件首先被转换为一个文件格式识别的NMRpipe格式和然后光谱进行傅立叶变换，相位校正，填充零。在一维的1 H NMR谱和2D 1 H-13 C NMR谱中所观察到的NMR峰被分配到特定的代谢物，用1 H和13 C化学位移分别为0.05和0.50 ppm时，公差，和麦迪逊代谢组学联合体数据库（MMCD ） <sup&gt; 5的BioMagResBank，6和人类代谢组数据库。具体来说，一维和二维NMR谱都是手工挑选的高峰，然后上传到人类代谢组数据库的核磁共振化学位移的峰值列表。人类代谢组数据库所确定的代谢物被分配基于两个最大化的数目匹配的NMR共振和属于代谢网络的NMR谱。每种化合物或代谢物通常有多个CH对和相应的多个NMR共振。因此，更多的这些核磁共振实验的NMR谱中所观察到的共振，就越有可能的代谢物是本。同样，识别多个代谢产物联营公司相同的途径的可能性增加了正确的任务。存在的代谢物和代谢途径进行了验证。9 结核分枝杆菌的基因和基因组（KEGG）8 MetaCyc数据库"京都百科全书smegmATIS是一个有用的模型系统，M.肺结核和其他分枝杆菌病原体。如其他地方，样品M.耻垢分枝杆菌也可能被超声破碎。 <img alt="图2" src="/files/ftp_upload/3673/3673fig2.jpg" /> 图2。1D 1 H NMR谱的结核分枝杆菌的细胞提取物。具有代表性的代谢产物的NMR峰标记。缩写如下:AMP，腺苷单磷酸，α-，α-酮戊二酸千克，谷氨酸，谷氨酸，丙氨酸和Ala。

<ol>
	<li>Larsen, M. H., Biermann, K., Tandberg, S., Hsu, T., Jacobs, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Manipulation+of+Mycobacterium+tuberculosis.">Genetic Manipulation of Mycobacterium tuberculosis.</a> Curr. Protoc. Microbiol. Chapter 10, 2 (2007).</li><li>Larsen, M. H., Biermann, K., Jacobs, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+Maintenance+of+Mycobacterium+tuberculosis.">Laboratory Maintenance of Mycobacterium tuberculosis.</a> Curr. Protoc. Microbiol. Chapter 10, 1 (2007).</li><li>Clarridge, J. E., Shawar, R. M., Shinnick, T. M., Plikaytis, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+use+of+polymerase+chain+reaction+for+detection+of+Mycobacterium+tuberculosis+in+a+routine+mycobacteriology+laboratory.">Large-scale use of polymerase chain reaction for detection of Mycobacterium tuberculosis in a routine mycobacteriology laboratory.</a> J. Clin. Microbiol. 31, 2049-2056 (1993).</li><li>Nguyen, B. D., Meng, X., Donovan, K. J., Shaka, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SOGGY:+solvent-optimized+double+gradient+spectroscopy+for+water+suppression.+A+comparison+with+some+existing+techniques.">SOGGY: solvent-optimized double gradient spectroscopy for water suppression. A comparison with some existing techniques.</a> J. Magn. Reson. 184, 263-274 (2007).</li><li>Cui, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolite+identification+via+the+Madison+Metabolomics+Consortium+Database.">Metabolite identification via the Madison Metabolomics Consortium Database.</a> Nat. Biotechnol. 26, 162-164 (2008).</li><li>Ulrich, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BioMagResBank.">BioMagResBank.</a> Nucleic Acids Res. 36, 402-408 (2008).</li><li>Wishart, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HMDB:+the+Human+Metabolome+Database.">HMDB: the Human Metabolome Database.</a> Nucleic Acids Res. 35, 521-526 (2007).</li><li>Kanehisa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KEGG+for+linking+genomes+to+life+and+the+environment.">KEGG for linking genomes to life and the environment.</a> Nucleic Acids Res. 36, 480-484 (2008).</li><li>Karp, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+the+BioCyc+collection+of+pathway/genome+databases+to+160+genomes.">Expansion of the BioCyc collection of pathway/genome databases to 160 genomes.</a> Nucleic Acids Res. 33, 6083-6089 (2005).</li><li>Halouska, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+NMR+metabolomics+to+analyze+the+targets+of+D-cycloserine+in+mycobacteria:+role+of+D-alanine+racemase.">Use of NMR metabolomics to analyze the targets of D-cycloserine in mycobacteria: role of D-alanine racemase.</a> J. Proteome. Res. 6, 4608-4614 (2007).</li><li>Boshoff, H. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcriptional+responses+of+Mycobacterium+tuberculosis+to+inhibitors+of+metabolism:+novel+insights+into+drug+mechanisms+of+action.">The transcriptional responses of Mycobacterium tuberculosis to inhibitors of metabolism: novel insights into drug mechanisms of action.</a> J. Biol. Chem. 279, 40174-40184 (2004).</li><li>Mehaffy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Descriptive+proteomic+analysis+shows+protein+variability+between+closely+related+clinical+isolates+of+Mycobacterium+tuberculosis.">Descriptive proteomic analysis shows protein variability between closely related clinical isolates of Mycobacterium tuberculosis.</a> Proteomics. 10, 1966-1984 (2010).</li><li>Schnappinger, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+Adaptation+of+Mycobacterium+tuberculosis+within+Macrophages:+Insights+into+the+Phagosomal+Environment.">Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages: Insights into the Phagosomal Environment.</a> J. Exp. Med. 198, 693-704 (2003).</li><li>Schnappinger, D., Schoolnik, G. K., Ehrt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+profiling+of+host+pathogen+interactions:+how+Mycobacterium+tuberculosis+and+the+macrophage+adapt+to+one+another.">Expression profiling of host pathogen interactions: how Mycobacterium tuberculosis and the macrophage adapt to one another.</a> Microbes. Infect. 8, 1132-1140 (2006).</li><li>Shui, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+proteomic+profiling+of+host-pathogen+interactions:+the+macrophage+response+to+Mycobacterium+tuberculosis+lipids.">Quantitative proteomic profiling of host-pathogen interactions: the macrophage response to Mycobacterium tuberculosis lipids.</a> J. Proteome. Res. 8, 282-289 (2009).</li><li>Talaat, A. M., Lyons, R., Howard, S. T., Johnston, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+temporal+expression+profile+of+Mycobacterium+tuberculosis+infection+in+mice.">The temporal expression profile of Mycobacterium tuberculosis infection in mice.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 4602-4607 (2004).</li><li>Forgue, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMR+metabolic+profiling+of+Aspergillus+nidulans+to+monitor+drug+and+protein+activity.">NMR metabolic profiling of Aspergillus nidulans to monitor drug and protein activity.</a> J. Proteome Res. 5, 1916-1923 (2006).</li><li>Goodacre, R., Vaidyanathan, S., Dunn, W. B., Harrigan, G. G., Kell, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolomics+by+numbers:+acquiring+and+understanding+global+metabolite+data.">Metabolomics by numbers: acquiring and understanding global metabolite data.</a> Trends Biotechnol. 22, 245-252 (2004).</li><li>Shin, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMR-based+Metabolomic+Profiling+in+Mice+Infected+with+Mycobacterium+tuberculosis.">NMR-based Metabolomic Profiling in Mice Infected with Mycobacterium tuberculosis.</a> J. Proteome Res. 10, 2238-2247 (2011).</li><li>Jaki, B. U., Franzblau, S. G., Cho, S. H., Pauli, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+extraction+method+for+mycobacterial+metabolome+analysis.">Development of an extraction method for mycobacterial metabolome analysis.</a> J. Pharm. Biomed. Anal. 41, 196-200 (2006).</li><li>de Carvalho, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolomics+of+Mycobacterium+tuberculosis+reveals+compartmentalized+co-catabolism+of+carbon+substrates.">Metabolomics of Mycobacterium tuberculosis reveals compartmentalized co-catabolism of carbon substrates.</a> Chem. Biol. 17, 1122-1131 (2010).</li><li>de Carvalho, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-based+metabolomic+profiling+of+enzymatic+function:+identification+of+Rv1248c+as+a+mycobacterial+2-hydroxy-3-oxoadipate+synthase.">Activity-based metabolomic profiling of enzymatic function: identification of Rv1248c as a mycobacterial 2-hydroxy-3-oxoadipate synthase.</a> Chem. Biol. 17, 323-332 (2010).</li><li>Marrero, J., Rhee, K. Y., Schnappinger, D., Pethe, K., Ehrt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gluconeogenic+carbon+flow+of+tricarboxylic+acid+cycle+intermediates+is+critical+for+Mycobacterium+tuberculosis+to+establish+and+maintain+infection.">Gluconeogenic carbon flow of tricarboxylic acid cycle intermediates is critical for Mycobacterium tuberculosis to establish and maintain infection.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 9819-9824 (2010).</li><li>Tang, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+metabolism+in+Mycobacterium+smegmatis+during+the+transition+from+O2-rich+to+O2-poor+conditions+as+studied+by+isotopomer-assisted+metabolite+analysis.">Central metabolism in Mycobacterium smegmatis during the transition from O2-rich to O2-poor conditions as studied by isotopomer-assisted metabolite analysis.</a> Biotechnol. Lett. 31, 1233-1240 (2009).</li><li>Kweon, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+polyomic+approach+to+elucidate+the+fluoranthene-degradative+pathway+in+Mycobacterium+vanbaalenii+PYR-1.">A polyomic approach to elucidate the fluoranthene-degradative pathway in Mycobacterium vanbaalenii PYR-1.</a> J. Bacteriol. 189, 4635-4647 (2007).</li><li>Hasan, M. R., Rahman, M., Jaques, S., Purwantini, E., Daniels, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+6-phosphate+accumulation+in+mycobacteria:+implications+for+a+novel+F420-dependent+anti-oxidant+defense+system.">Glucose 6-phosphate accumulation in mycobacteria: implications for a novel F420-dependent anti-oxidant defense system.</a> J. Biol. Chem. 285, 19135-19144 (2010).</li><li>Soga, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+metabolome+analysis+using+capillary+electrophoresis+mass+spectrometry.">Quantitative metabolome analysis using capillary electrophoresis mass spectrometry.</a> J. Proteome Res. 2, 488-494 (2003).</li><li>Metz, T. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+future+of+liquid+chromatography-mass+spectrometry+(LC-MS)+in+metabolic+profiling+and+metabolomic+studies+for+biomarker+discovery.">The future of liquid chromatography-mass spectrometry (LC-MS) in metabolic profiling and metabolomic studies for biomarker discovery.</a> Biomark Med. 1, 159-185 (2007).</li><li>Ludwig, C., Viant, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+J-resolved+NMR+spectroscopy:+review+of+a+key+methodology+in+the+metabolomics+toolbox.">Two-dimensional J-resolved NMR spectroscopy: review of a key methodology in the metabolomics toolbox.</a> Phytochem. Anal. 21, 22-32 (2010).</li><li>Simpson, R. J., Inglis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Proteins and Proteomics: A Laboratory Manual. , 425-595 (2003).</li><li>Beste, D. J., McFadden, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=System-level+strategies+for+studying+the+metabolism+of+Mycobacterium+tuberculosis.">System-level strategies for studying the metabolism of Mycobacterium tuberculosis.</a> Mol. Biosyst. 6, 2363-2372 (2010).</li><li>Rhee, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+carbon+metabolism+in+Mycobacterium+tuberculosis:+an+unexpected+frontier.">Central carbon metabolism in Mycobacterium tuberculosis: an unexpected frontier.</a> Trends Microbiol. , (2011).</li><li>Who. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Health+Organization.">Health Organization.</a> Anti-tuberculosis Drug Resistance in the World: Report No. 4. , (2008).</li><li>Jassal, M., Bishai, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensively+drug-resistant+tuberculosis.">Extensively drug-resistant tuberculosis.</a> Lancet Infect Dis. 9, 19-30 (2009).</li></ol>

没有利益冲突的声明。

一个相当多的研究分析，转录组和蛋白质组的配置文件的M.在体外和体内条件下各种肺结核 11-16最终，在基因表达和酶活性的变化导致在分子量小的分子的浓度的变化。的完整描述，这些化合物构成的代谢组。因此，药物和不同生长条件对代谢途径的影响可能其次是代谢组学分析。17,18很少有研究利用这种方法，研究代谢途径中的M.结核 （见下文）和其?...

<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> ADC富集</td><td> BD BBL米德尔</td><td> 212352 </td><td></td></tr><tr><td> BACS-120样品转换器</td><td>布鲁克</td><td></td><td></td></tr><tr><td> BRUKER AVANCE NMR </td><td>布鲁克</td><td></td><td> 500 MHz的</td></tr><tr><td>牛血清白蛋白</td><td> Fisher Scientific则</td><td> BP1600-100 </td><td>馏分V </td></tr><tr><td>离心分离</td><td> Beckman Coulter公司</td><td> Allegra的X-15R </td><td>台式</td></tr><tr><td>离心管</td><td>康宁</td><td> 430291 </td><td> 50毫升无菌聚丙烯</td></tr><tr><td>低温瓶</td><td>康宁</td><td> 430488 </td><td> 2.0毫升无菌聚丙烯</td></tr><tr><td> Cycloheximide </td><td> AG科学</td><td> C-1189 </td><td>有毒的</td></tr><tr><td> D（+） - 葡萄糖</td><td> ACROS </td><td> 41095-0010 </td><td></td></tr><tr><td>氧化氘</td><td> Sigma Aldrich公司</td><td> 617385 </td><td></td></tr><tr><td>锥形烧瓶</td><td> VWR </td><td> 89095-266 </td><td>不育，平底，聚碳酸酯，0.22微米PTFE膜通风帽</td></tr><tr><td>闪霜瓶</td><td> VWR </td><td> 82018-226 </td><td> 750毫升</td></tr><tr><td>冷冻干燥机</td><td> VWR </td><td> 82019-038 </td><td> 4.5 L台式</td></tr><tr><td>甘油</td><td> GibcoBRL </td><td> 15514-029 </td><td></td></tr><tr><td>恒温箱</td><td>新不伦瑞克省</td><td>伊诺40 </td><td>台式振动筛</td></tr><tr><td>裂解矩阵B </td><td> MP Biomedicals </td><td> 6911-100 </td><td></td></tr><tr><td>裂解机</td><td> MP Biomedicals </td><td> FastPrep-24 </td><td></td></tr><tr><td>微型离心机</td><td> Eppendorf公司</td><td> 5415D </td><td>台式</td></tr><tr><td>微型离心机</td><td> Beckman Coulter公司</td><td>微型离心22R </td><td>台式</td></tr><tr><td>米德尔7H9肉汤</td><td> Difco公司</td><td> 271310 </td><td></td></tr><tr><td> NMR管</td><td>诺莱尔</td><td> ST500-7 </td><td> 5mM的</td></tr><tr><td> OADC富集</td><td> BD BBL米德尔</td><td> 212351 </td><td></td></tr><tr><td>油酸</td><td>西格玛</td><td> O1008 </td><td></td></tr><tr><td>磷酸氢二钾</td><td> VWR </td><td> BDH0266 </td><td></td></tr><tr><td>磷酸二氢钾</td><td> VWR </td><td> BDH0268 </td><td></td></tr><tr><td> - 转子的离心22R </td><td> Beckman Coulter公司</td><td> F241.5P </td><td>密封和聚丙烯</td></tr><tr><td>转子 - Allegra的X-15R</td><td> Beckman Coulter公司</td><td> SX4750 </td><td>随着生物认证的封面</td></tr><tr><td>氯化钠</td><td> Fisher Scientific则</td><td> S271-3 </td><td></td></tr><tr><td>钠 - 3 - 三甲基甲硅烷基丙-2 ,2,3,3-D4 </td><td>剑桥同位素</td><td> DLM-48 </td><td></td></tr><tr><td>分光光度计</td><td> Beckman Coulter公司</td><td> DU-530 </td><td></td></tr><tr><td>分光光度计比色皿</td><td>生命线</td><td> LS-2410 </td><td> 1.5毫升聚苯乙烯，2明确双方</td></tr><tr><td>注射器</td><td> Becton Dickinson公司</td><td> 309585 </td><td>不育，3毫升鲁尔乐</td></tr><tr><td>针头式过滤器</td><td>的Nalgene </td><td> 190-2520 </td><td> 0.2微米的无菌醋酸纤维素</td></tr><tr><td>吐温80 </td><td> Fisher Scientific则</td><td> BP338-500 </td><td></td></tr></tbody></table>

sample preparation of mycobacterium tuberculosis extracts for nuclear magnetic resonance metabolomic studies

结核分枝杆菌是在全球范围内人类死亡的一个重要原因。两个多（MDR）和广泛耐药（XDR）株的出现，威胁要破坏当前疾病控制工作。因此，迫切需要开发比现有的，更有效的药物和疫苗。 分枝杆菌的基因组肺结核已超过10年，但在我们的基因的功能和重要性的认识有重要的差距。许多研究都因为基因表达的转录组和蛋白质组水平的分析，以确定影响的药物，氧化剂，对全球基因表达模式和生长条件。最终，这些变化的最终响应反映在代谢的细菌组合物，包括几千小分子量的化学品。比较野生型和突变株的代谢概况，无论是未经处理或者tr与一个特定的药物eated，可以有效地允许的目标识别和可能导致具有反结核活性的新型抑制剂的发展。同样地，对代谢组中的两个或多个条件的影响也可以被评估。核磁共振（NMR）是一个功能强大的技术，用于识别和量化的中间代谢产物。在这个协议中，程序编制M.结核病细胞提取物的NMR代谢组学分析进行了阐述。在适当条件下，所需的生物安全级别3遏制，1收获细胞培养物生长，并进行机械裂解，同时保持冷的温度，以最大限度地提高保存的代谢物。细胞裂解物中回收，过滤灭菌，并存储在超低温度下。从这些细胞提取物的等分试样镀米德尔7H9琼脂集落形成单位验证没有存活的细胞上。后两个月的孵育在37℃下，如果没有㈥能够观察到菌落，取出样品，从包容设施用于下游处理。萃取液冷冻干燥，再悬浮在氘化缓冲液中喷射的NMR仪器，捕获分光数据，然后将其进行统计分析。所描述的方法可以应用于两个一维（1D）的1 H NMR和二维（2D）的1 H-13 C-NMR分析。这种方法提供了更可靠的小分子量代谢物鉴定和色谱法比更可靠，更敏感的细胞提取液代谢成分的定量分析。描述的过程中的变化之后的细胞裂解步骤也可以适于并行蛋白质组学分析。

Watch this Scientific Journal Video about Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies at JoVE.com

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

代谢组学的档案<em&gt;结核分枝杆菌</em&gt;确定后在肉汤培养物的生长。条件可以改变，以测试的营养补充剂，氧化剂，抗结核剂的影响，对这种微生物的代谢的档案。提取物制剂的程序是适用于两个1D<sup&gt; 1</sup&gt; H和2D<sup&gt; 1</sup&gt; H-<sup&gt; 13</sup&gt; C NMR分析。

样品制备<em&gt;结核分枝杆菌</em提取物对核磁共振的代谢组学研究

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

sample-preparation-mycobacterium-tuberculosis-extracts-for-nuclear

Research

Education

JoVE Core

JoVE Journal

Analytical Chemistry

Immunology and Infection

Unit 1

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Principles of Nuclear Magnetic Resonance

SCIENTISTS IN ACTION

15.3K 次观看.  University of Nebraska-Lincoln. 代谢组学的档案结核分枝杆菌确定后在肉汤培养物的生长。条件可以改变，以测试的营养补充剂，氧化剂，抗结核剂的影响，对这种微生物的代谢的档案。提取物制剂的程序是适用于两个1D 1 H和2D 1 H- 13 C NMR分析。

视频: Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

增长结核分枝杆菌生物膜

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

科研

免疫与感染

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

监测树突状细胞迁移<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H磁共振成像

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

实验模型后的自然传播到研究结核病，疟疾混合感染结核分枝杆菌和伯氏疟原虫</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

展现着多药耐药<em&gt;结核分枝杆菌</em&gt;扩增基因芯片

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

关于功能研究的三维人体肺组织模型<em&gt;结核分枝杆菌</em&gt;感染

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

系统抑制剂靶向细胞内的疗效和毒性筛选<em&gt;结核分枝杆菌</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

高通量兼容检测，以评估对巨噬细胞传代药物疗效<em&gt;结核分枝杆菌</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

分析18葡萄糖 PET/CT 成像作为研究结核分枝杆菌感染和治疗非人类灵长类动物的工具

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

报告酶荧光法检测小鼠结核分枝杆菌的影像学

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

marinum 分枝杆菌感染成人斑马鱼结核的建模

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

样品制备

摘要

探索更多视频

此视频中的章节

增长结核分枝杆菌生物膜

监测树突状细胞迁移

实验模型后的自然传播到研究结核病，疟疾混合感染结核分枝杆菌和伯氏疟原虫

展现着多药耐药

关于功能研究的三维人体肺组织模型

系统抑制剂靶向细胞内的疗效和毒性筛选

高通量兼容检测，以评估对巨噬细胞传代药物疗效

分析¹⁸葡萄糖 PET/CT 成像作为研究结核分枝杆菌感染和治疗非人类灵长类动物的工具

报告酶荧光法检测小鼠结核分枝杆菌的影像学

marinum 分枝杆菌感染成人斑马鱼结核的建模