作者想感谢加里谢博士和赫伯特李马立克氏病的发展。我们也感谢博士。西蒙娜杜阿尔特，拉米罗村田制作所，在奎全度妍，杰奎琳Abranches，斯泰西格雷瓜尔女士对自己的技术和科学的分析工具盒组件的贡献。这项研究是支持的，从牙科和颅面研究所的国家研究所USPHS研究补助金DE018023的一部分。

1。步骤1 - 生物测定生物膜法采用齿替代光盘的羟基磷灰石（“房委会”）（克拉克森色谱产品，公司，南威廉波特，PA，美国;表面积= 2.7 ± 0.2厘米2）用唾液涂（模仿收购薄膜的存在），置于在一个垂直的位置4，5，8。 <ol><li> 生化分析。 <ol><li>生物膜是超声9匀浆（I）或（ii）保存完整， 为 8生化分析。匀浆悬浮生物膜可以用于测定生物量（干重），总蛋白，无机和胞外和胞内多糖成分/内容（见莱莫斯等细节。协议研究的8个口腔生物膜的生理）。完整的生物膜可以使用标准糖酵解的pH值下降，酸杀，质子通透性和F - ATP酶活性测定（ 莱莫斯等细节，协议研究的 8个口腔生物膜的生理）的生理反应进行分析。 </li></ol></li><li> 转录组学分析。 <ol><li>标准cDNA基因芯片和RT - qPCR的协议（例如http://pfgrc.jcvi.org/index.php/microarray/protocols.html）可以组合使用特定病原体（如变形链球菌 ）的转录反应的决心内生物膜的各种环境和治疗在各个发育阶段，6，7，10，11的挑战。在工具箱中，我们包括了两种方法，牙科生物膜的转录组分析的关键：1）RNA的质量（因为生物膜EPS矩阵的人口和存在的异质性），2）数据挖掘和解释（由于大型数据集由芯片产生的）。 <ul><li class="none"> RNA分离。为了解决从生物膜中提取RNA的质量问题，我们使用了一个优化的协议，专门制定了从细菌细胞的RNA分离和EPS 丰富的矩阵12（ http://dx.doi.org/10.1016/j纠缠纯化ab.2007.03.021）。该协议提供了RNA的完整性号码（RIN）大于8.5，这被认为是最佳转录使用RT - qPCR的分析和微阵列（例子，见6，11）。 </li></ul></li></ol></li><li> 荧光成像。生物膜的结构组织 。 <ol><li>文献共聚焦荧光成像协议大多侧重于微生物标签。 EPS作为生物膜的重要组成部分的分析已在很大程度上被忽视口腔生物膜涉及荧光成像技术的研究，除少数例外5，13， 14。我们已经开发出一种新的标签技术和重现的可视化和量化EPS，同时完整的生物膜内细菌细胞的特定的软件。阿米拉5（Visage的影像公司，德国柏林）用于生物膜的三维重建，同时COMSTAT（http://www.imageanalysis.dk）DUOSTAT（http://www.imageanalysis.dk）提供定量分析。 <ol><li>成像的EPS细菌在生物膜的结构组织。的Alexa Fluor 647标记的葡聚糖共轭用于可视化的EPS -矩阵。荧光标记的葡聚糖作为一个链球菌glucosyltransferases Gtfs底漆（尤其是GtfB和GtfC），可同时在生物膜的发展11过程中的胞外多糖基质合成期间成立。 每股收益的标签： <ol><li>过滤灭菌1H唾液大衣羟基磷灰石光盘，下面的“生物膜的制备”协议8。 </li><li>吸取2.8毫升培养液，每孔24孔板中，并带来一个黑暗的房间。 </li><li>到培养液中添加葡聚糖共轭的Alexa Fluor 1微米，到培养液中混合染料吹打（约10倍）。 </li><li>浸洗的唾液涂HA（沙田）光盘（孵育1 h后）两次在无菌AB缓冲区，并放置在含有葡聚糖共轭的Alexa Fluor介质。 </li><li>封面板用铝箔和孵化，在37 ° C，5％的CO 2。孵化时间的长短取决于每一个实验的设计。荧光标记的葡聚糖不染色浓度的细菌细胞，在此作为说11。 其他的EPS染色技术，如Calcofluor 14，可以组合使用。 </li></ol></li><li class="none"> 细菌的标签： <ol><li>在生物膜中的细菌组成部分是在使用标准的荧光核酸染色（如SYTO 9）生物膜形成结束标签;可以使用，当然其他的荧光技术（如物种特异荧光标记 抗体15或表达GFP的细胞16）检测一个或更多的细菌种类，在生物膜。图2显示了同步标签EPS（红色）在一个24小时的老南的三维图像和细菌/ microcolonies（绿色） 变形链球菌生物膜形成一个沙光盘表面上。 </li><li>扫描图像采集：每个生物膜是在5至10个随机选择的职位，以及Z系列光学切片，通过激光扫描共聚焦显微镜使用标准协议的立场产生。我们使用了奥林巴斯FV 1000双光子显微镜（奥林巴斯，日本东京，（卡尔蔡司）与X10配备;数值孔径0.45，工作距离3-4毫米）或X25（奥林巴斯LPlan N，NA 1.05，WD 2毫米）水浸泡目标。激发波长为810纳米，发射波长为祥发9过滤器（菌）540分之495OlyMPFC1过滤，而过滤器的Alexa Fluor 647（EPS）的HQ655/40M-2P过滤。保存和存储原始图像。 </li></ol></li></ol></li></ol></li></ol> 2。第2步 - 输入原始数据从生物化学和RT - qPCR的检测直接进入原始数据文件（RDF的MS Excel文件）输入的原始数据。微阵列数据，加载到JCVI Spotfinder（http://pfgrc.jcvi.org/index.php/bioinformatics.html）或类似软件扫描幻灯片的单通道图像。据JCVI规格，然后手动调整，以适应网格内的所有景点，创建专网。测量每个点的强度值，并保存到“。MeV的”文件，并存储为“原始的微阵列数据”。 3。第3步 - 数据处理组织RDF中的原始数据进行统计分析（生化和RT - qPCR的）。他们转移到“数据处理的文件”（DPF - MS Excel文件）。对于芯片和荧光成像分析，特定的软件（目前可用的和定制）是用来对数据进行处理。 <ol><li> 微阵列数据： <ol><li>提交数据正常化的步骤，使用特定的软件在第2步（存储到“原始的微阵列数据”）所产生的原始数据。规范化数据使用JCVI芯片数据分析软件MIDAS（http://www.tm4.org/midas.html）。使用默认设置LOWESS和标准差正规化，其次是在幻灯片复制分析。包含规范化的数据存储文件。在这个阶段（原始数据文件和规范化的数据文件准备），存款芯片在一个公共访问的数据库（如NCBI的基因表达的综合（GEO）的数据库http://www.ncbi.nlm.nih.gov/geo）的数据，并记录以供将来参考加入。 </li></ol></li><li> 荧光成像数据 <ol><li>生物膜结构定量分析。 <ol><li>每个生物膜的结构成分（EPS和细菌）相应的量化数据计算COMSTAT和新开发的DUOSTAT，其中在MATLAB 5.1编写的脚本。在软件上载原始荧光图像分析如下： <ul><li>使用COMSTAT计算生物量，厚度，层分布和其它参数量化和表征生物膜的立体结构（见http://www.imageanalysis.dk手册）。 </li><li>使用DUOSTAT计算的两个生物膜的组成部分，如EPS和细菌（或不同的微生物物种和/或细胞组成），共定位： <ol><li>设置在MATLAB 5.1 DUOSTAT路径，并打开图像文件夹，其中包括两个生物膜组成的图像。 </li><li>选择两个通道，将相关的分析。两个图像栈还必须在所有三个相同的像素尺寸大小（X，Y，Z），和相同数量的图​​像在每个堆栈。 </li><li>设置每个通道的阈值，根据该名男子UAL的COMSTAT。按照软件的操作界面（见视频文章; DUOSTAT http://www.imageanalysis.dk手册）的指令。输入到数码相框中获得的数据。参见图4.4的例子。 </li></ol></li></ul></li><li>三维生物膜重建。 <ol><li>生物膜的三维结构，可视化使用阿米拉。导入到“原始图像文件夹”到软件中存储的荧光图像，并使用voltex和ISO表面呈现在生物膜中的每个组件创建的3 - D效果图（见http://www.amira.com手册阿米拉/文档/手册和释放notes.html）。参见图4.4的例子。 </li></ol></li></ol></li></ol></li></ol> 4。第4步 - 数据分析生化，RT - qPCR的COMSTAT DUOSTAT检测在DPF的量化数据进行统计分析。进行统计分析后，图形和/或表可以构造（见“代表结果”一节）。 1。微阵列数据组织，使用软件的微阵列数据可视化工具（MDV）。 由于复杂性和大数据量的输出设置使用的芯片和多个实验条件时，我们设计了一个数据挖掘和组织软件名为微阵列数据可视化工具（MDV）7（http://www.oralgen.lanl.gov/提供） 。 使用BRB - ArrayTools（http://linus.nci.nih.gov/BRB-ArrayTools.html）为0.001截止P为类的预测值和类比较进行统计分析后，产生的数据可以提交马立克氏病，如下： <ol><li>使用Excel，广播事业检讨委员会的数据转换成MDV的制表符分隔的文本文件。删除所有的基因位点的标签号码的字母，例如：SMU.1423c，删除字母“C”。 </li><li>打开马立克氏病。转到文件，并选择“选择注释源”。选择“平面文件”。出现对话框时，使用“浏览”按钮选择文件，其中包含基因名称，基因本体论（GO）的数量，途径和功能分类数据的注释。 </li><li>转至文件，并导入每个项目1的文件。每个文件进行分析的实验条件。如果有多个条件进行分析，例如两个不同的治疗（治疗1和2）和控制（车）项目4。 </li><li>在这种情况下，可能比较：（一）治疗1比控制（差异表达基因治疗1），（B）2治疗与控制;（三）处理与治疗2。根据工作的前提，可以选择一套不同的基因。 </li><li>在比较选择唯一的差异表达的基因（独特的基因治疗1只影响）。在这种情况下，用从设置功能菜单中减去功能。从A减去比较乙基因，并保存结果。然后，减去彗星产生的数据。可以做相同的动作只得到B基因（仅适用于治疗2受影响的独特基因）。 </li><li>要选择仅在A和B检测基因，用从设置功能菜单联盟功能，结合A和B进行比较的结果，并保存结果。然后减去从结果数据的彗星。 </li><li>这些行动都可以可视化使用维恩图，马立克氏病，这是更直观的特色，并促进假说驱动的数据组织。维恩图数据输出将显示在屏幕上（见中图3的屏幕和视频文章视图）。 </li><li>数据保存为制表符分隔的文本文件，转到文件，并选择“导出”。 </li><li>导出制表符分隔的文本文件。使用Excel打开它们，并组织马立克氏病在表和/或隐蔽的图形显示的数据输出（参见图4.2，4.3和视频文章）。 </li></ol> 5。代表性的成果在这里，我们提供的分析工具盒如何集成与多个变量和实验条件的生​​物膜研究的各种分析的一个例子。 实验的情况： 变形链球菌在淀粉和蔗糖在生物膜发展7转录的动态。 背景： 主机唾液淀粉酶和链球菌glucosyltransferases饮食中的淀粉和蔗糖的相互作用可以增强 S的形成和毒性由增量生物膜内变形链球菌asing胞外多糖的合成，糖代谢和acidogenicity 11。这复杂的宿主 - 病原体饮食相互作用，可能调节相关龋齿病的致病生物膜的形成。我们进行了全面的生化和转录分析（包括全基因组分析），以进一步了解如何 S. 变形链球菌淀粉和蔗糖生物膜发展的不同阶段，在7淀粉酶的存在。 分析工具盒是用来帮助我们在整合各种实验条件和时间点下形成生物膜的生物化学和分子检测。总体数据输出使用的分析工具盒，提出在图4顺序的方式（4.1至4.4）。值得注意的是这里的主要焦点是要表现出实用的工具盒，而不是数据的解释和讨论。 <ol><li> 结合生物化学和RT - qPCR的检测，为进一步芯片分析选择的实验条件 ，起初，我们测试了6个不同的的蔗糖和5个不同的淀粉和蔗糖的组合的选择由S.生物膜发展的最佳浓度的碳水化合物使用我们的体外模型的变形链球菌。能力的考查，同时指导我们选择三个具体的浓度（在图4.1中显示的）的基础上（高架）不溶性胞外多糖量和生物膜（增强型）gtfB表达（不溶性葡聚糖合成）的生物测定数据输出。 </li><li> 微阵列分析。选定（三）饮食中碳水化合物的浓度被用来形成生物膜，这是反映了生物膜的发展过程中的各个阶段，在特定的时间点中删除。生物膜（分为3个实验组和4个时间点）结合BRB阵列工具和MDV全基因组分析（cDNA微阵列）的转录组学分析。 RNA的提取和纯化生物膜具体协议12，然后受到芯片分析分析工具盒，通过4个步骤的进程。图4.2说明的马立克氏病是如何帮助选择感兴趣的基因，根据我们的工作假说，蔗糖和淀粉的结合触发与增强毒力S.（致龋潜力）相关的特异性转录反应变形链球菌内生物膜。 BRB - ArrayTools（图4.2A）所产生的原始数据进行处理，马立克氏病病毒使用的维恩图（图4.2B）。在这项研究中，我们的假说有关的基因组比较差异表达的一个（0.5％蔗糖+ 1％的淀粉与1％蔗糖）和B（0.5％蔗糖+ 1％的淀粉与0.5％蔗糖），但不是在比较彗星的基因（0.5％蔗糖与1％蔗糖）（图4.2A和4.2B）。如图4.2c所示，马立克氏病大大减少总数的基因进行分析，同时筛选出结合蔗糖和淀粉的影响没有直接关系的基因。马立克氏病的输出数据也显示差异表达的基因的功能类中的淀粉（向上和向下调节）举办数据转换成图形显示+蔗糖生物膜（与蔗糖种植生物膜），在每个4个时间点（图4.3）。马立克氏病病毒软件是用户友好，促进了采矿/组织的庞大而复杂的的数据，从我们的微阵列实验套。 </li><li> 生物膜成像随之而来，生物膜的组织结构进行了分析，使用COMSTAT DUOSTAT，阿米拉包括在工具框中（参见图4.4淀粉的三维重建和定量分析的一个例子+蔗糖种植生物膜;也看到视频文章）。 EPS和microcolonies的形态，分布和结构关系，可以可视化使用阿米拉三维表面渲染。可以生成选定区域特写意见，这说明在微尺度特定细菌的EPS的结构关系（图4.4）。此外，同一组的共聚焦图像，可以处理COMSTAT生物质能/ microcolony测量，空间分布和细菌细胞共定位和EPS同时DUOSTAT。例如，从光盘表面流体相EPS和细菌的垂直分布是从每一个立体的共聚焦生物膜图像使用COMSTAT DUOSTAT的光部分（图4.4计算;看到视频文章）。数据显示，更高的比例比整个生物膜深入的细菌（绿线）EPS（红线）。此外，大多数细菌细胞与EPS（蓝线），特别是在中层和外层的生物膜。这一观察结果表明，淀粉和蔗糖种植的生物膜exopolymers，特别是在丰富的，这是enmeshing（在这样的组织结构）细菌细胞的最密切联系;提高生物膜 13的稳定和凝聚力。共聚焦图像的三维效果图和定量测量提供有关生物膜的结构，补充的生化和基因表达数据（增加GtfB型不溶性葡聚糖合成变形链球菌淀粉+蔗糖种植生物膜）的其他信息（更多的例子参见图5，6，11，13）。 分析工具框帮助我们获取，组织和整合的数据，从不同的生物测定，提供了一个全面的分析如何 S. 变形链球菌的反应可能在口腔中发现的饮食-宿主相互作用的结果作为一个复杂的环境变化 （见Klein等人，2009年11月 ;。Klein 等人，2010年7）。 </li></ol><img src="/files/ftp_upload/2512/2512fig1.jpg" alt="图1" /> 图1。流程图生物膜分析的分析工具盒。 <img src="/files/ftp_upload/2512/2512fig2.jpg" alt="图2" /> 图2。EPS和在生物膜中的细菌的共聚焦荧光成像。同时可视化的EPS（红色），在三维渲染沙光盘表面上形成的变形链球菌生物膜的细菌/ microcolonies（绿色）。 <img src="/files/ftp_upload/2512/2512fig3.jpg" alt="图3" /> 图3。芯片数据挖掘和组织使用的微阵列数据可视化工具（MDV）软件。维恩图功能，使用，选择感兴趣的基因，基因名称和功能的类注释此外。 <img src="/files/ftp_upload/2512/2512fig4.1.jpg" alt="图4.1" /> 图4.1由S.生物膜形成的评价 。使用生化分析（A）和RT - qPCR的（乙） 的变形链球菌。 （不溶性胞外多糖的INS）的数据以及相关 gtfB的表达模式，并与生物膜的生物量。在1％的蔗糖浓度最大移民局形成，gtfB的表达和对沙面的生物膜的积累，而0.5％的蔗糖是最佳生物膜发展所需的最低浓度在体外模型中使用由变形链球菌细胞中存在0.5％的蔗糖种植+ 1％的淀粉产生的生物量最高，并提出了增强 gtfB表达（B2）相关的其他生物膜，移民局超过。这些碳水化合物浓度被选作进一步的转录组学分析。 <img src="/files/ftp_upload/2512/2512fig4.2.jpg" alt="图4.2" /> 图4.2微阵列数据分析与马立克氏病病毒软件一起使用BRB -阵列工具。 a）代表的检测，在每个比较（A，B或C）和使用BRB -阵列工具评估的时间点差异表达的基因数目。 B）使用微阵列数据可视化工具（MDV）维恩图来选择感兴趣的基因。 C）基因的选择根据马立克氏病病毒的分析。 <img src="/files/ftp_upload/2512/2512fig4.3.jpg" alt="图4.3" /> 图4.3。S。 变形链球菌的基因差异表达淀粉+蔗糖生物膜功能类组织在不同的时间点（与蔗糖的生物膜）。基因注释的基础上的洛斯阿拉莫斯国家实验室（www.oralgen.lanl.gov）或发表的文献在同一网站提供的信息。 <img src="/files/ftp_upload/2512/2512fig4.4.jpg" alt="图4.4" /> 图4.4三维渲染和COMSTAT DUOSTAT分析淀粉+蔗糖生物膜。

<ol>
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C. u. r. y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+sucrose+in+cariogenic+dental+biofilm+formation--new+insight.">The role of sucrose in cariogenic dental biofilm formation--new insight.</a> J Dent Res. 85, 878-887 (2006).</li><li>Koo, H., Schobel, B. D., Scott-Annem, K., Watson, G., Bowen, W. H., Cury, J. A., Rosalen, P. L., Park, Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apigenin+and+tt-farnesol+with+fluoride+effects+on+S.+mutans+biofilms+and+dental+caries.">Apigenin and tt-farnesol with fluoride effects on S. mutans biofilms and dental caries.</a> J Dent Res. 84, 1016-1020 (2005).</li><li>Koo, H., Xiao, J., Klein, M. I., Jeon, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exopolysaccharides+produced+by+Streptococcus+mutans+glucosyltransferases+modulate+the+establishment+of+microcolonies+within+multispecies+biofilms.">Exopolysaccharides produced by Streptococcus mutans glucosyltransferases modulate the establishment of microcolonies within multispecies biofilms.</a> J Bacteriol. 192, 3024-3032 (2010).</li><li>Jeon, J. G., Klein, M. I., Xiao, J., Gregoire, S., Rosalen, P. L., Koo, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influences+of+naturally+occurring+agents+in+combination+with+fluoride+on+gene+expression+and+structural+organization+of+Streptococcus+mutans+in+biofilms.">Influences of naturally occurring agents in combination with fluoride on gene expression and structural organization of Streptococcus mutans in biofilms.</a> BMC Microbiol. 9, 228-228 (2009).</li><li>Klein, M. I., DeBaz, L., Agidi, S., Lee, H., Xie, G., Lin, A. H. M., Hamaker, B. R., Lemos, J. 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在此演示中，我们展示了两个分析工具盒（EPS /细菌成像和微阵列数据挖掘/处理），集成到系统中的各种检测的通用性和实用性的重要组成部分。显然，工具盒促进生物膜的生物化学，建筑和基因表达的各个方面的综合（比较），并同时分析，针对不同的实验条件下使用的体外模型。考虑的动态而复杂的变化组织结构，生理和转录反应由S.在生物膜中的变形链球菌（和其他病?...

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<td>Syto 9</td>
<td>&nbsp;</td>
<td>Invitrogen </td>
<td>S34854</td>
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<td>Syto 60</td>
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<td>Invitrogen </td>
<td>S11342</td>
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<td>Dextran conjugated alexa 647</td>
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<td>Invitrogen </td>
<td>D22914</td>
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<td>Olympus FV1000 two-photon laser scanning microscope</td>
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<td>Olympus, Tokyo, Japan</td>
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an analytical tool-box for comprehensive biochemical, structural and transcriptome evaluation of oral biofilms mediated by mutans streptococci

生物膜是高度动态的沉浸在一个变量的密度和组成1，2的细胞外基质的微生物细胞，组织和结构社区。在一般情况下，生物膜的发展从最初的微生物附着在表面形成细胞簇（或microcolonies）进一步发展和稳定的，它发生在一个复杂的胞外基质的microcolonies。大多数生物膜矩阵海港胞外多糖（EPS）和牙科生物膜也不例外，特别是那些与龋齿的疾病，其中大多是由变形链球菌3介导的相关。 EPS的合成微生物（ 变形链球菌 ，一个关键因素），由胞外酶的手段，如使用主要是蔗糖为底物3的glucosyltransferases。 在牙齿表面形成生物膜的研究是特别具有挑战性的，因为他们经常接触到复杂的饮食主机微生物的相互作用发生在口腔的环境挑战。口腔生物膜如何调节的致病性，更好地理解组织结构和矩阵组成，生理和转录/生物膜细胞的蛋白质组的个人资料，在应对这些复杂的相互作用的动态变化，将进一步推进现有的知识。因此，我们开发了一个分析工具框，以方便常用的和新颖的技术相结合，与定制的数据分析软件的生物膜在结构，生化和分子水平上的分析。标准分析（比色法，RT - qPCR的芯片）和新颖的荧光技术（同时细菌和EPS标签）进行了整合与特定的软件进行数据分析，以解决口腔生物膜研究的复杂性。 工具盒是由4个不同但相互关联的步骤（图1）：1）生物测定，2）原始数据输入，3）数据处理，以及4）数据分析。我们用在体外生物膜模型和具体的实验条件下的演示工具盒的实用性和灵活性。生物膜模型很简单，重现性好，多重复一次实验可同时完成4，5。此外，它允许时间的评估，各种微生物物种列入 5和不同的实验条件的影响的评估（如治疗6;敲除突变体与亲本菌株5的比较 ;碳水化合物可用性7）。在这里，我们描述了两个工具盒的具体组成部分，包括：（一）对芯片数据挖掘/组织（MDV）和荧光成像分析（DUOSTAT）的新的软件，以及（ii）在原位的EPS标签。我们还提供了一个实验性的案例，说明如何可以协助生物膜分析，数据组织，整合和解释工具盒。

Watch this Scientific Journal Video about An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci at JoVE.com

An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci

在牙齿表面形成的生物膜是非常复杂和暴露于恒定的先天和外生的环境挑战，调节它们的架构，生理和转录。我们制定了一个工具箱，研究的成分，组织结构和基因表达口腔生物膜，生物膜研究的其他领域可适应。

口腔生物膜的生物化学，结构和转录综合评价分析工具盒介导变形链球菌

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and ...

an-analytical-tool-box-for-comprehensive-biochemical-structural

Research

JoVE Journal

Immunology and Infection

17.6K 次观看.  University of Rochester Medical Center. 在牙齿表面形成的生物膜是非常复杂和暴露于恒定的先天和外生的环境挑战，调节它们的架构，生理和转录。我们制定了一个工具箱，研究的成分，组织结构和基因表达口腔生物膜，生物膜研究的其他领域可适应。

视频: An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci

Comprehensive &amp; Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical large scale diagnostic testing. In the low-income countries inexpensive ART has transformed the prospects for the survival of HIV seropositive patients but there are doubts whether there is a need for the laboratory monitoring of ART and at what costs - in situations when the overall quality of pathology services can still be very low. The appropriate answer is to establish economically sound services with better coordination and stricter internal quality assessment than seen in western countries. This video, photographed at location in the National Health Laboratory Services (NHLS-SA) at the Witwatersrand University, Johannesburg, South Africa, provides such a coordinated scheme expanding the original 2-color CD4-CD45 PanLeucoGating strategy (PLG). Thus the six modules of the video presentation reveal the simplicity of a 4-color flow cytometric assay to combine haematological, immunological and virology-related tests in a single tube. These video modules are: (i) the set-up of instruments; (ii) sample preparations; (iii) testing absolute counts and monitoring quality for each sample by bead-count-rate; (iv) the heamatological CD45 test for white cell counts and differentials; (v) the CD4 counts, and (vi) the activation of CD8+ T cells measured by CD38 display, a viral load related parameter. The potential cost-savings are remarkable. This arrangement is a prime example for the feasibility of performing &gt; 800-1000 tests per day with a stricter quality control than that applied in western laboratories, and also with a transfer of technology to other laboratories within a NHLS-SA network. Expert advisors, laboratory managers and policy makers who carry the duty of making decisions about introducing modern medical technology are frequently not in a position to see the latest technical details as carried out in the large regional laboratories with huge burdens of workload. Hence this video shows details of these new developments.

Anti-retroviral therapy to treat HIV/AIDS is monitored in South Africa on a large scale. Flow cytometry is combined for haematology (CD45), immunology (CD4) and viral-load linked CD38 assay. Recorded at NHLS-SA laboratories, Johannesburg, these modern methods are cost-efficient with heightened local internal quality control, serving as role-models for resource-limited diagnostics.

艾滋病毒/艾滋病的综合成本有效的实验室监测：非洲的角色模型

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical ...

科研

免疫与感染

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein complexes and assemblies. In fact, single particle electron microscopy1 and two-dimensional (2D) electron crystallography2 have become relatively routine methodologies and a large number of structures have been solved using these methods. At the same time, image processing and three-dimensional (3D) reconstruction of helical objects has rapidly developed, especially, the iterative helical real-space reconstruction (IHRSR) method3, which uses single particle analysis tools in conjunction with helical symmetry. Many biological entities function in filamentous or helical forms, including actin filaments4, microtubules5, amyloid fibers6, tobacco mosaic viruses7, and bacteria flagella8, and, because a 3D density map of a helical entity can be attained from a single projection image, compared to the many images required for 3D reconstruction of a non-helical object, with the IHRSR method, structural analysis of such flexible and disordered helical assemblies is now attainable.
In this video article, we provide detailed protocols for obtaining a 3D density map of a helical protein assembly (HIV-1 capsid9 is our example), including protocols for cryo-EM specimen preparation, low dose data collection by cryo-EM, indexing of helical diffraction patterns, and image processing and 3D reconstruction using IHRSR. Compared to other techniques, cryo-EM offers optimal specimen preservation under near native conditions. Samples are embedded in a thin layer of vitreous ice, by rapid freezing, and imaged in electron microscopes at liquid nitrogen temperature, under low dose conditions to minimize the radiation damage. Sample images are obtained under near native conditions at the expense of low signal and low contrast in the recorded micrographs. Fortunately, the process of helical reconstruction has largely been automated, with the exception of indexing the helical diffraction pattern. Here, we describe an approach to index helical structure and determine helical symmetries (helical parameters) from digitized micrographs, an essential step for 3D helical reconstruction. Briefly, we obtain an initial 3D density map by applying the IHRSR method. This initial map is then iteratively refined by introducing constraints for the alignment parameters of each segment, thus controlling their degrees of freedom. Further improvement is achieved by correcting for the contrast transfer function (CTF) of the electron microscope (amplitude and phase correction) and by optimizing the helical symmetry of the assembly.

This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.

HIV - 1衣壳大会通过低温电子显微镜的结构和迭代螺旋实时空间重构

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein ...

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 ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

与碘酸雪夫染色检测糖原的外周血单个核细胞

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

高通量实时定量RT-PCR检测方法的确定I型和III型干扰素亚型的表达谱

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their significance to both cell physiology and immunology, much remains to be discovered about novel lipoprotein forms, how they are synthesized, and the effect of the various forms on host immunity. To enable thorough studies on lipoproteins, this protocol describes a method for bacterial lipoprotein enrichment and preparation of N-terminal tryptic lipopeptides for structural determination by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Expanding on an established Triton X-114 phase partitioning method for lipoprotein extraction and enrichment from the bacterial cell membrane, the protocol includes additional steps to remove non-lipoprotein contaminants, increasing lipoprotein yield and purity. Since lipoproteins are commonly used in Toll-like receptor (TLR) assays, it is critical to first characterize the N-terminal structure by MALDI-TOF MS. Herein, a method is presented to isolate concentrated hydrophobic peptides enriched in N-terminal lipopeptides suitable for direct analysis by MALDI-TOF MS/MS. Lipoproteins that have been separated by Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) are transferred to a nitrocellulose membrane, digested in situ with trypsin, sequentially washed to remove polar tryptic peptides, and finally eluted with chloroform-methanol. When coupled with MS of the more polar trypsinized peptides from wash solutions, this method provides the ability to both identify the lipoprotein and characterize its N-terminus in a single experiment. Intentional sodium adduct formation can also be employed as a tool to promote more structurally informative fragmentation spectra. Ultimately, enrichment of lipoproteins and determination of their N-terminal structures will permit more extensive studies on this ubiquitous class of bacterial proteins.

The enrichment of bacterial lipoproteins using a non-ionic surfactant phase partitioning method is described for direct use in TLR assays or other applications. Further steps are detailed to prepare N-terminal tryptic lipopeptides for structural characterization by mass spectrometry.

质谱法富集细菌脂蛋白及 n-端脂肽的制备结构测定

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their ...

Robust Ligature-Induced Model of Murine Periodontitis for the Evaluation of Oral Neutrophils

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically modified strains, the vast number of analyses that can be performed on harvested soft and hard tissues. However, many of these systems are subject to procedural criticisms. As an alternative, the ligature-induced model of periodontal disease, driven by the localized development and retention of a dysbiotic oral microbiome, can be employed, which is rapidly induced and relatively reliable. Unfortunately, the variants of ligature-induced murine periodontitis protocol are isolated to focal regions of the periodontium and subject to premature avulsion of the installed ligature. This minimizes the amount of tissue available for subsequent analyses and increases the number of animals required for study. This protocol describes the precise manipulations required to place extended molar ligatures with improved retention and usage of a novel rinse technique to recover oral neutrophils in mice with an alternative approach that mitigates the aforementioned technical challenges.

This article presents a protocol for establishing a ligature-induced model of murine periodontitis involving multiple maxillary molars, resulting in larger areas of the involved gingival tissue and bone for subsequent analysis as well as reduced animal usage. A technique to assess oral neutrophils in a manner analogous to human subjects is also described.

用于口腔中性粒细胞病评价的鼠期牙周炎的强结合诱导模型

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically ...

Purification of a High Molecular Mass Protein in Streptococcus mutans

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has been disrupted. Loss of function following gene disruption is subsequently restored by exogenous addition of the product of the disrupted gene. This helps to determine the function of the gene. A method previously described involves generating a gtfC gene-disrupted Streptococcus mutans strain. Here, an undemanding method is described for purifying the gtfC gene product from the newly generated S. mutans strain following the gene disruption. It involves the addition of a polyhistidine-coding sequence at the 3&#8242; end of the gene of interest, which allows simple purification of the gene product using immobilized metal affinity chromatography. No enzymatic reactions other than PCR are required for the genetic modification in this method. The restoration of the gene product by exogenous addition after gene disruption is an efficient method for determining gene function, which may also be adapted to different species.

Purification of a High Molecular Mass Protein in Streptococcus mutans

Described here is a simple method for the purification of a gene product in Streptococcus mutans. This technique may be advantageous in the purification of proteins, especially membrane proteins and high molecular mass proteins, and can be used with various other bacterial species.

链球菌中高分子质量蛋白的纯化

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has ...

Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, periodontitis, mucosal infections and, most notably, early childhood caries. In all of these conditions, the pH in the biofilm matrix impacts microbe-host interactions and thus the disease progression. The present protocol describes a confocal microscopy-based method to monitor pH dynamics inside cross-kingdom biofilms comprising Candida albicans and Streptococcus mutans. The pH-dependent dual-emission spectrum and the staining properties of the ratiometric probe C-SNARF-4 are exploited to determine drops in pH in extracellular areas of the biofilms. Use of pH ratiometry with the probe requires a meticulous choice of imaging parameters, a thorough calibration of the dye, and careful, threshold-based post-processing of the image data. When used correctly, the technique allows for the rapid assessment of extracellular pH in different areas of a biofilm and thus the monitoring of both horizontal and vertical pH gradients over time. While the use of confocal microscopy limits Z-profiling to thin biofilms of 75 &#181;m or less, the use of pH ratiometry is ideally suited for the noninvasive study of an important virulence factor in cross-kingdom biofilms.

The protocol describes the cultivation of cross-kingdom biofilms consisting of Candida albicans and Streptococcus mutans and presents a confocal microscopy-based method for the monitoring of extracellular pH inside these biofilms.

使用共聚焦显微镜监测跨王国生物膜中的细胞外pH值

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, ...

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public health. Once ingested, the viruses must overcome the mosquito midgut barrier to reach the hemolymph, from where they might potentially spread to the salivary glands. When a mosquito bites, these viruses are spread to new vertebrate hosts. Similarly, the mosquito may pick up different viruses. In general, only a tiny portion of viruses may enter the salivary glands via the gut. The transmission efficiency of these viruses to the glands is be affected by the two physical barriers found in different mosquito species: midgut barriers and salivary glands barriers. This protocol presents a method for virus detection in salivary glands of Aedes aegypti&#39;s&#160;following oral feeding and intrathoracic injection infection. Furthermore, determining whether the guts and/or salivary glands hinder viral spread can aid in the risk assessments of MBVs transmitted by Aedes aegypti.

This methodology, which included oral feeding and intrathoracic injection infection, could effectively assess the influence of midgut and/or salivary gland barriers on arbovirus infection.

经口喂养和显微注射对成年蚊子进行实验性病毒感染

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public ...