Name: an analytical tool-box for comprehensive biochemical, structural and transcriptome evaluation of oral biofilms mediated by mutans streptococci
Uploaded: 2011-01-25T17:00:00
Description: 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

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...

<ol>
	<li>Costerton, J. W., Stewart, P. S., Greenberg, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilms:+a+common+cause+of+persistent+infections.">Bacterial biofilms: a common cause of persistent infections.</a> Science. 284, 1318-1322 (1999).</li><li>Branda, S. S., Vik, S., Friedman, L., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms:+the+matrix+revisited.">Biofilms: the matrix revisited.</a> Trends Microbiol. 13, 20-26 (2005).</li><li>Leme, P. a. e. s., Koo, A. F., Bellato, H., Bedi, C. M., G, J. A. 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. A., 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=Dynamics+of+Streptococcus+mutans+transcriptome+in+response+to+starch+and+sucrose+during+biofilm+development.">Dynamics of Streptococcus mutans transcriptome in response to starch and sucrose during biofilm development.</a> PLoS ONE. , 0013478-0013478 (2010).</li><li>Lemos, J. A., Abranches, J., Koo, H., Marquis, R. E., Burne, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+to+Study+the+Physiology+of+Oral+Biofilms.">Protocols to Study the Physiology of Oral Biofilms.</a> Methods Mol Biol. 666, 87-102 (2010).</li><li>Koo, H., Hayacibara, M. F., Schobel, B. D., Cury, J. A., Rosalen, P. L., Park, Y. K., Vacca-Smith, A. M., Bowen, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Streptococcus+mutans+biofilm+accumulation+and+polysaccharide+production+by+apigenin+and+tt-farnesol.">Inhibition of Streptococcus mutans biofilm accumulation and polysaccharide production by apigenin and tt-farnesol.</a> J Antimicrob Chemother. 52, 782-789 (2003).</li><li>Koo, H., Seils, J., Abranches, J., Burne, R. A., Bowen, W. H., Quivey, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+apigenin+on+gtf+gene+expression+in+Streptococcus+mutans+UA159.">Influence of apigenin on gtf gene expression in Streptococcus mutans UA159.</a> Antimicrob. Agents Chemother. 50, 542-546 (2006).</li><li>Klein, M. I., Duarte, S., Xiao, J., Mitra, S., Foster, T. H., 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=Structural+and+molecular+basis+of+the+role+of+starch+and+sucrose+in+Streptococcus+mutans+biofilm+development.">Structural and molecular basis of the role of starch and sucrose in Streptococcus mutans biofilm development.</a> Appl Environ Microbiol. 75, 837-841 (2009).</li><li>Cury, J. A., 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=Extraction+and+purification+of+total+RNA+from+Streptococcus+mutans+biofilms.">Extraction and purification of total RNA from Streptococcus mutans biofilms.</a> Anal Biochem. 365, 208-214 (2007).</li><li>Xiao, J., 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=Structural+organization+and+dynamics+of+exopolysaccharide+matrix+and+microcolonies+formation+by+Streptococcus+mutans+in+biofilms.">Structural organization and dynamics of exopolysaccharide matrix and microcolonies formation by Streptococcus mutans in biofilms.</a> J Appl Microbiol. 108, 2103-2113 (2010).</li><li>Thurnheer, T., Gmür, R., Shapiro, S., Guggenheim, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+transport+of+macromolecules+within+an+in+vitro+model+of+supragingival+plaque.">Mass transport of macromolecules within an in vitro model of supragingival plaque.</a> Appl Environ Microbiol. 69, 1702-1709 (2003).</li><li>Chalmers, N. I., Palmer, R. J. J. r., Du-Thumm, L., Sullivan, R., Shi, W., Kolenbrander, P. E. <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+quantum+dot+luminescent+probes+to+achieve+single-cell+resolution+of+human+oral+bacteria+in+biofilms.">Use of quantum dot luminescent probes to achieve single-cell resolution of human oral bacteria in biofilms.</a> Appl Environ Microbiol. 73, 630-636 (2007).</li><li>Deng, D. M., Hoogenkamp, M. A., Ten Cate, J. M. >., Crielaard, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+metabolic+activity+indicator+in+Streptococcus+mutans+biofilms.">Novel metabolic activity indicator in Streptococcus mutans biofilms.</a> J Microbiol Methods. 77, 67-71 (2009).</li></ol>

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>
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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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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

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