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

Авторы хотели бы поблагодарить д-ра Гэри Се и Герберта Ли за развитие MDV. Мы также благодарим д-ра. Симоне Дуарте, Рамиро Murata, Дже-Гю Чон, Жаклин Abranches, и г-жа Грегуар Стейси за их техническим и научным вкладом в аналитические компоненты набора инструментальных средств. Это исследование было поддержано частично USPHS Исследовательский грант DE018023 из Национального института стоматологических и черепно-лицевых исследований.

1. ШАГ 1 - биопробы Биопленки метод использует диски гидроксиапатита (ГА) в качестве суррогатной зуба (Кларксон хроматографии Products, Inc, Южной Уильямспорт, Пенсильвания, США; площадь поверхности = 2,7 ± 0,2 см 2) покрытый слюной (имитируя наличие приобрел пленку), размещаемы?...

<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>

В этой презентации мы показали два важных компонентов аналитического инструмента-Box (EPS / бактерий визуализации и анализа данных микрочипов / обработки), универсальность и полезность различных анализов интегрированы в систему. Очевидно, ящик для инструментов способствовало всеобъемлю...

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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

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and composition 1, 2. In general, biofilms develop from initial microbial attachment on a surface followed by formation of cell clusters (or microcolonies) and further development and stabilization of the microcolonies, which occur in a complex extracellular matrix. The majority of biofilm matrices harbor exopolysaccharides (EPS), and dental biofilms are no exception; especially those associated with caries disease, which are mostly mediated by mutans streptococci 3. The EPS are synthesized by microorganisms (S. mutans, a key contributor) by means of extracellular enzymes, such as glucosyltransferases using sucrose primarily as substrate 3.
Studies of biofilms formed on tooth surfaces are particularly challenging owing to their constant exposure to environmental challenges associated with complex diet-host-microbial interactions occurring in the oral cavity. Better understanding of the dynamic changes of the structural organization and composition of the matrix, physiology and transcriptome/proteome profile of biofilm-cells in response to these complex interactions would further advance the current knowledge of how oral biofilms modulate pathogenicity. Therefore, we have developed an analytical tool-box to facilitate biofilm analysis at structural, biochemical and molecular levels by combining commonly available and novel techniques with custom-made software for data analysis. Standard analytical (colorimetric assays, RT-qPCR and microarrays) and novel fluorescence techniques (for simultaneous labeling of bacteria and EPS) were integrated with specific software for data analysis to address the complex nature of oral biofilm research.
The tool-box is comprised of 4 distinct but interconnected steps (Figure 1): 1) Bioassays, 2) Raw Data Input, 3) Data Processing, and 4) Data Analysis. We used our in vitro biofilm model and specific experimental conditions to demonstrate the usefulness and flexibility of the tool-box. The biofilm model is simple, reproducible and multiple replicates of a single experiment can be done simultaneously 4, 5. Moreover, it allows temporal evaluation, inclusion of various microbial species 5 and assessment of the effects of distinct experimental conditions (e.g. treatments 6; comparison of knockout mutants vs. parental strain 5; carbohydrates availability 7). Here, we describe two specific components of the tool-box, including (i) new software for microarray data mining/organization (MDV) and fluorescence imaging analysis (DUOSTAT), and (ii) in situ EPS-labeling. We also provide an experimental case showing how the tool-box can assist with biofilms analysis, data organization, integration and interpretation.

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

Биопленки образуются на поверхности зубов, являются очень сложными и подвергаются постоянной врожденной и экзогенных экологических проблем, которые модулируют их архитектуре, физиологии и транскриптом. Мы разработали инструментарий для изучения состава, структурной организации и экспрессии генов устных биопленок, которые могут быть адаптированы к другим областям биопленки исследований.

Аналитический инструмент коробки комплексных биохимические, структурные и транскриптома Оценка устного Биопленки посредничестве Mutans стрептококков

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

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