Die Autoren bedanken sich bei Dr. Gary Xie und Herbert Lee für die Entwicklung des MDV danken. Wir danken auch Drs. Simone Duarte, Ramiro Murata, Jae-Gyu Jeon, Jacqueline Abranches und Frau Stacy Gregoire für ihre technischen und wissenschaftlichen Beitrag für die analytische Komponenten der Tool-Box. Diese Studie wurde zum Teil durch USPHS Forschungsstipendium DE018023 vom National Institute of Dental und kraniofaziale Forschung gefördert.

<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>

In dieser Präsentation haben wir gezeigt, zwei kritische Komponenten des Analytical Tool-Box (EPS / Bakterien Bildgebung und Microarray Data Mining / Verarbeitung), die Vielseitigkeit und Brauchbarkeit der verschiedenen Tests in das System integriert. Offensichtlich ist die Tool-Box das umfassende (Vergleich) und die gleichzeitige Analyse der verschiedenen Aspekte der Biofilme Biochemie, Architektur und Genexpression als Reaktion auf die verschiedenen experimentellen Bedingungen mit Hilfe eines in-vitro-Modell...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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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>
<td>&nbsp;</td>
<td>Invitrogen </td>
<td>S11342</td>
<td>&nbsp;</td>
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<td>Dextran conjugated alexa 647</td>
<td>&nbsp;</td>
<td>Invitrogen </td>
<td>D22914</td>
<td>&nbsp;</td>
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<td>Olympus FV1000 two-photon laser scanning microscope</td>
<td>&nbsp;</td>
<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

Biofilme sind hochdynamische, organisiert und strukturiert Gemeinden von mikrobiellen Zellen in eine extrazelluläre Matrix der variablen Dichte und Zusammensetzung 1, 2 verstrickt. In der Regel bilden sich Biofilme von der ersten mikrobiellen Anlage auf einer Fläche durch die Bildung von Zellverbänden (oder Mikrokolonien) und die weitere Entwicklung und Stabilisierung der Mikrokolonien, die in einer komplexen extrazellulären Matrix auftreten gefolgt. Die Mehrheit der Biofilm Matrizen Hafen Exopolysaccharide (EPS) und zahnärztliche Biofilme sind keine Ausnahme, vor allem diejenigen mit Karies Krankheit, die meist durch Streptococcus mutans 3 sind vermittelten verbunden. Die EPS werden von Mikroorganismen (S. mutans, einen wesentlichen Beitrag) durch extrazelluläre Enzyme, wie Glucosyltransferasen Verwendung von Saccharose in erster Linie als Substrat 3 synthetisiert. Studien von Biofilmen auf der Zahnoberfläche gebildet sind besonders anspruchsvoll aufgrund ihrer ständigen Belastung durch Umwelt-Herausforderungen mit komplexen Diät-host-mikrobielle auftretenden Wechselwirkungen in der Mundhöhle verbunden. Ein besseres Verständnis der dynamischen Veränderungen der strukturellen Organisation und Zusammensetzung der Matrix, Physiologie und Transkriptom / Proteom-Profil von Biofilm-Zellen als Antwort auf diese komplexen Wechselwirkungen würden weiter vorantreiben dem aktuellen Wissenstand, wie orale Biofilme modulieren Pathogenität. Deshalb haben wir ein analytisches Werkzeug-Box Biofilm Analyse bei strukturellen, biochemischen und molekularen Ebene zu erleichtern durch die Kombination von allgemein verfügbaren und neuen Techniken mit maßgeschneiderter Software für die Datenanalyse entwickelt. Standard-analytische (kolorimetrische Assays, RT-qPCR und Microarrays) und neuartige Fluoreszenz-Verfahren (für die gleichzeitige Kennzeichnung von Bakterien und EPS) wurden mit spezieller Software für die Datenanalyse, die komplexe Natur der oralen Biofilm-Forschung-Adresse integriert. Das Tool-Box besteht aus 4 verschiedenen, aber miteinander verbundenen Schritten (Abbildung 1) besteht aus: 1) Bioassays, 2) Raw Data Input, 3) Data Processing, und 4) Data Analysis. Wir nutzten unsere in vitro Biofilm-Modell und spezifischen experimentellen Bedingungen, den Nutzen und die Flexibilität der Tool-Box zu demonstrieren. Der Biofilm-Modell ist einfach, reproduzierbar und mehrere Replikate von einem einzigen Experiment kann gleichzeitig 4 erfolgen, 5. Darüber hinaus ermöglicht es zeitliche Auswertung, Einbindung verschiedener Mikroben-Spezies 5 und Beurteilung der Auswirkungen von unterschiedlichen experimentellen Bedingungen (z. B. Behandlungen 6; Vergleich von Knockout-Mutanten vs Elternstamm 5; Kohlenhydrate Verfügbarkeit 7). Hier beschreiben wir zwei spezifische Komponenten der Tool-Box, einschließlich (i) neue Software für Microarray Data Mining / Organisation (MDV) und Fluoreszenz-Imaging-Analyse (DUOSTAT), und (ii) in situ EPS-Kennzeichnung. Wir bieten auch eine experimentelle Fall zeigt, wie das Tool-Box kann mit Biofilmen Analyse, Daten-Organisation, Integration und Interpretation zu unterstützen.

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

Biofilme auf der Zahnoberfläche gebildet sind sehr komplex und in denen konstante angeborenen und exogene ökologischen Herausforderungen, die ihre Architektur modulieren, Physiologie und Transkriptom. Wir entwickelten eine Toolbox, um die Zusammensetzung, die strukturelle Organisation und Genexpression von oralen Biofilmen, die auf andere Bereiche der Biofilm-Forschung angepasst werden können zu prüfen.

Ein analytisches Werkzeug-Box für umfassende biochemische, strukturelle und Transkriptom Evaluation of Oral Biofilme von Mutans Streptokokken Mediated

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 Aufrufe.  University of Rochester Medical Center. Biofilme auf der Zahnoberfläche gebildet sind sehr komplex und in denen konstante angeborenen und exogene ökologischen Herausforderungen, die ihre Architektur modulieren, Physiologie und Transkriptom. Wir entwickelten eine Toolbox, um die Zusammensetzung, die strukturelle Organisation und Genexpression von oralen Biofilmen, die auf andere Bereiche der Biofilm-Forschung angepasst werden können zu prüfen.

Serie: 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.

Umfassende &amp; Kostengünstig Laboratory Überwachung von HIV / AIDS: eine afrikanische 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 ...

Forschung

Immunologie und Infektion

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.

Aufbau des HIV-1 Capsid Assemblies durch Kryo-Elektronenmikroskopie und Iterative Helical Real-Raum Wiederaufbau

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.

Wachstum von Mycobacterium tuberculosis Biofilme

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.

Erkennen von Glykogen in peripheren mononukleären Blutzellen mit Perjodsäure-Schiff-Färbung

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.

Hochdurchsatz-quantitative Echtzeit-RT-PCR-Assay zur Bestimmung Expressionsprofile der Typen I und III Interferon-Formationsglieder

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.

Bereicherung der bakterielle Lipoproteine und Vorbereitung der N-terminalen Lipopeptide Strukturaufklärung durch Massenspektrometrie

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.

Robuste Ligatur-induzierte Modell der Murine Parodontitis für die Bewertung von oralen Neutrophilen

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.

Reinigung eines hochmolekularen Proteins 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 ...

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.

Überwachung des extrazellulären pH-Werts in cross-kingdom Biofilmen mit Konfokalmikroskopie

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.

Experimentelle Virusinfektion bei adulten Stechmücken durch orale Fütterung und Mikroinjektion

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