저자 MDV의 발전을 위해 박사 게리 샤오와 허버트 리에게 감사를 표합니다. 우리는 또한 DRS 감사합니다. 시몬 두아르테, 라미 무라타, 재 - Gyu 전, 재클린 Abranches 및 공구 상자의 분석 구성 요소에 대한 자신의 기술과 과학에 대한 기여 미스 스테이시 그레 고아. 이 연구는 치과와 Craniofacial 연구의 국립 연구소에서 USPHS 연구 부여 DE018023에 의해 부분적으로 지원되었다.

<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>

관심 없음 충돌 선언하지 않습니다.

이 프레 젠 테이션에서, 우리는 분석 도구 - 박스 (EPS / 세균 이미징 및 microarray 데이터 마이닝 / 처리) 시스템에 통합된 다양한 assays의 다목적 및 유용의 두 중요한 구성 요소를 보여주었다. 분명히, 공구 상자 체외 모델을 사용하여 다양한 실험 조건에 대한 응답으로 biofilms 생화학, 아키텍처 및 유전자 발현의 다양한 측면의 종합적인 (비교) 및 동시 분석을 촉진. 7의 역동적이고 복?...

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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>
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<td>Dextran conjugated alexa 647</td>
<td>&nbsp;</td>
<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 변수 밀도 및 구성 1, 2의 세포외 매트릭스에 enmeshed 미생물 세포의 높은 동적 조직 및 구조 커뮤니티입니다. 일반적으로, biofilms는 세포 클러스터 (또는 microcolonies)과 복잡한 세포외 기질에서 발생하는 microcolonies의 발전과 안정의 형성에 의해 다음 표면에 초기 미생물의 첨부 파일에서 개발할 수 있습니다. biofilm의 매트릭스 하버 exopolysaccharides (EPS) 및 치과 biofilms의 대부분도 예외 없다, 특히 대부분의 mutans streptococci 3 중재 아르 카리 에스 질환과 관련된 사람. EPS는 등 주로 기판 3으로 자당을 사용 glucosyltransferases 같은 세포외 효소의 방법으로 미생물 (S. mutans의, 핵심적인 기여)에 의해 합성됩니다. 치아 표면에 형성 biofilms의 연구 특히 구강에서 발생하는 복잡한 식단 - 호스트 미생물 상호 작용과 관련된 환경 문제들의 지속적인 노출 때문에 도전하고 있습니다. 이러한 복잡한 상호 작용에 대한 응답으로 구조 조직과 모체의 조성, 생리학 및 transcriptome / biofilm - 세포의 프로테옴 프로파일의 역동적인 변화의 이해는 더 이상 구두 biofilms가 pathogenicity을 조절하는 방법의 현재 지식을 사전 것입니다. 따라서, 우리는 데이터 분석에 대한 사용자 정의 - 만든 소프트웨어를 사용하여 일반적으로 쉽게 구할 수 있고, 새로운 기술을 결합하여, 구조 생화학 및 분자 수준에서 biofilm 분석을 촉진하는 도구 박스 분석을 개발했습니다. 표준 분석 (colorimetric assays, RT - qPCR 및 microarrays)과 소설 형광 기법 (박테리아와 EPS의 동시 라벨)을 경구 biofilm 연구의 복잡한 성격을 해결하기 위해 데이터 분석을위한 특정 소프트웨어와 통합되었습니다. 공구 상자 4 별개지만, 서로 단계 (그림 1)으로 구성되어 있습니다 : 1) Bioassays, 2) 원시 데이터 입력, 3) 데이터 처리, 4) 데이터 분석. 우리는 공구 상자의 유용 성과 유연성을 입증하기 위해 체외 biofilm 모델과 구체적인 실험 조건에서 우리를 사용합니다. biofilm 모델은 간단하고 재현성 및 여러됩니다 것은 하나의 실험의 복제는 동시에 4 5을 수행할 수 있습니다. 또한, 그것은 수 있도록 시간적 평가, 각종 미생물 종의 포함 5 별개의 실험 조건의 영향 평가 (예 : 트리 트먼트 6, 녹아웃의 돌연변이의 비교 대 부모의 변형 5; 탄수화물 가능한 7). 여기, 우리는 microarray 데이터 마이닝 / 조직 (MDV)과 형광 이미징 분석 (DUOSTAT)에 대한 새로운 소프트웨어 (I)를 포함하여 공구 상자를 두 가지 특정 구성 요소를 설명하고, (2) EPS - 라벨 원위치 인치 우리는 또한 공구 상자가 biofilms 분석, 데이터 구성, 통합 및 해석 도와 수있는 방법을 보여주는 실험 사례를 제공합니다.

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

치아 표면에 형성 Biofilms는 매우 복잡하고 그들의 아키텍처를 조절 상수 타고난 및 외인성 환경 문제, 생리 및 transcriptome에 노출되어 있습니다. 우리는 조성, 구조 조직 및 biofilm 연구의 다른 분야에 적용할 수 있습니다 경구 biofilms의 유전자 발현을 검사하는 도구 상자를 개발했습니다.

구강 Biofilms의 종합, 생물 구조 및 Transcriptome 평가를위한 분석 도구 - 상자가 Mutans Streptococci의 중재

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 조회수.  University of Rochester Medical Center. 치아 표면에 형성 Biofilms는 매우 복잡하고 그들의 아키텍처를 조절 상수 타고난 및 외인성 환경 문제, 생리 및 transcriptome에 노출되어 있습니다. 우리는 조성, 구조 조직 및 biofilm 연구의 다른 분야에 적용할 수 있습니다 경구 biofilms의 유전자 발현을 검사하는 도구 상자를 개발했습니다.

비디오: 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.

HIV / AIDS의 종합 및 비용 효과적인 실험실 감시 : 아프리카 역할 모델

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

연구

면역학 및 감염병학

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.

Cryo - 전자 현미경에 의해 HIV - 1 Capsid 어셈블리의 구조와 반복 나선형 실제 공간 재건

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.

의 성장 결핵은 Mycobacterium Biofilms

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.

요오드 산 쉬프 염색 말초 혈액 단핵 세포에서 글리코겐을 감지

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.

높은 처리량 정량 실시간 RT-PCR 분석 유형 I과 III 인터페론 하위 유형의 발현 양상을 결정하는

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 ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

변조 C 디 GMP가에서 시그널링 그건 작은 분자를 스크리닝하기위한 높은 처리량 설정 구축<em&gt; 녹농균</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

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.

세균성 단백질 질량 분석에 의하여 구조 결심에 대 한 N 맨끝 Lipopeptides의 준비의 농축

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.

구강 호중구의 평가를 위한 뮤린 치주염의 강력한 합자 유도 모형

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.

연쇄상 구균 뮤탄에서 높은 분자 질량 단백질의 정화

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.