기금은 NIH / NIAID 부여 RO1 - AI - 63457와 아라 부여 보완 RO1 - AI - 63457 S1에 의해 제공되었다. 우리는 실험실 그룹과 공동 작업의 모든 현재 및 과거 회원의 기여를 인정합니다.

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapy+of+leprosy+for+control+programmes.">Chemotherapy of leprosy for control programmes.</a> Technical Report Series. 675, 18-22 (1982).</li><li>Cole, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massive+gene+decay+in+the+leprosy+bacillus.">Massive gene decay in the leprosy bacillus.</a> Nature. 409, 1007-1011 (2001).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leproma+Web+Server.">Leproma Web Server.</a> Leproma Web Server [Internet]. , (2004).</li><li>Groathouse, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+Polymorphic+Loci+for+Molecular+Typing+of+Strains+of+Mycobacterium+leprae.">Multiple Polymorphic Loci for Molecular Typing of Strains of Mycobacterium leprae.</a> J. of Clinical Microbiology. 42, 1666-1672 (2004).</li><li>Young, S. K. <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+Short+Tandem+Repeat+Sequences+to+Study+Mycobacterium+leprae+in+Leprosy+Patients+in+Malawi+and+India.">Use of Short Tandem Repeat Sequences to Study Mycobacterium leprae in Leprosy Patients in Malawi and India.</a> Public Library of Science: Neglected Tropical Diseases. 2, E214-E214 (2008).</li><li>Monot, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+Variable-Number+Tandem+Repeats+Appropriate+for+Genotyping+Mycobacterium+leprae.">Are Variable-Number Tandem Repeats Appropriate for Genotyping Mycobacterium leprae.</a> J. of Clinical Microbiology. 46, 2291-2297 (2008).</li><li>Kimura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Variable+Number+Tandem-Repeat+Genotyping+for+Mycobacterium+leprae+Clinical+Specimens.">Rapid Variable Number Tandem-Repeat Genotyping for Mycobacterium leprae Clinical Specimens.</a> J. of Clinical Microbiology. 47, 1757-1766 (2009).</li><li>Weng, X. i. a. o. m. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+Distribution+of+Mycobacterium+leprae+Genotypes+in+a+Region+of+High+Leprosy+Prevalence+in+China:+a+3-Year+Molecular+Epidemiological+Study.">Identification and Distribution of Mycobacterium leprae Genotypes in a Region of High Leprosy Prevalence in China: a 3-Year Molecular Epidemiological Study.</a> J. of Clinical Microbiology. 45, 1728-1734 (2007).</li><li>Weng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+of+leprosy+in+Qiubei+County,+Yunnan,+China:+Insights+from+an+eight+year+molecular+epidemiology+investigation.">Transmission of leprosy in Qiubei County, Yunnan, China: Insights from an eight year molecular epidemiology investigation.</a> Infection, Genetics and Evolution. , (2010).</li><li>Sakamuri, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Population-Based+Molecular+Epidemiology+of+Leprosy+in+Cebu,+Philippines.">Population-Based Molecular Epidemiology of Leprosy in Cebu, Philippines.</a> J. of Clinical Microbiology. 47, 2844-2854 (2009).</li><li>Fontes, A. N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+diversity+of+Mycobacterium+leprae+isolates+from+Brazilian+leprosy+patients.">Genetic diversity of Mycobacterium leprae isolates from Brazilian leprosy patients.</a> Leprosy Review. 80, 302-315 (2009).</li></ol>

관심 없음 충돌 선언하지 않습니다.

나병 환자의 피부 샘플 컬렉션 피부 진료소에서 일하는 숙련된 임상 또는 기술자가 필요합니다. 이 샘플을 처리 실험실 종사자 실험실 외투, 장갑 및 보호 눈 착용을 착용하고 인간이나 아르마 딜로 조직의 감염 샘플을 처리하는 바이오 안전 캐비넷에서 작업에 큰주의를해야합니다. 표면 및 도구의 소독도 중요합니다. 깨끗하고 살균 바이오 안전 캐비넷에서 작업하는 것은 DNA 샘플의 오염을 피하는 것이 중요합니다. DNA 추출은 Qiagen 같은 회사에서 추출 키트의 발전에 상대적으로 쉽게 덕분되고 있습니다. 약도는 신중하게 따라야합니다. 사용하지 않을 때는 모든 샘플은 추운 보관해야합니다. 샘플에 대한 반복, 극단적인 온도 변화를 피하십시오. 이 작품에 사용되는 DNA의 primers는이 문서의 끝부분에 문학 참조 목록에서 인용되었습니다 여러 업체에서 주문하실 수 있습니다. 케어는 오염을 방지하기 위해 DNA 무료 환경에서 primers 작업로 이동해야합니다. Primers가 건조 분말로 와서 TE와 혼합 및 100μM 농도로 희석해야 다음 작은 양이 (그림 2A)로 구분됩니다. primers의 작업 솔루션은 더욱 10μM 농도로 희석하고 있습니다. 다시 말하지만, 영역 / primers를 사용하고 준비 후즈에서 DNA 샘플을 사용하지 마십시오. PCR 제품도 사용하지 않을 때 감기에 보관해야합니다. 3 % 아가로 오스 겔 준비 더 DNA 밴드 분리를 제공하지만, 실행하는 데 오래 걸립니다. 순수하게 정성을 위해, 2 %는 일반적으로 충분합니다. 아가로 오스 젤에서 DNA를 얼룩에 사용 Ethidium의 브로마이드 솔루션은 피부를 통해 흡수되어 매우 활성화 genotoxin입니다. 이 솔루션과 아주 잘 사용하고 항상 장갑을 착용해야되는 스테인드 젤을 처리합니다. 모든 DNA 샘플이나 ethidium 브로마이드 자료를 처리 후에는 깨끗이 손을 씻으십시오. ethidium의 브로마이드 얼룩 솔루션의 폐기, 기관 지침에 따라 세척 및 젤류. 젤이 정기적으로 필요하지 않습니다, FLA 방법이 설립되면이 단계는 제거하실 수 있습니다. 그것은 시간과 시약이 소요됩니다. FLA에 대한 샘플을 준비에 사용되는 포름 아미드 솔루션은 또한 매우 유독 신경을 써서 처리한다. 의 사용 후 손을 씻으십시오. 기관 지침에 따라 그것의 처분. 피크 스캐너 소프트웨어를 사용하여 FLA의 결과를 읽는 것은 도전하실 수 있습니다. allele 전화 (탠덤 반복의 수)의 기본적인 세트 CSU (표 4-7)에서 개발되었습니다. (M. 다른 변종이 leprae 연구 있으므로 그것은 4-7 테이블 모두 포함되지 않을 수 있다고 지적한다. 나열된 범위 밖 복사 번호 대립 유전자를 찾을 수 있습니다.) 하나 특정 과제는 &#39;말을 더듬&#39;봉우리를 포함합니다. 이들은 특히 그러한 10 (TA)로 2-3 기본 쌍 반복을 포함 STRs의 봉우리의 가족입니다. 때때로 읽고 올바른 최고를 선택하는 것은 어렵습니다 (그림 7). 이 그림에서, 190 BP의 긍정적인 통제 정상가 주봉이다. 2아르 높이 낮은 봉우리, 4, 또는 6 기본 쌍 큰 또는 주봉보다 작은 &#39;못들었의 봉우리.라고합니다 A - 미행도 혼란을 일으킬 수 있습니다. A - 미행은 텍스트와 그림 8의 앞부분에서 설명한 것입니다. 마지막으로, PCR 제품 샘플에 매우 풍부하는 경우, 봉우리는 이중 스파이크와 함께 나타날 수 있습니다. 이 경우, 정상 형태의 센터를 참조하십시오. (그림 6, 환자 6 참조). 데이터 관리 작업이 유형에 대한 강력한 작업이 될 수 있습니다. 작업 또는 절차의 날짜, 운영자, 스트립 / 플레이트지도, FLA 주문, PCR 조건과 요리법, 젤류 및 사진의 날짜, 저장 온도, DNA 템플릿 dilutions, FLA 모든 관련 등의 모든 실험에 대한 정보를 기록하는 것이 중요하다 전자 파일의 저장 위치 등 좋은 조직 및 데이터 관리는 미래의 정보의 특정 부분을 찾는 동안 시간의 시간을 절약할 수 있습니다. 여기에 설명된 실험실 작업은 콜로라도 주립 대학에서 그리고 전세계 다른 지역에서 몇 년 동안 계속되어왔다. 수집된 데이터의 모든 수단과 방법은 나중에 사용할 수있을 어떤의 대형 사진이 등장하기 시작합니다. 수치 10A와 10B는 이러한 DNA 지문이 다른 M.을 구분하는 데 사용할 수있는 방법을 보여줍니다 국가 (10A) 또는도 간의 가족 (10B) 사이 leprae 변종. 가족 및 지역 사회 연계 케이스 M. 휴대 표시되었습니다 leprae 유사하거나 동일한 VNTR 스트레인 종류의. 희망은 전송 네트워크의 조기 발견 시스템이 사람 MOS 위해 개발된 수 있도록 나병 전송 모드 (S)에 대한 통찰력을 얻을 수있을 수있다영구적인 신경 손상과 피부과이 완료되기 전에 위험하고 치료 약물 치료에 t가 시작할 수 있습니다.

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 </th></tr><tr><td> DNeasy 혈액 및 조직 키트 </td><td> Qiagen </td><td> 69,504 </td><td></td></tr><tr><td> 멀티 플렉스 PCR 키트 </td><td> Qiagen </td><td> 206143 </td><td></td></tr><tr><td> DNA Primers </td><td> 여러 </td><td></td><td></td></tr><tr><td> 아가로 오스 겔 </td><td> 여러 </td><td></td><td></td></tr><tr><td> 5 배 또는 배의 젤 로딩 버퍼 </td><td> 뉴 잉글랜드 Biolabs </td><td> B7021S </td><td> 자신의 *를 사용할 수 요리법 </td></tr><tr><td> Ethidium의 브로마이드 솔루션 </td><td> 여러 </td><td></td><td> 죄수의 희석. </td></tr><tr><td> 안녕하세요 - 디 포름 아미드 솔루션 </td><td> 응용 BioSystems </td><td> 4,311,320 </td><td></td></tr><tr><td> 진 스캔 -500 리즈 </td><td> 응용 BioSystems </td><td> 4,322,682 </td><td></td></tr></tbody></table> <a href="http://biowww.net/buffer-reagent/6X-Gel-loading-buffer-bromophenol-blue-sucrose.html">* http://biowww.net/buffer-reagent/6X-Gel-loading-buffer-bromophenol-blue-sucrose.html</a> <table border="1"><tbody><tr><th> 장비 </th><th> 댓글 </th></tr><tr><td> 2 냉장고 </td><td> 4 ° C : DNA 샘플 1, 1 primers 및 멀티 플렉스 시약에 대한 </td></tr><tr><td> 1 전자 레인지 </td><td> 아가로 오스와 물이 젤를 만들기 위해 가열 또는 적절한 방법 </td></tr><tr><td> 1 압력솥 </td><td> 자료를 살균 또는 압력 밥솥 </td></tr><tr><td> PCR 기계 </td><td> Thermocycler </td></tr><tr><td> pipettes 2 세트 </td><td> 0.5-10 μl, 10-100 μl, 20-200 μl, primers 1000 μl 1 세트, DNA 1 세트 </td></tr><tr><td> 잠수함 겔 전기 영동 상자 </td><td> 준비 젤류에 대한 양식과 빗과 함께. </td></tr><tr><td> 전기 전원 공급 장치 </td><td> 겔 상자에 연결하기위한 케이블 </td></tr><tr><td> 열 블록 </td><td> Eppendorf 튜브에 대한 </td></tr><tr><td> 원심 분리기 </td><td> Eppendorf 튜브 / 스트립의 경우 </td></tr><tr><td> 모세관 전기 영동 시스템 (유전자 분석기) </td><td> 또는 분석을 수행할 수 있습니다 기관에 대한 액세스 권한 </td></tr><tr><td> 플라스틱 </td><td> 일회용 에어로졸 피펫 팁, Eppendorf 튜브, 8 - 잘 스트립, 96 - 웰 플레이트, 광학 품질의 일부 </td></tr><tr><td> 인터넷 연결과 컴퓨터 </td><td></td></tr></tbody></table>

dna fingerprinting of mycobacterium leprae strains using variable number tandem repeat (vntr) - fragment length analysis (fla)

나병의 전송의 연구는 원인이되는 대리인 이후, Mycobacterium leprae은, 실험실에서 배양해 수 없습니다 특히 어렵습니다. 박테리아의 유일한 소스는 나병 환자, 그리고 실험적으로 감염된 딜 및 누드 마우스입니다. 따라서 현대적인 역학에서 사용되는 방법 많은 나병 연구에 대해 사용할 수 없습니다. WHO 1 구현된 나병에 대한 광범위한 글로벌 약물 치료 프로그램에도 불구하고, 나병 약 250,000 새로운 가지 경우 매년 많은 국가에서 발병 남아 있습니다. 2 전체 M.가 leprae (마이크로 및 minisatellites라고도 함) 게놈은 3,4를 매핑되어 많은 loci는 2 개 이상의 기본 쌍 세그먼트를 반복 것으로 확인되었습니다. M. 5 임상 계통 leprae 이러한 loci의 많은에 탠덤 반복 세그먼트의 개수 (짧은 탠덤 반복, STR)에 따라 다를 수 있습니다. 5,6,7 가변 번호 탠덤 반복 (VNTR) 5 분석 나병 bacilli의 다른 변종을 구별하기 위해 사용되었습니다. loci 중 일부는 다른 사람이 같은 환자에서 때로는 더 빠르게 변경하는 것 동안 반복 숫자가 적은 변화를 보여주는, 다른 사람보다 더 안정적인 것 같습니다. 특정 VNTRs의 다양성은 스트레인 입력 7, 8, 9에 대한 그들의 적합성에 관한 질문을 가져왔습니다 반면, 신흥 데이터들의 안정성에 다양한 여러 loci을 분석, 가치 역학 도구로 사용할 수있는 것이 좋습니다. 여러 로커스 VNTR 분석 (MLVA) 10 중국 11,12, 말라위 8, 필리핀 10,13, 브라질 14 등 여러 국가에서 나병의 진화 및 전송을 연구하는 데 사용되었습니다. MLVA 여러 단계를 포함한다. 첫째, 박테리아 DNA는 임상 biopsies 또는 슬릿 피부 얼룩 (SSS)에서 호스트 조직 DNA와 함께 추출됩니다. 10 원하는 loci는 다음 중합 효소의 연쇄 반응 (PCR)를 통해 추출 DNA에서 증폭됩니다. 4-5 다른 loci에 대한 찬란 - 표시 primers 그 다음 10 PCR 제품을 원하는 DNA 세그먼트의 존재를 확인하기 위해 아가로 오스 겔 전기를 받게 될 수도 있습니다. 18 loci 네개가 반응의 총 증폭되는, 반응 당 사용, 그리고 모세관 전기 영동을 사용하여 형광 조각 길이 분석 (FLA)에 제출. 아르마 딜로의 DNA는 긍정적인 컨트롤로 사용되는 각 로커스에 대한 반복 복사 알려진 번호 박테리아를 passaged. FLA의 chromatograms 그러면 피크 스캐너 소프트웨어와 조각의 길이가 VNTR 사본 (allele)의 숫자로 변환을 사용하여 검사하고 있습니다. 마지막으로, VNTR의 haplotypes는 패턴 분석하고 있으며, 환자 임상 데이터와 결합하면이 변형 유형의 분포를 추적하는 데 사용할 수 있습니다.

Watch this Scientific Journal Video about DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat VNTR - Fragment Length Analysis FLA at JoVE.com

DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat VNTR - Fragment Length Analysis FLA

에 의한 나병,<emleprae&gt; Mycobacterium</em&gt; 많은 장소에서 아직도 발병합니다. 나병의 전염의 확산과 모드에 대한 자세한 내용을 보려면 위해서는, 결정하는 것이 중요합니다 어떤 변형의<em&gt; M. leprae</em&gt;은 환자를 감염하고 있습니다. 탠덤 반복의 변수 숫자 (VNTR) 입력은 하나의 방법입니다.

의 DNA 지문<emleprae> Mycobacterium</em단편 길이 분석 (FLA) - 가변 번호 탄뎀 반복 (VNTR)를 사용하기> 레인스

The study of the transmission of leprosy is particularly difficult since the causative agent, Mycobacterium leprae, cannot be cultured in the laboratory.  ...

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Research

JoVE Journal

Immunology and Infection

DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat (VNTR) - Fragment Length Analysis (FLA)

27.0K 조회수.  Colorado State University. 에 의한 나병, Mycobacterium 많은 장소에서 아직도 발병합니다. 나병의 전염의 확산과 모드에 대한 자세한 내용을 보려면 위해서는, 결정하는 것이 중요합니다 어떤 변형의 M. leprae은 환자를 감염하고 있습니다. 탠덤 반복의 변수 숫자 (VNTR) 입력은 하나의 방법입니다.

비디오: DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat VNTR - Fragment Length Analysis FLA

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

의 샘플 준비<em&gt; Mycobacterium 결핵</em핵 자기 공명 Metabolomic의 연구&gt; 압축을 풉니 다

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

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면역학 및 감염병학

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 ...

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Genetic Manipulation in &lt;em&gt;&amp;Delta;ku80&lt;/em&gt; Strains for Functional Genomic Analysis of &lt;em&gt;Toxoplasma gondii&lt;/em&gt;

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

유전 조작<em&gt; Δku80</em의 기능 게놈 분석을위한&gt; 균주<em&gt; 톡소 포자충</em

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

Optimized Protocols for Mycobacterium leprae Strain Management: Frozen Stock Preservation and Maintenance in Athymic Nude Mice

Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. There are currently no known methods for growing M. leprae in vitro, presenting a major obstacle in the study of this pathogen in the laboratory. Therefore, the maintenance and growth of M. leprae strains are preferably performed in athymic nude mice (NU-Foxn1nu). The laboratory conditions for using mice are readily available, easy to perform, and allow standardization and development of protocols for achieving reproducible results. In the present report, we describe a simple protocol for purification of bacilli from nude mouse footpads using trypsin, which yields a suspension with minimum cell debris and with high bacterial viability index, as determined by fluorescent microscopy. A modification to the standard method for bacillary counting by Ziehl-Neelsen staining and light microscopy is also demonstrated. Additionally, we describe a protocol for freezing and thawing bacillary stocks as an alternative protocol for maintenance and storage of M. leprae strains.

Optimized Protocols for Mycobacterium leprae Strain Management: Frozen Stock Preservation and Maintenance in Athymic Nude Mice

Mycobacterium leprae, the causative agent of leprosy, does not grow in vitro. We describe an easy to follow protocol to prepare a bacillary suspension to ensure the maintenance of large quantities of M. leprae for a variety of applications. Protocols for propagation by mouse footpad inoculation, evaluation of viability, freezing and thawing bacillary stock are described in detail.

에 최적화 된 프로토콜<em&gt;은 Mycobacterium leprae</em&gt; 스트레인 관리 : 무 흉선 누드 마우스에서 냉동 보관 보존 ​​및 유지 관리

Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

설립<em&gt; 체외</em&gt; 시스템의 세포 내 행동을 연구하는<em&gt; 칸디다 글라 브라</em&gt; 인간 THP-1 식세포에

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

밀도 Transposon 삽입 라이브러리를 사용 하 여 세균성 활용 Enterobacterial 종자와 같은 대장균 또는 Shigella flexneri 의 창조

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

18FDG PET/CT 영상 공부 결핵균 감염 및 비 인간 영장류에서 치료 도구로 서의 분석

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

기자 효소 형광와 쥐에서 이미징 결핵균

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability to multiply inside such cells. These virulence traits render M. abscessus pathogenic, especially in vulnerable hosts with underlying structural lung disease, such as cystic fibrosis, bronchiectasis or tuberculosis. How patients become infected with M. abscessus remains unclear. Unlike many mycobacteria, M. abscessus is not found in the environment but might reside inside amoebae, environmental phagocytes that represent a potential reservoir for M. abscessus. Indeed, M. abscessus is resistant to amoebal phagocytosis and the intra-amoeba life seems to increase M. abscessus virulence in an experimental model of infection. However, little is known about M. abscessus virulence in itself. To decipher the genes conferring an advantage to M. abscessus intracellular life, a screening of a M. abscessus transposon mutant library was developed. In parallel, a method of RNA extraction from intracellular Mycobacteria after co-culture with amoebae was developed. This method was validated and allowed the sequencing of whole M. abscessus transcriptomes inside the cells; providing, for the first time, a global view on M. abscessus adaptation to intracellular life. Both approaches give us an insight into M. abscessus virulence factors that enable M. abscessus to colonize the airways in humans.

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

Here, we present two protocols to study Phagocyte-Mycobacterium abscessus interactions: the screening of a transposon mutant library for bacterial intracellular deficiency and the determination of bacterial intracellular transcriptome from RNA sequencing. Both approaches provide insight into the genomic advantages and transcriptomic adaptations enhancing intracellular bacteria fitness.

식 세포에서 세포내 복제에 대 한의 진 균 abscessus 독성 마커의 식별

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability ...

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon.

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

We describe here the in vitro generation of HBV DNA via a Hepatitis B virus infection system and the highly sensitive detection of its (1&#8211;2 copies) integration using inverse nested PCR.

낮은 복사의 번호 통합된 바이러스 성 DNA 생체 외에서 B 형 간염 감염에 의해 형성 된

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally ...

의 DNA 지문 Mycobacterium 레인스