Finansman NIH / NIAID hibe RO1-AI-63457 ve arra hibesine ek RO1-AI-63457 S1 tarafından sağlanmıştır. Biz laboratuvar grubu ve işbirlikçilerinin tüm mevcut ve geçmiş üyeleri katkıları kabul etmiş sayılırsınız.

<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>

Lepra hastalardan alınan deri örneklerinin toplanması cilt kliniklerinde çalışan yetenekli klinisyenler veya teknisyenler gerektirir. Bu örnekleri ele Laboratuar çalışanları, laboratuvar mont, eldiven ve koruyucu gözlük takın ve enfekte insan veya armadillo doku örnekleri ele alırken bir biyo-güvenlik kabini içinde çalışmak çok dikkat etmelidir. Dezenfeksiyon yüzeyler ve araçları da önemlidir. DNA örnekleri kirlenmesini önlemek için temiz, steril bir biyo-güvenli kabine çalışmak önemlidir. DNA ekstraksiyonu Qiagen gibi şirketlerden ekstraksiyon kitleri geliştirme sayesinde nispeten kolay hale gelmiştir. Yol dikkatle takip edilmelidir. Kullanılmadığı zaman Tüm numuneler soğuk tutulmalıdır. Örnekleri için tekrarlanan, aşırı sıcaklık değişimleri kaçının. Bu çalışmada kullanılan DNA primerleri Bu yazının sonunda literatür referansları listesinde atıf almıştır çeşitli şirketlerden sipariş edilebilir. Kontaminasyonu önlemek için primerler ile DNA özgür bir ortamda çalışması için önlemler alınmalıdır. Astarlar kuru toz olarak gelir ve TE ile karıştırılır ve bir 100μM konsantrasyona seyreltilmelidir, sonra da küçük miktarlarda (Şekil 2a) ayrılır . Astar Çalışma çözümleri 10μM konsantrasyonları daha seyreltilir. Yine, alanları / primerler kullanılır ve hazırlanan davlumbaz DNA örnekleri kullanmayın. PCR ürünleri kullanılmadığı zaman da soğuk muhafaza edilmelidir. % 3 agaroz jel hazırlanması daha iyi DNA bant ayrımı vermek, ama çalıştırmak için uzun sürer. Tamamen niteliksel amaçlar için,% 2 genellikle yeterlidir. Agaroz jel DNA boyama için kullanılan etidyum bromür çözüm son derece aktif genotoxin deri yoluyla emilir. Bu çözüm ve büyük bir özenle ve eldiven giymek için her zaman emin olmak boyanmış jeller taşıyınız. Herhangi bir DNA numune veya etidyum bromür malzemeleri dokunduktan sonra ellerinizi iyice yıkayın. Etidyum bromür boyama çözüm atın, kurumsal kurallarına göre yıkama ve jeller. Jeller rutin olarak gerekli değildir; FLA yöntemleri kurulduktan sonra bu adımı ortadan kaldırılabilir. Zaman ve reaktif alıcıdır. FLA için numune hazırlamada kullanılan formamid çözüm de son derece toksik ve dikkatle ele alınmalıdır. Kendi kullanımdan sonra ellerinizi yıkayınız. Kurumsal yönergeleri izleyerek atın. Tepe Tarayıcı yazılımı kullanarak FLA sonuçları okunması zor olabilir. Allel aramaları (tandem tekrarlar sayısı) bir temel set CSU (Tablo 4-7) geliştirilmiştir. (M. diğer suşları leprae çalışılmaktadır 4-7 Tablolar herşey dahil olmayabilir dikkat edilmelidir. Listelenen aralıkları dışında kopya sayıları ile allel olabilir.) Bir nevi meydan okumadır &#39;kekeme&#39; doruklarına içerir. Bu aileler, özellikle sadece böyle 10 (TA) 2-3 baz çifti tekrarlar içeren Şüpheli İşlem zirvelerinden. Bazen okumak için doğru tepe seçmek zordur (Şekil 7). Bu şekilde, 190 bp pozitif kontrol tepe ana zirve. 2 yüksekliği düşük Peaks, 4, hatta 6 baz çiftinden daha büyük veya daha küçük ana zirve &#39;kekeme doruklarına.&#39; Denir A-atık da karışıklığa neden olabilir. A-atık önceki metin ve Şekil 8&#39;de açıklanmıştır. Son olarak, PCR ürünlerinin örnekleri oldukça bol, zirveleri çift sivri görünebilir. Bu durumda, tepe formu merkezi okuyun. (Bkz: Şekil 6, Hasta 6 ) Veri yönetimi, bu tür çalışma için zorlu bir görev olabilir. Bir görev ya da işlem tarihinden, operatör, şerit / plaka haritalar, FLA siparişleri, PCR koşulları ve yemek tarifleri, jeller ve fotoğraflar tarihleri, depolama sıcaklıkları, DNA örneğinin dilüsyonları, FLA: tüm ilgili gibi tüm deneyler için bilgi kaydetmek için önemlidir. elektronik dosya depolama yerleri, vb İyi organizasyon ve veri yönetimi, gelecekte belirli bilgi parçaları için harcanan zaman saatlerce kaydedebilirsiniz. Burada açıklanan laboratuar çalışması, Colorado State Üniversitesi ve dünyadaki diğer yerlerde birkaç yıldır devam etmektedir. Toplanan verilerin her türlü vasıta ve ileride kullanmak nasıl olabilir ne büyük resim ortaya çıkmaya başlıyor. Şekiller 10A ve 10B bu DNA parmak izi farklı M. ayırt etmek için nasıl kullanılabileceğini göstermek ülkelerde (10A) ve hatta arasında aile (10B) leprae suşları arasında. Aile ve toplum bağlantılı durumlarda M. taşımak için gösterilmiştir. leprae benzer veya aynı VNTR zorlanma türleri. Umut iletim şebekeleri erken bir algılama sistemi, bu insanlar ecek için geliştirilmiş olabilir cüzam iletim modu (ler) böylece daha fazla fikir edinmek mümkün olabilir.t risk ve tedavi edici ilaç tedavisi, kalıcı nörolojik ve dermatolojik hasar yapılmadan önce başlayabilir.

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar </th></tr><tr><td> DNeasy Kan ve Doku Seti </td><td> Qiagen </td><td> 69504 </td><td></td></tr><tr><td> Multiplex PCR seti </td><td> Qiagen </td><td> 206143 </td><td></td></tr><tr><td> DNA Astarlar </td><td> Çeşitli </td><td></td><td></td></tr><tr><td> Agaroz Jel </td><td> Çeşitli </td><td></td><td></td></tr><tr><td> 5x veya 6x Jel Yükleniyor Tampon </td><td> New England Biolabs </td><td> B7021S </td><td> Kendi * yapmak için kullanılabilir Tarifler </td></tr><tr><td> Ethidium Bromür çözümü </td><td> Çeşitli </td><td></td><td> Con ile seyreltilir. </td></tr><tr><td> Hi-Di Formamide Çözüm </td><td> Applied Biosystems </td><td> 4311320 </td><td></td></tr><tr><td> Gene Tarama -500 Liz </td><td> Applied Biosystems </td><td> 4322682 </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> Ekipman </th><th> Yorumlar </th></tr><tr><td> 2 Buzdolapları </td><td> 4 ° C: DNA örnekleri için 1, 1, astar ve Multiplex reaktifler </td></tr><tr><td> 1 adet mikrodalga fırın </td><td> Agaroz ve su jeller yapmak için ısıtma veya uygun bir şekilde </td></tr><tr><td> 1 otoklav </td><td> Malzemelerin sterilizasyon veya düdüklü tencere </td></tr><tr><td> PCR makinesi </td><td> PCR </td></tr><tr><td> 2 takım pipet </td><td> 0.5-10 ul, 10-100 ul, 20-200 ul, astarlar için 1000 ul 1 set, 1 set DNA </td></tr><tr><td> Denizaltı jel elektroforezi kutusu </td><td> Formlar hazırlanması jeller ve taraklar. </td></tr><tr><td> Elektroforez Güç kaynağı </td><td> Jel kutusu bağlantı kabloları ile </td></tr><tr><td> Isı blok </td><td> Eppendorf tüpleri için </td></tr><tr><td> Santrifüj </td><td> Eppendorf tüpleri / şeritler için </td></tr><tr><td> Kapiler elektroforez sistemi (genetik analizör) </td><td> Veya bu analiz, bir kurumun erişim </td></tr><tr><td> Plastik </td><td> Tek kullanımlık aerosol pipet uçları, Eppendorf tüpleri, 8-iyi şeritler, 96-iyi plakaları, optik kalitesi </td></tr><tr><td> İnternet bağlantısı olan bilgisayar </td><td></td></tr></tbody></table>

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

Cüzam iletim çalışma etkeni bu yana, Mycobacterium leprae, laboratuvar ortamında kültüre edemez özellikle zordur . Bakteriler tek kaynakları cüzzam hastaları ve deneysel olarak enfekte armadillos ve çıplak farelerin. Böylece, modern epidemiyolojisi kullanılan yöntemlerden birçok cüzam çalışma mevcut değildir. Lepra için DSÖ 1 tarafından uygulanan kapsamlı bir küresel uyuşturucu tedavi programına rağmen, cüzzam, yaklaşık 250.000 yeni vaka, her yıl pek çok ülkede endemik olarak kalır. 2 tüm M. leprae (mikro-ve minisatellites olarak da adlandırılır) genom 3,4 eşlenen olmuştur ve birçok lokusların 2 veya daha fazla baz çiftinden segmentleri tekrarlanan olduğu tespit edilmiştir. M. 5 Klinik suşları leprae bu lokuslar birçok tandem tekrarlanan segmentlerinin sayısı (kısa tandem tekrarlar, STR) değişebilir. 5,6,7 Değişken numarası tandem repeat (VNTR) 5 analizi, cüzzam basilinin farklı suşları ayırt etmek için kullanılır olmuştur . Lokusların bazıları diğerlerine göre daha istikrarlı, diğerleri aynı hasta, bazen daha hızlı bir şekilde değiştirmek gibi görünüyor ise, tekrar sayısı daha az varyasyon gösteren görünür. Belirli VNTRs değişkenliği gerginlik yazarak 7,8,9 için uygunluğuna ilişkin sorular getirdi, ortaya çıkan veriler, istikrar içinde farklı birden fazla lokusların, analiz etmek, değerli bir epidemiyolojik araç olarak kullanılabileceğini göstermektedir. Birden fazla lokus VNTR analizi (MLVA) 10 Çin 11,12, Malavi 8, Filipinler 10,13, 14 ve Brezilya da dahil olmak üzere birçok ülkede cüzzam evrim ve iletim incelemek için kullanılır olmuştur. MLVA birden fazla adım içerir. İlk olarak, bakteriyel DNA klinik biyopsilerin veya yarık cilt smear (SSS) konak doku DNA ile birlikte ayıklanır 10 istenilen lokusların sonra çıkarılan DNA polimeraz zincir reaksiyonu (PCR) ile güçlendirilir. 4-5 farklı lokusların için floresan etiketli primerler sonra istenen DNA segmentleri varlığını doğrulamak için 10 PCR ürünleri agaroz jel elektroforezi tabi olabilir. Dört reaksiyon toplam 18 lokusların amplifiye olan, reaksiyon başına kullanılan ve kapiller elektroforez kullanarak floresan parçası uzunluğu analizi (FLA) için sunulmuştur. Armadillo gelen DNA pozitif bir kontrol olarak kullanılan her bir lokus için tekrar kopya sayısı ile bilinen bir bakteri geçişli. FLA kromatogramlar sonra Tepe Tarayıcı yazılımı ve parça uzunluğu VNTR kopya (alleli) sayısı dönüştürülür kullanılarak incelenmektedir. Son olarak, VNTR haplotipleri kalıpları için analiz edilir ve hastanın klinik veriler ile bir araya geldiğinde gerginlik türlerinin dağılımını izlemek için kullanılabilir.

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

Neden Lepra<emLeprae&gt; Mycobacterium</em&gt;, Birçok yerde hala endemiktir. Yayılması ve cüzam iletim modu hakkında bilgi edinmek için, bunu belirlemek için önemlidir suşu<em&gt; M. leprae</em&gt; Bir hasta enfekte. Değişken sayıda tandem tekrarlar (VNTR) yazarak, böyle bir yöntem.

DNA parmak izi<emLeprae> Mycobacterium</emFragment Lenght Analizi (FLA) - Değişken Numarası Tandem Repeat (VNTR)> Suşlarının

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

dna-fingerprinting-mycobacterium-leprae-strains-using-variable-number

Research

JoVE Journal

Immunology and Infection

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

27.0K Görüntüleme.  Colorado State University. Neden Lepra Mycobacterium, Birçok yerde hala endemiktir. Yayılması ve cüzam iletim modu hakkında bilgi edinmek için, bunu belirlemek için önemlidir suşu M. leprae Bir hasta enfekte. Değişken sayıda tandem tekrarlar (VNTR) yazarak, böyle bir yöntem.

Video: 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.

Numune Hazırlama<em&gt; Mycobacterium tuberculosis</emNükleer Manyetik Rezonans metabolomik Araştırmaları&gt; özler

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

İmmünoloji ve Enfeksiyon

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.

Büyüme Mycobacterium tuberculosis Biyofilmler

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.

Genetik Manipülasyon<em&gt; Δku80</emFonksiyonel Genomik Analiz&gt; Suşlarının<em&gt; Toxoplasma gondii</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.

Için optimize Protokoller<em&gt; Mycobacterium leprae</em&gt; Gerilme Yönetimi: Atimik Çıplak Farelerde Dondurulmuş Hazır Koruma ve Bakım

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.

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.

Bir yoğun Transposon ekleme kütüphane kullanarak bakteriyel konjugasyon Escherichia Coli Enterobacterial suşları gibi veya Shigella flexneri oluşturulması

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.

Analiz 18FDG PET/BT uzmanı Mycobacterium tüberküloz enfeksiyon ve insan olmayan primatlarda tedavisi için bir araç olarak görüntüleme

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.

Mycobacterium tüberküloz ile muhabir enzim Floresans farelerde görüntüleme

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.

Mycobacterium abscessus virülans işaretleri tanımlaması fagositler hücre içi çoğaltma için

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.

Algılama düşük kopya sayısı Vitro hepatit B enfeksiyonu tarafından kurulan entegre Viral DNA

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

DNA parmak izi Mycobacterium Suşlarının