資金は、NIH / NIAIDの助成金RO1 - AI - 63457およびARRA助成金補助RO1 - AI - 63457 S1によって提供されていました。我々は、実験グループと共同研究者のすべての現在および過去のメンバーの貢献を認める。

<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>

利害の衝突は宣言されません。

ハンセン病の患者から皮膚サンプルの収集は、皮膚クリニックで働く熟練した臨床医や技術者が必要です。これらのサンプルを扱う研究室の労働者は、白衣、手袋、保護メガネを着用を着用し、人間やアルマジロ組織の感染サンプルを取り扱う際にはバイオセーフティキャビネット内で動作するように細心の注意を払う必要があります。面とツールの消毒も重要です。清潔、滅菌バイオセーフキャビネットでの作業は、DNAサンプルの汚染を回避するために重要です。 DNA抽出は、キアゲンのような企業から抽出キットの開発に比較的容易に感謝となっています。指示に注意深く従っている必要があります。使用しないときは全てのサンプルは低温に保たれるべきである。サンプルの繰り返し、極端な温度変化は避けてください。 本研究で使用したDNAプライマーは、本論文の末尾に参考文献のリストに引用されている様々な企業から注文することができます。ケアは、汚染を避けるために、DNAのない環境でプライマーを用いて動作するように注意が必要です。プライマーは、少量（ 図2a）に分け、乾燥粉末として来るとTEと混合し、100μMの濃度に希釈する必要があります。プライマーのワーキング溶液をさらに10μMの濃度に希釈されています。再び、プライマーが使用され、用意されている地域/フードでDNAサンプルを使用しないでください。 使用しないときは、PCR産物はまた寒さに保つ必要があります。 3％アガロースゲルの調製は、より良いDNAのバンドの分離を与えるが、実行に時間がかかりますでしょう。純粋に定性的な目的のために、2％は通常は十分です。アガロースゲルでDNAを染色するために使用される臭化エチジウム溶液は、皮膚から吸収されている非常にアクティブな遺伝毒性物質である。このソリューションは、それは細心の注意を使用して、常に手袋を着用しているで染色したゲルを取り扱う。任意のDNAサンプルやエチジウムブロマイドの材料を取扱い後はよく手を洗う。エチジウムブロマイド染色液を廃棄する際、制度的なガイドラインに従って洗浄し、ゲル。ゲルは日常的に必須ではありません。FLA方法が確立された後、このステップを排除することができます。それは時間と試薬のかかる作業です。 FLAのための試料の調製に用いホルムアミド溶液はまた、非常に毒性があり、取り扱いには注意してください。その使用後はよく手を洗う。制度的なガイドラインに従って、それを処分する。 ピークスキャナのソフトウェアを使用してFLAの結果を読み込むことは困難な場合があります。アレルコール（タンデムリピートの数）の基本セットは、CSU（ 表4-7）で開発されています。 （M.の他の株がらい研究を行っているとして、それは4月7日テーブルはすべての包括的ではないかもしれないことに留意すべきである。、記載されている範囲outsideのコピー数を持つ対立遺伝子が発見されることがあります。）一つの特定の課題は&quot;スタッター&#39;のピークを伴う。これらは、特に唯一、10（TA）として2〜3塩基対の繰り返しを、含むSTRをのピークの家族、です。時には読み取るための正しいピークを選択することは困難である（ 図7）。この図では、190 bpの陽性対照のピークが主ピークである。 2である高さの低いピークは、4、あるいは6塩基対は、大規模なまたは主ピークよりも小さい&quot;スタッターピーク&quot;と呼ばれているテーリングも混乱を引き起こす可能性があります。テーリングは、以前のテキストで、 図8に記載されています。最後に、PCR産物は、サンプル中の非常に豊富である場合、ピークはダブルスパイクで表示されることがあります。この場合、ピークフォームの中央を読んで。 （ 図6、患者6を参照してください。） データ管理は、この種の作業のための手ごわい作業になる場合があります。タスクまたはプロシージャの日付、演算子、ストリップ/プレートのマップ、FLAの受注、PCR条件とレシピ、ゲルや写真の日付、保存温度、DNAテンプレートの希釈、FLA：などのすべての実験のすべての関連情報を記録することが重要です。電子ファイルの保管場所、等グッド組織とデータの管理は、時間の時間を節約することができます将来、特定の情報の断片を探して過ごした。 ここで説明する実験室での作業は、コロラド州立大学で、世界中の他の場所で数年前から続いている。収集されたデータのすべてを意味し、どのように将来の使用の可能性があるものの拡大写真が現れ始めている。 図10Aおよび図10Bは、これらのDNAフィンガープリントが異なるM.を区別するために使用できる方法を示しています家族（10B）との間であっても国（10A）または間にらい菌。家族やコミュニティリンクされているケースはM.を運ぶことが示されているらい類似または同一のVNTRの歪みの種類の。希望は、それは、伝送ネットワークの早期発見のシステムがそれらの人々のMOSのために開発されるように、ハンセン病の伝送のモード（S）さらなる洞察を得ることが可能かもしれないということです永続的な神経学的および皮膚損傷が行われる前に、リスク、およびその治療薬物療法ではtが開始することがあります。

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント</th></tr><tr><td> DNeasy血液と組織のキット</td><td>キアゲン</td><td> 69504 </td><td></td></tr><tr><td>マルチプレックスPCRキット</td><td>キアゲン</td><td> 206143 </td><td></td></tr><tr><td> DNAプライマー</td><td>様々な</td><td></td><td></td></tr><tr><td>アガロースゲル</td><td>様々な</td><td></td><td></td></tr><tr><td> 5倍または6倍ゲルローディングバッファー</td><td>ニューイングランドバイオラボ</td><td> B7021S </td><td>あなた自身の*を作るために利用できるレシピ</td></tr><tr><td>臭化エチジウム溶液</td><td>様々な</td><td></td><td> CONから希釈する。 </td></tr><tr><td>こんにちは - ジホルムアミド溶液</td><td>アプライドバイオシステムズ</td><td> 4311320 </td><td></td></tr><tr><td>ジーンスキャン-500リズ</td><td>アプライドバイオシステムズ</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>機器</th><th>コメント</th></tr><tr><td> 2冷蔵庫</td><td> 4 ° C：プライマーおよび多重化試薬のためのDNAサンプルの1、1 </td></tr><tr><td> 1電子レンジ</td><td>ゲルを作るためにアガロースと水を加熱するためのまたは適切な方法</td></tr><tr><td> 1オートクレーブ</td><td>材料を殺菌するためのまたは圧力鍋</td></tr><tr><td> PCRマシン</td><td>クラー</td></tr><tr><td>ピペットの2セット</td><td> 0.5から10μlを、10〜100μlを、20〜200μlを、プライマー1000μlの1セット、DNAの1セット</td></tr><tr><td>潜水艦ゲル電気泳動ボックス</td><td>準備ゲル用のフォームや櫛ている。 </td></tr><tr><td>電気泳動用電源</td><td>ゲルボックスへの接続用ケーブルを使用</td></tr><tr><td>ヒートブロック</td><td>エッペンドルフチュー​​ブ用</td></tr><tr><td>遠心</td><td>エッペンドルフチュー​​ブ/ストリップ用</td></tr><tr><td>キャピラリー電気泳動システム（遺伝アナライザ） </td><td>またはこの分析を実行できる機関へのアクセス</td></tr><tr><td>プラスチック</td><td>使い捨てエアロゾルピペットチップ、エッペンドルフチュー​​ブ、8ウェルストリップ、96ウェルプレート、光学的品質のいくつか</td></tr><tr><td>インターネットに接続されたコンピュータ</td><td></td></tr></tbody></table>

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

ハンセン病の伝送の研究は、病原以来、 結核菌はらい 、実験室で培養することができない特に困難です。細菌の唯一の源は、ハンセン病の患者、および実験的に感染アルマジロやヌードマウスです。従って、現代の疫学で使用されているメソッドの多くは、ハンセン病の研究のため使用できません。 WHO 1で実装されたハンセン病のための広範なグローバルな薬物治療プログラムにもかかわらず、ハンセン病は約25万の新たな症例は毎年多くの国では風土病のまま2全体M.らい菌ゲノムは、3,4をマッピングされており、多くの遺伝子座は、（マイクロおよびminisatellitesと呼ばれる）2つ以上の塩基対のセグメントを繰り返していることが確認されている。M. 5臨床菌株は、 らいこれらの遺伝子座の多くでタンデム繰り返しセグメント（ショートタンデムリピート、STR）の数が異なる場合があります。5,6,7可変数のタンデムリピート（VNTR）5分析は、ハンセン病菌の異なる菌株を区別するために使用されています。遺伝子座のいくつかは他の人が同じ患者でもある、より急速に変化するように見える一方、繰り返し数であまり変化を示す、他のものより安定しているようです。特定のVNTRsの変動は歪タイピング7,8,9のための適性に関する質問を育てているが、新興のデータは、その安定性で多様化している複数の遺伝子座を、分析し、貴重な疫学的ツールとして使用できることを示唆している。複数の遺伝子座VNTR分析（MLVA）10が中国11,12、マラウイ8、フィリピン10,13、およびブラジル14を含むいくつかの国でハンセン病の進化と伝達を研究するために使用されています。 MLVAは、複数の手順が含まれます。最初に、細菌のDNAは、臨床生検またはスリットの皮膚スメア（SSS）から宿主組織のDNAと一緒に抽出されます。10は 、目的の遺伝子座は、その後、ポリメラーゼ連鎖反応（PCR）を経由して抽出したDNAから増幅されています。 4月5日別の遺伝子座のための蛍光標識プライマーを10 PCR産物が所望のDNAセグメントの存在を確認するためにアガロースゲル電気泳動に供されることがあります。18遺伝子座は4つの反応の合計で増幅されていると、反応ごとに使用、とされていますキャピラリー電気泳動を用いた蛍光断片長解析（FLA）のために提出。各遺伝子座の繰り返しコピーの数がわかっているアルマジロ継代細菌からのDNAをポジティブコントロールとして使用されます。 FLAのクロマトグラムは、 ピークスキャナのソフトウェアと断片の長さを用いて検討しているVNTRのコピー数（対立遺伝子）に変換されます。最後に、VNTRのハプロタイプは、パターンが分析され、そして患者の臨床データと組み合わせることで、歪みの種類の分布を追跡するために使用することができます。

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

によって引き起こされるハンセン病、<emらい&gt;結核</em&gt;、多くの場所ではまだ流行している。ハンセン病の伝送の普及とモードについて学ぶために、それはどの菌株のかを決定することが重要です。<em&gt; M.らい</em&gt;は、患者を感染している。縦列反復の変数番号（VNTR）タイピングは、そのようなメソッドです。

のDNAフィンガープリント<emらい>結核</em可変数のタンデムリピート（VNTR）を使用>菌株 - 断片長解析（FLA）

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)

26.9K ビュー.  Colorado State University. によって引き起こされるハンセン病、結核、多くの場所ではまだ流行している。ハンセン病の伝送の普及とモードについて学ぶために、それはどの菌株のかを決定することが重要です。 M.らいは、患者を感染している。縦列反復の変数番号（VNTR）タイピングは、そのようなメソッドです。

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

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

ハイドロキシアパタイトクロマトグラフィーを用いた環境ウイルスのコミュニティから一本鎖DNA、二本鎖DNAとRNAの分離

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

免疫・感染学

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.

の成長結核バイオフィルム

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.

の遺伝的操作<em&gt;Δku80</emの機能ゲノム解析のための&gt;株<em&gt;トキソプラズマ</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.

のための最適化されたプロトコル<em&gt;らい菌</em&gt;ひずみ管理：無胸腺ヌードマウスにおける凍結ストック保全とメンテナンス

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.

設立<em&gt;インビトロ</em&gt;システムの細胞内挙動を研究するために、<em&gt;カンジダ·グラブラタ</em&gt;人間のTHP-1マクロファージにおける

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

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

多剤耐性を実証<em&gt;結核菌</em&gt;増幅マイクロアレイ

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

細胞内の標的阻害剤の有効性及び細胞毒性スクリーニングのためのシステム<em&gt;結核菌</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

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.

密なトランスポゾン挿入ライブラリを使用して細菌活用腸内細菌系統など、大腸菌や赤痢菌の作成

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 イメージング勉強結核感染と非ひと霊長類における治療のためのツールとしての解析

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

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.

貪食細胞における細胞内のレプリケーションの結核菌 abscessusの病原性マーカーの同定

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

のDNAフィンガープリント結核菌株 - 断片長解析（FLA）