O financiamento foi fornecido pelo NIH / NIAID conceder RO1-AI-63457 e conceder ARRA suplemento RO1-AI-63457 S1. Reconhecemos as contribuições de todos os membros atuais e anteriores do grupo de laboratório e colaboradores.

<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>

Não há conflitos de interesse declarados.

A coleta de amostras de pele de pacientes com hanseníase exige clínicos qualificados ou técnicos que trabalham em clínicas da pele. Pessoal de laboratório manipulando essas amostras devem tomar muito cuidado para vestir jalecos, luvas e óculos de proteção e de trabalhar em um gabinete de bio-segurança ao manusear amostras de tecido humano infectado ou tatu. Desinfecção de superfícies e ferramentas também é crítica. Trabalhando em um ambiente limpo, gabinete bio-segurança estéril é importante para evitar a contaminação de amostras de DNA. Extração de DNA tornou-se relativamente fácil, graças ao desenvolvimento de kits de extração de empresas como a Qiagen. Direções devem ser seguidas rigorosamente. Todas as amostras devem ser mantidas frio quando não em uso. Evite a repetição, mudanças extremas de temperatura para as amostras. Primers DNA utilizado neste trabalho pode ser encomendado a partir de várias empresas que foram citadas na lista de referências bibliográficas no final deste artigo. Cuidados devem ser tomados para trabalhar com primers de DNA em um ambiente livre, a fim de evitar a contaminação. Primers vir na forma de pó seco e deve ser misturado com TE e diluído a uma concentração 100μM, em seguida, separada em quantidades menores (Figura 2a). Soluções de trabalho de primers são ainda mais diluído em concentrações 10μM. Mais uma vez, não usam amostras de DNA em áreas / capuzes onde primers são usados ​​e preparados. Os produtos de PCR também deve ser mantido frio quando não em uso. A 3% agarose gel preparação dará melhor separação da banda DNA, mas demora mais tempo para ser executado. Para fins puramente qualitativa, 2% é geralmente suficiente. Solução de brometo de etídio utilizado para a coloração do DNA em gel de agarose é um genotoxin altamente ativa que é absorvida através da pele. Lidar com esta solução e géis corados com ele usando muito cuidado e sempre tendo a certeza de usar luvas. Lave bem as mãos após manusear qualquer DNA de amostras ou materiais brometo de etídio. Dispor de etídio solução corante brometo, lave e géis de acordo com as diretrizes institucionais. Géis não são rotineiramente necessárias; esta etapa pode ser eliminada de uma vez métodos FLA foram estabelecidas. É tempo e consumindo reagente. A solução formamida utilizados na preparação de amostras para FLA também é altamente tóxico e deve ser manuseado com cuidado. Lavar as mãos após o uso. Descartá-lo seguindo as diretrizes institucionais. Leitura dos resultados da FLA usando o software de scanner de pico pode ser desafiador. Um conjunto básico de chamadas alelos (número de repetições em tandem) foi desenvolvido no CSU (Tabelas 4-7). (Note-se que os quadros 07/04 pode não ser tudo incluído. Como outras cepas de M. leprae são estudados, os alelos com números de cópia fora dos intervalos listados podem ser encontrados.) Um desafio particular envolve &#39;gaguejar&#39; picos. São famílias de picos, especialmente dos STRs que envolvem apenas repete 2-3 pares de base, tais como (TA) 10. Às vezes, selecionando o pico correto de ler é difícil (Figura 7). Nesta figura, o pico no controle positivo em 190 bp é o pico principal. Picos mais baixos de altura que são 2, 4 ou mesmo 6 pares de bases maiores ou menores do que o pico principal são chamados &quot;picos de gaguejar.&quot; A-rejeito também pode causar confusão. A-rejeito é descrito anteriormente no texto e na Figura 8. Finalmente, se produtos de PCR são muito abundantes nas amostras, os picos podem aparecer com um pico duplo. Neste caso, leia o centro do pico de forma. (Veja a Figura 6, paciente 6). Gerenciamento de dados pode ser uma tarefa formidável para esse tipo de trabalho. É importante registrar todas as informações pertinentes para todos os experimentos, tais como: data de uma tarefa ou procedimento, a operadora, tira / chapa de mapas, ordens FLA, as condições de PCR e receitas, datas de géis e fotografias, temperaturas de armazenamento, diluições modelo de DNA, FLA locais de armazenamento de arquivo eletrônico, etc Uma boa organização e gerenciamento de dados podem salvar horas de tempo gasto à procura de informações específicas no futuro. O trabalho de laboratório descritas aqui vem acontecendo há vários anos na Colorado State University e em outras localidades ao redor do mundo. O grande retrato do que todos os dados coletados significa e como ela pode ser de uso futuro está começando a emergir. Figuras 10A e 10B demonstram como estas impressões digitais de DNA pode ser usado para distinguir diferentes M. leprae tensões entre países (10A) ou mesmo entre as famílias (10B). Família e na comunidade casos relacionados foram mostrados para transportar M. leprae de tipos de tensão semelhante ou idêntico VNTR. A esperança é que isso pode ser possível obter mais conhecimentos sobre o modo (s) de transmissão da hanseníase para que um sistema de detecção precoce de redes de transmissão pode ser desenvolvida para aqueles mos pessoast em risco, e que a terapia de droga curativa pode começar antes que os danos neurológicos e dermatológicos permanente é feito.

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários </th></tr><tr><td> Sangue Dneasy e Kit Tissue </td><td> Qiagen </td><td> 69504 </td><td></td></tr><tr><td> Multiplex PCR Kit </td><td> Qiagen </td><td> 206143 </td><td></td></tr><tr><td> Primers do DNA </td><td> Vários </td><td></td><td></td></tr><tr><td> Gel de agarose </td><td> Vários </td><td></td><td></td></tr><tr><td> Tampão de carregamento 5x ou 6x Gel </td><td> New England Biolabs </td><td> B7021S </td><td> Receitas disponíveis para fazer a sua própria * </td></tr><tr><td> Solução de brometo de etídio </td><td> Vários </td><td></td><td> Diluir de con. </td></tr><tr><td> Oi-Di Solução Formamida </td><td> Applied Biosystems </td><td> 4311320 </td><td></td></tr><tr><td> Gene digitalização -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> Equipamento </th><th> Comentários </th></tr><tr><td> 2 Refrigeradores </td><td> 4 ° C: 1 para amostras de DNA, 1 de primers e reagentes Multiplex </td></tr><tr><td> Um forno de microondas </td><td> Ou uma maneira adequada para o aquecimento agarose e água para fazer géis </td></tr><tr><td> Uma autoclave </td><td> Ou panela de pressão para esterilizar materiais </td></tr><tr><td> Máquina de PCR </td><td> Termociclador </td></tr><tr><td> 2 conjuntos de pipetas </td><td> 0,5-10 mL, 10-100 mL, 2-20 mL, 1000 set 1 ml de primers, um conjunto de DNA </td></tr><tr><td> Caixa de eletroforese em gel de submarinos </td><td> Com formas e pentes para preparar géis. </td></tr><tr><td> Fonte de alimentação de eletroforese </td><td> Com cabos para ligação à caixa de gel </td></tr><tr><td> Bloco de aquecimento </td><td> Para tubos Eppendorf </td></tr><tr><td> Centrifugador </td><td> Para tubos Eppendorf / tiras </td></tr><tr><td> Sistema de eletroforese capilar (analisador de genética) </td><td> Ou o acesso a uma instituição que pode realizar esta análise </td></tr><tr><td> Plásticos </td><td> Descartáveis ​​aerosol ponteiras, tubos Eppendorf, tiras de 8 poços, placas de 96 poços, alguns de qualidade óptica </td></tr><tr><td> Computador com ligação à Internet </td><td></td></tr></tbody></table>

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

O estudo da transmissão da hanseníase é particularmente difícil, pois o agente causador, o Mycobacterium leprae, não podem ser cultivadas em laboratório. As únicas fontes de bactérias são pacientes de hanseníase, e tatus infectados experimentalmente e camundongos nude. Assim, muitos dos métodos utilizados em epidemiologia moderna não estão disponíveis para o estudo da hanseníase. Apesar de um programa de tratamento global de drogas extensa para a lepra implementado pela OMS 1, a hanseníase ainda é endêmica em muitos países, com aproximadamente 250.000 casos novos a cada ano. 2 A M. inteira leprae genoma foi mapeado 3,4 e muitos loci foram identificados que têm repetido segmentos de 2 ou mais pares de bases (chamada de micro e minissatélites). 5 cepas clínicas de M. leprae podem variar no número de segmentos em tandem repetidas (repetições em tandem curtas, STR) em muitos destes loci. 5,6,7 tandem repeat número variável (VNTR) 5 análise tem sido utilizada para distinguir diferentes cepas do bacilo da lepra. Alguns dos loci parecem ser mais estáveis ​​do que outros, mostrando uma menor variação no número de repetir, enquanto outros parecem mudar mais rapidamente, às vezes em um mesmo paciente. Enquanto a variabilidade dos VNTRs certas trouxe à tona questões sobre a sua adequação para a digitação tensão 7,8,9, os dados emergentes sugerem que a análise de múltiplos locais, que são diferentes em sua estabilidade, pode ser usado como uma valiosa ferramenta epidemiológica. Múltiplos lócus VNTR análise (MLVA) 10 tem sido usado para estudar a evolução da hanseníase e de transmissão em vários países, incluindo China 11,12, Malawi 8, Filipinas 10,13, e Brasil 14. MLVA envolve várias etapas. Primeiro, o DNA bacteriano é extraído juntamente com o DNA do hospedeiro de tecido de biópsias clínicas ou manchas da pele de fenda (SSS). 10 O loci desejado são amplificados a partir do DNA extraído por meio de reação em cadeia da polimerase (PCR). Primers fluorescentes marcado por 4-5 loci diferentes são usados ​​por reação, com 18 loci sendo amplificado em um total de quatro reações. 10 Os produtos de PCR pode ser submetido a eletroforese em gel de agarose para verificar a presença dos segmentos de DNA desejado, e então submetidos para análise fluorescentes fragmento de comprimento (FLA), utilizando eletroforese capilar. DNA de bactérias tatu várias passagens com um número conhecido de cópias de repetição para cada locus é usado como um controle positivo. Os cromatogramas FLA são então analisados ​​usando o software de scanner de pico e de comprimento de fragmentos é convertido em número de cópias VNTR (alelo). Finalmente, os haplótipos VNTR são analisados ​​por padrões, e quando combinados com os dados clínicos do paciente pode ser usado para controlar distribuição dos tipos de tensão.

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

Hanseníase, causada pelo<em&gt; Mycobacterium leprae</em&gt;, Ainda é endêmica em muitos lugares. A fim de aprender sobre a propagação e modo de transmissão da hanseníase, é importante para determinar qual a tensão de<em&gt; M. leprae</em&gt; Contaminou um paciente. Número variável de repetições em tandem (VNTR) a digitação é um tal método.

Impressões Digitais de DNA de Mycobacterium leprae Estirpes Usando Repita Tandem número variável (VNTR) - Análise Fragmento (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 visualizações.  Colorado State University. Hanseníase, causada pelo Mycobacterium leprae, Ainda é endêmica em muitos lugares. A fim de aprender sobre a propagação e modo de transmissão da hanseníase, é importante para determinar qual a tensão de M. leprae Contaminou um paciente. Número variável de repetições em tandem (VNTR) a digitação é um tal método.

Vídeo: 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.

Separação de Single-stranded DNA, Double-stranded DNA e RNA de uma Comunidade Ambiental Viral Usando Cromatografia hidroxiapatita

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

Imunologia e Infecção

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.

Crescimento de Mycobacterium tuberculosis Biofilmes

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.

Manipulação genética em<em&gt; Δku80</em&gt; Cepas para Análise Genômica Funcional de<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.

Protocolos otimizados para<em&gt; Mycobacterium leprae</em&gt; Gestão de Strain: congelada Stock Preservação e Manutenção em atímicos Nudez Ratos

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.

Estabelecimento de um<em&gt; In vitro</em&gt; Sistema para estudar o comportamento intracelular de<em&gt; Candida glabrata</em&gt; Em humanos THP-1 macrófagos

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.

Demonstrando um multi-resistente<em&gt; Mycobacterium tuberculosis</em&gt; Ampliação Microarray

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.

Sistema de Eficácia e citotoxicidade Rastreio de inibidores Segmentação intracelular<em&gt; Mycobacterium tuberculosis</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.

Criação de um denso Transposon inserção biblioteca usando bacteriana conjugação em entérobactérienne cepas tais como Escherichia Coli ou 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 ...

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.

Análise de 18FDG PET-CT Imaging como uma ferramenta para estudar infecção por Mycobacterium tuberculosis e tratamento em primatas não-humanos

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 tuberculosis em camundongos com fluorescência de enzima do repórter de imagem

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