El financiamiento fue proporcionado por el NIH / NIAID subvención RO1-AI-63457 y concesión de suplemento de ARRA SR1-AI-63457 S1. Reconocemos las contribuciones de todos los miembros actuales y pasados ​​del grupo de laboratorios y 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>

La recogida de muestras de piel de pacientes con lepra requiere médicos clínicos especializados o técnicos que trabajan en las clínicas de la piel. Los trabajadores de laboratorio que manipula las muestras deben tener mucho cuidado al usar batas, guantes y protección para los ojos y trabajar en un gabinete de bioseguridad al manipular muestras de tejido humano infectado o armadillo. Desinfección de superficies y herramientas también es fundamental. Trabajar en un lugar limpio, estéril bio-seguridad del gabinete es importante para evitar la contaminación de las muestras de ADN. La extracción de ADN se ha convertido en relativamente fácil gracias al desarrollo de kits de extracción de empresas como Qiagen. Instrucciones deben ser seguidas cuidadosamente. Todas las muestras deben mantenerse en frío, cuando no esté en uso. Evitar la repetición, cambios extremos de temperatura para las muestras. Cebadores de ADN utilizado en este trabajo se pueden pedir de varias empresas que han sido citados en la lista de referencias bibliográficas al final de este documento. Se debe tener cuidado al trabajar con los primers en un ambiente libre de ADN con el fin de evitar la contaminación. Cebadores vienen en forma de polvo seco y se debe mezclar con TE y se diluye a una concentración de 100μM, se separa en cantidades más pequeñas (Figura 2a). Las soluciones de trabajo de los cebadores se vuelve a diluir a concentraciones de 10μM. Una vez más, no utilizan las muestras de ADN en las áreas / campanas que se utilizan cebos y preparados. Los productos de PCR también deben mantenerse en frío, cuando no esté en uso. Un 3% de agarosa preparación del gel le dará una mejor separación de las bandas de ADN, pero tarda más en ejecutarse. Con fines puramente cualitativa, el 2% es suficiente. Solución de bromuro de etidio utilizado para teñir el ADN en geles de agarosa es una genotoxina muy activo que se absorbe por la piel. Manejar esta solución y geles teñidos con él usando mucho cuidado y estar siempre asegúrese de usar guantes. Lávese las manos minuciosamente después de manipular el ADN de muestras o materiales de bromuro de etidio. Disponer de una solución de tinción con bromuro de etidio, lavar y geles de acuerdo con las directrices institucionales. Geles no son habitualmente requeridos, este paso puede ser eliminada una vez métodos FLA se han establecido. Es mucho tiempo y reactivos. La solución de formamida utilizados en la preparación de muestras para la FLA es también muy tóxico y debe ser manejado con cuidado. Lávese las manos después de su uso. Disponer de ella siguiendo las directrices institucionales. Lectura de los resultados de la FLA utilizando el software del escáner pico puede ser un reto. Un conjunto básico de llamadas alelos (número de repeticiones en tándem) ha sido desarrollado en CSU (Tablas 4-7). (Cabe señalar que los cuadros 7.4 no sea definitiva. Como otras cepas de M. leprae se estudian, los alelos con el número de copias fuera de los rangos mencionados pueden ser encontrados.) Uno de los retos en particular es &quot;tartamudeo&quot; picos. Estas son las familias de los picos, sobre todo de los ROS que sólo implican 2-3 repeticiones de pares de bases, tales como (TA) 10. A veces la selección de la punta correcta para leer es difícil (Figura 7). En esta figura, el pico en el control positivo de 190 pb es el pico principal. Picos más bajos de altura, que son 2, 4, o incluso 6 pares de bases de mayor o menor que el pico principal se denominan &quot;picos de tartamudear. A-cola también puede causar confusión. A-cola se ha descrito anteriormente en el texto y en la Figura 8. Por último, si los productos de PCR son muy abundantes en las muestras, los picos pueden aparecer con un aumento doble. En este caso, lea el centro de la forma de pico. (Ver Figura 6, los pacientes 6.) Gestión de datos puede ser una tarea formidable para este tipo de trabajo. Es importante registrar toda la información pertinente para todos los experimentos, tales como: fecha de una tarea o procedimiento, el operador tira / la placa de los mapas, las órdenes de FLA, las condiciones de PCR y las recetas, las fechas de los geles y las fotografías, las temperaturas de almacenamiento, las diluciones de ADN de la plantilla, FLA lugares de almacenamiento de archivos electrónicos, etc organización y una gestión de datos puede ahorrar horas de tiempo dedicado a buscar información específica en el futuro. El trabajo de laboratorio se describe aquí ha estado ocurriendo desde hace varios años en la Universidad Estatal de Colorado y en otros lugares alrededor del mundo. La imagen grande de lo que todos los datos recogidos medios y la forma en que pueden ser de uso en el futuro está empezando a surgir. Figuras 10A y 10B muestran cómo estas huellas de ADN se pueden utilizar para distinguir diferentes M. leprae entre los países (10A) o incluso entre las familias (10B). Casos de la familia y la comunidad vinculada Se ha demostrado que para llevar a M. leprae de tipos de cepas similares o idénticos VNTR. La esperanza es que tal vez sea posible para obtener una mayor comprensión del modo (s) de transmisión de la lepra, para que un sistema de detección precoz de las redes de transmisión puede ser desarrollado para los meses la gentet en situación de riesgo, y que el tratamiento farmacológico curativo puede comenzar antes de daño neurológico permanente y dermatológicos que se hace.

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios </th></tr><tr><td> Sangre y DNeasy Tissue Kit </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> Cartilla de ADN </td><td> Varios </td><td></td><td></td></tr><tr><td> En gel de agarosa </td><td> Varios </td><td></td><td></td></tr><tr><td> Gel de 5x o 6x tampón de carga </td><td> New England Biolabs </td><td> B7021S </td><td> Recetas disponibles para hacer su propia * </td></tr><tr><td> Solución de bromuro de etidio </td><td> Varios </td><td></td><td> Diluir en contra. </td></tr><tr><td> Hi-Di formamida Solución </td><td> Applied Biosystems </td><td> 4311320 </td><td></td></tr><tr><td> Gene exploración -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> Equipo </th><th> Comentarios </th></tr><tr><td> 2 refrigeradores </td><td> 4 ° C: 1 para las muestras de ADN, uno de los cebadores y reactivos Multiplex </td></tr><tr><td> Un horno de microondas </td><td> O la manera adecuada para la calefacción de agarosa y agua para hacer geles </td></tr><tr><td> Un autoclave </td><td> O de olla a presión para la esterilización de los materiales </td></tr><tr><td> PCR máquina </td><td> Termociclador </td></tr><tr><td> 2 juegos de pipetas </td><td> 0,5-10 l, l 10-100, 20-200 l, 1000 l 1 juego de primers, 1 juego de ADN </td></tr><tr><td> Gel de electroforesis submarina caja </td><td> Con las formas y peines para la preparación de los geles. </td></tr><tr><td> Electroforesis de la fuente de alimentación </td><td> Con cables para la conexión a la caja de gel </td></tr><tr><td> Bloque de calor </td><td> Para tubos Eppendorf </td></tr><tr><td> Centrífugo </td><td> Para los tubos Eppendorf / tiras </td></tr><tr><td> Sistema de electroforesis capilar (analizador genético) </td><td> O el acceso a una institución que puede realizar este análisis </td></tr><tr><td> Plásticos </td><td> Aerosol desechables puntas de pipeta, tubos Eppendorf, tiras de 8 pocillos, placas de 96 pozos, algunos de calidad óptica </td></tr><tr><td> Ordenador con conexión a Internet </td><td></td></tr></tbody></table>

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

El estudio de la transmisión de la lepra es particularmente difícil, ya que el agente causal, Mycobacterium leprae, no se pueden cultivar en el laboratorio. Las únicas fuentes de las bacterias son los enfermos de lepra, y armadillos infectados experimentalmente y los ratones desnudos. Así, muchos de los métodos utilizados en la epidemiología moderna no están disponibles para el estudio de la lepra. A pesar de un extenso programa global de tratamiento de drogas para la lepra implementado por la OMS 1, la lepra sigue siendo endémica en muchos países, con aproximadamente 250.000 nuevos casos cada año. 2 Toda la M. leprae genoma ha sido mapeado 3,4 y muchos loci han sido identificados que han repetido los segmentos de dos o más pares de bases (llamados micro y minisatélites). 5 cepas clínicas de M. leprae puede variar en el número de segmentos repetidos en tándem (repeticiones cortas en tándem, STR) en muchos de estos lugares. 5,6,7 repetir tándem número variable (VNTR) 5 de análisis se ha utilizado para distinguir las diferentes cepas del bacilo de la lepra. Algunos de los lugares parecen ser más estables que otros, que muestran una menor variación en el número de repeticiones, mientras que otros parecen cambiar más rápidamente, a veces en el mismo paciente. Mientras que la variabilidad de ciertas VNTRs ha planteado preguntas acerca de su idoneidad para la tipificación de cepas 7,8,9, los datos emergentes sugieren que el análisis de múltiples loci, que son diversos en su estabilidad, se puede utilizar como una herramienta epidemiológica útil. Múltiples locus VNTR análisis (MLVA) 10 se ha utilizado para estudiar la evolución de la lepra y la transmisión en varios países incluyendo a China 11,12, 8 Malawi, Filipinas 10,13, y Brasil 14. MLVA implica varios pasos. En primer lugar, el ADN bacteriano se extrae junto con el ADN del huésped de tejido de biopsias clínicas o manchas raja la piel (SSS). El 10 loci deseados se amplifican a partir del ADN extraído a través de la reacción en cadena de polimerasa (PCR). Cebadores fluorescente marcada por 4-5 diferentes loci se utilizan por reacción, con 18 loci que se amplifica en un total de cuatro reacciones. 10 Los productos de PCR puede ser sometido a electroforesis en gel de agarosa para verificar la presencia de los segmentos de ADN deseada, y luego presentado para el análisis de fluorescencia de longitud de fragmentos (FLA) mediante electroforesis capilar. ADN de las bacterias armadillo pasajes con un número conocido de copias de repetición para cada locus se utiliza como control positivo. Los cromatogramas FLA son examinados usando el software del escáner de pico y la longitud del fragmento se convierte en el número de copias VNTR (alelo). Finalmente, los haplotipos VNTR se analizan los patrones, y cuando se combina con los datos clínicos del paciente puede ser usado para rastrear la distribución de los tipos de cepa.

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

La lepra, causada por<em&gt; Mycobacterium leprae</em&gt;, Sigue siendo endémica en muchos lugares. Con el fin de aprender acerca de la propagación y el modo de transmisión de la lepra, es importante determinar qué cepa del<em&gt; M. leprae</em&gt; Ha infectado a un paciente. Un número variable de repeticiones en tándem (VNTR) escribir es uno de esos métodos.

La huella de ADN de Mycobacterium leprae Uso de las cepas Repita Variable Tandem Número (VNTR) - Análisis de longitud de fragmentos (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 vistas.  Colorado State University. La lepra, causada por Mycobacterium leprae, Sigue siendo endémica en muchos lugares. Con el fin de aprender acerca de la propagación y el modo de transmisión de la lepra, es importante determinar qué cepa del M. leprae Ha infectado a un paciente. Un número variable de repeticiones en tándem (VNTR) escribir es uno de esos métodos.

Video: 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.

La separación de una sola hebra de ADN, el ADN de doble cadena y el ARN de una comunidad ambiental viral mediante cromatografía de hidroxiapatita

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

Investigación

Inmunología e Infección

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.

Crecimiento de Mycobacterium tuberculosis Las biopelículas

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.

La manipulación genética en<em&gt; Δku80</em&gt; Las cepas de Análisis Genómico 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 optimizados para<em&gt; Mycobacterium leprae</em&gt; Gestión Strain: Congelado Stock Conservación y Mantenimiento en ratones desnudos atímicos

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.

Establecimiento de un<em&gt; In vitro</em&gt; Sistema para estudiar el comportamiento intracelular de<em&gt; Candida glabrata</em&gt; En 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.

Demostración de un resistente a múltiples fármacos<em&gt; Mycobacterium tuberculosis</em&gt; Microarray Amplificación

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 para la eficacia y la citotoxicidad de detección de inhibidores de focalización 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.

Creación de un denso Transposon inserción biblioteca usando Conjugación bacteriana en cepas enterobacterianas tales como Escherichia Coli o 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.

Análisis de 18FDG PET/CT la proyección de imagen como una herramienta para estudiar la tuberculosis del Mycobacterium infección y tratamiento en Primates no humanos

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.

Identificación de marcadores de virulencia de Mycobacterium abscessus para la replicación intracelular de fagocitos

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