Este estudio fue apoyado por el Servicio de Salud Pública de subvención AI-34 431 (a DAH) del Instituto Nacional de Alergias y Enfermedades Infecciosas y VA Fondos de Investigación Médica (a DAH).

1. La fijación de Leptospira a las diapositivas de cristal <ol><li> Leptospira interrogans es una clase BSL2 patógeno. Trabajar con células vivas que requiere un manejo adecuado, como guantes, bata de laboratorio y medidas de pipeteo en una campana estéril. </li><li> Crecer Leptospira en EMJH medium11, complementada con suero de conejo al 1% a 30 ° C hasta llegar a mediados o finales de la fase de inicio de sesión (la densidad de 5 x 10 7 5 x 10 8 células / ml) durante aproximadamente 6 días. </li><li> La cosecha del cultivo por centrifugación a 2000 xg durante ~ 7 minutos a temperatura ambiente. </li><li> Eliminar el sobrenadante por aspiración y suavemente resuspender el precipitado en tampón fosfato salino (PBS) -5 mM MgCl 2, pH 7.2 a una concentración final de 5 x 10 8 células / ml. </li><li> Añadir 1 ml de suspensión celular a cada pocillo de dos y la cámara de diapositivas de vidrio y se incuba a 30 ° C durante 80 minutos para permitir que las células se adhieran. </li><li> Retire con cuidado el líquido que contiene las células no unidas por la aspiración. </li><li> Fix restantes bacterias intactas a portaobjetos de vidrio mediante la adición de 1 ml / pocillo de 2% de paraformaldehído en PBS-5 de MgCl 2 mM. Incubar durante 40 min a 30 ° C. Estas diapositivas serán para la evaluación de la superficie expuesta proteínas. </li><li> Para evaluar el control de diapositivas bajo la superficie las proteínas, permeabilizar la membrana externa mediante la fijación de 1 ml / pocillo de 100% helado de metanol. Se incuba a 20 ° C durante 20 min. El metanol actúa de varias maneras: permeabilizes la membrana externa, denaturates las proteínas y las células correcciones a las diapositivas de cristal. </li><li> Eliminar la fijación de los agentes por aspiración. </li></ol> 2. Etiquetado con anticuerpos específicos <ol><li> Bloquear la unión no específica mediante la adición de 1 ml / pocillo de tampón de bloqueo (Difco Leptospira enriquecimiento EMJH). Se incuba a 30 ° C durante 90 min. </li><li> Diluir el anticuerpo específico (sueros de conejo inmune o los anticuerpos monoclonales de ratón, el suero de conejo aquí policlonales específicos para OmpL54 10, FlaA1 12 y 13 OmpL1) y pre-inmune de conejo líquido ascético sueros o un ratón que no contiene anticuerpos (cuando se utiliza como controles negativos) en tampón de bloqueo. Diluciones para cada anticuerpo tiene que ser determinada empíricamente en función de análisis de anticuerpos, antígeno-anticuerpo reactividad y la abundancia de proteínas en la célula. El rango normal es de 1:50 a 1:600. </li><li> Retire el amortiguador de bloqueo por aspiración y se añade 1 ml / pocillo de anticuerpos primarios diluidos. </li><li> Se incuba a 30 ° C durante 1 hora. </li><li> Eliminar el líquido mediante la aspiración y lavar los pocillos tres veces con PBS (1 ml / pocillo). </li></ol> 3. Visualización de las leptospiras <ol><li> Añadir 1 ml / pocillo de Alexa Fluor 488-anticuerpos secundarios marcados (ya sea de cabra anti-IgG de conejo o cabra anti-ratón IgG) diluido 1:2000 y el ácido nucleico mancha fluorescente, 4&#39;6-diamidino-2-fenil-indol dihidrocloruro ( DAPI) diluido a una concentración final de 0,25 mg / ml en tampón de bloqueo. Este paso asegura la detección de la unión de los anticuerpos y la presencia de todas las espiroquetas independiente de la unión de los anticuerpos, respectivamente. </li><li> Incubar los portaobjetos a 30 ° C durante 45 min. </li><li> Eliminar el líquido mediante la aspiración y lavar los pocillos dos veces con PBS y una vez con agua destilada (1 ml / pocillo). </li><li> Quitar las cámaras y la cinta adhesiva del portaobjetos de vidrio y el aire seco de aproximadamente 10 min. </li><li> Añadir prolongar Oro anti-fade medio de montaje (2 x 20 l por diapositiva) y un 24 x 50 mm cubierta antideslizante. </li><li> Incubar toda la noche a temperatura ambiente en la oscuridad. Este paso es necesario para permitir que el medio de montaje para curar (endurecer). </li><li> Sellar la hoja de la cubierta con esmalte de uñas. </li><li> Visualizar las manchas por microscopía de fluorescencia utilizando un filtro de detección cian / azul para DAPI y un filtro verde para la detección de Alexa Fluor 488. Para asegurarse de que una representación exacta de las muestras se hace, asegúrese de evaluar el área de la cámara por completo para cada muestra. </li><li> Registrar los datos de un campo de imágenes representativas de cada muestra. Cuando la imagen Alexa Fluor 488 de fluorescencia, utilice el mismo tiempo de exposición para todas las muestras. El uso de un tiempo de exposición constante permitirá una comparación más precisa de los resultados con los antígenos de prueba frente a los controles. </li></ol> 4. Los resultados representativos: Ejemplos de los resultados de la superficie de IFA con Leptospira interrogans células utilizando anticuerpos contra la superficie y del subsuelo, las proteínas se muestran en la Figura 2. Una prueba se considera positiva cuando se detecta fluorescencia claramente en las muestras con leptospiras intacto para una proteína de interés que es la superficie expuesta (Figura 2A). Por lo general, un resultado positivo (en este caso, la superficie expuesta OmpL54), la intensidad de fluorescencia aproximadamente el mismo que se observa tanto en las células intactas y permeabilized (Fig. 2A) Es imprescindible incluir un control negativo para la integridad de la membrana externa, el uso de anticuerpos contra un subsuelo , preferiblemente periplasmic proteínas (por ejemplo, la leptospirosis FlaA1, fig. 2B). Sólo cuando los datos muestran que la membrana externa se ha mantenido intacta during el experimento, es decir, anticuerpos contra una proteína de sub-superficie no reaccionan a las células intactas (no fluorescencia o fluorescencia mucho más débil si se compara con la muestra permeabilized como en la fig. 2B), se puede concluir que la proteína (s) de interés superficie expuesta. La inclusión de los experimentos con suero pre-inmune (en el caso de anticuerpos policlonales) como control negativo adicional es esencial para los datos sin ambigüedades y no deben producir fluorescencia claramente en las células intactas o permeabilized (Fig. 2A, 2C). Tinción DAPI es necesario visualizar leptospiras cuando los resultados negativos (no Alexa Fluor 488 fluorescencia) se observan. Contratinción con yoduro de propidio es una alternativa aceptable a la DAPI. Este método es más cualitativa que cuantitativa, como la fluorescencia más brillante puede ser debida a la superficie expuesta múltiples epítopos antigénicos de ser reconocido en un solo antígeno en relación con otros antígenos de igual abundancia, pero con menos superficie expuesta epítopos. Por lo tanto la intensidad de fluorescencia sólo puede ser utilizado para comparar la reactividad de los mismos anticuerpos, tales como las células intactas frente permeabilized y la intensidad de fluorescencia no entre diferentes proteínas. Esta distinción es importante cuando un resultado ambiguo que se obtiene de fluorescencia mucho menos se observa en las células intactas que en las células permeabilizadas (Fig. 2C). Tal resultado indica que, o bien la proteína está en un lugar debajo de la superficie (con algunas manchas de fondo no específico de las células intactas), los anticuerpos son vinculantes preferentemente a los no nativos epítopos que son expuestos después de la permeabilización de metanol o una proteína puede tener múltiples sitios de localización como se describe para Erp y elp proteínas de otra espiroqueta, Borrelia burgdorferi 14. En cualquier caso, un resultado ambiguo IFA de este tipo requiere de enfoques alternativos, tales como biotinilación superficie o proteólisis de superficie para determinar si la proteína de interés es la superficie expuesta o no 10. <img src="/files/ftp_upload/2805/2805fig1.jpg" alt="Figura 1"/> Figura 1. Diagrama de flujo para la superficie de ensayo de inmunofluorescencia de Leptospira interrogans espiroquetas. En primer lugar, 1 ml de suspensión celular se añade a cada pocillo de la cámara de diapositivas de dos así (al menos dos láminas para cada experimento) y se incubó durante las leptospiras a que se adhieran. A continuación, las leptospiras se fijan a la cámara de diapositivas mediante la adición de 1 ml / pocillo de 2% de paraformaldehído (PFA) para las muestras con membrana intacta externa (OM) y 1 ml / pocillo de 100% de metanol frío para muestras con permeabilized OM. A continuación, las leptospiras son incubadas con anticuerpos que reconocen proteínas de superficie expuesta, junto con el control de anticuerpos para las proteínas del subsuelo (Primaria AB) a 30 ° C durante 90 min. A continuación, los anticuerpos primarios se detectan mediante la incubación con 1 ml / pocillo de Alexa Fluor 488 anticuerpos secundarios conjugados. Finalmente, las células se visualizan por microscopía de fluorescencia. <img src="/files/ftp_upload/2805/2805fig2.jpg" alt="Figura 2"/> Figura 2. Superficie inmunofluorescencia análisis de las proteínas de leptospirosis. Leptospira interrogans espiroquetas se probaron con sueros inmunes y pre-inmune, y los resultados negativos se combina con una contratinción DAPI para demostrar la presencia de espiroquetas. Un filtro verde fluorescencia se utilizan para detectar Alexa Fluor 488 anticuerpo secundario marcado la unión a las proteínas de la superficie expuesta y azul / cian filtro de fluorescencia para detectar la unión al ADN de células DAPI. A) Las leptospiras probaron con anticuerpos que reconocen una proteína de superficie expuesta, OmpL54 10. B) Las células probaron con anticuerpos contra una proteína periplasmic subsuelo, FlaA1 (control negativo). C) Las células probaron con anticuerpos contra la leptospira porina, OmpL1. La unión de suero de conejo a las leptospiras se detectaron con Alexa Fluor 488 conjugado de cabra anti-conejo IgG fragmentos. Todas las imágenes son tomadas después de 4 segundos de exposición largo.

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La superficie de nuestra técnica de IFA es similar a la utilizada para demostrar la superficie de exposición de los diversos borrelial 15, 16 y leptospirosis 12, 17 proteínas. Los detalles de la superficie, método IFA descritos aquí están diseñados para reducir al mínimo la manipulación de células en un esfuerzo por mantener la integridad de la membrana externa de tiempo que se aprovechan de la tendencia de las células de Leptospira a adherirse a las diapositivas de cristal. El método c...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (opcional) </th></tr><tr><td> Dos, así la cámara de diapositivas de vidrio </td><td> Lab-Tek </td><td> 177380 </td><td></td></tr><tr><td> Suero de conejo </td><td> Rockland inmunoquímicos </td><td> D209-00-0100 </td><td></td></tr><tr><td> Leptospira enriquecimiento EMJH </td><td> BD Difco </td><td> 279510 </td><td></td></tr><tr><td> Alexa Fluor 488 cabra anti-conejo IgG (H + L) </td><td> Invitrogen / sondas moleculares </td><td> A-11034 </td><td></td></tr><tr><td> Alexa Fluor 488 cabra anti-ratón IgG (H + L) </td><td> Invitrogen / sondas moleculares </td><td> A-11029 </td><td></td></tr><tr><td> 4&#39;6-diamidino-2-fenil-indol dihidrocloruro (DAPI) </td><td> Invitrogen / sondas moleculares </td><td> D1306 </td><td></td></tr><tr><td> Prolongar Oro anti-fade medio de montaje </td><td> Invitrogen / sondas moleculares </td><td> P36930 </td><td> Alcancen la temperatura ambiente antes de su uso </td></tr><tr><td> Fisherfinest prima de vidrio cubierta </td><td> Pescador </td><td> 12-544-14 </td><td></td></tr><tr><td> Microscopio de fluorescencia </td><td> Zeiss </td><td> Axioskop 40 </td><td></td></tr></tbody></table>

immuno-fluorescence assay of leptospiral surface-exposed proteins

La superficie de las proteínas bacterianas están involucradas en contacto directo con las células huésped y en la absorción de nutrientes del medio ambiente 1. Por esta razón, la localización celular puede ayudar a comprender el papel funcional de las proteínas bacterianas. Localización de la superficie de las proteínas bacterianas es un paso clave hacia la identificación de factores de virulencia que participan en los mecanismos de patogenicidad. Métodos de fraccionamiento 2.5 membranas leptospira puede ser selectiva para una cierta clase de proteínas de la membrana externa (OMP), como las lipoproteínas vs OMPs transmembrana, y por lo tanto, llevar a errores de clasificación. Esto probablemente es debido a las diferencias estructurales y la forma en que están asociados a la membrana externa. Las lipoproteínas se asocia con membranas a través de una interacción hidrofóbica entre la fracción lipídica N-terminal (tres ácidos grasos) y los fosfolípidos bicapa lipídica 6, 7. Por el contrario, OMPs transmembrana suelen estar integrados en la bicapa lipídica por anfipáticas β-hojas dispuestas en una estructura de barril-como 8, 9. Además, la presencia de una proteína en la membrana externa, no garantiza necesariamente que la proteína o sus dominios están expuestos en la superficie. Espiroquetas membranas externas se sabe que son métodos frágil y por tanto, requiere la participación manipulación suave de las células y la inclusión de proteínas de sub-superficie de control para evaluar la integridad de la membrana externa. A continuación, presentamos un ensayo de inmunofluorescencia (IFA) para evaluar directamente la exposición de la superficie de las proteínas en las leptospiras intacto. Este método se basa en el reconocimiento de proteínas de la superficie de leptospiras por el antígeno de anticuerpos específicos. En esto, los anticuerpos específicos para OmpL54 10 son detetcted aftero vinculante a los nativos epítopos de la superficie, expuestos. Comparación de la reactividad de anticuerpos frente a células intactas permeabilized permite la evaluación de la distribución de celulares y si una proteína está presente en la superficie de forma selectiva leptospirosis. La integridad de la membrana externa deben ser evaluados utilizando anticuerpos contra una o más proteínas del subsuelo, preferentemente en el periplasma. El método de la superficie de la IFA se puede utilizar para analizar la exposición de la superficie de cualquier proteína de leptospira a que los anticuerpos específicos disponibles. Tanto la utilidad y las limitaciones del método depende de si los anticuerpos empleados son capaces de unirse a epítopos nativos. Dado que los anticuerpos a menudo se levantan contra las proteínas recombinantes, los epítopos de los nativos, expuestas en la superficie las proteínas no pueden ser reconocidos. Sin embargo, el método de la superficie de IFA es una herramienta valiosa para el estudio de los componentes de la superficie bacteriana intacta. Este método se puede aplicar no sólo para las leptospiras, pero también otras espiroquetas y bacterias gram-negativas. Para conclusiones más sólidas sobre la superficie de exposición de OMP, un enfoque integral que implique a varios métodos de localización celular se recomienda 10.

Watch this Scientific Journal Video about Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins at JoVE.com

Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

Un método eficaz para evaluar la superficie de exposición de las proteínas de la leptospirosis se describe. El método está diseñado específicamente para evitar la interrupción de la frágil membrana externa de las células de leptospira. Esta técnica requiere el empleo de varios controles negativos para evaluar la integridad de la membrana externa y la especificidad de la reacción de los anticuerpos.

Inmunofluorescencia ensayo de leptospiras superficie expuesta proteínas

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment 1. For this reason, cellular ...

immuno-fluorescence-assay-of-leptospiral-surface-exposed-proteins

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Immunology and Infection

15.7K vistas.  University of California, Los Angeles. Un método eficaz para evaluar la superficie de exposición de las proteínas de la leptospirosis se describe. El método está diseñado específicamente para evitar la interrupción de la frágil membrana externa de las células de leptospira. Esta técnica requiere el empleo de varios controles negativos para evaluar la integridad de la membrana externa y la especificidad de la reacción de los anticuerpos.

Video: Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

Una técnica de microscopía TIRF en tiempo real, imágenes simultáneas de la TCR y sus proteínas asociadas de señalización

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

Investigación

Inmunología e Infección

Plaque Assay for Murine Norovirus

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages 1. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay 1. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques 3.
Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay 3, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers 4. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50) 3. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration 5. However, its limit of detection is higher compared to a plaque assay 4.
In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples 1, 4, 6. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample 3.

Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV&#x2019;s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV.

Placa de Ensayo para Norovirus murino

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

Metodología Para La Generación Eficiente De Proteínas Del Virus Vaccinia Marcadas Fluorescentemente

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

El uso de proteínas fluorescentes de Seguimiento glicosoma Dinámica en el tripanosoma africano

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

Fenómica fagos: enfoques fisiológicos para caracterizar Novela Virales Proteínas

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

Ensayo muy sensible para la medición del Adjunto de células arenavirus

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral ...

Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

Optimización de un ensayo cuantitativo micro-neutralización

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

Identificación de secuencias de localización Plasmodesmal en proteínas en Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

Dirigida a tioles de la cisteína para in Vitro específica glicosilación de proteínas recombinantes

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.