Nos gustaría agradecer al Dr. Philippe Erbs para que nos proporciona la secuencia de los genes FCU1. Este trabajo fue apoyado por el NIH subvención AI 26649.

1. FCU sistema de selección negativa en Entamoeba histolytica <ol><li> La línea de control de vectores (HM1 / pKT3M) y la línea experimental (HM1/pKT3M-FCU1) fueron generados por 1,2 se ha descrito anteriormente técnicas. Semillas de las líneas de estudio de los tubos de valores en T-25 frascos y mantener la selección mediante la adición de antibiótico apropiado (12 mg / ml de G418) en TYI-S-33 3 medianas. Los frascos deben ser del 70-80% confluente después de 16-18 horas de crecimiento a 37 ° C. </li><li> Prepare una solución fresca de valores de 50 mm de 5-fluorocitosina (5-FC, Sigma) en agua tibia TYI-S-33. Vortex rigurosamente durante varios minutos para disolver 5-FC por completo. Filtro a esterilizar la solución stock de pasar por un filtro de 0,22 micras. </li><li> Trofozoítos de la cosecha mediante la colocación de las botellas en hielo durante 3-5 minutos y la recolección de las células por centrifugación a 200xg durante 5 minutos a temperatura ambiente. Resuspender el precipitado en medio fresco TYI y el recuento de las células. </li><li> Diseño de un experimento típico utiliza 0,5 x 10 4 células por pocillo de una placa de 48 y en un volumen final de 1 ml, y se repite por triplicado para cada condición de prueba. Además, para facilitar las mediciones de fluorescencia, un plato aparte se utiliza para cada cepa por cada punto del tiempo. </li><li> Semillas de 0,5 x 10 4 células por pocillo para cada prueba 5-FC concentración, por triplicado. Mantener la presión de selección de antibióticos en el medio TYI. Ahora agregue la cantidad necesaria de 5-FC solución madre y trofozoítos a cada pocillo. </li><li> Las placas se incuban en una bolsa anaeróbica a 37 º C. </li></ol> 2. Determinación de la eficiencia de la selección <ol><li> Prepare 2 mg / ml de solución de archivo de la célula de seguimiento verde CMFDA (Invitrogen) en DMSO. Para hacer la solución de trabajo diluir la acción de 1000 veces en medio libre de suero TYI. </li><li> Se elimina el medio totalmente de placas de cultivo de trofozoíto y reemplazar con 150 l de suero libre de CMFDA TYI contiene. Continuar la incubación a 37 ° C durante 1 hora. </li><li> Retire la solución de colorante de los pozos y leer la placa en la espectrofluorómetro Spectramax M2 microplacas. La longitud de onda de excitación se debe establecer a 492 nm y la emisión de fluorescencia leer a 517 nm. </li><li> Comparar los valores de fluorescencia para el control de las células frente a los transfectantes de prueba. </li><li> Seguir leyendo las placas en cada intervalo de tiempo. Uso positivo adecuado (TYI solamente) y negativos (25 mg / ml de higromicina) pozos de control en cada plato. </li></ol> 3. Resultados representante La E. transfectantes histolytica mostró sólida expresión de codón optimizado FCU1 recombinante (Figura 1). Cuando este protocolo se sigue correctamente el resultado es la eliminación selectiva de E. histolytica células que expresan FCU1 en presencia de 0,5 mM 5-fluorocitosina. Control de las células, por el contrario seguirá creciendo normalmente en concentraciones de hasta 5 mm de 5-FC y llegar a la confluencia entre el 72 y 96 horas (Figura 2-a). El FCU1 células portadoras a las 48 horas está completamente lisadas en los pozos de tratamiento se observan bajo el microscopio. Estos también muestran una disminución de la fluorescencia cuando se tiñen con los colorantes vitales CMFDA, que se utiliza en los estudios cuantitativos participación de gran número de muestras (figura 2-b). El sistema FCU1/5-FC es una herramienta de selección negativa eficaz y potente para E. histolytica trofozoítos. <img src="/files/ftp_upload/2410/2410fig1.jpg" alt="Figura 1" /> Figura 1. Generación de E. histolytica HM1 transfectantes que expresan marcador de selección negativa (A) proteínas recombinantes FCU1 se detectó en un Western blot utilizando anticuerpos anti-c-Myc (panel superior). Como era de esperar, una banda de 42kDa podrían ser detectados (que se muestra con una flecha) en el lisado total de los transfectantes FCU, pero no en el vector solo transfectantes control. La imagen inferior muestra la misma mancha probaron con anticuerpos contra la actina como control de carga. (B) Los resultados de la PCR cuantitativa en tiempo real que muestra la expresión de ARNm específicos FCU1 normalizado a lgl4 control. <img src="/files/ftp_upload/2410/2410fig2.jpg" alt="Figura 2" /> Figura 2. Fármacos in vitro la sensibilidad de E. histolytica transfectantes que expresan marcador de selección negativa Las curvas de supervivencia de los transfectantes control (a) y (b) transfectantes FCU1 expresar cultivadas en 48 y placa. Las células fueron cultivadas en presencia de concentraciones crecientes del profármaco-5-fluorocitosina, la supervivencia celular se cuantificó por tinción CMFDA en cada intervalo de tiempo indicado en el eje X. El eje Y representa unidades de fluorescencia y es un reflejo directo de la población de células sobrevivientes. Tenga en cuenta que la FCU1 transfectantes que expresan mostró una sensibilidad significativa en la concentración de 0,5 mM profármaco en comparación con el control. No se observaron células en el punto 120 horas de tiempo en estos pozos como comcomparación con el crecimiento confluente visto en los pozos de control correspondiente al microscopio (datos no mostrados). Hygro denota 25μg / ml de higromicina utilizó como control negativo.

<ol>
	<li>Olvera, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+transfection+of+Entamoeba+histolytica+trophozoites+by+lipofection.">Stable transfection of Entamoeba histolytica trophozoites by lipofection.</a> Arch. Med. Res. , 49-51 (1997).</li><li>Saito-Nakano, Y., Yasuda, T., Nakada-Tsukui, K., Leippe, M., Nozaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab5-associated+vacuoles+play+a+unique+role+in+phagocytosis+of+the+enteric+protozoan+parasite+Entamoeba+histolytica.">Rab5-associated vacuoles play a unique role in phagocytosis of the enteric protozoan parasite Entamoeba histolytica.</a> J. Biol. Chem. 279, 49497-49507 (2004).</li><li>Diamond, L. S., Harlow, D. R., Cunnick, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+medium+for+the+axenic+cultivation+of+Entamoeba+histolytica+and+other+Entamoeba.">A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba.</a> Trans. R. Soc. Trop. Med. Hyg. 72, 431-432 (1978).</li><li>Clark, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anaerobic parasitic protozoa : genomics and molecular biology. , (2010).</li><li>Linford, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+hairpin+RNA-mediated+knockdown+of+protein+expression+in+Entamoeba+histolytica.">Short hairpin RNA-mediated knockdown of protein expression in Entamoeba histolytica.</a> BMC Microbiol. 9, 38-38 (2009).</li><li>Solis, C. F., Guill&#233;n, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+genes+by+RNA+interference+in+the+protozoan+parasite+Entamoeba+histolytica.">Silencing genes by RNA interference in the protozoan parasite Entamoeba histolytica.</a> Methods Mol. Biol. 442, 113-128 (2008).</li><li>Solis, C. F., Santi-Rocca, J., Perdomo, D., Weber, C., Guill&#233;n, N. <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+bacterially+expressed+dsRNA+to+downregulate+Entamoeba+histolytica+gene+expression.">Use of bacterially expressed dsRNA to downregulate Entamoeba histolytica gene expression.</a> PLoS ONE. 4, e8424-e8424 (2009).</li><li>MacFarlane, R. C., Singh, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+dsRNA-based+gene+silencing+in+Entamoeba+histolytica:+implications+for+approaches+to+genetic+analysis.">Loss of dsRNA-based gene silencing in Entamoeba histolytica: implications for approaches to genetic analysis.</a> Exp. Parasitol. 119, 296-300 (2008).</li><li>Dhar, S. K., Vines, R. R., Bhattacharya, S., Petri, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosomal+DNA+fragments+enhance+the+stability+of+transfected+DNA+in+Entamoeba+histolytica.">Ribosomal DNA fragments enhance the stability of transfected DNA in Entamoeba histolytica.</a> J. Eukaryot. Microbiol. 45, 656-660 (1998).</li><li>Erbs, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+cancer+gene+therapy+by+adenovirus-mediated+transfer+of+a+bifunctional+yeast+cytosine+deaminase/uracil+phosphoribosyltransferase+fusion+gene.">In vivo cancer gene therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion gene.</a> Cancer Res. 60, 3813-3822 (2000).</li><li>Braks, J. A. M., Franke-Fayard, B., Kroeze, H., Janse, C. J., Waters, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+application+of+a+positive-negative+selectable+marker+system+for+use+in+reverse+genetics+in+Plasmodium.">Development and application of a positive-negative selectable marker system for use in reverse genetics in Plasmodium.</a> Nucleic Acids Res. 34, e39-e39 (2006).</li><li>Maier, A. G., Braks, J. A. M., Waters, A. P., Cowman, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+selection+using+yeast+cytosine+deaminase/uracil+phosphoribosyl+transferase+in+Plasmodium+falciparum+for+targeted+gene+deletion+by+double+crossover+recombination.">Negative selection using yeast cytosine deaminase/uracil phosphoribosyl transferase in Plasmodium falciparum for targeted gene deletion by double crossover recombination.</a> Mol. Biochem. Parasitol. 150, 118-121 (2006).</li></ol>

La producción de este video fue patrocinado por Sigma Aldrich, Inc., que produce algunos de los reactivos utilizados en este artículo.

Aunque ha habido avances sustanciales en la Entamoeba biológica herramientas moleculares que no hay métodos actualmente disponibles para eliminar o alterar los genes 4. Derribo de la expresión de genes específicos se ha logrado mediante el uso de 5 ARN horquilla, pequeños ARN de interferencia bacteriana 6 y expresó dsRNA 7. Sin embargo, estos métodos si bien son eficaces para el objetivo genes respectivos, tienen varias limitaciones, incluyendo el uso de productos químicos costosos, la selección continua de los antibióticos y la necesidad de validación constante debido a la alta incidencia de reversión que ocurre en el tiempo (Alicia Linford, comunicación personal) 8. Cualquier plásmido puede replicarse en Entamoeba, ya que aparentemente no es un requisito secuencia estricta de los orígenes de replicación del ADN 9, y así una vez transfectadas, por lo general es difícil de curar un plásmido. Esto limita el posible uso de plásmidos intactos en los experimentos de recombinación de genes y por nocaut. Marcadores negativos de selección son componentes clave de los métodos de manipulación genética, ya que se puede utilizar para eliminar subpoblaciones recombinante. Hemos expresado con éxito un codón optimizado FCU1 gen en Entamoeba histolytica y probado su potencial como marcador de selección negativa. Esta fusión de genes ha sido utilizada para la terapia génica suicida de células tumorales humanas y metaboliza el profármaco 5-FC en productos derivados tóxicos, provocando la inhibición de la síntesis de ARN y ADN 10. FCU1 recientemente ha sido utilizada como un marcador negativo seleccionable en Plasmodium, otro parásito protozoario. Plasmodium berghei las células que expresan FCU1 se encontraron casi 1000 veces más sensible que el profármaco in vitro e in vivo del tratamiento con 5-FC mató a más de 99,9% de los infectando los parásitos recombinantes en el modelo de ratón eritrocítica etapas de la malaria 11. E. Las células que expresan histolytica FCU1 sólo mostró una sensibilidad 30 veces mayor que el profármaco 5-FC, en contraste con la sensibilidad observada en P. berghei. Las posibles razones para esto podría ser una gran piscina de nucleótidos disponibles en el medio de cultivo en competencia con los metabolitos tóxicos. En tanto P. berghei y P. falciparum en el marcador FCU1 se ha utilizado en la interrupción de genes específicos. estudios 11,12. Nuestro objetivo es manipular el genoma de la Entamoeba usando recombinación homóloga. La validación del sistema de selección negativa FCU1/5-FC es un paso importante hacia este objetivo.

<table border="1">
<tr>
<td>Material Name</td>
<td>Type</td>
<td>Company</td>
<td>Catalogue Number</td>
<td>Comments</td>
</tr>
<tr>
<td>TYI-S-33 medium</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>&nbsp;</td>
<td>Reference included in the text</td>
</tr>
<tr>
<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=F7129|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC">5-Fluorocytosine</a></td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F7129</td>
<td>&nbsp;</td>
<tr>
<td>Cell tracker green CMFDA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>C2925</td>
<td>&nbsp;</td>
<tr>
<td>Spectra Max M2 plate-reader</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		
</table>

development of a negative selectable marker for entamoeba histolytica

Entamoeba histolytica es el agente causal de la amibiasis e infecta hasta un 10% de la población mundial. Las técnicas moleculares que han permitido a la arriba y abajo-regulación de la expresión génica se basan en la transfección de plásmidos mantenido de forma estable. Si bien estos han aumentado nuestra comprensión de los factores de virulencia de Entamoeba, la capacidad de integrar ADN exógeno en el genoma, lo que permitiría revertir experimentos genéticos, sería una ventaja significativa en el estudio de este parásito. Los desafíos presentados por este organismo incluyen la incapacidad para seleccionar a los eventos de recombinación homóloga y la dificultad para curar ADN plásmido episomal de trofozoítos transfectadas. Los resultados más adelante en un contexto de alta de ADN exógeno, un problema importante en la identificación de trofozoitos en el que una bona fide caso de la integración genómica se ha producido. Se presenta el desarrollo de un sistema de selección negativa basada en la expresión transgénica de una citosina deaminasa levadura y uracilo fosforribosil transferasa quimera (FCU1) y la selección con el profármaco 5-fluorocitosina (5-FC). La enzima convierte FCU1 no tóxico 5-FC en tóxico 5-fluorouracilo y 5-fluorouridina-5&#39;-monofosfato de E.. líneas histolytica expresar FCU1 resultaron ser 30 veces más sensibles a los profármaco comparación con la cepa control.

Watch this Scientific Journal Video about Development of a Negative Selectable Marker for Entamoeba histolytica at JoVE.com

Development of a Negative Selectable Marker for Entamoeba histolytica

Se presenta el desarrollo de un sistema de selección negativa en<em&gt; E. histolytica</em&gt; Basado en la expresión transgénica de una proteína quimérica (FCU1) y la selección con el profármaco 5-fluorocitosina. La proteína FCU1 es una fusión de la levadura deaminasa citosina y uracilo fosforribosiltransferasa. Expresión de FCU1 traducido en un aumento<em&gt; E. histolytica</em&gt; Sensibilidad hacia la 5-fluorocitosina.

Desarrollo de un marcador negativo seleccionable para Entamoeba histolytica</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

development-of-a-negative-selectable-marker-for-entamoeba-histolytica

Research

JoVE Journal

Immunology and Infection

Development of a Negative Selectable Marker for Entamoeba histolytica

12.3K vistas.  University of Virginia Health System. Se presenta el desarrollo de un sistema de selección negativa en E. histolytica Basado en la expresión transgénica de una proteína quimérica (FCU1) y la selección con el profármaco 5-fluorocitosina. La proteína FCU1 es una fusión de la levadura deaminasa citosina y uracilo fosforribosiltransferasa. Expresión de FCU1 traducido en un aumento E. histolytica Sensibilidad hacia la 5-fluorocitosina.

Video: Development of a Negative Selectable Marker for Entamoeba histolytica

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 (Ehmeth) Enzyme

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of the three most common causes of death from parasitic disease. The agent of amebiasis is the amoeba parasite Entamoeba histolytica that exists under two stages: the infective cyst found in food or water and the invasive trophozoite living in the intestine. The clinical manifestations of amebiasis range from being asymptomatic to colitis, dysentery or liver abscesses. E. histolytica is one of the rare unicellular parasite with 5-methylcytosine (5mC) in its genome. 1, 2 It contains a single DNA methyltransferase, Ehmeth, that belongs to the Dnmt2 family. 2 A role for Dnmt2 in the control of repetitive elements has been established in E. histolytica, 3 Dictyostelium discoideum 4,5 and Drosophila. 6 Our recent work has shown that Ehmeth methylates tRNAAsp, and this finding indicates that this enzyme has a dual DNA/tRNAAsp methyltransferase activity. 7 This observation is in agreement with the dual activity that has been reported for D. discoideum and D. melanogaster. 8 The functional significance of the DNA/tRNA specificity of Dnmt2 enzymes is still unknown. To address this question, a method to determine the tRNA methyltransferase activity of Dnmt2 proteins was established. In this video, we describe a straightforward approach to prepare an adequate tRNA substrate for Dnmt2 and a method to measure its tRNA methyltransferase activity.

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 Ehmeth Enzyme

This protocol describes the preparation of a synthetic tRNA substrate for the Entamoeba histolytica DNA/tRNA methyltransferase 2 (Dnmt2) homolog Ehmeth and the measure of its methyltransferase activity. This experimental approach can be used for investigating the activity of other Dnmt2 proteins.

Ensayo in vitro de metilación del ADN tRNA con Entamoeba histolytica y Dnmt2 tRNA metiltransferasa (Ehmeth) Enzima

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of ...

Investigación

Inmunología e Infección

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

El examen de selección tímica positivo y negativo por citometría de flujo

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Usando Click Química para medir el efecto de la infección viral en el Host-Cell ARN Síntesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

La identificación rápida de bacterias gram negativas de Cultura sangre caldo de cultivo utilizando MALDI-TOF espectrometría de masas

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

Análisis del daño epitelial Producido por<em&gt; Entamoeba histolytica</em&gt; Infección

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will die from the disease. The human leukocyte antigen (HLA) class I samples peptides derived from proteasomal degradation of cellular proteins and presents these fragments on the cell surface for interrogation by circulating cytotoxic T lymphocytes (CTL). Generation of T-cell receptor mimic (TCRm) monoclonal antibodies (mAbs) which recognize breast cancer specific peptide/HLA-A*02:01 complexes such as those derived from macrophage migration inhibitory factor (MIF19-27) and NY-ESO-1157-165 enable detection and destruction of breast cancer cells in the absence of an effective anti-tumor CTL response. Intact class I HLA/peptide complexes are shed by breast cancer cells and represent potentially relevant cancer biomarkers. In this work, a breakthrough biomarker screening system for cancer diagnostics incorporating T-cell receptor mimic monoclonal antibodies combined with a novel, label-free biosensor utilizing guided-mode resonance (GMR) sensor technology is presented. Detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated. The impact of this work could revolutionize personalized medicine through development of companion disease diagnostics for targeted immunotherapies.

Intact class I HLA/peptide complexes are shed by cancer cells, representing a potential relevant cancer biomarker. Utilizing label-free sensor technology and T-cell receptor mimicking monoclonal antibodies, detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated, enabling development of a novel cancer diagnostic platform.

La detección del antígeno leucocitario humano biomarcadores en cáncer de mama Utilizando-Label gratuita tecnología de biosensores

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

La visualización de los Efectos de esputo en el desarrollo del biofilm mediante un modelo de Septadas cubreobjetos

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Desarrollo de un sistema reportero virus de la hepatitis B para supervisar las primeras etapas del ciclo de replicación

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents. The thin surface is easily damaged, leaving the sample difficult or impossible to image. Infectious viruses must be handled in a biosafety cabinet (BSC) and some require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL)-3 and -4 is especially challenging because these environments are more turbulent and technicians are required to wear personal protective equipment (PPE), which decreases dexterity. 
In this study, we evaluated a new device to assist in negative staining viruses in biocontainment. The device is a capsule that works as a specialized pipette tip. Once grids are loaded into the capsule, the user simply aspirates reagents into the capsule to deliver the virus and stains to the encapsulated grid, thus eliminating user handling of grids. Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.

This protocol provides instruction for negative staining virus samples which can easily be used in BSL-2, -3, or -4 laboratories. It includes the use of an innovative processing capsule, which protects the transmission electron microscopy grid and provides the user easier handling in the more turbulent environments within biocontainment.

Utilización de cápsulas para la tinción negativa de muestras víricas en biocontención

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

Simultaneous Distinction of Monospecific and Mixed DFS70 Patterns During ANA Screening with a Novel HEp-2 ELITE/DFS70 Knockout Substrate

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies available for ANA screening, indirect immunofluorescence (IIF) using Human epithelial cells-2 (HEp-2) substrate remains the primary and recommended method because of its superior sensitivity. HEp-2 substrates can detect a multitude of patterns resulting from autoantibody binding to various protein and nucleic acid autoantigens distributed throughout the nucleus and cytoplasm of the cells. The great diversity of monospecific and mixed patterns resulting from positive reactions on HEp-2 substrate also complicate the interpretation and accuracy of reporting. One specific example which received utmost attention recently is the dense fine speckled 70 (DFS70) pattern resulting from autoantibodies that specifically bind to a protein called lens epithelium derived growth factor (LEDGF). Lack of clear association with a specific systemic autoimmune disease and high prevalence in healthy populations have made accurate interpretation of DFS70 pattern important. Accurate distinction of DFS70 pattern from disease-associated patterns using conventional HEp-2 substrate is challenging. Moreover, frequent co-occurrence of DFS70 pattern along with disease-associated patterns such as homogeneous, speckled, and mixed homogeneous-speckled patterns complicate the IIF interpretation. The goal of this paper is to demonstrate the utility of a novel engineered HEp-2 IIF substrate that retains all advantages of conventional HEp-2 substrate while simultaneously providing the ability to distinguish DFS70 pattern with high confidence in both monospecific and mixed ANA positive examples. The new substrate is further able to unmask disease-associated ANA patterns previously concealed by DFS70 pattern.

DFS70 autoantibodies mimic common disease-associated antinuclear antibody patterns making accurate interpretation challenging when using conventional HEp-2 substrates. The protocol describes advantages of novel engineered HEp-2 substrate over conventional HEp-2 in ANA screening and distinguishing DFS70 patterns with high confidence in both monospecific and mixed ANA positive cases.

Distinción simultánea de monoespecíficas y patrones mixtos DFS70 durante la proyección de ANA con un sustrato de la novela nocaut en ELITE/DFS70 HEp-2

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies ...