Nous tenons à remercier le Dr Philippe Erbs pour nous avoir fourni la séquence du gène FCU1. Ce travail a été soutenu par le NIH subventions AI 26649.

1. Système FCU sélection négative dans Entamoeba histolytica <ol><li> La ligne de la lutte antivectorielle (HM1 / pKT3M) et la ligne expérimentale (HM1/pKT3M-FCU1) ont été générés par 1,2 les techniques décrites précédemment. Graine les lignes à l&#39;étude à partir des tubes d&#39;actions dans T-25 flacons et conserver la sélection par l&#39;ajout des antibiotiques appropriés (12 pg / ml de G418) dans TYI-S-33 3 moyen. Les flacons doivent être 70-80% confluentes après 16-18 heures de croissance à 37 ° C. </li><li> Préparer une solution stock de frais de 50mm de la 5-fluorocytosine (5-FC, Sigma) dans chaude TYI-S-33 à moyen terme. Vortex rigoureuse pendant plusieurs minutes à se dissoudre complètement la 5-FC. Filtre-stériliser la solution stock en le passant à travers un filtre de 0,22 um. </li><li> Trophozoïtes récolte en plaçant les flacons sur la glace pendant 3-5 minutes et recueillir les cellules par centrifugation à 200xg pendant 5 minutes à température ambiante. Reprendre le culot dans TYI milieu frais et compter les cellules. </li><li> Conception d&#39;une expérience typique utilise 0,5 x 10 4 cellules par puits d&#39;une plaque de 48 puits dans un volume final de 1 ml, et répète trois exemplaires pour chaque condition d&#39;essai. En outre, pour faciliter les mesures fluorescente, une plaque séparée est utilisée pour chaque souche par point dans le temps. </li><li> Graine de 0,5 x 10 4 cellules par puits pour chaque test de concentration de 5-FC, en trois exemplaires. Maintenir la pression de sélection antibiotique dans le milieu TYI. Maintenant, ajoutez la quantité requise de 5-FC solution stock et des trophozoïtes à chaque puits. </li><li> Incuber les plaques dans un sac anaérobie à 37 ° C. </li></ol> 2. Détermination de l&#39;efficacité de sélection <ol><li> Préparer 2 mg / ml solution stock de Cell Tracker CMFDA vert (Invitrogen) dans le DMSO. Pour rendre la solution de travail diluer le stock de 1000 fois dans un milieu sans sérum TYI. </li><li> Retirez le support complètement de plaques de culture trophozoïte et la remplacer par 150 ul sans sérum contenant CMFDA TYI. Continuer d&#39;incubation à 37 ° C pendant 1 heure. </li><li> Retirer la solution de colorant à partir de puits et de lire la plaque dans le spectrofluorimètre microplaques Spectramax M2. La longueur d&#39;onde d&#39;excitation doit être réglé à 492 nm et l&#39;émission de fluorescence lue à 517 nm. </li><li> Comparez les valeurs de fluorescence pour les cellules de contrôle par rapport au test de transfectants. </li><li> Continuer la lecture des plaques à chaque intervalle de temps. Utilisez positifs appropriés (TYI seulement) et négatifs (25 ug / ml d&#39;hygromycine) puits de contrôle dans chaque assiette. </li></ol> 3. Les résultats représentatifs Le E. transfectants histolytica a montré une expression robuste de codon FCU1 recombinante optimisée (figure 1). Lorsque ce protocole est suivi correctement il en résulte l&#39;élimination sélective des E. cellules exprimant FCU1 histolytica en présence de 0,5 mM de 5-fluorocytosine. Cellules de contrôle, en revanche continuer à croître normalement à un maximum de 5 mM de concentration de 5-FC et atteindre la confluence entre 72 et 96 heures (figure 2-a). Les cellules porteuses FCU1 à 48 heures sont complètement lysées lorsque le puits traités sont considérés sous un microscope. Ces montrent également une diminution de fluorescence lorsqu&#39;elles sont colorées avec le colorant vital CMFDA, qui est utilisé dans les études quantitatives impliquant grand nombre d&#39;échantillons (figure 2-b). Le système FCU1/5-FC est un outil de sélection efficace et puissant négatifs pour E. histolytica trophozoïtes. <img src="/files/ftp_upload/2410/2410fig1.jpg" alt="Figure 1" /> Figure 1. Génération de E. histolytica HM1 transfectants exprimant marqueur de sélection négative (A) FCU1 protéine recombinante a été détecté sur un western blot à l&#39;aide d&#39;anticorps anti-c Myc (panneau supérieur). Comme prévu, une bande 42kDa a pu être détecté (indiqué par une flèche) dans le lysat total de la transfectants FCU, mais pas dans le vecteur seul transfectants contrôle. Le panneau du bas montre la même blot sondé avec l&#39;anticorps anti-actine comme contrôle du chargement. (B) Résultats de la PCR en temps réel quantitative montrant l&#39;expression des ARNm spécifiques FCU1 normalisée à lgl4 contrôle. <img src="/files/ftp_upload/2410/2410fig2.jpg" alt="Figure 2" /> Figure 2. Sensibilité aux médicaments in vitro de E. transfectants exprimant histolytica marqueur de sélection négative Les courbes de survie des transfectants de contrôle (a) et (b) des transfectants exprimant FCU1 cultivées dans 48 puits assiette. Les cellules ont été cultivées en présence de concentrations croissantes de la prodrogue-5-fluorocytosine, la survie cellulaire a été quantifiée par une coloration CMFDA à chaque intervalle de temps que notée sur le X-axe. L&#39;axe Y représente les unités de fluorescence et est le reflet direct de la population de cellules survivantes. S&#39;il vous plaît noter que les transfectants exprimant FCU1 a montré une sensibilité significative à la concentration 0,5 mM prodrogue par rapport au témoin. Pas de cellules ont été observées au point dans le temps 120h dans ces puits comme comcomparé à la croissance confluente vu dans le puits de contrôle correspondants sous le microscope (données non présentées). Hygro représente 25μg / ml d&#39;hygromycine utilisé comme contrôle négatif.

<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 production de cette vidéo a été parrainé par Sigma Aldrich, Inc, qui produit certains des réactifs utilisés dans cet article.

Bien qu&#39;il y ait eu des progrès substantiels dans Entamoeba outils de biologie moléculaire il n&#39;existe pas de méthodes actuellement disponibles pour supprimer ou perturber les gènes 4. Knockdown de l&#39;expression génique spécifique a été réalisé par l&#39;utilisation d&#39;ARN en épingle à cheveux 5, 6 petits ARN interférents et exprimées par la bactérie ARNdb 7. Cependant, ces méthodes tout en étant efficace pour les gènes cibles respectifs, ont plusieurs limites dont l&#39;utilisation des produits chimiques coûteux, la sélection continue contre les antibiotiques et la nécessité pour la validation constante due à l&#39;incidence élevée de réversion survenant au cours du temps (Alicia Linford, communication personnelle) 8. Toute plasmide peut se répliquer dans les Entamoeba car il n&#39;est apparemment pas une exigence stricte pour la séquence origines de réplication de l&#39;ADN 9, et donc une fois transfectées, il est généralement difficile à guérir un plasmide. Ceci limite l&#39;utilisation possible des plasmides intacts dans des expériences de recombinaison et knockout du gène. Marqueurs de sélection négative sont des éléments clés de ciblage génique méthodes, car ils peuvent être utilisés pour éliminer les sous-populations recombinantes. Nous avons exprimé avec succès un codon optimisé FCU1 gène dans E. histolytica et testé son potentiel en tant que marqueur sélectionnable négatif. Ce gène de fusion a été utilisé pour la thérapie génique suicide de cellules tumorales humaines et métabolise le promédicament 5-FC en produits toxiques aval résultant de l&#39;inhibition de la synthèse d&#39;ARN et l&#39;ADN 10. FCU1 a été récemment utilisé comme un marqueur sélectionnable négatif au Plasmodium, un autre parasite protozoaire. Plasmodium berghei cellules exprimant FCU1 ont été trouvés pour être près de 1000 fois plus sensible à la prodrogue in vitro et in vivo avec un traitement de 5-FC a tué plus de 99,9% du infectant des parasites recombinants dans le modèle de souris au stade érythrocytaire du paludisme 11. E. cellules exprimant FCU1 histolytica a montré qu&#39;une sensibilité de 30 fois plus élevé au promédicament 5-FC par opposition à la sensibilité observée dans P. berghei. Les raisons possibles pour cela pourrait être important bassin de nucléotides disponibles dans le milieu de croissance en compétition avec les métabolites toxiques. Dans les deux P. berghei et P. falciparum le marqueur FCU1 a été utilisé dans disruption génique ciblée. Des études 11,12. Notre objectif est de manipuler le génome Entamoeba utilisant la recombinaison homologue. La validation du système de sélection négative FCU1/5-FC est une étape importante vers cet objectif.

<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 est l&#39;agent causal de l&#39;amibiase et infecte jusqu&#39;à 10% de la population mondiale. Les techniques moléculaires qui ont permis le haut et le bas-régulation de l&#39;expression génique reposent sur la transfection de plasmides maintenu de manière stable. Alors que ceux-ci ont amélioré notre compréhension des facteurs de virulence Entamoeba, la capacité d&#39;intégrer l&#39;ADN exogène dans le génome, ce qui permettrait inverse expériences génétique, serait un avantage significatif dans l&#39;étude de ce parasite. Les défis présentés par cet organisme comprennent l&#39;incapacité de choisir pour les événements de recombinaison homologue et de la difficulté à guérir épisomale ADN plasmidique de trophozoïtes transfectées. Les résultats plus tard dans un bruit de fond élevé d&#39;ADN exogène, un problème majeur dans l&#39;identification des trophozoites dans lequel une bonne foi évènement intégration génomique s&#39;est produite. Nous rapportons le développement d&#39;un système de sélection négative basée sur l&#39;expression transgénique d&#39;une cytosine désaminase de la levure et l&#39;uracile phosphoribosyl transférase chimère (FCU1) et la sélection avec prodrogue 5-fluorocytosine (5-FC). L&#39;enzyme convertit FCU1 non toxique 5-FC en 5-fluorouracile toxiques et 5-fluoro-5&#39;-monophosphate. E. histolytica lignes exprimant FCU1 ont été trouvés à 30 fois plus sensible à la prodrogue par rapport à la souche témoin.

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

Nous rapportons le développement d&#39;un système de sélection négative dans<em&gt; E. histolytica</em&gt; Basé sur l&#39;expression transgénique d&#39;une protéine chimérique (FCU1) et la sélection avec la prodrogue 5-fluorocytosine. La protéine FCU1 est une fusion de la cytosine désaminase levure et uracile phosphoribosyltransférase. Expression de FCU1 entraîné une augmentation<em&gt; E. histolytica</emSensibilité&gt; vers la 5-fluorocytosine.

Développement d&#39;un marqueur sélectionnable négatif pour les 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

12.4K vues.  University of Virginia Health System. Nous rapportons le développement d'un système de sélection négative dans E. histolytica Basé sur l'expression transgénique d'une protéine chimérique (FCU1) et la sélection avec la prodrogue 5-fluorocytosine. La protéine FCU1 est une fusion de la cytosine désaminase levure et uracile phosphoribosyltransférase. Expression de FCU1 entraîné une augmentation E. histolytica vers la 5-fluorocytosine.

Vidéo: 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.

Test in vitro avec la méthylation de l&#39;ADN ARNt avec Entamoeba histolytica et ARNt Dnmt2 méthyltransférase (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 ...

Recherche

Immunologie et infection

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.

L&#39;examen de sélection positive et négative thymique par cytométrie en flux

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.

Utilisation de Click Chemistry pour mesurer l&#39;effet de l&#39;infection virale sur Host-Cell Synthèse des ARN

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.

Identification rapide des bactéries à Gram négatif de sang Bouillon de culture à l&#39;aide MALDI-TOF spectrométrie de masse

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.

Analyse de l&#39;altération de l&#39;épithélium Produit par<em&gt; Entamoeba histolytica</em&gt; Maladies infectieuses

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.

Détection de l&#39;antigène leucocytaire humain biomarqueurs du cancer du sein Utilisant la technologie des biocapteurs Sans étiquette

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.

Visualisation des effets des expectorations sur le développement du biofilm Utilisation d&#39;un modèle Chambered lamelle couvre-objet

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.

Développement d&#39;un système de virus de l&#39;hépatite B Reporter pour surveiller les premiers stades du cycle de réplication

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.

Utilisation de capsules pour la coloration négative des échantillons viraux dans le cadre du suivi biologique

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.

Distinction simultanée de monospécifiques et modèles mixtes DFS70 cours ANA de dépistage avec un substrat de roman HEp-2 ELITE/DFS70 Knockout

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