Nous sommes reconnaissants à Junko Wada pour son excellente assistance technique. Nous sommes reconnaissants au Prof Akio Nomoto d&#39;avoir bien voulu fournir un vecteur d&#39;expression baculovirus pour le PVR-IgG2a. Cette étude a été financée en partie par des subventions en aide à la promotion de l&#39;éradication de la poliomyélite et de la recherche sur les maladies émergentes et ré-émergentes Maladies infectieuses du ministère de la Santé, du Travail et des Affaires sociales.

1. Moyennes Préparation du milieu de maintenance (MM) et la croissance (GM) Pour la préparation de MM et GM, s&#39;il vous plaît consulter un manuel de laboratoire de la poliomyélite de l&#39;OMS 1. Milieu de Dulbecco modifié Eagle (DMEM) supplémenté avec 2% sérum de veau foetal (FCS) et 10% de FCS pourrait être utilisés comme substituts de MM et GM, respectivement. 2. Préparation des anticorps anti-PV <ol><li> Reconstituer des anticorps anti-PV (RIVM PV tapant antisérum) par adjonction d&#39;eau (0,5 ml par flacon) pour chaque flacon d&#39;anti-PV anticorps (de type 1, 2 ou 3). Ajouter cette solutions d&#39;anticorps reconstitué (0,5 ml chacun) de 4,5 mL de MM dans des tubes étiquetés séparément. Remarque: Certains lots de l&#39;anti-PV anticorps sont fournis en tant que forme reconstituée (0,5 ml par flacon). Pour ce lot d&#39;anti-PV anticorps, ajout d&#39;eau pour la reconstitution n&#39;est pas nécessaire. </li><li> Mélanger un volume égal de reconstituer anti-PV anticorps. Pour les anti-PV1 2, 2 3, 1 ou 3: Mélanger 1,8 ml de chaque anticorps reconstitué (1,8 ml + 1,8 ml = 3,6 ml pour chaque mélange). Pour les anti-PV1 2 3: Mélanger 1,2 ml de chaque anticorps reconstitué (1,2 ml + 1,2 ml + 1,2 ml = 3,6 ml). Remarque: Les échantillons de PV pourraient contenir un mélange de différents sérotypes de PV. Pour identifier seul sérotype de PV dans les échantillons avec un mélange de PV, les piscines de l&#39;anti-PV anticorps (c&#39;est à dire 1 +2, 2 +3, 3 +1, 1 +2 +3) est nécessaire. </li><li> Ajouter le volume 1 / 5 de FCS (0,72 ml) à chaque anti-PV (1 +2, 2 +3, 3 +1, ou 1 +2 +3) d&#39;anticorps (3,6 ml chacune) (dernière 18,2% de FCS). Remarque: Ajout de FCS est pour la réduction de la non-spécifiques agglutination de particules de gélatine causée par des anticorps. </li><li> Gardez les piscines d&#39;anticorps à -20 ° C jusqu&#39;à leur utilisation dans le dosage de l&#39;AP. </li></ol> 3. Reconstitution de particules de gélatine sensibilisées <ol><li> Ajouter 1,5 mL de tampon de reconstitution inclus dans le kit d&#39;un flacon de particules de gélatine sensibilisées (à condition que poudre lyophilisée). </li><li> Mélangez bien en tapotant légèrement </li><li> Gardez la particule reconstituée sensibilisés à 4 ° C. Reconstitué particules de gélatine pourrait être actif au moins 2 semaines conservés à 4 ° C, mais doit être préparé juste avant le dosage. </li></ol> Note importante: Ne mélangez pas beaucoup différents de particules de gélatine sensibilisées, car l&#39;image non-agglutination de particules de gélatine sensibilisées peuvent être différents par le nombre de particules. Pour chaque expérience, un seul lot de particules de gélatine sensibilisées doivent être utilisés. 4. PA test d&#39;identification pour le PV <ol><li> Préparer une plaque de micro-titrage. Pour l&#39;identification d&#39;un échantillon de virus, nous avons besoin de 5 puits + au moins un contrôle négatif (particule sensibilisée sans virus et les anticorps, ainsi n ° 6 ci-dessous. See No de contrôle des virus et des anticorps dans l&#39;exemple). Nombre total de puits est de 5 échantillons x + au moins un contrôle dans le dosage. Note importante: S&#39;il vous plaît prenez au moins un virus et pas de contrôle d&#39;anticorps dans le dosage. Un flacon de particule sensibilisée est d&#39;environ 10 pour l&#39;identification des échantillons de virus. </li><li> Ajouter 12 ul / puits d&#39;anticorps anti-PV d&#39;anticorps ou de GM à la plaque de titration de micro-. L&#39;agencement de la plaque est comme suit (Voir aussi l&#39;exemple ci-dessous). Dans les échantillons sans anticorps (puits # 1 et 6), ajouter 12 ul de GM au lieu de l&#39;anti-PV anticorps. Note importante: Si de nombreux échantillons PV doivent être testés dans cet essai, pipette multicanaux ou un distributeur serait utile pour cette étape. <table border="1"><tbody><tr><td> Eh bien # 1 </td><td> Eh bien # 2 </td><td> Eh bien # 3 </td><td> Eh bien # 4 </td><td> Eh bien # 5 </td><td> Eh bien # 6 </td></tr><tr><td> GM </td><td> anti-P1 +2 +3 </td><td> anti-P1 2 </td><td> anti-P2 3 </td><td> Ani-P1 3 </td><td> GM </td></tr></tbody></table></li><li> Ajouter 12 ul / puits d&#39;échantillon du virus. Dans un échantillon sans virus (puits # 6), ajouter 12 ul de GM au lieu d&#39;une solution de virus. Remarque: l&#39;échantillon de virus est surnageant de culture de cellules infectées, les cellules ou les cellules L20B généralement RD. Note importante: Si de nombreux échantillons PV doivent être testés dans cet essai, pipette multicanaux serait utile pour cette étape. <table border="1"><tbody><tr><td> Eh bien # 1 </td><td> Eh bien # 2 </td><td> Eh bien # 3 </td><td> Eh bien # 4 </td><td> Eh bien # 5 </td><td> Eh bien # 6 </td></tr><tr><td> échantillon de virus </td><td> échantillon de virus </td><td> échantillon de virus </td><td> échantillon de virus </td><td> échantillon de virus </td><td> GM </td></tr></tbody></table></li><li> Prenez la solution reconstituée particule sensibilisée à partir de 4 ° C à la température ambiante, et tapotez doucement à suspendre totalement les particules. Note importante: Après la suspension complète des particules en tapotant légèrement, les particules doivent être ajoutés immédiatement aux plaques avant la précipitation des particules en suspensionte et la forme non homogène solution. </li><li> Ajouter 25 ul / puits de particule sensibilisée à tous les puits. Note importante: Après l&#39;ajout de particules sensibilisées, la plaque doit être immédiatement soumis à la plaque de mélange pour démarrer la réaction uniforme des échantillons. Par conséquent, l&#39;ajout de particules sensibilisées doit être effectuée en utilisant distributeur de minimiser un décalage entre les échantillons de la plaque. </li><li> Mettez la plaque de titration de micro-sur une plaque de mélangeur et mélanger la plaque pendant 30 sec. </li><li> Mettez la plaque de titration de micro-sur papier blanc (pour la commodité de l&#39;observation visuelle) sur une table à la température ambiante (15 ~ 30 ° C) pendant 2 h. Note importante: Ne pas déplacer la plaque pendant et après la réaction pour l&#39;observation des résultats et de prendre des photos. L&#39;agglutination formé n&#39;est pas stable et facilement perturbé par le déplacement des plaques. </li><li> Prenez des photos de la plaque. Note importante: Vue partielle des photos ne pouvait pas donner suffisamment de données pour examen ultérieur et l&#39;examen des résultats d&#39;interprétation. Plusieurs photos doivent être prises pour couvrir tous les puits des plaques en se concentrant sur l&#39;image d&#39;agglutination des puits. </li></ol> 5. Interprétation des résultats <ol><li> Identification des puits positifs et négatifs. Comparez l&#39;agglutination dans les échantillons avec celle du Pas de contrôle du virus et d&#39;anticorps (précipitation de particules de gélatine, non-agglutination) (figure 1 et 2). Images d&#39;agglutination qui sont clairement distingués des Pas de virus et pas de contrôle d&#39;anticorps (non-agglutination, témoin négatif) doit être considéré comme positif, et les images d&#39;agglutination qui sont similaires au témoin négatif devrait être jugé comme négatif. Les échantillons qui montrent clairement l&#39;agglutination, mais seulement partiel pouvait être qualifié de positif, mais le bilan comme «positive (agglutination partielle). </li><li> Identification des PV. Interpréter les résultats basés sur des puits positifs et négatifs avec des anti-PV anticorps pour identifier les sérotypes PV. Principe de l&#39;interprétation du résultat est similaire à celle du test de neutralisation du système de culture cellulaire (par exemple, positifs pour l&#39;anti-P1 deux anticorps indique que l&#39;échantillon contient de type 3 PV). </li></ol> 6. Les résultats représentatifs Résultat représentatif de PA test d&#39;identification PV est montré dans la figure 1. Toutes les souches PV formé agglutination de particules sensibilisées gélatine (Aucun anticorps des échantillons). En présence de correspondants anti-PV anticorps, les précipitations de particules de gélatine (non-agglutination) semblable à aucun virus et aucun contrôle des anticorps (témoin négatif) a été observée (par exemple, PV1 (Sabin) dans la présence d&#39;anticorps anti-P1 +2 ou anti-P3 1 anticorps). <img src="/files/ftp_upload/2824/2824fig1.jpg" alt="Figure 1" /> Figure 1. Virus solutions d&#39;identification par analyse de PV PA. De PV1 (Sabin), PV2 (Sabin) et PV3 (Sabin) (titres de virus de 1,2 x 10 8 CCID 50 / 12 uL, 8,2 x 10 7 CCID 50 / 12 uL, et 3,5 x 10 7 CCID 50 / 12 uL, respectivement) ont été utilisés pour l&#39;identification par test PA. Pas de virus et aucun contrôle des anticorps a montré la précipitation de particules de gélatine (non-agglutination, témoin négatif). <img src="/files/ftp_upload/2824/2824fig2.jpg" alt="Figure 2" /> Figure 2. Interprétation des puits positifs et négatifs. Virus des solutions de PV1 (Sabin), PV2 (Sabin) et PV3 (Sabin) (titres de virus de 2,4 x 10 8 CCID 50 / 25 uL, 1,7 x 10 8 CCID 50 / 25 uL, et 7,3 x 10 7 CCID 50 / 25 uL, respectivement) ont été dilués 1 / 20-1 / 320, puis 25 uL d&#39;échantillons dilués ont été examinés par un dosage PA sans anti-PV anticorps.

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Polio Laboratory Manual (4th edition) WHO/IVB/04.10. , (2004).</li><li>Arita, M., Masujima, S., Wakita, T., Shimizu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+particle+agglutination+method+with+soluble+virus+receptor+for+identification+of+poliovirus.">Development of a particle agglutination method with soluble virus receptor for identification of poliovirus.</a> J. Clin. Microbiol. 48, 2698-2702 (2010).</li><li>Bibb, J. A., Witherell, G., Bernhardt, G., Wimmer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+poliovirus+with+its+cell+surface+binding+site.">Interaction of poliovirus with its cell surface binding site.</a> Virology. 201, 107-115 (1994).</li><li>Koike, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+poliovirus+receptor+protein+is+produced+both+as+membrane-bound+and+secreted+forms.">The poliovirus receptor protein is produced both as membrane-bound and secreted forms.</a> Embo J. 9. , 3217-3224 (1990).</li><li>Mendelsohn, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+a+human+poliovirus+receptor+gene+into+mouse+cells.">Transformation of a human poliovirus receptor gene into mouse cells.</a> Proc. Natl. Acad. Sci. U S A. 83, 7845-7849 (1986).</li><li>Mendelsohn, C. L., Wimmer, E., Racaniello, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+receptor+for+poliovirus:+molecular+cloning,+nucleotide+sequence,+and+expression+of+a+new+member+of+the+immunoglobulin+superfamily.">Cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily.</a> Cell. 56, 855-865 (1989).</li><li>Arita, M., Koike, S., Aoki, J., Horie, H., Nomoto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+poliovirus+with+its+purified+receptor+and+conformational+alteration+in+the+virion.">Interaction of poliovirus with its purified receptor and conformational alteration in the virion.</a> J. Virol. 72, 3578-3586 (1998).</li><li>McDermott, B. M., Rux, A. H., Eisenberg, R. J., Cohen, G. H., Racaniello, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+distinct+binding+affinities+of+poliovirus+for+its+cellular+receptor.">Two distinct binding affinities of poliovirus for its cellular receptor.</a> J. Biol. Chem. 275, 23089-23096 (2000).</li><li>Pipkin, P. A., Wood, D. J., Racaniello, V. R., Minor, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+L+cells+expressing+the+human+poliovirus+receptor+for+the+specific+detection+of+polioviruses+in+vitro.">Characterisation of L cells expressing the human poliovirus receptor for the specific detection of polioviruses in vitro.</a> J. Virol. Methods. 41, 333-340 (1993).</li><li>Avoort, H. G. v. a. n. d. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+five+methods+for+intratypic+differentiation+of+polioviruses.">Comparative study of five methods for intratypic differentiation of polioviruses.</a> J. Clin. Microbiol. 33, 2562-2566 (1995).</li><li>Kilpatrick, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+group-,+serotype-,+and+vaccine+strain-specific+identification+of+poliovirus+isolates+by+real-time+reverse+transcription-PCR+using+degenerate+primers+and+probes+containing+deoxyinosine+residues.">Rapid group-, serotype-, and vaccine strain-specific identification of poliovirus isolates by real-time reverse transcription-PCR using degenerate primers and probes containing deoxyinosine residues.</a> J. Clin. Microbiol. 47, 1939-1941 (2009).</li></ol>

Souji Masujima est un employé d&#39;un nouveau produit de conception Département, Fujirebio Inc

L&#39;unique point de ce test PA est l&#39;utilisation de la molécule au lieu PVR de l&#39;anti-PV anticorps pour la détection de PV 2. Ceci permet une interaction spécifique des particules de gélatine sensibilisées avec PV avec une affinité uniforme à tous les trois sérotypes du PV 3-6. Nous avons utilisé une forme immunoadhésine de PVR (PVR-IgG2a) pour la sensibilisation des particules de gélatine, car les formes monomères de PVR ont seulement des affinités modérées au site de liaison unique ...

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires </th></tr><tr><td> Sensibilisé-gélatine particules </td><td> Fujirebio Inc </td><td></td><td> Test Kit </td></tr><tr><td> Reconstitution de tampon </td><td> Fujirebio Inc </td><td></td><td> Test Kit </td></tr><tr><td> Plaque de microtitration </td><td> Fujirebio Inc </td><td></td><td> Test Kit </td></tr><tr><td> Anti-PV d&#39;anticorps </td><td> RIVM </td><td></td><td></td></tr></tbody></table>

particle agglutination method for poliovirus identification

Dans le Global Polio Eradication Initiative, le diagnostic de laboratoire joue un rôle crucial en isolant et en identifiant PV à partir des échantillons de selles de paralysie flasque aiguë (PFA). À l&#39;Organisation mondiale de la Santé (OMS) Global Polio Laboratory Network PV isolement et l&#39;identification sont actuellement effectuées en utilisant le système de culture cellulaire et en temps réel de RT-PCR, respectivement. Dans l&#39;ère post-éradication de la PV, les procédures d&#39;identification simple et rapide serait utile pour la confirmation rapide des cas de polio dans les laboratoires nationaux. Dans la présente étude, nous allons montrer la procédure du roman de PA test développé pour l&#39;identification PV. Ce test utilise PA interaction du PV du récepteur numérique (PVR) et la molécule de virion qui est une affinité spécifique et uniforme pour tous les sérotypes de PV. La procédure est simple (une procédure étape en plaques de réaction) et rapide (les résultats peuvent être obtenus dans les 2 h de réaction), et le résultat est visuellement observé (observation de l&#39;agglutination des particules de gélatine).

Watch this Scientific Journal Video about Particle Agglutination Method for Poliovirus Identification at JoVE.com

Particle Agglutination Method for Poliovirus Identification

Une particule récemment développé une agglutination roman (PA) de dosage utilisant molécule réceptrice du virus a permis une identification rapide et facile de poliovirus (PV). Dans cet article, nous allons montrer la procédure pour le dosage de PA pour l&#39;identification PV.

Méthode d&#39;agglutination de particules pour l&#39;identification des poliovirus

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute ...

particle-agglutination-method-for-poliovirus-identification

Research

JoVE Journal

Immunology and Infection

15.2K vues.  National Institute of Infectious Diseases. Une particule récemment développé une agglutination roman (PA) de dosage utilisant molécule réceptrice du virus a permis une identification rapide et facile de poliovirus (PV). Dans cet article, nous allons montrer la procédure pour le dosage de PA pour l'identification PV.

Vidéo: Particle Agglutination Method for Poliovirus Identification

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

Une procédure de 1,5 heure pour l&#39;identification des Enterococcus Espèces directement à partir d&#39;hémocultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

Recherche

Immunologie et infection

Peptide-based Identification of Functional Motifs and their Binding Partners

Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.

Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef&#x2019;s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

Identification de Motifs Fonctionnelles et leurs PARTENAIRES DE LIAISON Peptide-basée sur

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

Les tests pour l&#39;identification de nouveaux antiviraux contre la fièvre catarrhale Virus

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.

VIGS-Mediated dépistage génétique avant pour l&#39;identification des gènes impliqués dans la résistance non hôte


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

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 ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Moyenne de la glycoprotéine d&#39;enveloppe virale Pointes de Electron Cryotomography reconstructions utilisant Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

Capsulaire sérotypage<em&gt; Streptococcus pneumoniae</em&gt; Par agglutination au latex

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

Isolement, identification et la purification de cellules murines thymique épithéliales

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

Early Viral entrée dosages pour l&#39;identification et l&#39;évaluation des composés antiviraux

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Cytométrie en flux Analyse de la particule liée Bet v 1 Allergène en PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...