Siamo grati a Junko Wada per la sua eccellente assistenza tecnica. Siamo grati al Prof. Akio Nomoto per la gentile fornendo un vettore di espressione baculovirus per PVR-IgG2a. Questo studio è stato sostenuto in parte da sovvenzioni-in-Aid per la promozione della eradicazione della polio e della Ricerca sulle emergenti e riemergenti Malattie Infettive del Ministero della Salute, del Lavoro e del Welfare.

1. Preparazione del terreno di mantenimento (MM) e una crescita media (GM) Per la preparazione di MM e GM, consultare un manuale Polio laboratorio dell&#39;OMS 1. Medio Dulbecco modificato Eagle (DMEM) arricchito con il 2% di siero fetale bovino (FCS) e il 10% di FCS potrebbero essere utilizzati come sostituti di MM e GM, rispettivamente. 2. Preparazione di anticorpi anti-PV <ol><li> Ricostituire anticorpi anti-PV (PV RIVM digitando antisiero) con l&#39;aggiunta di acqua (0,5 ml per flaconcino) per ogni flaconcino di anticorpi anti-PV (tipo 1, 2 o 3). Aggiungi questo anticorpo soluzioni ricostituite (0,5 ml ciascuno) a 4,5 ml di MM in tubi separati etichettati. Nota: alcuni lotti di anticorpi anti-PV sono forniti come forma ricostituito (0,5 ml per flaconcino). Per il lotto di tali anticorpi anti-PV, aggiunta di acqua per la ricostituzione non è necessaria. </li><li> Mescolare uguale volume di ricostituito anticorpi anti-PV. Per anti-PV1 +2, 2 +3 o 3 +1: Mescolare 1,8 ml di ciascun anticorpo ricostituito (1,8 ml + 1,8 ml = 3,6 mL per ciascuna miscela). Per anti-PV1 +2 +3: Mescolare 1,2 ml di ciascun anticorpo ricostituito (1,2 ml + 1,2 ml + 1,2 ml = 3,6 mL). Nota: i campioni PV potrebbe contenere una miscela di diversi sierotipi del fotovoltaico. Per identificare il sierotipo singolo del fotovoltaico in campioni con miscela di PV, pool di anticorpi anti-PV (cioè 1 +2, 2 +3, 3 +1, 1 +2 +3) è necessario. </li><li> Aggiungere il volume 1 / 5 di FCS (0,72 ml) per ogni anti-PV (1 +2, 2 +3, 3 +1 o 1 +2 +3) anticorpi (3,6 mL) (finale 18,2% FCS). Nota: L&#39;aggiunta di FCS sia per la riduzione della non-specifici agglutinazione di particelle di gelatina causata da anticorpi. </li><li> Mantenere le piscine degli anticorpi a -20 ° C fino all&#39;utilizzo nei test PA. </li></ol> 3. Ricostituzione di gelatina sensibilizzata particelle <ol><li> Aggiungere 1,5 mL di tampone ricostituzione inclusi nel kit di un flaconcino di particelle di gelatina sensibilizzata (fornito come polvere liofilizzata). </li><li> Mescolare bene picchiettando leggermente </li><li> Mantenere la particella ricostituito sensibilizzato a 4 ° C. Ricostituito particelle gelatina potrebbe essere attiva almeno 2 settimane a 4 ° C, ma deve essere preparato poco prima del test. </li></ol> Nota importante: Non mescolare molto diverso di particelle di gelatina sensibilizzata, perché il non-agglutinazione immagine di particelle di gelatina sensibilizzata potrebbe essere diverso dal lotto delle particelle. Per ogni esperimento, un singolo lotto di gelatina sensibilizzata particella dovrebbe essere usato. 4. PA test per l&#39;identificazione PV <ol><li> Preparare una micro-titolazione piatto. Per l&#39;identificazione di un campione di virus, abbiamo bisogno di 5 pozzi + almeno un controllo negativo (particelle sensibilizzati senza virus e anticorpi, ben n. 6 di seguito. See No controllo del virus e anticorpi in esempio). Numero totale dei pozzi è di 5 x campioni + almeno un controllo nel saggio. Nota importante: Si prega di prendere almeno un non controllo del virus e anticorpi nel saggio. Un flaconcino di particelle sensibilizzati circa l&#39;identificazione di 10 campioni del virus. </li><li> Aggiungere 12 microlitri / pozzetto di anticorpi anti-PV o GM al micro-titolazione piatto. La disposizione del piatto è la seguente (Vedi anche Esempio di seguito). Nei campioni senza anticorpi (camera n. 1 e 6), aggiungere 12 ml di GM invece di anticorpi anti-PV. Nota importante: Se molti campioni PV dovrebbero essere testati in questo saggio, pipetta multicanale o distributore sarebbe utile per questo passo. <table border="1"><tbody><tr><td> Bene # 1 </td><td> Bene # 2 </td><td> Bene # 3 </td><td> Bene # 4 </td><td> Bene # 5 </td><td> Bene # 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> Aggiungere 12 microlitri / pozzetto di campione virus. In un campione senza virus (o meglio # 6), aggiungere 12 ml di GM al posto della soluzione di virus. Nota: campione di virus è supernatante della coltura delle cellule infettate, le cellule di solito L20B o cellule RD. Nota importante: Se molti campioni PV dovrebbero essere testati in questo saggio, pipetta multicanale sarebbe utile per questo passo. <table border="1"><tbody><tr><td> Bene # 1 </td><td> Bene # 2 </td><td> Bene # 3 </td><td> Bene # 4 </td><td> Bene # 5 </td><td> Bene # 6 </td></tr><tr><td> virus campione </td><td> virus campione </td><td> virus campione </td><td> virus campione </td><td> virus campione </td><td> GM </td></tr></tbody></table></li><li> Prendere soluzione ricostituita sensibilizzati particelle da 4 ° C a temperatura ambiente, e picchiettare delicatamente di sospendere completamente le particelle. Nota importante: Dopo la sospensione completa delle particelle picchiettando leggermente, le particelle dovrebbero essere aggiunti immediatamente ai piatti prima della particelle sospese precipitazionite e la forma non omogenea soluzione. </li><li> Aggiungere 25 microlitri / pozzetto di particelle sensibilizzati a tutti i pozzetti. Nota importante: Dopo l&#39;aggiunta di particelle sensibilizzate, la piastra deve essere immediatamente sottoposto a piastra di miscelazione per iniziare la reazione uniforme dei campioni. Pertanto, l&#39;aggiunta di particelle sensibili deve essere effettuata utilizzando dosatore per minimizzare un intervallo di tempo tra i campioni nel piatto. </li><li> Mettere il micro-titolazione piastra su un piatto e mixer miscelare la piastra per 30 sec. </li><li> Mettere il micro-titolazione piastra su carta bianca (per comodità di osservazione visuale) su un tavolo a temperatura ambiente (15 ~ 30 ° C) per 2 ore Nota importante: Non spostare la piastra durante e dopo la reazione per l&#39;osservazione dei risultati e fotografare. L&#39;agglutinazione formato non è stabile e facilmente disturbati dal movimento delle piastre. </li><li> Scatta foto del piatto. Nota importante: Veduta parziale della foto non poteva dare dati sufficienti per il successivo esame e la revisione dei risultati per l&#39;interpretazione. Diverse foto dovrebbe essere preso per coprire tutti i pozzetti delle piastre di messa a fuoco l&#39;immagine agglutinazione dei pozzi. </li></ol> 5. Interpretazione dei risultati <ol><li> L&#39;identificazione dei pozzetti positivi e negativi. Confrontare le agglutinazione nei campioni con quella di nessun controllo dei virus e degli anticorpi (precipitazione di particelle di gelatina, non agglutinazione) (Figura 1 e 2). Immagini di agglutinazione che sono chiaramente distinti dalle Nessun virus e nessun controllo di anticorpi (non agglutinazione, controllo negativo) dovrebbe essere giudicata positivamente, e le immagini di agglutinazione che sono simili al controllo negativo deve essere giudicato negativo. I campioni che mostrano agglutinazione evidente, ma solo parziale potrebbe essere giudicata positivamente, ma record come &#39;positivo (agglutinazione parziale)&#39;. </li><li> Identificazione del fotovoltaico. Interpretare i risultati sulla base di pozzetti positivi e negativi con anticorpi anti-PV per identificare i sierotipi fotovoltaico. Principio della interpretazione del risultato è simile a quella del test di neutralizzazione del sistema di coltura cellulare (ad esempio positivo per anti-P1 due anticorpi indica che il campione contiene tipo 3 PV). </li></ol> 6. Rappresentante Risultati Risultato rappresentativo della PA test PV identificazione è mostrata in Figura 1. Tutti i ceppi PV formata agglutinazione di particelle di gelatina sensibilizzata (senza anticorpi campioni). In presenza di corrispondenti anticorpi anti-PV, la precipitazione di particelle di gelatina (non-agglutinazione) simile a No virus e nessun controllo di anticorpi (controllo negativo) è stato osservato (ad esempio, PV1 (Sabin) in presenza di anti-P1 +2 o anti-P3 1 anticorpi). <img src="/files/ftp_upload/2824/2824fig1.jpg" alt="Figura 1" /> Figura 1. Le soluzioni antivirus identificazione del fotovoltaico mediante test PA. Di PV1 (Sabin), PV2 (Sabin), e PV3 (Sabin) (titoli virus di 1,2 x 10 8 CCID 50 / 12 microlitri, 8,2 x 10 7 CCID 50 / 12 microlitri, e 3,5 x 10 7 CCID 50 / 12 microlitri, rispettivamente) sono stati utilizzati per l&#39;identificazione mediante test PA. Nessun virus e nessun controllo di anticorpi hanno mostrato precipitazione di particelle di gelatina (non-agglutinazione, controllo negativo). <img src="/files/ftp_upload/2824/2824fig2.jpg" alt="Figura 2" /> Figura 2. Interpretazione dei pozzi positivi e negativi. Soluzioni antivirus di PV1 (Sabin), PV2 (Sabin), e PV3 (Sabin) (titoli virus di 2,4 x 10 8 CCID 50 / 25 microlitri, 1,7 x 10 8 CCID 50 / 25 microlitri, e 7,3 x 10 7 CCID 50 / 25 microlitri, rispettivamente) sono stati diluiti 1 / 20 a 1 / 320, e poi 25 ml di campioni diluiti sono stati esaminati mediante test PA senza anti-PV anticorpi.

<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 è un dipendente di New Product Design Department, Fujirebio Inc.

L&#39;unico punto di questo test PA è l&#39;utilizzo della molecola PVR invece di anticorpi anti-PV per il rilevamento di PV 2. Questo consente una specifica interazione delle particelle di gelatina sensibilizzata con PV con una affinità uniforme per tutti e tre i sierotipi di PV 3-6. Abbiamo usato una forma immunoadhesin di PVR (PVR-IgG2a) per la sensibilizzazione delle particelle di gelatina, in quanto forme monomeriche di PVR hanno solo affinità moderata al singolo sito vincolanti per le virione (costan...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti </th></tr><tr><td> Gelatina sensibilizzata-particelle </td><td> Fujirebio Inc. </td><td></td><td> Kit per il test </td></tr><tr><td> Ricostituzione del buffer </td><td> Fujirebio Inc. </td><td></td><td> Kit per il test </td></tr><tr><td> Piastra di microtitolazione </td><td> Fujirebio Inc. </td><td></td><td> Kit per il test </td></tr><tr><td> Anticorpi anti-PV </td><td> RIVM </td><td></td><td></td></tr></tbody></table>

particle agglutination method for poliovirus identification

Nel Global Polio Eradication Initiative, diagnosi di laboratorio svolge un ruolo critico, isolando e identificando PV dai campioni di feci di paralisi flaccida acuta (AFP) casi. Nel Organizzazione Mondiale della Sanità (OMS) Global Polio Laboratory Network, PV isolamento e l&#39;identificazione sono attualmente in corso, utilizzando la cultura sistema di celle e real-time RT-PCR, rispettivamente. Nell&#39;era post-eradicazione era del fotovoltaico, procedure di identificazione semplice e rapida sarebbe utile per una rapida conferma di casi di polio presso i laboratori nazionali. Nel presente studio, mostreremo la procedura di test romanzo PA sviluppato per l&#39;identificazione fotovoltaico. Questo test PA utilizza l&#39;interazione del recettore PV (PVR) e virione molecola che è specifica affinità e uniforme a tutti i sierotipi del fotovoltaico. La procedura è semplice (una procedura passo in lastre di reazione) e rapido (risultati possono essere ottenuti entro 2 h di reazione), e il risultato è visivamente osservato (osservazione di agglutinazione di particelle di gelatina).

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

Particle Agglutination Method for Poliovirus Identification

Un recente sviluppo di particelle romanzo agglutinazione (PA) test utilizzando molecola recettore del virus ha permesso l&#39;identificazione rapida e facile dei poliovirus (PV). In questo articolo mostreremo la procedura per il saggio PA per l&#39;identificazione fotovoltaico.

Metodo di particelle agglutinazione per l&#39;identificazione 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 Views.  National Institute of Infectious Diseases. Un recente sviluppo di particelle romanzo agglutinazione (PA) test utilizzando molecola recettore del virus ha permesso l'identificazione rapida e facile dei poliovirus (PV). In questo articolo mostreremo la procedura per il saggio PA per l'identificazione fotovoltaico.

Video: 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).

Una procedura 1,5 ore per l&#39;identificazione di Enterococcus Specie Direttamente da emocolture

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

Ricerca

Immunologia e infezioni

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.

Identificazione Peptide-based di Motivi funzionali e dei loro partner di legame

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.

Saggi per l&#39;individuazione di nuovi antivirali contro virus della Bluetongue

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 Forward Genetica screening per la identificazione di geni coinvolti nella resistenza Nonhost


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

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.

Della media di Viral Busta Glicoproteina Spikes da Electron Cryotomography Ricostruzioni con 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.

Capsulare Sierotipizzazione di<em&gt; Streptococcus pneumoniae</em&gt; Da agglutinazione al lattice

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.

Isolamento, identificazione e purificazione di cellule murine timica epiteliali

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.

All&#39;inizio virali ingresso saggi per l&#39;identificazione e la valutazione di composti antivirali

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

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

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

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

Purificazione di DNA virale per l'identificazione delle proteine virali e cellulari associate

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

Formazione di Biofilm indotta da sali biliari in patogeni enterici: tecniche di identificazione e quantificazione

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...