1. Virus Inoculazione e purificazione VLP da piante di Nicotiana benthamiana <ol><li> Produrre ricoperti T7-RNA trascritti da Virus X della patata (PVX) basati su plasmidi vettori che trasportano MRFV wild-type (WT) e proteina di rivestimento Cys-mutato (CP) geni 12, utilizzando Ambion il T7-mMessage mmachine kit. </li><li> Per ogni reazione di trascrizione T7, inoculare due foglie completamente espanse di N. benthamiana con 10 reazioni e incubare le piante per 10 giorni in serra, al 60% di umidità per 16 ore di luce (25.000-30.000 lux) a 25 ° C e 8 ore buio a 20 ° C. </li><li> Harves t N. benthamiana infettate da virus foglie di 10 giorni dopo l&#39;inoculazione (dpi), pesare il tessuto vegetale (50 grammi) e posto sul ghiaccio. </li><li> Omogeneizzare il materiale vegetale in un frullatore in presenza di 100 ml di una soluzione fredda di 0,5 M Na-citrato, pH 5,5 (uso 2 ml di tampone per grammo di tessuto vegetale). </li><li> Filtrare omogeneizzato attraverso 3 strati di cheeseclOTH e chiarire mediante centrifugazione a 27.000 xg per 30 min. Trasferire il surnatante in un cilindro graduato refrigerati, misurarne il volume, e poi trasferire in un recipiente freddo sterile. Eliminare il pellet. </li><li> Elaborare il surnatante aggiungendo glicole polietilenico 8000 10% (PEG 8000), agitare per 15 min a 4 ° C, e incubare per altri 45 min a 4 ° C per precipitare le particelle simili a virus (VLPs)-PEG matrice. </li><li> Centrifugare il campione a 27.000 xg per 20 min a 4 ° C e rimuovere il surnatante. </li><li> Elaborare il pellet aggiungendo 8 ml di 0,05 M Na-citrato, 5,5 contenente 2% Triton X-100 pH. Swirl tubi la centrifuga bene a rilasciare il pellet. Trasferire il pellet e buffer a un beaker refrigerati sterile. Sciacquare i tubi immediatamente con 2 ml dello stesso tampone e aggiungere al becher. Agitare la sospensione a 4 ° C fino a quando i pellet vengono disciolti (generalmente 1 ora). </li><li> Chiarificare mediante centrifugazione per 5 minuti a 27.000 xg, scartare il pellet, e pPROCESSO surnatante mediante centrifugazione a 140.000 xg per 2 ore a 4 ° C. </li><li> Risospendere il pellet in 1 ml di 0,05 M Na-citrato, pH 5,5 e posizionare la sospensione su un gradiente di saccarosio 10-40% made in 0,05 M Na-citrato, pH 5,5. </li><li> Centrifugare il gradiente per 3 ore a 140,000 xg a 4 ° C. </li><li> Accendere una luce sopra la parte superiore della provetta e raccogliere la luce-dispersione banda superiore a 4 cm dalla sommità del tubo, diluire con 0,05 M Na-citrato, pH 5,5, e centrifugare per 3 ore a 140.000 xg a 4 ° C. </li><li> Risospendere il pellet in 0,5 ml di acqua sterile e conservare a 4 ° C per un massimo di sei mesi. </li><li> Quantificare la concentrazione VLPs eseguendo un saggio di proteina Bradford. La resa di VLPs è tra 1 e 3 mg / g di tessuto vegetale. </li></ol> 2. Fluoresceina-5-maleimide-etichettatura Reazioni di VLP <ol><li> Preparare una soluzione 1 mM di fluoresceina-5-maleimide (427,37 g / mole) in 50 mM di sodio Phosphate tampone, pH 7,0, 1 mM EDTA. Mescolare con il vortice e verificare la completa dissoluzione. Proteggere il tubo dalla luce con un foglio di alluminio. </li><li> Aggiungi fluoresceina-5-maleimide a VLPs prodotte nel passaggio 1 e mescolare. Utilizzare di 25 volte l&#39;eccesso molare di fluoresceina-5-maleimide per quantità molare di sulfidrili. Generalmente, utilizzando 30 ml di VLPs alla concentrazione di 1 pg / pl e 60 pl della fluoresceina-5-maleimide soluzione produce risultati accettabili. </li><li> Incubare la reazione per 2 ore a temperatura ambiente o per una notte a 4 ° C. </li><li> Terminare la reazione mediante aggiunta di ditiotreitolo (DTT) ad una concentrazione finale di 50 mM. </li><li> Rimuovere non reagito fluorescina utilizzando una colonna dissalazione spin (Thermo Scientific Inc, Rockford IL) e centrifugazione per 2 min a 1500 xga temperatura ambiente. </li><li> Per analisi SDS-PAGE, aggiungere 10 ml di 2 x SDS-PAGE tampone Laemmli a 10 pl di ciascuna reazione. Memorizzare la proteina marcata protetto dalla luce a 4 ° C per un mese omonouso in aliquote a -20 ° C fino a 3 mesi. </li></ol> 3. Etichettatura di reazione e la determinazione di Costituzione Biotina <ol><li> Utilizzare spin column desalificazione (Thermo Scientific Inc., Rockford IL) per eseguire uno scambio di buffer dall&#39;acqua al tampone fosfato salino (PBS), pH 7,0 delle VLPs prodotte nel passo 1. </li><li> Preparare una soluzione stock 20 mM di maleimide-PEG2-biotina aggiungendo 190 microlitri di PBS in n-maleimide-PEG2 Pesare-biotina e mescolare pipettando su e giù. </li><li> Aggiungi maleimide-PEG2-biotina alle VLP e mescolare. Utilizzare un 10-fold eccesso molare di reagente per soluzioni VLPs ≤ 2 mg / ml. </li><li> Incubare la reazione a temperatura ambiente per 4 h. </li><li> Rimuovere non reagito maleimide-PEG2-biotina utilizzando una colonna di spin dissalazione (Thermo Scientific Inc, Rockford IL) e centrifugazione per 2 min a 1500 xga temperatura ambiente. </li><li> Determinare le moli di biotina per moli di VLP utilizzando la Pierce Biotina quantificazione Kit (Thermo Scientific) e following istruzioni del produttore. </li></ol> 4. Cross-linking di Cys 2-VLP e F Peptide <ol><li> VLPs che portano a residui di cisteina disponibili per reticolazione nella fase 2 (2-Cys VLPs) vengono utilizzati nel passaggio seguente. Preparare un nuovo cross-linking soluzione madre (28,63 mM) sciogliendo 10 mg di NHS-PEG4-maleimide reticolante in 680 ml di dimetilsolfossido. </li><li> Sciogliere un 17 (F)-amino peptide acido lunga (LLPNMOKDKEACAKAPL) in 50 pl di tampone di coniugazione (1 x PBS, pH 7,2, 1 mM EDTA) ad una concentrazione di 0,1 mM. </li><li> Aggiungere 4 ml di reticolante a 50 microlitri del peptide disciolto (proteina-NH2) ad una concentrazione di 1 mM finale (che è di 10 volte l&#39;eccesso molare di 0,1 mM di soluzione di peptide). </li><li> Incubare la reazione per 2 ore a 4 ° C. </li><li> Purificare reticolato peptide utilizzando una colonna dissalazione (Thermo Scientific Inc., Rockford IL) equilibrata con tampone di coniugazione (1 x PBS, pH 7,2, 1 mM EDTA) per centrifugazione a 1500 xgper 1 min a temperatura ambiente. </li><li> Impostare un mix di reazione combinando VLPs (proteina-SH) e reticolato peptide (proteina-NH2) in un rapporto molare determinata dal numero di tioli e ammine attivati ​​coinvolte nella reazione e la differenza di peso molecolare tra VLP e peptidi. </li><li> Incubare la miscela di reazione a temperatura ambiente per 2 ore a 4 ° C. </li><li> Per l&#39;analisi Western blot, miscelare il prodotto risultante coniugato 1:1 con tampone di Laemmli, bollire per 10 min, e procedere con SDS-PAGE su un pre-cast 10-20% Tris-glicina gel (Invitrogen), seguita da elettroblotting su una membrana di nitrocellulosa (Invitrogen). Bloccare le membrane con 1 x PBS, pH 7,2 contenente 0,05% Tween-20 (PBS-Tween) e 5% di latte scremato in polvere e sonda con peptide anticorpi specifici seguiti dal appropriata fosfatasi-anticorpo secondario marcato. </li></ol>

<ol>
	<li>Barnhill, H., Reuther, R., Ferguson, P. L., Dreher, T. W., Wang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turnip+yellow+mosaic+virus+as+a+chemoaddressable+bionanoparticle.">Turnip yellow mosaic virus as a chemoaddressable bionanoparticle.</a> Bioconj. Chem. 18, 852-859 (2007).</li><li>Chapman, S., Kavanagh, T., Baulcombe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potato+virus+X+as+a+vector+for+gene+expression+in+plants.">Potato virus X as a vector for gene expression in plants.</a> Plant J. 2, 549-557 (1992).</li><li>Chen, C., Kwak, E. S., Stein, B., Kao, C. C., Dragnea, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Packaging+of+gold+particles+in+viral+capsids.">Packaging of gold particles in viral capsids.</a> J. Nanosci. Nanotechnol. 5, 2029-2033 (2005).</li><li>Fowler, C. E., Shenton, W., Stubbs, G., Mann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+mosaic+virus+liquid+crystals+as+templates+for+the+interior+design+of+silica+mesophases+and+nanoparticles.">Tobacco mosaic virus liquid crystals as templates for the interior design of silica mesophases and nanoparticles.</a> Advanced Materials. 13, 1266-1269 (2001).</li><li>Gazit, E. <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+biomolecular+templates+for+the+fabrication+of+metal+nanowires.">Use of biomolecular templates for the fabrication of metal nanowires.</a> FEBS. J. 274, 317-322 (2007).</li><li>Gillitzer, E., Wilts, D., Young, M., Douglas, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+modification+of+a+viral+cage+for+multivalent+presentation.">Chemical modification of a viral cage for multivalent presentation.</a> Chem. Commun. , 2390-2391 (2002).</li><li>Hammond, R. W., Hammond, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maize+rayado+fino+virus+capsid+proteins+assemble+into+virus-like+particles+in+Escherichia+coli.">Maize rayado fino virus capsid proteins assemble into virus-like particles in Escherichia coli.</a> Virus Res. 147, 208-215 (2010).</li><li>Hermamson, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioconjugate techniques. , (1991).</li><li>Kaiser, C. R., Flenniken, M. L., Gillitzer, E., Harmsen, A. L., Harmsen, A. G., Jutila, M. A., Douglas, T., Young, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodistribution+studies+of+protein+cage+nanoparticles+demonstrate+broad+tissue+distribution+and+rapid+clearance+in+vivo.">Biodistribution studies of protein cage nanoparticles demonstrate broad tissue distribution and rapid clearance in vivo.</a> Int. J. Nanomed. 2, 715-733 (2007).</li><li>Knez, M., Bittner, A. M., Boes, F., Wege, C., Jeske, H., Maisse, E., Kern, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotemplate+synthesis+of+3-nm+nickel+and+cobalt+nanowires.">Biotemplate synthesis of 3-nm nickel and cobalt nanowires.</a> Nano Lett. 3, 1079-1082 (2003).</li><li>Lee, S. Y., Culver, J. N., Harris, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+CuCl2+concentration+on+the+aggregation+and+mineralization+of+Tobacco+mosaic+virus+biotemplate.">Effect of CuCl2 concentration on the aggregation and mineralization of Tobacco mosaic virus biotemplate.</a> J. Colloid. Interface. Sci. 297, 554-560 (2006).</li><li>Lvov, Y., Haas, H., Decher, G., Mohwald, H., Mikhailov, A., Mtchedlishvily, B., Morgunova, E., Vainshtein, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successive+deposition+of+alternate+layers+of+polyelectrolytes+and+a+charged+virus.">Successive deposition of alternate layers of polyelectrolytes and a charged virus.</a> Langmuir. 10, 4232-4236 (1994).</li><li>Natilla, A., Hammond, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maize+rayado+fino+virus+virus-like+particles+expressed+in+tobacco+plants:+a+new+platform+for+cysteine+selective+bioconjugation+peptide+display.">Maize rayado fino virus virus-like particles expressed in tobacco plants: a new platform for cysteine selective bioconjugation peptide display.</a> J. Virol. Methods. 178, 209-215 (2011).</li><li>Rae, C. S., Khor, I. W., Wang, Q., Destito, G., Gonzalez, M. J., Singh, P., Thomas, D. M., Estrada, M. N., Powell, E., Finn, M. G., Manchester, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+trafficking+of+plant+virus+nanoparticles+in+mice+via+the+oral+route.">Systemic trafficking of plant virus nanoparticles in mice via the oral route.</a> Virology. 343, 2224-2235 (2005).</li><li>Raja, K. S., Wang, Q., Gonzalez, M. J., Manchester, M., Johnson, J. E., Finn, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+virus-polymer+materials.+Synthesis+and+properties+of+PEG-decorated+Cowpea+mosaic+virus.">Hybrid virus-polymer materials. Synthesis and properties of PEG-decorated Cowpea mosaic virus.</a> Biomacromolecules. 4, 472-476 (2003).</li><li>Royston, E., Lee, S. Y., Culver, J. N., Harris, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+silica-coated+Tobacco+mosaic+virus.">Characterization of silica-coated Tobacco mosaic virus.</a> J. Colloid Interface Sci. 298, 706-712 (2006).</li><li>Schlick, T. L., Ding, Z., Kovacs, E. W., Francis, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-surface+modification+in+the+Tobacco+mosaic+virus.">Dual-surface modification in the Tobacco mosaic virus.</a> J. Am. Chem. Soc. 127, 3718-3723 (2005).</li><li>Young, M., Willits, D., Uchida, M., Douglas, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+viruses+as+biotemplates+for+materials+and+their+use+in+nanotechnology.">Plant viruses as biotemplates for materials and their use in nanotechnology.</a> Annu. Rev. Phytopathol. 46, 361-384 (2008).</li></ol>

Menzione dei nomi commerciali dei prodotti commerciali nella pubblicazione è al solo scopo di fornire informazioni specifiche e non implica raccomandazione o approvazione da parte del Dipartimento dell&#39;Agricoltura degli Stati Uniti.

Il metodo qui presentato permette una analisi molto sensibile e rapido delle cisteine ​​reattive presenti sulla superficie delle piante prodotte VLPs nonché su complessi di proteine. Maleimmidi sono tiolo-specifici reagenti, che reagiscono con liberi sulfidrilici contenenti molecole di formare legami stabili tioetere. Questo metodo disegna sulla specificità delle maleimmidi di reagire con i gruppi sulfidrilici non coinvolti in interazioni con altri aminoacidi. Mantenendo la struttura nativa delle VLPs è molto imp...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Thinwall, Ultra-Clear Tubi </td><td> Beckman </td><td> 344059 </td><td></td></tr><tr><td> mMessage mmachine T7 Kit </td><td> Vita Tecnologie </td><td> AM1344M </td><td></td></tr><tr><td> Fluoresceina-5-Maleimide </td><td> Thermo Scientific Life Technologies </td><td> 46130 F150 </td><td> 46130 non è sostituto ordine con F150 </td></tr><tr><td> Pierce Biotina quantificazione Kit </td><td> Thermo Scientific </td><td> 28005 </td><td></td></tr><tr><td> EZ-Link Maleimide-PEG2-biotina, No-Pesare Formato </td><td> Thermo Scientific </td><td> 21901 </td><td></td></tr><tr><td> SM (PEG) n Reticolanti </td><td> Thermo Scientific </td><td> 22107 </td><td></td></tr><tr><td> 10-20% Tris-glicina gel </td><td> Invitrogen </td><td> EC61352 </td><td></td></tr><tr><td> Laemmli buffer </td><td> Bio-Rad </td><td> 1610737 </td><td></td></tr><tr><td> Tris glicina SDS tampone di corsa </td><td> Invitrogen </td><td> LC2675 </td><td></td></tr><tr><td> Tris glicina Transfer Buffer </td><td> Invitrogen </td><td> LC3675 </td><td></td></tr><tr><td> Nitrocellulosa membrana del filtro di carta Sandwich </td><td> Invitrogen </td><td> LC2001 </td><td></td></tr><tr><td> Fosfatasi affinità etichetta Purificato Anticorpi anti-IgG di coniglio </td><td> Kirkegaard e Laboratori Perry </td><td> 0751516 </td><td></td></tr><tr><td> NBT / BCIP Fosfatasi substrato </td><td> Kirkegaard e Laboratori Perry </td><td> 508107 </td><td></td></tr></tbody></table>

analysis of the solvent accessibility of cysteine residues on maize rayado fino virus virus-like particles produced in nicotiana benthamiana plants and cross-linking of peptides to vlps

Imitare e sfruttando le proprietà e le caratteristiche fisico-chimiche dei virus e fisico promette di fornire soluzioni ad alcune delle sfide più pressanti del mondo. La gamma pura e tipi di virus insieme con le loro proprietà potenzialmente intriganti dare infinite opportunità per le applicazioni in tecnologie basate su virus. Virus hanno la capacità di auto-assemblano in particelle di forma e dimensioni discrete, specificità di simmetria, polivalenza e proprietà stabili in un&#39;ampia gamma di condizioni di temperatura e di pH. Non sorprendentemente, con una notevole gamma di proprietà, i virus sono proposti per l&#39;uso in vaccini biomateriali 9, 14, 15, materiali elettronici, strumenti chimici e molecolare elettronica contenitori 4, 5, 10, 11, 16, 18, ​​12. Al fine di utilizzare i virus in nanotecnologie, devono essere modificati dalle loro forme naturali per impartire nuove funzioni. Questo impegnativo process può essere effettuato attraverso meccanismi diversi tra modificazione genetica del genoma virale e attaccare chimicamente molecole estranee o desiderato per i gruppi reattivi delle particelle virali 8. La possibilità di modificare un virus dipende principalmente dalle proprietà fisico-chimiche e fisiche del virus. Inoltre, le modifiche genetiche o fisico-chimiche devono essere eseguite senza alterare la struttura originaria del virus e la funzione virus. Mais Rayado Fino virus (MRFV) proteine ​​di rivestimento auto-assemblano in Escherichia coli che producono VLP stabili e vuoti che sono stabilizzati da proteina-proteina interazioni e che possono essere utilizzati in applicazioni basate su virus tecnologie 8. VLPs prodotte in piante di tabacco sono stati esaminati da impalcatura su cui una varietà di peptidi possono essere covalentemente visualizzato 13. Qui, descriviamo la procedura per 1) determinare quale dei solventi accessibili cisteine ​​in un capside del virus sono disponibili per modifizione, e 2) Bioconjugate peptidi alle capsidi modificati. Utilizzando nativi o mutationally-inserito residui di aminoacidi e tecnologie standard di accoppiamento, una grande varietà di materiali sono stati visualizzati sulla superficie del virus vegetali come, virus del mosaico Brome 3, virus della screziatura Carnation 12, Cowpea clorotiche mottle virus 6, mosaico del tabacco virus 17, Rapa virus del mosaico giallo 1, e MRFV 13.

Espressione transitoria di cappotto mutanti MRFV proteine ​​(CP) geni in N. benthamiana piante in un PVX basati VLPs vettore producono è descritto nella Figura 1. Il mantello modificato MRFV proteina gene viene amplificato mediante PCR e quindi posto sotto il controllo trascrizionale del promotore subgenomico duplicato CP in un PVX-based vettore, pP2C2S 2, (un dono di D. Baulcombe, Sainsbury Laboratories, Norwich, Inghilterra). In vitro la trascrizione di RNA produce tr...

Watch this Scientific Journal Video about Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Un metodo per analizzare l&#39;accessibilità al solvente del gruppo tiolo della cisteina residui di<em&gt; Mais Rayado Fino virus</em&gt; (MRFV)-particelle simili a virus (VLPs) seguiti da un peptide reazione di reticolazione è descritto. Il metodo sfrutta la disponibilità di gruppi chimici diversi sulla superficie delle VLPs che possono essere bersagli per reazioni specifiche.

Analisi della accessibilità al solvente dei residui di cisteina in<em&gt; Mais Rayado Fino virus</em&gt; Particelle virus-simili Prodotto in<em&gt; Nicotiana benthamiana</em&gt; Piante e Cross-linking di peptidi a VLP

Mimicking  and exploiting virus properties and physicochemical and physical  characteristics holds promise to provide solutions to some of  the world's ...

analysis-solvent-accessibility-cysteine-residues-on-maize-rayado-fino

Research

JoVE Journal

Immunology and Infection

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

11.0K Views.  Agricultural Research Service, United States Department of Agriculture. Un metodo per analizzare l'accessibilità al solvente del gruppo tiolo della cisteina residui di Mais Rayado Fino virus (MRFV)-particelle simili a virus (VLPs) seguiti da un peptide reazione di reticolazione è descritto. Il metodo sfrutta la disponibilità di gruppi chimici diversi sulla superficie delle VLPs che possono essere bersagli per reazioni specifiche.

Video: Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Isolation and Genome Analysis of Single Virions using &#039;Single Virus Genomics&#039;

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

Isolamento e analisi del genoma di virioni singole utilizzando &quot;Genomica singolo virus &#39;

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

Ricerca

Immunologia e infezioni

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.

Analisi del danno epiteliale Prodotto da<em&gt; Entamoeba histolytica</em&gt; Infezione

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

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

In Analisi Vitro di Myd88 mediata Cellular Immune Response to West Nile virus mutante Strain Infezione

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or specific treatments for the disease caused by these viruses, and work with infectious Ebola viruses is restricted to biosafety level 4 laboratories, significantly limiting the research on these viruses. Lifecycle modeling systems model the virus lifecycle under biosafety level 2 conditions; however, until recently such systems have been limited to either individual aspects of the virus lifecycle, or a single infectious cycle. Tetracistronic minigenomes, which consist of Ebola virus non-coding regions, a reporter gene, and three Ebola virus genes involved in morphogenesis, budding, and entry (VP40, GP1,2, and VP24), can be used to produce replication and transcription-competent virus-like particles (trVLPs) containing these minigenomes. These trVLPs can continuously infect cells expressing the Ebola virus proteins responsible for genome replication and transcription, allowing us to safely model multiple infectious cycles under biosafety level 2 conditions. Importantly, the viral components of this systems are solely derived from Ebola virus and not from other viruses (as is, for example, the case in systems using pseudotyped viruses), and VP40, GP1,2 and VP24 are not overexpressed in this system, making it ideally suited for studying morphogenesis, budding and entry, although other aspects of the virus lifecycle such as genome replication and transcription can also be modeled with this system. Therefore, the tetracistronic trVLP assay represents the most comprehensive lifecycle modeling system available for Ebola viruses, and has tremendous potential for use in investigating the biology of Ebola viruses in future. Here, we provide detailed information on the use of this system, as well as on expected results.

Work with infectious Ebola viruses is restricted to biosafety level 4 laboratories. Tetracistronic minigenome-containing replication and transcription-competent virus like particles (trVLPs) represent a lifecycle modeling system that allows us to safely model multiple infectious cycles under biosafety level 2 conditions, relying exclusively on Ebola virus components.

Modellazione il ciclo di vita Ebola Virus Sotto livello di biosicurezza 2 condizioni con particelle virus-simili contenenti Tetracistronic Minigenomes

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrofili Isolamento e analisi per determinare il loro ruolo nel linfoma a cellule sensibilità agli agenti terapeutici

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Lo sviluppo di cellule staminali derivate antigene-specifiche cellule T regolatorie contro autoimmunità

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

L&#39;utilizzo della tecnica di sovrapposizione Soft-agar per lo screening composti inibitori bacterially Prodotto

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low titers in their host or cannot be mechanically inoculated to another host are difficult to propagate to achieve a greater titer of infectious material. Infected leaves are ground in a mild buffer with optimal pH and ionic composition recommended for purifying most bacilliform Para retroviruses. Urea is used to break up inclusion bodies that trap virions and to dissolve cellular components. Differential centrifugation provides further separation of virions from plant contaminants. Then proteinase K treatment removes the capsids. Then the viral DNA is concentrated and used for next-generation sequencing (NGS). The NGS data are used to assemble contigs which are submitted to NCBI-BLASTn to identify a subset of virus sequences in the generated dataset. In a parallel pipeline, RNA is isolated from infected leaves using a standard column-based RNA extraction method. Then ribosome depletion is carried out to enrich for a subset of mRNA and virus transcripts. Assembled sequences derived from RNA sequencing (RNA-seq) were submitted to NCBI-BLASTn to identify a subset of virus sequences in this dataset. In our study, we identified two related full-length badnavirus genomes in the two datasets. This method is preferred to another common approach which extracts the aggregate population of small RNA sequences to reconstitute plant virus genomic sequences. This latter metagenomic pipeline recovers virus related sequences that are retro-transcribing elements inserted into the plant genome. This is coupled to biochemical or molecular assays to further discern the actively infectious agents. The approach documented in this study, recovers sequences representative of replicating viruses that likely indicate active virus infection.

Here we present a new approach to identify plant viruses with double-strand DNA genomes. We use standard methods to extract DNA and RNA from infected leaves and carry out next-generation sequencing. Bioinformatic tools assemble sequences into contigs, identify contigs representing virus genomes and assign genomes to taxonomic groups.

Combinando l'analisi di DNA in un'estrazione di greggio virione con l'analisi del RNA dalle foglie infette per scoprire nuovi Virus genomi

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Istituzione dell'infezione virale e analisi dell'interazione ospite-Virus in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&#947;R Cross-linking

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in response to various immune stimuli. ROS generated during immune activation exerts potent antimicrobial activity primarily through the ability of ROS to damage DNA and proteins, causing death of microorganisms. Being able to measure ROS production reproducibly and with ease is necessary in order to assess the contribution of various pathways and molecules to this mechanism of host defense. In this paper, we demonstrate the use of fluorescent probes and flow cytometry to detect ROS production. Although widely used, fluorescent measurement of ROS is notoriously problematic, especially with regards to measurement of ROS induced by specific and not mitogenic stimuli. We present a detailed methodology to detect ROS generated as a result of specific Fc&#947;R stimulation beginning with macrophage generation, priming, staining, Fc&#947;R cross-linking, and ending with flow cytometric analysis.

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&amp;#947;R Cross-linking

This study demonstrates the use of flow cytometry to detect reactive oxygen species (ROS) production resulting from activation of the Fc&#947;R. This method can be used to assess changes in the antimicrobial and redox signaling function of phagocytes in response to immune complexes, opsonized microorganisms, or direct Fc&#947;R cross-linking.

Misurazione del flusso Cytometric di produzione di ROS nei macrofagi In risposta a FcγR cross-linking

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in ...