Name: protein misfolding cyclic amplification of prions
Uploaded: 2012-11-07T12:00:00
Description: Watch this Scientific Journal Video about Protein Misfolding Cyclic Amplification of Prions at JoVE.com

Vi vill tacka Dr Vesper Fe Marie Ramos för kritisk läsning av manuskriptet. Detta arbete stöddes av National Center for Research Resources (P20 RR0115635-6, C06 RR17417-01 och G20RR024001) och Institutet för neurologiska sjukdomar och stroke (2R01 NS052609).

1. Förbereda utrustning <ol><li> Använd en Misonix 3000 eller Misonix 4000 sonikator (Farmingdale, NY) kopplad till en Thermo Electron Neslab EX-7 vattenbad (Newington, NH) för att hålla en konstant temperatur på 37 ° C. Sonikera proverna i 200 ^ tunnväggiga PCR-rör band med välvda lock som erhållits från Thermo Scientific (Waltham, MA). </li><li> En helt ny sonikator kräver en &quot;break-in&quot; period av kontinuerlig drift 9. En två månaders inkörningsperiod består av en 40-sek ultraljud...

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+sources+of+scrapie+prions.">Environmental sources of scrapie prions.</a> J. Virol. 84, 11560-11562 (2010).</li><li>Nichols, T. A., Pulford, B., Wyckoff, A. C., Meyerett, C., Michel, B., Gertig, K., Hoover, E. A., Jewell, J. E., Telling, G. C., Zabel, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+protease-resistant+cervid+prion+protein+in+water+from+a+CWD-endemic+area.">Detection of protease-resistant cervid prion protein in water from a CWD-endemic area.</a> Prion. 3, 171-183 (2009).</li><li>Prusiner, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+proteinaceous+infectious+particles+cause+scrapie.">Novel proteinaceous infectious particles cause scrapie.</a> Science. 216, 136-144 (1982).</li><li>Saborio, G. P., Permanne, B., Soto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+detection+of+pathological+prion+protein+by+cyclic+amplification+of+protein+misfolding.">Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding.</a> Nature. 411, 810-813 (2001).</li><li>Saunders, S. E., Bartz, J. C., Vercauteren, K. C., Bartelt-Hunt, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzymatic+treatment+of+soil-bound+prions+effectively+inhibits+replication.">An enzymatic treatment of soil-bound prions effectively inhibits replication.</a> Appl. Environ. Microbiol. 77, 4313-4317 (2011).</li><li>Saunders, S. E., Shikiya, R. A., Langenfeld, K., Bartelt-Hunt, S. L., Bartz, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+efficiency+of+soil-bound+prions+varies+with+soil+type.">Replication efficiency of soil-bound prions varies with soil type.</a> Journal of virology. , (2011).</li><li>Schutt, C. R., Bartz, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+interference+with+multiple+prion+isolates.">Prion interference with multiple prion isolates.</a> Prion. 2, 61-63 (2008).</li><li>Shikiya, R. A., Ayers, J. I., Schutt, C. R., Kincaid, A. E., Bartz, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-infecting+prion+strains+compete+for+a+limiting+cellular+resource.">Co-infecting prion strains compete for a limiting cellular resource.</a> Journal of. 84, 5706-5714 (2010).</li><li>Shikiya, R. A., Bartz, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+generation+of+high+titer+prions.">In vitro generation of high titer prions.</a> Journal of virology. , (2011).</li><li>Weber, P., Giese, A., Piening, N., Mitteregger, G., Thomzig, A., Beekes, M., Kretzschmar, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+genuine+prion+infectivity+by+serial+PMCA.">Generation of genuine prion infectivity by serial PMCA.</a> Veterinary microbiology. 123, 346-357 (2007).</li></ol>

Utmaningar för förstärkning smittsamma prionproteiner är långa inkubationstider och kostnader för in vivo-studier. Den PMCA tekniken är ett kostnadseffektivt sätt att förstärka smittsamma prion ämnen. Flera laboratorier har bekräftat förmågan hos PMCA att noggrant förstärka prion stammar in vitro 7, 9, 12, 14, 19,24. Prionsjukdomar kan överföras mellan arter. Bessen och Marsh har i praktiken inokulerade hamstrar med överförbar mink encefalopat...

<table border="1"><tbody><tr><td> Reagens / utrustning </td><td> Tillverkare </td><td> Kat. Nummer </td></tr><tr><td> Misonix 3000 </td><td> Misonix </td><td> S-3000 </td></tr><tr><td> Misonix 4000 </td><td> Misonix </td><td> S-4000 </td></tr><tr><td> Tenbroeck Tissue Grinder </td><td> Kontes </td><td> 885000-0007 </td></tr><tr><td> Neslab EX-7 Vattenbad </td><td> Thermo Electron </td><td> Neslab EX-7 </td></tr><tr><td> 0,2 ml PCR-rör Strips </td><td> Thermo Scientific </td><td> AB-0451 </td></tr><tr><td> Triton X-100 </td><td> Sigma Aldrich </td><td> T9284-100ML </td></tr><tr><td> Komplett Protease Inhibitor </td><td> Roche </td><td> 11 697 498 001 </td></tr><tr><td> EDTA </td><td> JT Baker </td><td> 4040-00 </td></tr><tr><td> DPBS </td><td> Mallinckrodt Baker Mediatech </td><td> 21-031-CV </td></tr><tr><td> Versi-Dry Lab Soakers </td><td> Fisher Scientific </td><td> 14 206 28 </td></tr><tr><td> Repti Therm Värmare </td><td> Zoo Med Laboratories, Inc. </td><td> RH-4 </td></tr></tbody></table>

protein misfolding cyclic amplification of prions

Prioner är smittämnen som orsakar oundvikligen dödlig transmissibel spongiform encefalopati (TSE) hos djur och människor 9,18. Prionproteinet har två distinkta isoformer, icke-infektiösa värd-kodade proteinet (PrP ^) och infektiöst protein (PrP ^), en onormalt vikta isoformen av PrP ^ 8. En av utmaningarna med att arbeta med prion medel är den långa inkubationstiden före utvecklingen av kliniska tecken efter värd ympning 13. Detta uppdrag traditionellt långa och dyra djurförsök bioassay. Dessutom är de biokemiska och biofysiska egenskaper hos PrP dåligt karakteriseras på grund av deras ovanliga konformation och aggregering stater. PrP kan utsäde omvandlingen av PrP ^ till PrP ^ in vitro 14. PMCA är en in vitro-teknik som tar Advantage av denna förmåga med hjälp sonikering och inkubering cykler för att producera stora mängder PrP ^, i en ökande takt, från ett system innehållande överskottsmängder av PrP ^ och små mängder av PrP Sc frö 19. Denna teknik har visat sig effektivt rekapitulera art och stam specificitet PrP omvandling från PrP ^, för att emulera interferens prionstam, och för att förstärka mycket låga nivåer av PrP från infekterade vävnader, vätskor, och miljöprover 6,7,16, 23. Detta dokument specificerar PMCA protokoll, inklusive rekommendationer för att minimera kontaminering, genererar konsekventa resultat och kvantifiera dessa resultat. Vi diskuterar också flera PMCA program, inklusive produktion och karakterisering av smittsamma prion stammar, prionstam störningar samt upptäckt av prioner i miljön.

Protein felveckning cyklisk amplifiering (PMCA) används för att amplifiera PrP in vitro 7, 12, 14, 19, 24. En framgångsrik PrP ​​amplifiering visas genom en ökning i bandintensitet på Western-blottar av PK-resistent prionprotein (migrera mellan 19 och 30 kDa för hamster-härledda prion stammar) som visas i figur 3. Efter Ökningen i bandintensitet PMCA indikerar amplifiering av PK-PrP Sc material. Framgångsrik amplifiering av hamster-härle...

Watch this Scientific Journal Video about Protein Misfolding Cyclic Amplification of Prions at JoVE.com

Protein Misfolding Cyclic Amplification of Prions

Protein felveckning cyklisk förstärkning (PMCA) är en in vitro-analys för att studera prioner konvertering och påfrestningar och arter hinder. Den kan också användas som en prion detektionsanalys.

Protein felveckning Cyklisk amplifiering av prioner

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

Research

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En komparativ metod för att karakterisera Landskap av värdpatogen Protein-protein interaktioner

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Rutiner för att identifiera smittsamma prioner efter passage genom matsmältningssystemet av en fågelart

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Demonstration av en multiresistent<em&gt; Mycobacterium tuberculosis</em&gt; Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Loop-medierad Isothermal Amplification (LAMP) Analyser för de artspecifika Upptäckt av<em&gt; Eimeria</em&gt; Som infekterar Kycklingar

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

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Utvecklingen av lyofiliserad Loop-medie Isothermal amplifieringsreagenserna för detektion av<em&gt; Coxiella burnetii</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

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Realtid skakande-inducerad konvertering analys för detektion av CWD prioner i fekalt Material

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant ...

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Bygga upp en High-throughput Screening plattform att bedöma heterogenitet HER2 genen amplifiering i bröstcancer

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast ...

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Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

Dissekera multi signalering proteinkomplex av Bimolecular komplettering affinitet rening (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

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Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

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Generation, amplifiering och titrering av rekombinant respiratoriska Syncytial virus

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...