Gostaríamos de agradecer ao Dr. Vesper Fe Marie Ramos para a leitura crítica do manuscrito. Este trabalho foi financiado pelo Centro Nacional de Pesquisa de Recursos (P20 RR0115635-6, C06 RR17417-01 e G20RR024001) e do Instituto Nacional de Distúrbios Neurológicos e Derrame (2R01 NS052609).

1. Preparando o Equipamento <ol><li> Usar um 3000 Misonix ou sonicador Misonix 4000 (Farmingdale, NY) ligado a um banho de Thermo Electron Neslab EX-7 água (Newington, NH) para manter uma temperatura constante de 37 ° C. Sonicar as amostras em 200 uL de paredes finas tiras de tubos PCR com tampas abobadadas obtidos a partir de Thermo Scientific (Waltham, MA). </li><li> A sonicador novo requer um &quot;break-in&quot; período de operação contínua 9. Uma quebra-in período de dois meses é composto por uma onda de ultra-sons de 40 seg e ciclos de 10 minutos de incubação com o nível de amplitude definida para 10. Deve-se observar a erosão ao longo da superfície da microplaca titânio corno (Figura 1, painel B). A circulação de água vai ficar turva branca, devido à erosão da microplaca de titânio chifre. Substituir essa água por dia. </li><li> A amplificação da PrP Sc irá variar ao longo da trompa de microplacas. A melhor eficiência de amplificação da PrP Sc pode ser obtida dentro deum raio de 5 cm a partir do centro do feixe de microplacas (Ver Figura 1, Painel C). </li><li> Uma rodada de PMCA consiste 144 ciclos. Cada ciclo é constituído por cinco segundos de sonicação e 10 minutos de incubação. Isso vai implicar cerca de 24 horas por rodada de PMCA. </li><li> A saída de energia de ultra-sons, exibido no painel de controle do sonicador, varia de acordo com o número de tubos de PCR presentes na prateleira do sonicador e a temperatura da água de circulação do banho de água. Certifique-se de que a água é equilibrado a 37 ° C, e não encha mais do que 30% de cremalheira do sonicador do tubo (Figura 1, painel C). Espalhe os tubos de PCR através do chifre microplaca. Ter pelo menos uma linha de espaço vazio entre as tiras de tubos de PCR e permitir não mais do que quatro tubos ligados entre si por tiras (Figura 1, painel C). </li><li> A força de sonicação é crítica para uma amplificação bem sucedida de prião estirpe. Enquanto sonicando, ajustar a amplitude de ter uma saída de potênciavariando entre 155 ~ 170 watts. Em nosso laboratório o nível de energia está definido para seis e sete para o 3000 Misonix e Misonix 4000, respectivamente. </li><li> Uso de água destilada em banho de água para evitar a acumulação manchas de água dura. Substituir este semanário água. Durante os ciclos de incubação e de sonicação, a condensação de água irá ocorrer no interior da tampa de chifre de microplacas. Para evitar a condensação, coloque um bloco de calor pequeno aquário para o topo da caixa do sonicador do invólucro, conforme mostrado na Figura 1, o painel A. </li></ol> 2. Preparação das amostras <ol><li> Todos os procedimentos envolvendo animais foram aprovados pelo Animal Care Institucional e Creighton University Use Comissão e cumprir com o Guia para o Cuidado e Uso de Animais de Laboratório. Antes de qualquer recolha de tecido, o animal deve ser anestesiado e transcardially perfundidos com fosfato arrefecido com gelo, com solução salina tamponada com EDTA 5 mM, pH 7,4, para remover o sangue do cérebro e evitar a inibição daPMCA reação por sangue 7. Coletar tecidos animais rapidamente, usando técnica asséptica e ter instrumentos de dissecação dedicados para cada cepa príon. Após a dissecação, colocar o tecido em um tubo de microcentrífuga de 1,5 ml, flash congelá-lo em gelo seco e armazená-lo a -80 ° C até à homogeneização. </li><li> Para preparar a solução de substrato PMCA (PrP C), homogeneizar o cérebro de hamster não infectadas (UN BH) para uma solução a 10% peso / volume (w / v) com tampão de gelo frio de conversão (ver parte 3), utilizando um triturador de tecidos Tenbroeck. Enquanto homogeneização, tome cuidado para não entrar ar dentro do moedor para evitar o excesso de espuma da solução. Clarificar a solução por centrifugação a 500 xg durante 30 seg e aliquota do sobrenadante em tubos de microcentrífuga de 1,5 ml. Armazenar a -80 ° C até à sua utilização. </li><li> Para preparar a semente PMCA (PrP Sc) de solução, homogeneizar um material infeccioso (gânglios cerebrais, baço, linfa, ou outros tecidos ou solo contaminado), a uma solução a 10% w / v de gelo frio water e alíquota da solução em tubos de microcentrífuga de 0,6 ml. Armazenar a -80 ° C até à sua utilização. </li></ol> 3. Preparando buffer de conversão <ol><li> Pesar 0,5093 g de Triton X-100, num balão volumétrico de 50 ml (resultado é uma concentração final de 1% de Triton X-100) (Sigma-Aldrich GmbH; Steinheim, Alemanha). </li><li> Adicionar um inibidor de protease completo Tablet (Roche Diagnostics GmbH, Mannheim, Alemanha). </li><li> Adicionar 0,0931 g de EDTA (isto irá resultar numa concentração final de 5 mM de EDTA) (Mallinckrodt Baker Inc.; Phillipsburg, NJ). </li><li> Ajustar o pH para 7,4 com NaOH 1N. </li><li> Ajustar o volume a 50 ml com fosfato salino tamponado de Dulbecco (DPBS) (Mediatech Inc., Manassas, VA). </li><li> Filtrar a solução através de uma membrana de nitrocelulose de 0,45 um, com um aparelho de filtração sob vácuo. O buffer de conversão pode ser armazenado a 4 ° C durante não mais de um mês. </li></ol> 4. Amplificação de Cepas Prion Usando PMCA <ol><li>Dilui-se a semente para dentro do substrato PMCA PMCA numa proporção de 1:20 (mistura ou seja, 5 ul da semente PMCA e 95 ul do substrato PMCA) para um tubo de PCR. Remover e armazenar-se uma alíquota de 15 uL a -80 ° C para o controle unsonicated; este controlo unsonicated será utilizada para quantificar a PrP Sc amplificação através de proteinase K de digestão (PK), seguido por análise Western blot (WB). Um controlo adicional pode ser gerado unsonicated colocando uma aliquota 15 segundo uL a 37 ° C durante a duração da reacção PMCA. Um mínimo de três repetições é necessária para fins estatísticos. </li><li> Objecto do restante da amostra (70 ul) para um ciclo de PMCA como representado na Figura 1, Painel A. Agitação tubos cada hora 5 para evitar a condensação de água na tampa abaulada do tubo de PCR. </li><li> Tubos da amostra deve ser girado para baixo e gentilmente agitadas (tendo o cuidado de que a solução não vão para a tampa abobadada) antes de abrir depois de qualquer procedimento para garantir homogeneity e evitar a contaminação potencial. </li><li> Para PMCA serial (sPMCA) de estirpes de incubação curtos períodos (isto é, HY TME, HaCWD ou 263K), dilui-se o material sonicado a uma proporção de 1:20 em transferir 5 ul da amostra sonicada de uma rodada em 95 ul de uninfected fresco homogenato cerebral (Figura 2, painel B). </li><li> Para sPMCA de estirpes de incubação longos (por exemplo, período de DY TME, 139H, 22CH, 22AH, ou ME7H), dilui-se o material sonicado a uma proporção de 1:1, transferindo 50 ul da amostra sonicada da rodada em 50 ul de uninfected fresco homogenato cerebral (Figura 2, painel B). </li><li> Remover duas alíquotas de 15 ul da mistura previamente diluída sonicado (a partir de etapas de 4,4 ou 4,5), e armazenar uma delas a -80 ° C e o outro a 37 ° C (solução de controlo para unsonicated PMCA dois redondos). Submeter os restos de uma rodada PMCA dois. </li><li> Este procedimento pode ser repetido indefinidamente. Em nosso laboratório, nóster realizado até 15 rodadas PMCA, com as propriedades de tensão permaneçam inalteradas. </li><li> PK digerir as amostras. Para um típico WB, misturar 5 ul de PK pi 80 ug / ml a 5 de amostra (esta solução irá ter uma concentração final de PK de 40 ug / ml) e incubar a 37 ° C durante uma hora. Adicionar 10 ul de tampão de carga de gel. Ferver esta solução durante 10 min. Deixar a solução arrefecer e 10 ul carregar no gel. </li><li> Para calcular o sucesso da amplificação realizar uma análise de WB nas amostras PK-digeridas para estimar a quantidade de PrP Sc amplificado comparando-os com um ou outro, os controles unsonicated 20,21 Figura 2, painel C ou a quantidades conhecidas de PK-recombinante não digerido PrP. </li></ol> 5. Evitando a contaminação cruzada Passos críticos têm de ser tomadas medidas para minimizar a contaminação cruzada e a ocorrência de falsos positivos devido a formação de novo de produtos resistentes a protease. </p&gt; <ol><li> Use um conjunto dedicado de instrumentos de dissecação (fórceps ou seja, pinças, tesouras) para coleta de amostras para cada estirpe príon. </li><li> Antes homogeneizando cérebro não infectados a serem utilizados como substrato PMCA, limpe as pipetas com lixívia a 1%. Mergulhe o rack sonicador do tubo de PCR, homogeneizadores, tecidos e racks de armazenamento de tubos de PCR com água sanitária a 1% por 10 minutos e enxaguar com água destilada. </li><li> Cobrir a área de trabalho com um Soaker Lab fresco Versi-Dry cada vez que uma nova amostra deve ser homogeneizada, numa experiência PMCA novo é para ser preparado, ou quando entre alíquotas sPMCA rodadas. </li><li> Use luvas novas a cada vez novas amostras são manipulados (especialmente quando a transferência de alíquotas entre as rodadas sPMCA). </li><li> Use rajadas curtas de sonicação para cada ciclo PMCA. Ciclos de 5 segundos de sonicação combinado com 10 min de incubação é suficiente para amplificar os priões, sem gerar de novo PrP Sc 1,20,21,23,24. Como outros laboratórios têm demonstrado, aumentando atempo de sonicação, o aumento da amplitude de sonicação, e estendendo-se o número de ciclos de PMCA aumenta a probabilidade de para PrP Sc novo de formação 2. </li></ol>

<ol>
	<li>Ayers, J. I., Schutt, C. R., Shikiya, R. A., Aguzzi, A., 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=The+strain-encoded+relationship+between+PrP+replication,+stability+and+processing+in+neurons+is+predictive+of+the+incubation+period+of+disease.">The strain-encoded relationship between PrP replication, stability and processing in neurons is predictive of the incubation period of disease.</a> PLoS pathogens. 7, e1001317 (2011).</li><li>Barria, M. A., Mukherjee, A., Gonzalez-Romero, D., Morales, R., 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=De+novo+generation+of+infectious+prions+in+vitro+produces+a+new+disease+phenotype.">De novo generation of infectious prions in vitro produces a new disease phenotype.</a> PLoS Pathog. 5, e1000421 (2009).</li><li>Bessen, R. A., Marsh, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+physical+properties+of+the+prion+protein+from+two+strains+of+the+transmissible+mink+encephalopathy+agent.">Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent.</a> J. Virol. 66, 2096-2101 (1992).</li><li>Bessen, R. A., Marsh, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+PrP+properties+suggest+the+molecular+basis+of+strain+variation+in+transmissible+mink+encephalopathy.">Distinct PrP properties suggest the molecular basis of strain variation in transmissible mink encephalopathy.</a> J. Virol. 68, 7859-7868 (1994).</li><li>Bessen, R. A., Marsh, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+two+biologically+distinct+strains+of+transmissible+mink+encephalopathy+in+hamsters.">Identification of two biologically distinct strains of transmissible mink encephalopathy in hamsters.</a> J. Gen. Virol. 73, 329-334 (1992).</li><li>Castilla, J., Gonzalez-Romero, D., Saa, P., Morales, R., De Castro, J., 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=Crossing+the+species+barrier+by+PrP(Sc)+replication+in+vitro+generates+unique+infectious+prions.">Crossing the species barrier by PrP(Sc) replication in vitro generates unique infectious prions.</a> Cell. 134, 757-768 (2008).</li><li>Castilla, J., Morales, R., Saa, P., Barria, M., Gambetti, P., 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=Cell-free+propagation+of+prion+strains.">Cell-free propagation of prion strains.</a> EMBO J. 27, 2557-2566 (2008).</li><li>Caughey, B., Raymond, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+scrapie-associated+form+of+PrP+is+made+from+a+cell+surface+precursor+that+is+both+protease-+and+phospholipase-sensitive.">The scrapie-associated form of PrP is made from a cell surface precursor that is both protease- and phospholipase-sensitive.</a> J. Biol. Chem. 266, 18217-18223 (1991).</li><li>Deleault, N. R., Harris, B. T., Rees, J. R., Supattapone, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+native+prions+from+minimal+components+in+vitro.">Formation of native prions from minimal components in vitro.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 9741-9746 (2007).</li><li>Dickinson, A. G., Fraser, H., Meikle, V. M., Outram, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Competition+between+different+scrapie+agents+in+mice.">Competition between different scrapie agents in mice.</a> Nat. New Biol. 237, 244-245 (1972).</li><li>Gonzalez-Romero, D., Barria, M. A., Leon, P., Morales, R., 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=Detection+of+infectious+prions+in+urine.">Detection of infectious prions in urine.</a> FEBS Lett. 582, 3161-3166 (2008).</li><li>Green, K. M., Castilla, J., Seward, T. S., Napier, D. L., Jewell, J. E., Soto, C., Telling, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+high+fidelity+prion+amplification+within+and+across+prion+species+barriers.">Accelerated high fidelity prion amplification within and across prion species barriers.</a> PLoS Pathog. 4, e1000139 (2008).</li><li>Hadlow, W. J., Race, R. E., Kennedy, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+distribution+of+transmissible+mink+encephalopathy+virus+in+mink+inoculated+subcutaneously.">Temporal distribution of transmissible mink encephalopathy virus in mink inoculated subcutaneously.</a> J. Virol. 61, 3235-3240 (1987).</li><li>Kocisko, D. A., Come, J. H., Priola, S. A., Chesebro, B., Raymond, G. J., Lansbury, P. T., Caughey, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+formation+of+protease-resistant+prion+protein.">Cell-free formation of protease-resistant prion protein.</a> Nature. 370, 471-474 (1994).</li><li>Kurt, T. D., Telling, G. C., Zabel, M. D., Hoover, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-species+amplification+of+PrP(CWD)+and+correlation+with+rigid+loop+170N.">Trans-species amplification of PrP(CWD) and correlation with rigid loop 170N.</a> Virology. 387, 3235-3240 (2009).</li><li>Maddison, B. C., Baker, C. A., Terry, L. A., Bellworthy, S. J., Thorne, L., Rees, H. C., Gough, K. 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>

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Desafios da amplificação príons infecciosos são os longos períodos de incubação e as despesas de experimentos in vivo. A técnica PMCA é um meio rentável para ampliar agentes príon infeccioso. Vários laboratórios confirmaram a capacidade de amplificar PMCA com precisão as estirpes de priões in vitro 7, 9, 12, 14, 19,24. Doenças de priões, pode ser transmitida entre as espécies. Bessen e Marsh efetivamente inoculados hamsters com encefalo...

<table border="1"><tbody><tr><td> Reagente / Equipamento </td><td> Fabricante </td><td> Gato. Número </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 Grinder Tissue </td><td> Kontes </td><td> 885000-0007 </td></tr><tr><td> Neslab Bath EX-7 Água </td><td> Thermo Electron </td><td> Neslab EX-7 </td></tr><tr><td> Tubo 0,2 ml Tiras de PCR </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> Inibidor de Protease Completo </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 Soakers Lab </td><td> Fisher Scientific </td><td> 14 206 28 </td></tr><tr><td> Aquecedor Repti Therm </td><td> Zoo Med Laboratories, Inc. </td><td> RH-4 </td></tr></tbody></table>

protein misfolding cyclic amplification of prions

Os príons são os agentes infecciosos que causam a encefalopatia espongiforme inevitavelmente fatal transmissível (EET) em animais e seres humanos 9,18. A proteína de prião tem duas isoformas distintas, a não infecciosa da proteína codificada hospedeira (PrP C) e a proteína infecciosa (PrP Sc), uma isoforma anormal de dobrado PrP C 8. Um dos desafios de trabalhar com agentes prião é o longo período de incubação antes do desenvolvimento de sinais clínicos após a inoculação de acolhimento 13. Isto tradicionalmente, imposto estudos bioensaio longas e caras animais. Além disso, as propriedades bioquímicas e biofísicas da PrP Sc ainda são pouco caracterizados devido à sua conformação invulgar e estados de agregação. PrPSc pode semear a conversão da PrP C e PrP Sc in vitro 14. PMCA é uma técnica in vitro, em que toma advantage desta capacidade utilizando ciclos de sonicação e a incubação para a produção de grandes quantidades de PrP Sc, a uma taxa acelerada, a partir de um sistema contendo quantidades em excesso de PrP C e pequenas quantidades de sementes a PrP Sc 19. Esta técnica provou ser eficaz para recapitular a especificidade de espécie e estirpe de PrP Sc conversão da PrP C, para emular interferência estirpe do prião, e para amplificar os níveis muito baixos de PrP Sc em tecidos infectados, fluidos e amostras ambientais 6,7,16, 23. Este trabalho detalha o protocolo PMCA, incluindo recomendações para minimizar a contaminação, gerando resultados consistentes, e quantificar esses resultados. Também discutimos várias aplicações PMCA, incluindo a geração e caracterização de cepas de príon infeccioso, interferência tensão prião, e na detecção de príons no ambiente.

Proteína de amplificação cíclica misfolding (PMCA) é usado para amplificar a PrP Sc in vitro 7, 12, 14, 19, 24. Uma bem sucedida PrP Sc amplificação é mostrado por um aumento na intensidade da banda em Western blots da proteína do prião PK-resistente (migrando entre 19 e 30 kDa para as estirpes derivadas do hamster prion), como mostrado na Figura 3. O aumento da intensidade da banda após a sua PMCA indica a amplificação do material resistente ao PK ...

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

Protein Misfolding Cyclic Amplification of Prions

Proteína de amplificação cíclica misfolding (PMCA) é um ensaio in vitro em para o estudo da conversão de prião e as barreiras de tensão e espécies. Também pode ser utilizado como um ensaio de detecção de prião.

Proteína amplificação cíclica misfolding de príons

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

protein-misfolding-cyclic-amplification-of-prions

Research

JoVE Journal

Immunology and Infection

19.4K visualizações.  University of Nebraska at Lincoln. Proteína de amplificação cíclica misfolding (PMCA) é um ensaio in vitro em para o estudo da conversão de prião e as barreiras de tensão e espécies. Também pode ser utilizado como um ensaio de detecção de prião.

Vídeo: Protein Misfolding Cyclic Amplification of Prions

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

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

Procedimentos para identificar Infecciosas príons após a passagem através do sistema digestivo de uma espécie de aves

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

Imunologia e Infecção

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis 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 environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

Demonstrando um multi-resistente<em&gt; Mycobacterium tuberculosis</em&gt; Ampliação 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

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

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

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

Isothermal amplificação (lâmpada), Ensaios mediada por loop para detecção específica da espécie os de<em&gt; Eimeria</em&gt; Que infectam Galinhas

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

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

O Desenvolvimento da liofilizado Loop-mediadas isotérmicas de amplificação Reagentes para a detecção de<em&gt; Burnetii Coxiella</em

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

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal 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 prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with &#7526;-sheet rich proteins. Thus, amyloid formation can be detected in real time. We attempted to develop a reliable non-invasive screening test to detect chronic wasting disease (CWD) prions in fecal extract. Here, we have specifically adapted the RT-QuIC technique to reveal PrPSc seeding activity in feces of CWD infected cervids. Initially, the seeding activity of the fecal extracts we prepared was relatively low in RT-QuIC, possibly due to potential assay inhibitors in the fecal material. To improve seeding activity of feces extracts and remove potential assay inhibitors, we homogenized the fecal samples in a buffer containing detergents and protease inhibitors. We also submitted the samples to different methodologies to concentrate PrPSc on the basis of protein precipitation using sodium phosphotungstic acid, and centrifugal force. Finally, the feces extracts were tested by optimized RT-QuIC which included substrate replacement in the protocol to improve the sensitivity of detection. Thus, we established a protocol for sensitive detection of CWD prion seeding activity in feces of pre-clinical and clinical cervids by RT-QuIC, which can be a practical tool for non-invasive CWD diagnosis.

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

Em tempo real a tremer induzida por conversão ensaio para detecção de príons CWD em Material Fecal

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

Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast cancers. However, a minority of cases displays a heterogeneous distribution of HER2-positive cells, which generates major clinical challenges. To date, no reliable and standardized protocols for the characterization and quantification of HER2 heterogeneous gene amplification in large cohorts have been proposed. Here, we present a high-throughput methodology to simultaneously assess the HER2 status across different topographic areas of multiple breast cancers. In particular, we illustrate the laboratory procedure to construct enhanced tissue microarrays (TMAs) incorporating a targeted mapping of the tumors. All TMA parameters have been specifically optimized for the silver in situ hybridization (SISH) of formalin-fixed paraffin-embedded (FFPE) breast tissues. Immunohistochemical analysis of the prognostic and predictive biomarkers (i.e., ER, PR, Ki67, and HER2) should be performed using automated procedures. A customized SISH protocol has been implemented to allow a high-quality molecular analysis across multiple tissues that underwent different fixation, processing, and storage procedures. In this study, we provide a proof-of-principle that specific DNA sequences could be localized simultaneously in distinct topographic areas of multiple and heterogeneously processed breast cancers using an efficient and cost-effective method.

Heterogeneous distribution of HER2-positive cells can be observed in a subset of breast cancers and generates clinical dilemmas. Here, we introduce a reliable and cost-effective protocol to define, quantify, and compare HER2 intra-tumor genetic heterogeneity in a large series of heterogeneously processed breast cancers.

Construindo uma plataforma High-throughput Screening para avaliar a heterogeneidade da amplificação do Gene HER2 no câncer de mama

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

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

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

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

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

Dissecando a proteínas multi sinalização complexos pela purificação da afinidade de complementação Bimolecular (BiCAP)

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

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

Análise comportamental de grande c. elegans populações usando um amplo campo de visão de rastreamento plataforma automatizada

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

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

Geração, amplificação e titulação do vírus sincicial respiratórios recombinantes

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

Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Specific and Rapid Detection of Tilapia Lake Virus

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the aquaculture industry that has resulted in the mass morbidity and mortality of tilapia in many parts of the world. An effective, rapid, and accurate diagnostic assay for TiLV infection is therefore necessary to detect the initial infection and to prevent the spread of the disease in aquaculture farming. In this study, a highly sensitive and practical reverse transcription loop-mediated isothermal amplification (RT-LAMP) method is presented to detect tilapia lake virus in fish tissue. A comparison of the RT-qPCR and RT-LAMP assays of infected samples revealed positive results in 63 (100%) and 51 (80.95%) samples, respectively. Moreover, an analysis of uninfected samples showed that all 63 uninfected tissues yielded negative results for both the RT-qPCR and RT-LAMP assays. The cross-reactivity with five pathogens in tilapia was evaluated using RT-LAMP, and all the tests showed negative results. Both the liver and mucus samples obtained from infected fish showed comparable results using the RT-LAMP method, suggesting that mucus can be used in RT-LAMP as a nonlethal assay to avoid killing fish. In conclusion, the results demonstrated that the presented RT-LAMP assay provides an effective method for TiLV detection in tilapia tissue within 1 h. The method is therefore recommended as a screening tool on farms for the rapid diagnosis of TiLV.

Reverse Transcription Loop-Mediated Isothermal Amplification RT-LAMP Assay for the Specific and Rapid Detection of Tilapia Lake Virus

We present an RT-LAMP assay for the detection of TiLV in tilapia fish using simple instruments over a relatively short period of time compared to conventional RT-PCR techniques. This protocol may help control the epidemic spread of TiLVD, especially in developing countries.