We thank John Olsen (Wisconsin Department of Natural Resources), Tom Cooley, Daniel O'Brien and Steve Schmitt (Michigan Department of Natural Resources) and Dr. Daniel Walsh (USGS National Wildlife Health Center) for assistance with tissue acquisition. We also thank the Wisconsin State Laboratory of Hygiene for diagnostic testing services, and Dr. Tonie Rocke and her staff (USGS National Wildlife Health Center) for use of equipment. Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the U.S. Government.

Trabalho animal realizado no USGS Centro Nacional de Saúde da Vida Selvagem foi realizada de acordo com o Escritório de NIH diretrizes Laboratory Animal Welfare e sob cuidados com os animais institucional e comissão uso protocolo # EP080716. Os tecidos de animais colhidas-caçadores estavam presentes do Wisconsin ou Michigan Departamentos de Recursos Naturais. 1. Preparação de Solução NOTA: As soluções listados abaixo são necessários para a preparação do substrato ensaio CER na Seção 2 abaixo. Preparar cada uma destas soluções a partir de soluções de estoque de concentração mais elevados; concentrações recomendadas para soluções de estoque será dada entre parênteses após cada respectivo nome químico listado. Prepare todas as soluções frescas sobre a preparação do substrato e armazenar a 4 ° C até ser utilizado. <ol><li> Para tampão de lise, prepare uma solução de o seguinte em 10 mM Tris, pH 7,5 (1 M Tris, pH 7,5 solução estoque recomendado): 100 mm scloreto de ódio (NaCl, 1 M de solução de estoque), 10 mM de ácido etilenodiaminotetracético (EDTA, EDTA 500 mM, pH 8,0, solução de estoque), NP-40 (solução de reserva a 10%) 0,5%, 0,5% de desoxicolato (DOC, solução estoque de 10% ). </li><li> Para conversão tampão, preparar uma solução de dodecil sulfato de sódio a 0,05% (SDS, solução de estoque a 10%) e 0,5% de Triton X-100 (solução a 10% de estoque) em solução salina tamponada com fosfato 1 (PBS, 10, pH 7,4 solução estoque). </li><li> Para soluções caotrï¿½icas, preparar dois 3 M soluções de guanidina (GdnHCl, 8 M de ações) em 1x PBS (solução estoque 10x). Ajustar o pH de uma das soluções a 3,5 ± 0,05 utilizando ácido clorídrico concentrado (HCl). Verificar se o pH da segunda solução se mantém a um pH de 7,4 ± 0,05 e ajustar usando HCl concentrado ou hidróxido de sódio (NaOH), se necessário. </li></ol> 2. Prepare Pairs CER ensaio de substrato <ol><li> Obter, o tecido cerebral não-TSE-infectado saudável a partir de espécies de interesse e preparar um volume peso-por-10%(W / v) homogeneizado em tampão de lise utilizando qualquer um dos seguintes métodos: Dounce, grânulo-moinho, ou homogeneização almofariz e pilão. Refinar resultando homogeneizados submetendo-os a seringa e agulha homogeneização, utilizando agulhas de aumento de calibre até substrato pode ser aspirado e expulso com facilidade através de uma seringa de agulha 27 G. <ol><li> Para substratos preparados a partir de roedores e outros pequenos mamíferos, homogeneizar cérebro inteiro (s); para substratos preparados a partir de mamíferos maiores, usar óbex (tronco cerebral) para preparar homogenatos de tecido. Ao seleccionar a quantidade de homogenato de cérebro, preparar não mais do que 50% da capacidade da centrifugadora usada para a precipitação de proteína no passo 2.6. </li><li> Se a qualidade do tecido cerebral adquirida é questionável, avaliar os níveis de PrP C por SDS-PAGE e imunotransferência 16 17 antes da preparação do substrato. </li></ol></li><li> Divida homogeneizado de cérebro em dois volumes iguais em tubos cônicos separados rotulados de plástico. Adicionar GdnHCl (3 M em soluções de 1 PBS) a pH 3,5 ou 7,4 ou a cada tubo em uma proporção de 1: 1 de volume de homogenato de cérebro. </li><li> Verificar os valores de pH das duas soluções. Ajustar o pH a pH 3,5 ± substrato a pH 0,05 utilizando ácido clorídrico concentrado. Certifique-se de que o pH do outro substrato permanece a um pH de 7,4 ± 0,05 e ajustar o pH, se necessário, com HCl ou NaOH, conforme necessário. Evite ultrapassar os valores de pH pela adição criterioso de ácido ou base. </li><li> Rodar soluções em um misturador de extremidade-a-extremidade, à temperatura ambiente (TA) durante 5 h. </li><li> Precipitar a proteína por adição de quatro volumes de metanol para as soluções de substrato em cada tubo cónico, vortex para misturar bem e incuba-se a -20 ° C durante 16-18 horas. </li><li> As amostras de sedimento por centrifugação a 13.000 x g durante 30 min a 4 ° C. Cuidadosamente decantar e descartar os sobrenadantes e permitir que o metanol se evaporar a partir dos peletes. Use aplicadores com ponta de algodão para ajudar na absorção de metanol agarrado no interior do tubo. Não permita pelotas para o excesso de dry e uma vez que o metanol não é mais visível, passe para a próxima etapa. </li><li> Pelotas de proteína Ressuspender em tampão de conversão. Usar uma quantidade igual de tampão de conversão para a quantidade de material de partida homogenato de cérebro distribuído por cada tubo no passo 2.2. NOTA: É importante que o tampão de conversão utilizado para ressuspender peletes de proteína a partir de tanto um pH de 3,5 e preparações de substrato 7,4-tratados é a solução definido no passo 1.2 acima (o qual contém níveis baixos de detergentes e um pH fisiológico). </li><li> Soluções Resumidamente sonicate substrato num sonicator cuphorn (10 seg a ~ 30% de potência máxima), alíquota em 0,5-1,0 ml volumes em rotulados 1,5 ml microtubos. Realizar uma imunotransferência de controlo de qualidade (passo 2.9), numa alíquota de cada substrato. Guarde outras alíquotas a -80 ° C até que esteja pronto para realizar ensaio CER (Seção 4). </li><li> Executar o controlo de qualidade sobre os pares de substratos CER antes da utilização (Figura 2). Estas medidas asseguram que os substratos CER provide ótimos resultados em estudos de conversão. <ol><li> Certifique-se de quantidades comparáveis ​​de PrP C em dois substratos. Comparar os níveis de PrP em 10-25 mL de cada substrato por SDS-PAGE 16 e 17 de immunoblotting. Se os níveis de proteína em que o pH 7,4 e 3,5 são substratos dentro de ~ 10% uma da outra, os pares de substratos são apropriados para utilização em estudos de CER. NOTA: Os immunoblots PrP não devem mostrar evidências de uma forte degradação PrP C. A imunorreactividade da PrP devem ser principalmente&gt; 20 kDa. Bandas inferiores a essa massa molecular podem ser produtos de degradação. Padrões de bandas deve ser mais ou menos equivalente entre os pares de substrato. </li><li> À medida que a PrP C em ambos os substratos devem ser sensíveis PK, tratar 10-25 ul de cada substrato com PK a uma concentração final de 100 ug / ml e digerir as amostras a 1000 rpm a 37 ° C durante 1 h na termo-agitador. Avaliar qualquer sinal PrP restante por SDS-PAGE 16 e 17 de immunoblotting.Substratos com res PrP não são apropriados para utilização em estudos de CER. </li></ol></li></ol> 3. Prepare Seeds TSE Agente <ol><li> Obter tecido cerebral TSE-infectados a partir de espécies de interesse e preparar a 10% (w / v) homogeneizado em 1x PBS pH 7,4, utilizando os métodos listados na etapa 2.1 acima. NOTA: As sementes também podem ser produzidos a partir de outros tecidos infectados com EET fora do sistema nervoso central, no entanto, a eficiência de conversão de substratos cerebrais derivadas de sementes derivadas de tecidos periféricos infectadas por EET ainda não foi experimentalmente avaliada na literatura publicada. </li><li> Aliquota homogenato resultante (s) em 100-300 ul volumes de 0,5 ml em tubos de microcentrífuga e armazenar a -80 ° C até estar pronto para executar o ensaio de CER. </li></ol> 4. CER Assay NOTA: O ensaio CER envolve a adição de uma quantidade relativamente pequena de PrP TSE (fornecido no &quot;semente&quot;) a partir de uma espécieem um excesso relativo de PrP C (fornecida no cérebro infectado &quot;substrato&quot;) a partir de uma outra espécie que foi parcialmente desnaturada a pH 3,5 ou 7,4 ou. Após um período de incubação prolongado agitação, a conversão baseada em modelos de PrP C por PrP TSE em cada reacção é avaliada pelo sinal densitométrica da PrP remanescente após a digestão res PK e detecção de imunotransferência. Comparando os níveis res PrP nos substratos previamente desnaturadas a pH 3,5 versus 7,4 fornece uma medida da barreira das espécies. Uma visão geral experimental deste procedimento de ensaio é fornecida na Figura 1. <ol><li> Lentamente descongelar pares substrato CER não infectados (ensaio requer volumes iguais de substratos previamente desnaturado, tanto pH 3,5 e 7,4) e soluções de sementes TSE-infectadas, colocando-os em cima de uma cama de gelo (cerca de 1 hr tempo degelo). </li><li> Uma vez completamente descongelado, e depois aspirado expulsar cada solução através de um substrato27 g de agulhas de seringa várias vezes para homogeneizar re-lo. Resumidamente sonicate cada solução semente TSE-infectados por 10 s em ~ 30% da potência máxima. Mantenha todas as soluções em gelo enquanto se prepara reações de conversão. </li><li> Etiqueta 5 individuais baixa ligação, de paredes finas tubos de PCR por agente TSE está sendo testado. </li><li> Prepare reacções de conversão em destes tubos por adição de 5 uL de solução de sementes infectadas por EET 10% a 95 ul de (a) substrato CER preparados a pH 7,4 e (b) substrato CER preparado a pH 3,5. <ol><li> Para cada amostra experimental, prepare reações paralelas em pares substrato CER previamente desnaturado, tanto a pH 3,5 e 7,4. </li><li> Otimizar os rácios de sementes agente TSE para substrato cérebro não infectado através da determinação empírica. Semente comum: rácios de substrato empregues manter um volume de reacção total de 100 uL e incluem ul 1:99, 2:98 e 5:95 ul ul. Para a maioria das aplicações, a semente 5:95 ul: proporção em volume de substrato e é apropriado é o que é apresentado no thé protocolo. </li></ol></li><li> Preparar três amostras de controlo por agente da EET serem testados como se segue: (a) adicionam-se 5 ml de solução de sementes infectadas por EET 10% a conversão de 95 ul de tampão como um controlo para a entrada res PrP; adicionar 5 uL de tampão de conversão de 95 ul de substrato preparada em (b) pH 3,5 e (c) a pH 7,4 como controlos para a conversão não-específico, não modelada. </li><li> Resumidamente vortex cada amostra em seu tubo de PCR fechado para misturar sementes e substrato bem. Siga com um pulso momentânea numa baixa velocidade de topo de bancada de mini-centrifugadora para remover qualquer volume da mistura de reacção preso na tampa do tubo de PCR. </li><li> Amostras de Carga em tubos de PCR-rotulados individualmente em cima da bancada thermo-shaker equipados para aceitar tubos PCR. Agitar as amostras a 1000 rpm a 37 ° C durante 24 horas. </li><li> Após agitação, adicionar sarcosil a uma concentração final de 2% (w / v) e PK a uma concentração final de 100 ug / ml. Digest amostras a 1000 rpm a 37 ° C durante 1 h na termo-agitador. NOTA: A adição de sarcosil a amostras auxiliares pós-conversão na digestão PK de PrP C. Sinais positivos falsos em amostras unseeded (passo 4.5) são praticamente eliminadas pela adição de Sarkosyl. </li><li> Adicionar tampão de amostra de SDS-PAGE, as amostras de calor a 95 ° C durante 5 minutos e res PrP resolver restantes em cada amostra por SDS-PAGE 16 seguida de imunotransf erência 17. NOTA: Após a adição de tampão de amostra e de aquecimento, as amostras podem ser processadas imediatamente ou amostras podem ser armazenadas a -20 ° C ou -80 ° C antes da imunomancha. Todos os dados aqui apresentados foram obtidos usando os sistemas de gel e de transferência Novex NuPAGE. As amostras individuais de ensaio CER foram tratadas com tampão de amostra e o agente redutor de acordo com as instruções do fabricante. As amostras foram, subsequentemente, aquecida a 95 ° C durante 5 min e 30 ul de cada amostra foi carregada em 12% Bis-Tris géis pré-moldados. </li><li> Calcule o índice de eficiência de conversão (CER) como uma medidade barreira das espécies. Avalie os níveis de res PrP por densitometria 17 e dividir a densidade total da amostra pH 7,4 por que do pH 3,5 amostra. Multiplique por 100 para produzir uma porcentagem. </li><li> Compilar dados de réplicas experimentais independentes para gerar uma média CER ± desvio-padrão para uma espécie com um determinado agente TSE. </li></ol>

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The authors have nothing to disclose.

Para a conclusão deste protocolo, a atenção deve ser dada aos níveis de PrP C no tecido cerebral infectado utilizado para a preparação do substrato (passo 2.1.2) e as sementes ao substrato razão para reações de conversão (etapa 4.4.2). Na nossa experiência, o cérebro pode ser extraída para uso como substrato CER depois de um período substancial de pós-mortem, enquanto a PrP C está presente por imunotransferência (Figura 4). Na verdade, alguns autólise foi observad...

Encefalopatias espongiformes transmissíveis (EET, doenças de príon) são um grupo de doenças neurodegenerativas fatais com longos períodos de incubação que afetam uma variedade de animais e seres humanos. O agente etiológico da EET putativo é composto por um isómero enrolamento incorrecto da proteína prião de acolhimento (PrP), que é capaz de se auto-propagação por conversão baseada em modelos da forma celular normal da PrP (PrP C) para uma doença infecciosa, doença associada formulário (PrP TSE) que se acumula nos tecidos do sistema nervoso central do hospedeiro infectado 1. Príons infecciosos mamíferos geralmente transmitem a partir de-host-to anfitrião de uma forma específica da espécie, o que deu origem ao conceito da &quot;barreira das espécies TSE&quot; limitando eventos de transmissão entre espécies 2. Os determinantes biológicos da barreira das espécies de prião não são bem compreendidos. Similaridade de sequência de aminoácidos entre o infecciosa PrP TSE eo anfitrião PrP C cum influenciam fortemente se a conversão ocorre 3-5, mas continua a ser insuficiente para explicar todos os eventos de transmissão de prion observados in vivo 6,7. Assim, a caracterização da barreira de espécie TSE foi amplamente invocado estudos de provocação em animais: exposição dos animais ingênuos de uma determinada espécie de prions de outro e medindo o tempo de incubação resultante para o início da doença e taxa de ataque como indicadores de eficiência de transmissão. Ratos que expressam PrP C a partir de espécies heterólogas são também utilizados para este tipo de estudo de 8. Os custos associados à produção de camundongo transgênico e bioensaios prião prolongadas, bem como considerações éticas do uso de animais, são obstáculos para a investigação experimental de espécies TSE barreiras. Avaliação da barreira da espécie humana às TSE conta com ratos geneticamente modificados para expressar humano PrP C. Estes ratos exigem longos períodos de incubação de sucumbir às TSE humanos ou modificações a Tele humana molécula PrP C para início rápido da doença 9. Os períodos de incubação das EET não humana nesses camundongos pode estender-se para além do tempo de vida normal do mouse dificultando a interpretação dos resultados negativos desafiadoras. Os primatas não humanos também têm sido utilizados como proxies para estudar barreiras espécie humana, mas estes estudos são preocupantes com os mesmos desafios que outros tipos de experimentos com animais e primatas não-humanos não podem recapitular precisa da doença, uma vez que prossegue no hospedeiro humano. Bioensaios em animais continuam a ser o método &quot;padrão ouro&quot; para medir a susceptibilidade de uma espécie para a TSE, mas os obstáculos, custos e ética destes estudos em animais vivos têm obrigado investigação sobre alternativas. Um certo número de ensaios in vitro, com base na avaliação da conversão da PrP C hospedeiro para uma proteinase K (PK) estado -resistente (res PrP) quando semeadas por PrP TSE, foram desenvolvidos e utilizados para investigar TSE spECIES barreiras 10-12. Exemplos de ensaios in vitro incluem ensaios livres de células de conversão, proteína misfolding amplificação cíclica (CMAP), e o índice de eficiência de conversão (CER) ensaio 10-14. Embora nenhum destes ensaios de levar em conta fatores periféricos envolvidos em barreiras das espécies após a infecção natural, tudo pode ser útil para identificar os anfitriões potencialmente sensíveis para as EET. Aqui apresenta-se o protocolo para o ensaio de CER, em que dois substratos desnaturados PrP C derivadas de homogenatos cerebrais normais são utilizados em reacções de conversão de topo de bancada de prião (Figura 1) 13. PrP C no substrato desnaturada a pH 7,4 só pode ser convertido em res PrP por PrP TSE semeadas em a reacção na ausência de uma barreira 14 espécies. Em contraste, a desnaturação da PrP C no outro substrato a pH 3,5 permite que seja convertido ao PrP res após incubaçãocom PrP TSE de quaisquer espécies e serve como um controlo para a conversão. A proporção de conversão da PrP C e PrP no res de pH 7,4 em relação ao substrato que o substrato de pH 3,5 dá uma medida da barreira das espécies. Descobrimos que o ensaio CER prediz conhecido espécies barreiras de ratos de laboratório para vários EET e ter usado o ensaio em esforços para prever a barreira das espécies de numerosas espécies de mamíferos, incluindo carneiros selvagens, a doença debilitante crônica (CWD) e outras EET 14, 15. Os investigadores interessados ​​em uma ferramenta que permite a verificação rápida de espécies TSE barreiras ou avaliação da conversão PrP C -para-PrP res vai encontrar esta metodologia útil.

<table border="0" cellpadding="0" cellspacing="0" width="944">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Name of Material/ Equipment</td>
			<td style="width:205px;">Company</td>
			<td style="width:157px;">Catalog Number</td>
			<td style="width:287px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">12% Bis-Tris SDS-PAGE gels</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NP0342</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">0.5 mm zirconium oxide beads</td>
			<td style="width:205px;">Next Advance</td>
			<td style="width:157px;">ZROB05&#160;</td>
			<td style="width:287px;">Other varieties of beads are also effective</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:296px;">Antibodies</td>
			<td style="width:205px;">various suppliers</td>
			<td style="width:157px;"></td>
			<td style="width:287px;">Select appropriate primary and secondary antibodies for immunoblot detection of PrPres from species of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Bead homogenizer</td>
			<td style="width:205px;">Next Advance</td>
			<td style="width:157px;">BBY24M</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Centrifuge&#160;</td>
			<td style="width:205px;">Beckman Coulter</td>
			<td style="width:157px;">369434</td>
			<td style="width:287px;">High speed with temperature control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Conical tubes</td>
			<td style="width:205px;">any brand</td>
			<td style="width:157px;"></td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Cotton-tipped applicator</td>
			<td style="width:205px;">Uline</td>
			<td style="width:157px;">S-18991</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Cuphorn sonicator</td>
			<td style="width:205px;">Heat Systems-Ultrasonics</td>
			<td style="width:157px;">W-380</td>
			<td style="width:287px;">Heat Systems-Ultrasonics, Inc. is now Qsonica, LLC</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:296px;">Densitometry software program</td>
			<td style="width:205px;">UVP</td>
			<td style="width:157px;">Vision Works LS Image Acquisition and Analysis software</td>
			<td style="width:287px;">Other programs, such as NIH ImageJ, will also work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Dounce homogenizer</td>
			<td style="width:205px;">Kimble Chase</td>
			<td style="width:157px;">885300</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">End-over-end mixer</td>
			<td style="width:205px;">Labnet International</td>
			<td style="width:157px;">H5600</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylenediaminetetra acetic acid</td>
			<td style="width:205px;">Boston Bioproducts</td>
			<td style="width:157px;">P-770</td>
			<td style="width:287px;">Hazardous chemical: eye irritation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Guanidine hydrochloride, 8M</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td style="width:157px;">24115</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, skin irritation, eye irritation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating block</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">11-718</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrochloric acid</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">435570</td>
			<td style="width:287px;">Hazardous chemical: strong acid</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Lithium dodecyl sulfate sample buffer, 4X</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NP0008</td>
			<td style="width:287px;">Hazardous chemical: skin &#38; respiratory irritation, serious eye damage, flammable solid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Methanol</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">A454-4</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, flammable liquid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge tubes</td>
			<td style="width:205px;">any brand</td>
			<td style="width:157px;"></td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-centrifuge</td>
			<td style="width:205px;">Labnet International</td>
			<td style="width:157px;">C1301</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">N-lauroyl-sarcosine (sarkosyl)</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">L-5125</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, skin irritation, eye damage</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Nonidet P-40</td>
			<td style="width:205px;">Amresco</td>
			<td style="width:157px;">M158</td>
			<td style="width:287px;">Hazardous chemical: skin irritation, eye damage</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:296px;">SDS-PAGE gel system</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NuPAGE electrophoresis system</td>
			<td style="width:287px;">Other SDS-PAGE systems will also work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">PCR tubes (low-binding)</td>
			<td style="width:205px;">Axygen</td>
			<td style="width:157px;">PCR-02-L-C</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Pestle homogenizer</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">03-392-106</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">pH meter</td>
			<td style="width:205px;">Sentron</td>
			<td style="width:157px;">SI600</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Polyvinyldifluoride membrane</td>
			<td style="width:205px;">Millipore</td>
			<td style="width:157px;">IPVH00010</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Proteinase K</td>
			<td style="width:205px;">Promega</td>
			<td style="width:157px;">V3021</td>
			<td style="width:287px;">Hazardous chemical: skin &#38; eye irritation, respiratory sensitisation, organ toxicity</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:296px;">Reducing agent for SDS-PAGE samples, 10X</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NP0009</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sodium Chloride</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">7647-14-5</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sodium deoxycholate</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">D6750</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Sodium dodecyl sulfate</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td style="width:157px;">28364</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, skin irritation, eye damage, flammable solid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sodium hydroxide</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">S5881</td>
			<td style="width:287px;">Hazardous chemica; strong base</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Syringe</td>
			<td style="width:205px;">BD Biosciences</td>
			<td style="width:157px;">various</td>
			<td style="width:287px;">Use syringe size appropriate to volumes of substrate to be homogenized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Syringe needles</td>
			<td style="width:205px;">BD Biosciences</td>
			<td style="width:157px;">various</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Thermoshaker (PCR tube shaker)</td>
			<td style="width:205px;">Hangzhou All Sheng Instruments</td>
			<td style="width:157px;">MS-100</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Tris base</td>
			<td style="width:205px;">Bio Basic&#160;</td>
			<td style="width:157px;">77-86-1</td>
			<td style="width:287px;">Hazardous chemical: skin, eye, respiratory irritation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Triton X-100</td>
			<td style="width:205px;">Integra Chemical Company</td>
			<td style="width:157px;">T756.30.30</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, eye irritation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Vortexer</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">12-812</td>
			<td style="width:287px;"></td>
		</tr>
	</tbody>
</table>

assessing transmissible spongiform encephalopathy species barriers with an in vitro prion protein conversion assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

A utilização bem sucedida do ensaio CER é em grande parte dependente da qualidade dos pares de substratos utilizados em reacções de conversão. Por esta razão, de acordo com procedimentos para preparar pares de substrato CER, uma imunotransferência de controlo de qualidade deve ser realizado (Figura 2). Para ambos substratos, ensaio 10-25 ul por imunotransferência. PrP C deve ser facilmente detectável em cada substrato e os níveis de PrP C deve ser aproximadamente a mesma...

Watch this Scientific Journal Video about Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay at JoVE.com

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

Avaliando Encefalopatia Espongiforme Transmissível Espécies Barreiras com uma<em&gt; In Vitro</em&gt; Prion Conversão Protein Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

assessing-transmissible-spongiform-encephalopathy-species-barriers

Research

JoVE Journal

Medicine

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

7.7K visualizações.  USGS National Wildlife Health Center. Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

Vídeo: Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarcadores em um modelo animal para Revelando correlatos neurais, hematológica e Comportamental do TEPT

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Medicina

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Avaliar as alterações teratogênicos em um modelo de Zebrafish de exposição fetal ao álcool

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform PrPSc 1. PrPC is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrPSc forms detergent-insoluble aggregates and is partially resistant to PK2-6. The conversion of PrPC to PrPSc is known to involve a conformational transition of &alpha;-helical to &beta;-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrPSc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain7.
Using a combination of biophysical and biochemical approaches, we identified insoluble PrPC aggregates (designated iPrPC) from uninfected mammalian brains and cultured neuronal cells8, 9. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms10, and PK-treatment. The combination of these approaches isolates not only insoluble PrPSc and PrPC aggregates but also soluble PrPC oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrPSc from infected brains and iPrPC from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrPC are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrPSc that shares the immunoreactive behavior and fragmentation with iPrPC 11, 12. Moreover, we recently demonstrated that iPrPC is the main species that interacts with amyloid-&beta; protein in Alzheimer disease13. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer's disease13, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.

A new species of cellular prion protein (PrPC) has recently been identified in uninfected human brains using the methods described here. These methods can be used to isolate various PrP species, while some of them are also useful in isolating other misfolded protein aggregates from human brains.

Isolamento de Oligómeros PrP solúveis e insolúveis no cérebro humano normal

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform ...

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the pathophysiological role of neuroplasticity in GI disorders remains unknown. Novel experimental models which allow simulation and modulation of GI neuroplasticity may enable enhanced appreciation of the contribution of neuroplasticity in particular GI diseases such as pancreatic cancer (PCa) and chronic pancreatitis (CP). Here, we present a protocol for simulation of pancreatic neuroplasticity under in vitro conditions using newborn rat dorsal root ganglia (DRG) and myenteric plexus (MP) neurons. This dual-neuron approach not only permits monitoring of both organ-intrinsic and -extrinsic neuroplasticity, but also represents a valuable tool to assess neuronal and glial morphology and electrophysiology. Moreover, it allows functional modulation of supplied microenvironmental contents for studying their impact on neuroplasticity. Once established, the present neuroplasticity assay bears the potential of being applicable to the study of neuroplasticity in any GI organ.

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuronal plasticity is an increasingly recognized, but insufficiently understood feature of the gastrointestinal (GI) tract. Here, in the example of human pancreatic disorders, we present an in vitro neuroplasticity assay for the study of neuronal plasticity in the GI tract at both morphological and functional level.

Simulando pâncreas Neuroplasticidade:<em&gt; In Vitro</em&gt; Dual-neurônio Plasticidade Assay

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

Usando vírus adeno-associado como uma ferramenta para o estudo da retina Barreiras na Doença

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

Um Modelo de Leitão Neonatal encefalopatia hipóxico-isquêmica

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Modelagem Encefalopatia da Prematuridade Usando pré-natal hipóxia-isquemia com Lipopolysaccharide intra-amniótica em Ratos

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

Assessing Specificity of Anticancer Drugs In Vitro

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key element is the quantitative determination of a tumor-specific genetic alteration in relation to a universal sequence using a dual-probe digital PCR assay and the subsequent calculation of the proportion of tumor cells. The assay is carried out on a culture containing tumor cells of an established line and spiked-in non-tumor cells. The mixed culture is treated with a test drug at various concentrations. After the treatment, DNA is prepared directly from the survived adhesive cells in wells of 96-well plates using a simple and inexpensive method, and subjected to a dual-probe digital PCR assay for measuring a tumor-specific genetic alteration and a reference universal sequence. In the present demonstration, a heterozygous deletion of the NF1 gene is used as the tumor-specific genetic alteration and a RPP30 gene as the reference gene. Using the ratio NF1/RPP30, the proportion of tumor cells was calculated. Since the dose-dependent change of the proportion of tumor cells provides an in vitro indication for specificity of the drug, this genetic and cell-based in vitro assay will likely have application potential in drug discovery. Furthermore, for personalized cancer-care, this genetic- and cell-based tool may contribute to optimizing adjuvant chemotherapy by means of testing efficacy and specificity of candidate drugs using primary cultures of individual tumors.

Assessing Specificity of Anticancer Drugs In Vitro

The goal of this protocol is to assess specificity of anticancer drugs in vitro using mixed cultures containing both tumor and non-tumor cells.

Avaliando Especificidade de Anticancer Drugs<em&gt; In Vitro</em

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Inchaço na Organóides intestinal induzida por forscolina: An<em&gt; In Vitro</em&gt; Ensaio para a Avaliação de Medicamentos Response na Fibrose Cística Os pacientes

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

Técnica de Explante Coclear Murino Neonatal como Ferramenta de Triagem In Vitro na Pesquisa Auditiva

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...