Y.N.T. would like to acknowledge the Agency for Science, Technology and Research (A*STAR), Singapore for the financial support under the JCO CDA grant 13302FG063.

Atenção: Por favor, consultar as fichas de segurança (SDS) de todos os produtos químicos envolvidos antes de usar. A experiência de rastreio de drogas envolve a síntese e manipulação de nanomateriais, que podem ter riscos adicionais em comparação com o seu homólogo grandes quantidades. Por favor, garantir todas as medidas de controlo necessárias para ser praticado durante todo o experimento, incluindo o uso de controles de engenharia (extractor de fumo) e equipamentos de proteção individual (EPI, por exemplo, calças de comprimento de segurança, sapatos fechados, luvas resistentes a produtos químicos, e óculos de segurança). 1. Preparação de reagentes químicos para Rastreio de drogas <ol><li> Os precursores para a síntese de Au CNs <ol><li> Dissolve-se 30 mg de uma solução de ouro (III), cloreto (99,99% base metais vestigiais, 30 p.% Em ácido clorídrico diluído) em 6,9 ml de água ultrapura para preparar 15 mM de (III) solução de cloreto de ouro. Cuidado: solução de cloreto de ouro é corrosivo e irritante. Utilizar EPI adequado para evitar o contato direto com os olhos ea pele. </li><li> Dissolve 74 mg de HSA ou de BSA em 1 ml de água ultrapura para preparar a 74 mg / ml de solução-mãe de proteínas. </li></ol></li><li> As soluções de fármaco <ol><li> Dissolve-se a quantidade desejada de droga, por exemplo, 1,9 mg de (um) de ibuprofeno, 2,8 mg de (b), a varfarina, 2,3 mg de (c), fenitoína, e 1,5 mg de (d) sulfanilamida em 20 ul de DMSO para preparar a 450 mM das soluções de droga. Nota: DMSO é seleccionado como solvente porque estas drogas são hidrofóbicos e têm baixa solubilidade em água. </li></ol></li><li> Outros reagentes <ol><li> Dissolve-se 600 mg de pastilhas de NaOH em 10 ml de água ultrapura para preparar a 1,5 M de solução de NaOH. Dissolve-se 2,4 g de ureia em 2 ml de água ultrapura para preparar a 20 M de solução de ureia. </li></ol></li></ol> 2. Síntese de Templated-Protein Au NCs <ol><li> HSA-templated Au NCs (HSA-Au CNs) <ol><li> Coloque dois frascos de vidro, cada uma contendo um micro barra magnética nele, em dois de temperatura separado de agitação magnética controlávelRER. </li><li> Ajustar a temperatura de um agitador magnético a 60 ° C, e Rotular o frasco de vidro em cima dele como &quot;60 ° C&quot;; enquanto o outro agitador magnético é mantida à temperatura ambiente (RT), em que o frasco de vidro em cima do que é rotulado como &quot;RT&quot;. </li><li> Adicionar 200 ul de solução de HSA, 200 ul de água ultrapura, e 200 ul de solução de ouro (III), cloreto de para cada frasco, sob agitação constante (definida como 360 rpm, a menos que especificado de outro modo) para permitir o encapsulamento de Au iões dentro do modelo de proteína. </li><li> 2 minutos mais tarde, adicionar 20 ul de uma solução de NaOH a cada frasco, de modo a activar a capacidade redutora do conjugado de HSA na formação de Au CNs e começar a gravar o tempo de reacção como 0 min. </li><li> Cada 20 minutos, extrair 50 mL de soluções de cada da amostra para uma placa preta de 384 cavidades e medir o espectro de emissão com um leitor de microplacas. A configuração de varredura típico: λ ex = 370 nm, em = λ 410-850 nm. </li><li> Parar o agitador magnético após 100 min. </li><li> Arrefecer os frascos de vidro com água corrente em uma pia. Atenção: Os frascos de vidro estiverem quentes. Usar luvas resistentes ao calor para evitar a queima. </li><li> Traça-se a espectros de fotoemissão em todo o tempo, para cada amostra para obter a cinética de formação de Au CNs em diferentes condições de temperatura. </li></ol></li><li> BSA-templated Au NCs (BSA-Au CNs) <ol><li> Colocar num frasco de vidro contendo um micro barra de agitação magnética no topo de um agitador magnético. Deixar a temperatura como a temperatura ambiente. </li><li> Misturar 200 ml de solução de BSA, 200 ul de ureia, e 200 ul de solução de ouro (III), cloreto de dentro do recipiente de vidro sob agitação constante. </li><li> 2 minutos mais tarde, adicionar 20 ul de uma solução de NaOH para activar a capacidade redutora de BSA para formar Au CNs e começar a gravar o tempo de reacção como 0 min. Medir o espectro de fotoemissão da mistura de reacção por hora. Nota: O espectro de fotoemissão de BSA-Au NCs é medidomenos frequentemente porque a taxa de formação de BSA-au CNs é mais lento do que o de HSA-Au, à mesma temperatura, devido à menor eficácia de ureia para desdobrar a proteína. </li><li> Parar o agitador magnético após 7 h. Traçar o espectro de fotoemissão em todos os tempos para ver a cinética de formação de Au NCs por uréia desnaturação. </li></ol></li></ol> 3. Apresentação de pequeno Drogas Molecular <ol><li> Tela afinidade de ligação relativa das diferentes drogas moleculares pequenos para HSA <ol><li> Coloque cinco frascos de vidro, cada uma contendo uma barra de agitação magnética, em que, na parte superior de uma temperatura controlável multiponto agitador magnético. Rotular estes frascos como a, b, c e d, respectivamente, para cada fármaco, ou seja, o ibuprofeno, a varfarina, fenitoína, e sulfanilamida. Rotular a um puro com HSA como controle. </li><li> Adicionar 200 mL de HSA a cada frasco. Adicionar 1 ml de solução de fármaco para os quatro frascos correspondentes. Adicionar 1 ml de DMSO com o controlo. Ligue o agitador e definir a velocidade de rotação para 360rpm e incubar durante 1 h para permitir a ligação à ASH de drogas para completar. </li><li> 1 h depois, adicionar 200 mL de água Milli-Q e 200 ul de solução de ouro (III), cloreto de para cada frasco, sob agitação constante. Ajustar a temperatura para 60 ° C, sob agitação constante durante 10 min. Adicionar 20 mL de NaOH 1,5 M a cada frasco e começar a gravar o tempo de reação como 0 min. </li><li> Desenhar se rapidamente 50 ml de solução a partir de cada frasco para uma placa preta de 384 cavidades e medir o espectro de emissão (resultados rotulado como 0 min). Grave os espectros de emissão de cada amostra a cada 10 min. Recolher pelo menos quatro espectros no tempo diferente de quatro, que é 0, 10, 20, e 30 min. </li><li> Repita os passos 3.1.1 - 3.1.4 várias vezes para se obter resultados com uma tendência consistente. Parar o agitador magnético. </li><li> Traçar o espectro de fotoemissão resolvida no tempo para cada amostra (a - d, e controle). Identificar o pico de intensidade de todas as amostras e conspiração contra o tempo para comparar a cinética de formação de Au NCs em dimodelos fferent proteína carregado de drogas. </li></ol></li><li> Medir a constante de ligação de um fármaco específico para HSA <ol><li> Repita os passos 3.1.1 - 3.1.4. Substitua a solução de um fármaco com soluções de ibuprofeno em quatro concentrações diferentes (incluindo uma amostra de controlo usando HSA só que sem a carga de droga). Rotular o frasco de vidro de acordo com a concentração do fármaco correspondentemente. </li><li> 10 min mais tarde, arrefecer os frascos de vidro com água corrente em uma pia. Atenção: Os frascos de vidro estiverem quentes. Usar luvas resistentes ao calor para evitar a queima. </li><li> Desenhar 50 ul das soluções acima a partir de cada frasco para uma placa preta de 384 cavidades e medir o espectro de emissão. </li><li> Repita os passos 3.2.1 - 3.2.3 mais duas vezes para se obter mais dois conjuntos de resultados em diferentes concentrações da droga. </li><li> Parar o agitador magnético. Traçar o espectro de fotoemissão e analisar os resultados. <ol><li> Para os dados brutos obtidos em cada lote individual traçar o espectro de fotoemissão de cadaamostra em software como o software OriginPro e descobrir a intensidade de pico. </li><li> Calcular e representar graficamente a intensidade relativa de fluorescência [(I 0 - I) / I 0] contra a concentração de fármaco, e onde I I 0 referem-se a intensidade de fluorescência de Au CNs formado no carregadas com droga HSA e HSA puro, respectivamente. </li><li> Calcula-se a constante de ligação através do ajuste dos dados a um modelo único local de ligação utilizando a equação de Michaelis-Menten: Y = Rmax × C / (K D + C), onde R max indica o sinal de resposta máxima, C é a concentração do ligando e K D a constante de ligação é. Execute isso em software como OriginPro. Selecione o menu Análise Fitting Nonlinear Curve Fit, em seguida, selecione a função monte de Crescimento / categoria Sigmoidal. No Settings: página Seleção de Função, clique em Ajustar a mostrar-se os resultados embutidos. </li></ol></li></ol></li></ol>

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S., Montich, G. G., Fidelio, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Stability+Induced+by+Ligand+Binding+Correlates+with+Changes+in+Protein+Flexibility.">Protein Stability Induced by Ligand Binding Correlates with Changes in Protein Flexibility.</a> Protein Sci. 12, 1496-1506 (2003).</li><li>Layton, C. J., Hellinga, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamic+Analysis+of+Ligand-Induced+Changes+in+Protein+Thermal+Unfolding+Applied+to+High-Throughput+Determination+of+Ligand+Affinities+with+Extrinsic+Fluorescent+Dyes.">Thermodynamic Analysis of Ligand-Induced Changes in Protein Thermal Unfolding Applied to High-Throughput Determination of Ligand Affinities with Extrinsic Fluorescent Dyes.</a> Biochemistr. 49, 10831-10841 (2010).</li><li>Yu, Y., New, S. Y., Xie, J., Su, X., Tan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-Based+Fluorescent+Metal+Nanoclusters+for+Small+Molecular+Drug+Screening.">Protein-Based Fluorescent Metal Nanoclusters for Small Molecular Drug Screening.</a> Chem. Commun. 50, 13805-13808 (2014).</li><li>Shortridge, 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=.">.</a> Nuclear Magnetic Resonance Affinity Screening Methods for Functional Annotation of Proteins and Drug Discover. , (2010).</li></ol>

The authors have nothing to disclose.

Há vários passos críticos que precisam ser destacadas neste método. No protocolo de rastreio da afinidade de ligação relativa das diferentes drogas moleculares pequenos, Passos 3.1.2, 3.1.3, 3.1.4 e são críticas para se obter bons resultados que mostram tendência consistente para a força de ligação relativa. Nestas etapas, as acções de adição de produtos químicos e de desenho soluções de reacção para a medição devem ser tão rapidamente quanto possível para minimizar o efeito de atraso no tempo e...

Albuminas do soro tais como albumina do soro humano (HSA) e de albumina de soro bovino (BSA) são a proteína mais abundante no plasma e desempenham um papel fundamental na manutenção da pressão osmótica do compartimento de sangue. Eles também são reconhecidos como proteínas veículo para moléculas pequenas de baixa solubilidade em água, tais como esteróides, ácidos gordos, hormonas da tiróide, e uma vasta variedade de drogas. A propriedade de ligação (por exemplo, locais de ligação, a afinidade de ligação ou a resistência) dessas moléculas de soro de albuminas forma um tópico importante na farmacocinética. 1-4 vários métodos analíticos foram desenvolvidos para estudar as propriedades de ligação de drogas diferentes para as albuminas do soro, tal como cristalografia de raios-X, 5,6 ressonância magnética nuclear (RMN), 7-11 e de ressonância de plasmon de superfície (SPR), 12,13 etc. No entanto, estes métodos estão limitados por qualquer um processo de análise tediosa e consumidora de tempo (por exemplo, crescimento de cristal único para Crystallo de raios-Xestudo gráfico), a exigência de equipamento especializado e caro (SPR), ou em necessidade de marcação isotópica dispendioso (RMN) para a detecção. Por conseguinte, é altamente desejável desenvolver formas alternativas de pequena seleção da droga molecular de uma maneira rápida, simples e direta, e custo-eficiente. Nanopartículas de ouro (Au NCs) são um tipo especial de nanomaterial, que contêm várias dezenas de átomos de metal com tamanhos inferiores a 2 nm. 14-17 Eles têm atraído interesses de investigação extensas, devido à sua estrutura eletrônica discreta e dependentes de tamanho, 18, 19 e molecular como absorções e emissões. 20-23 Tais propriedades de materiais exclusivos, em particular a forte fluorescência, foram encontradas diversas aplicações, tais como detecção e visualização em sistemas biológicos. 24-32 ultrasmall fluorescente Au NCs pode ser sintetizado usando proteínas funcionais, tais como albuminas do soro, como molde. 33 Numa síntese típica da modelada por proteínas<html> Au CNs, uma certa quantidade de sais de Au são encapsulados no interior da primeira proteína e subsequentemente reduzido pela própria proteína. A capacidade de reduzir a proteína é atribuída a constitutivos funcionais resíduos de aminoácidos (por exemplo, tirosina) que podem ser activados pelo aumento do pH da solução para alcalino. Desdobramento da estrutura da proteína é considerada como um passo crítico para a formação de Au NCs. Isto porque, em uma proteína desdobrada, os grupos funcionais mais redutores podem ser expostas a A UA sais encapsulados. Proteína se desdobrar pode ser alcançada por tratamento com calor ou a exposição a agentes desnaturantes. Introdução de pequenas drogas moleculares também pode afetar o processo de desdobramento, ou seja, modificação da temperatura de desnaturação do ponto médio e a entalpia de desdobramento. 34,35 O efeito de todos esses fatores, por sua vez, pode ser refletido pela cinética de formação de fluorescente Au NCs e manifestado em a intensidade de fluorescência resultantes Au NCs. 36 e_content &quot;&gt; Este vídeo demonstra o método de rastreio de fármacos através da síntese de Au CN de proteínas de albumina carregadas com fármaco a uma temperatura mais elevada (60 ° C) ou na presença de agentes desnaturantes (por exemplo, ureia). A intensidade da fluorescência resultante Au CNs é a leitura do sinal. Em primeiro lugar, Au CNs são sintetizados em moldes HSA e BSA tratadas a 60 ° C ou na presença de ureia para mostrar como a proteína se desdobrar (induzida por tratamento térmico ou desnaturantes) afecta a cinética de formação de Au NCs. Em segundo lugar, Au CNs são sintetizados em moldes de proteína pré-carregadas com drogas diferentes, e o efeito de carga da droga sobre as intensidades de fluorescência relativas dos resultante Au CNs é estudada, que fornece a medida da força de ligação relativa. Finalmente, o protocolo de rastreio Au NC-droga é modificada para medição quantitativa de proteína-droga constante de ligação (K D) variando o conteúdo de medicamento pré-carregado na proteína de uma concentração fixa.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Gold (III) chloride solution, 30%</td>
			<td>Sigma-Aldrich</td>
			<td>484385</td>
			<td>Corrosive, irritant</td>
		</tr>
		<tr>
			<td>Human serum albumin, 96%</td>
			<td>Sigma-Aldrich</td>
			<td>A1887</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum albumin, 96%</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Ibuprofen, 98%</td>
			<td>Sigma-Aldrich</td>
			<td>I4883&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>warfarin, 98%</td>
			<td>Sigma-Aldrich</td>
			<td>A2250</td>
			<td></td>
		</tr>
		<tr>
			<td>phenytoin</td>
			<td>Sigma-Aldrich</td>
			<td>PHR1139</td>
			<td></td>
		</tr>
		<tr>
			<td>sulphanilamide, 99%</td>
			<td>Sigma-Aldrich</td>
			<td>S9251</td>
			<td></td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>urea</td>
			<td>Sigma-Aldrich</td>
			<td>U5128</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>221465</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stirrer</td>
			<td>IKA</td>
			<td>RT5</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Tecan</td>
			<td>Infinite M200</td>
			<td></td>
		</tr>
		<tr>
			<td>384-well plate</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL air displacement pipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1000 mL air displacement pipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL air displacement pipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5000 mL Eppendorf tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1000 mL Eppendorf tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL Eppendorf tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL micro tube</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL glass vial with screw cap</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 mL glass vial with screw cap</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a rapid and quantitative fluorimetric method for protein-targeting small molecule drug screening

Nós demonstramos um novo método de rastreio de fármacos para a determinação da afinidade de ligação de pequenas moléculas de droga a uma proteína-alvo através da formação de nanopartículas de ouro (Au fluorescentes CN) dentro da proteína carregadas com fármaco, com base no sinal diferencial fluorescência emitida pela UA CN. Proteínas, tais como albumina de soro de albumina humana (HSA) e de albumina de soro bovino (BSA) são seleccionados como as proteínas modelo. Quatro pequenas drogas moleculares (por exemplo, ibuprofeno, a varfarina, fenitoína, e sulfanilamida) de diferentes afinidades de ligação para as proteínas de albumina são testados. Verificou-se que a taxa de formação de Au CNs fluorescentes no interior do fármaco carregado proteína albumina sob condições desnaturantes (por exemplo, 60 ° C ou na presença de ureia) é mais lento do que se formou na proteína primitiva (sem droga). Além disso, a intensidade de fluorescência do SAE-formado como se encontra a ser inversamente correlacionadas com as afinidades de ligação destas drogas para as proteínas de albumina. Particularmente,quanto maior a afinidade da proteína de ligação de drogas, mais lenta é a taxa de formação de Au CNs, e, assim, uma intensidade de fluorescência menor do Au CNs resultante é observado. A intensidade de fluorescência das resultantes Au CNs, por conseguinte, fornece uma medida simples da força de ligação relativa de diferentes fármacos testados. Este método também é extensível para medir a constante de ligação específica à proteína droga (K D) variando simplesmente o conteúdo de medicamento pré-carregado na proteína a uma concentração fixa de proteína. Os resultados medidos correspondem bem com os valores obtidos usando outros métodos de prestígio mas mais complicados.

Proteína desdobramento é um procedimento importante para a formação de proteínas modeladas por Au CNs porque os grupos funcionais mais reactivos (por exemplo, resíduos de tirosina de uma proteína) pode ser exposto a reduzir os iões Au encapsulado e, assim, acelerar a velocidade de formação de Au NCs. Os agentes de desnaturação e de aquecimento externo são dois meios comuns para promover o processo de desdobramento da proteína. A Figura 1 demonstra o efeito do aquecimento e adiçã...

Watch this Scientific Journal Video about A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening at JoVE.com

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

Uma rápida e quantitativa método fluorimétrico para-Targeting proteína pequena molécula Rastreio de drogas

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

a-rapid-quantitative-fluorimetric-method-for-protein-targeting-small

Research

JoVE Journal

Bioengineering

9.9K visualizações.  Agency for Science, Technology and Research (A*STAR). A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

Vídeo: A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

Visualização melhorada e Análise Quantitativa dos Efeitos de Drogas Usando Células micropatterned

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Bioengenharia

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

Candida albicans biofilme Chip (Ca BChip) para seleção de alto rendimento antifúngico

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Investigando Sistemas receptor-ligante do Celulossoma com base-AFM única molécula Força Espectroscopia

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

Induzida por drogas Sensibilização de adenililciclase: Racionalização Ensaio e miniaturização para pequenas moléculas e siRNA Triagem Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

Rápido Mix Preparação de Bioinspirada Nanoscale hidroxiapatita para aplicações biomédicas

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

Automated Análise contração do Coração Humano engenharia de tecidos para Screening Cardiac Drug Safety

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

Microtissues 3D para terapia regenerativa injetável e triagem de drogas de alto rendimento

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Reprimir a transcrição do Gene redirecionando maquinaria celular com modificadores epigenéticas químicas

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Purificação e análise de um anticorpo monoclonal de células de ovário de hamster chinês usando um sistema de Microbioreator automatizado

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Applications for Open Source Microplate-Compatible Illumination Panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well as large-scale high-throughput screening (HTS) campaigns. Though laboratory automation has greatly increased the utility of microplates there remain instances where automation-based instrumentation is not feasible, cost-effective or compatible with microplate formatting needs. In these cases, microplates must be manually prepared. Problematic to manual microplate manipulations is that a number of difficulties can arise pertaining to the accurate tracking of sample operations, data record keeping and quality control (QC) inspection for well artifacts or formatting errors. As microplate well densities increase (i.e., 96-well, 384-well, 1536-well) the potential for introducing errors also drastically increases.&#160; Moreover, for small bench-top laboratory operations there exists a need to improve the ease and accuracy of sample handling in a cost-effective fashion. Herein, we describe a system that acts as a semi-automated pipetting guide referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.). &#160;M.A.P.L.E. has multiple uses for supporting compound hit-picking and microplate preparation for assay development in high-throughput screening or laboratory benchtop operations, as well as QC/quality assurance (QA) diagnostic evaluation of microplate quality or visualizing well formatting errors.

Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.) is a computer-driven device that systematically illuminates microtiter wells to provide guidance for the manual preparation of microplates.&#160; M.A.P.L.E. improves the accuracy of microplate preparation while automating data recordkeeping. &#160;In addition, it can assist with examining microplate quality or aid in the detection of errors.&#160; &#160;

Aplicações para painéis de iluminação compatíveis com microplate de código aberto

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...