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.

Precaución: Por favor, consulte las hojas de datos de seguridad (FDS) de todos los productos químicos implicados antes de su uso. El experimento de detección de drogas implica la síntesis y la manipulación de los nanomateriales, que puede tener riesgos adicionales en comparación con su contraparte granel. Por favor, garantizar todas las medidas de control necesarias para practicar durante todo el experimento, incluyendo el uso de controles de ingeniería (campana extractora de gases) y equipo de protección personal (PPE, por ejemplo, los pantalones de la longitud de seguridad, zapatos cerrados, guantes resistentes a productos químicos y gafas protectoras). 1. Preparación de los reactivos químicos para Pruebas De Drogas <ol><li> Los precursores para la síntesis de Au CN <ol><li> Disolver 30 mg de cloruro de solución de oro (III) (99,99% base metales traza, 30 wt.% En HCl diluido) en 6,9 ml de agua ultrapura para preparar 15 mM de oro (III) solución de cloruro. Precaución: solución de cloruro de oro es corrosivo e irritante. Use PPE adecuado para evitar el contacto directo con los ojos y la piel. </li><li> Dissolve 74 mg de HSA o BSA en 1 ml de agua ultrapura para preparar 74 mg / ml de solución de proteína de valores. </li></ol></li><li> Soluciones de drogas <ol><li> Disolver la cantidad deseada de fármaco, por ejemplo, 1,9 mg para (a) el ibuprofeno, 2,8 mg de (b) la warfarina, 2,3 mg de (c) la fenitoína, y 1,5 mg de (d) sulfanilamida en 20 l de DMSO para preparar 450 mM de soluciones de drogas de las acciones. Nota: se selecciona DMSO como disolvente ya que estos medicamentos son hidrófobos y tienen una mala solubilidad en agua. </li></ol></li><li> Otros reactivos <ol><li> Disolver 600 mg de gránulos de NaOH en 10 ml de agua ultrapura para preparar 1,5 M de solución de NaOH. Disolver 2,4 g de urea en 2 ml de agua ultrapura para preparar 20 M de solución de urea. </li></ol></li></ol> 2. Síntesis de Proteína-Templated Au CN <ol><li> Con plantilla-HSA Au CN (HSA-Au CN) <ol><li> Coloque dos viales de vidrio, conteniendo cada uno una micro barra de agitación magnética en ella, en dos de temperatura independiente de agitación magnética controlableRPR. </li><li> Ajuste la temperatura de un agitador magnético a 60 ° C, y la etiqueta del frasco de vidrio en la parte superior de la misma como &quot;60 ° C&quot;; mientras que el otro agitador magnético se mantiene a temperatura ambiente (RT) donde el vial de vidrio en la parte superior de ella se etiqueta como &quot;RT&quot;. </li><li> Añadir 200 l de solución de HSA, 200 l de agua ultrapura, y 200 l de solución de oro cloruro de (III) a cada vial bajo agitación constante (establecido como 360 rpm a menos que se especifique lo contrario) para permitir la encapsulación de Au iones dentro de la plantilla de proteínas. </li><li> 2 min después, añadir 20 l de solución de NaOH a cada vial de manera que para activar la capacidad reductora de HSA en la formación de Au NCs y empezar a grabar el tiempo de reacción como 0 min. </li><li> Cada 20 min, dibujar 50 l de soluciones de cada uno de la muestra a una placa de color negro de 384 pocillos y medir el espectro de emisión con un lector de microplacas. La configuración de exploración típica: λ ex = 370 nm, λ em = 410-850 nm. </li><li> Detenga el agitador magnético después de 100 min. </li><li> Enfriar los frascos de vidrio con agua corriente en un fregadero. Precaución: Los viales de vidrio están calientes. Use guantes resistentes al calor para evitar la quema. </li><li> Trazar los espectros de fotoemisión en todo momento para cada muestra para adquirir la cinética de formación de Au NCs en diferentes condiciones de temperatura. </li></ol></li><li> Con plantilla-BSA Au CN (BSA-Au CN) <ol><li> Colocar un vial de vidrio que contiene un micro barra de agitación magnética en la parte superior de un agitador magnético. Deja la temperatura como la temperatura ambiente. </li><li> Mezclar 200 l de solución de BSA, 200 l de urea, y 200 l de solución de cloruro de oro (III) en el vial de vidrio con agitación constante. </li><li> 2 min después, añadir 20 l de solución de NaOH para activar la capacidad reductora de BSA para formar Au NCs y empezar a grabar el tiempo de reacción como 0 min. Medir el espectro de fotoemisión de la mezcla de reacción por hora. Nota: se mide el espectro de fotoemisión de BSA-Au CNcon menos frecuencia debido a que la tasa de formación de BSA-au NCs es más lenta que la de HSA-Au a la misma temperatura debido a la menor eficacia de urea para desplegar la proteína. </li><li> Detenga el agitador magnético después de 7 h. Trazar los espectros de fotoemisión en todo momento para ver la cinética de formación de Au CN por desnaturalización urea. </li></ol></li></ol> Proyección 3. Pequeño Molecular de Drogas <ol><li> Pantalla afinidad de unión relativa de los diferentes fármacos moleculares pequeños a HSA <ol><li> Coloque cinco viales de vidrio, conteniendo cada uno una barra de agitación magnética en ella, en la parte superior de un agitador magnético multipunto temperatura controlable. Etiqueta de estos viales como A, B, C y D, respectivamente, para cada fármaco, es decir, ibuprofeno, warfarina, fenitoína y sulfanilamida. Etiqueta de la una con HSA puro como control. </li><li> Añadir 200 l de HSA a cada vial. Añadir 1 l de solución de fármaco a las cuatro viales correspondientes. Añadir 1 l de DMSO al control. Encender el agitador y ajustar la velocidad de giro de 360rpm e incubar durante 1 hora para permitir la unión a HSA para completar la droga. </li><li> 1 hr después, añadir 200 l de agua Milli-Q y 200 l de solución de cloruro de oro (III) a cada vial bajo agitación constante. Ajuste la temperatura a 60 ° C bajo agitación constante durante 10 min. Añadir 20 l de NaOH 1,5 M a cada vial y empezar a grabar el tiempo de reacción como 0 min. </li><li> Dibujar rápidamente 50 l de solución de cada vial a un plato negro 384 pocillos y medir el espectro de emisión (resultados etiquetados como 0 min). Registre los espectros de emisión de cada muestra cada 10 min. Recoge al menos cuatro espectros a cuatro tiempo diferentes, es decir 0, 10, 20, y 30 min. </li><li> Repita los pasos 3.1.1 - 3.1.4 varias veces para obtener resultados con una tendencia constante. Detenga el agitador magnético. </li><li> Trazar los espectros de fotoemisión resuelta en el tiempo para cada muestra (a - d, y control). Identificar la intensidad máxima de todas las muestras y complot contra el tiempo para comparar la cinética de formación de Au CN en diferentes plantillas de proteínas cargadas con fármaco. </li></ol></li><li> Medir la constante de unión de un fármaco específico para HSA <ol><li> Repita los pasos 3.1.1 - 3.1.4. Reemplazar la solución de fármaco con soluciones de ibuprofeno a cuatro concentraciones diferentes (incluyendo una muestra de control usando HSA sólo sin carga de fármaco). Etiquetar el vial de vidrio de acuerdo con la concentración de fármaco correspondientemente. </li><li> 10 minutos más tarde, enfriar los viales de vidrio con agua corriente en un fregadero. Precaución: Los viales de vidrio están calientes. Use guantes resistentes al calor para evitar la quema. </li><li> Dibujar 50 l de las soluciones anteriores de cada vial a un plato negro 384 pocillos y medir el espectro de emisión. </li><li> Repita los pasos 3.2.1 - 3.2.3 dos veces más para obtener otros dos conjuntos de resultados a diferentes concentraciones del fármaco. </li><li> Detenga el agitador magnético. Trazar los espectros de fotoemisión y analizar los resultados. <ol><li> Para los datos brutos obtenidos en cada lote individual, trazar los espectros de fotoemisión de cadamuestra en el software como el software OriginPro y averiguar la intensidad máxima. </li><li> Calcular y representar gráficamente la intensidad de fluorescencia relativa [(I 0 - I) / I 0] en contra de la concentración de fármaco, donde I y I 0 refiero a la intensidad de fluorescencia de Au NCs formado en HSA y HSA pura cargada de fármaco, respectivamente. </li><li> Calcula la constante de unión por ajuste de los datos a un modelo único sitio de unión usando la ecuación de Michaelis-Menten: Y = R max × C / (K D + C), donde R max indica la señal de respuesta máxima, C es la concentración de ligando y K D es la constante de unión. Realice esto en software como OriginPro. Seleccione el menú de ajuste no lineal Análisis de la curva de ajuste, a continuación, seleccione la función Hill Crecimiento / categoría sigmoidal. En la configuración: página Selección de Función, haga clic en Ajustar para mostrar los resultados de armarios. </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.

Hay varios pasos críticos que deben ser destacados en este método. En el protocolo de la detección de la afinidad de unión relativa de los diferentes fármacos moleculares pequeños, Etapas 3.1.2, 3.1.3, 3.1.4 y son críticos para obtener buenos resultados que muestran tendencia constante para la fuerza de unión relativa. En estos pasos, las acciones de adición de productos químicos y el dibujo soluciones de reacción para la medición deben ser lo más rápidamente posible para minimizar el efecto tiempo de reta...

Albúminas séricas tales como albúmina de suero humano (HSA) y albúmina de suero bovino (BSA) son la proteína más abundante en el plasma y juegan un papel vital en el mantenimiento de la presión osmótica del compartimiento de sangre. También son reconocidos como proteínas portadoras para las pequeñas moléculas de baja solubilidad en agua, tales como esteroides, ácidos grasos, hormonas tiroideas, y una amplia variedad de fármacos. La propiedad de unión (por ejemplo, sitios de unión, afinidad o fuerza de unión) de estas moléculas a suero albúminas forma un tema importante en la farmacocinética. 1-4 Varios métodos analíticos se han desarrollado para estudiar las propiedades de unión de diferentes fármacos al suero albúminas, tales como cristalografía de rayos X, 5,6 resonancia magnética nuclear (RMN), 7-11 y resonancia de plasmón superficial (SPR), 12,13 etc. Sin embargo, estos métodos están limitados ya sea por un proceso de análisis tedioso y requiere mucho tiempo (por ejemplo, el crecimiento del cristal único para Crystallo de rayos Xestudio gráfico), requerimiento de equipo especializado y caro (SPR), o en necesidad de etiquetado de isótopos costosa (RMN) para la detección. Por tanto, es altamente deseable desarrollar métodos alternativos para la detección de drogas molecular pequeña de una manera rápida y directa, y rentable. Nanoclusters oro (Au CN) son un tipo especial de nanomaterial, que contienen varias decenas de átomos de metal con tamaños menores de 2 nm. 14-17 Ellos han atraído amplios intereses de investigación debido a su estructura electrónica discreta y dependiente del tamaño, 18, 19 y molecular como absorciones y emisiones. 20-23 Tales propiedades materiales únicos, en particular, la fuerte fluorescencia, han encontrado diversas aplicaciones tales como detección y proyección de imagen en los sistemas biológicos. ultrapequeño de 24-32 fluorescente Au NCs se puede sintetizar utilizando proteínas funcionales, tales como albúminas séricas, como plantilla. 33 En un típico síntesis de proteínas con plantilla de<html> Au NCs, una cierta cantidad de sales de Au están encapsulados dentro de la primera proteína y posteriormente reducida por la propia proteína. La capacidad reductora de la proteína se atribuye a Constituyente residuos de aminoácidos funcionales (por ejemplo, tirosina) que se pueden activar mediante el aumento del pH de la solución a alcalino. Despliegue de la estructura de la proteína se considera como un paso fundamental para la formación de Au CN. Esto es porque en una proteína desplegada, los grupos funcionales más reductores pueden estar expuestos a las sales de Au encapsulados. Desplegamiento de la proteína se puede lograr mediante tratamiento térmico o la exposición a agentes desnaturalizantes. La introducción de pequeños drogas moleculares también puede afectar el proceso de desarrollo, es decir, la modificación de la temperatura de desnaturalización punto medio y la entalpía de despliegue. 34,35 El efecto de todos estos factores, a su vez, puede ser reflejada por la cinética de formación de fluorescente Au CN y se manifiesta en la intensidad de fluorescencia de las resultantes Au CN. 36 e_content &quot;&gt; Este video demuestra el método de cribado de fármacos mediante la síntesis de Au NCs en las proteínas de albúmina cargadas con fármaco a una temperatura más alta (60 ° C) o en presencia de agentes desnaturalizantes (por ejemplo, urea). La intensidad de fluorescencia de resultante Au NCs es la lectura de la señal. En primer lugar, Au NCs se sintetizan en HSA y BSA plantillas tratadas a 60 ° C o en presencia de urea para mostrar cómo desplegamiento de la proteína (inducida por tratamiento térmico o desnaturalizantes) afecta a la cinética de formación de Au NCs. En segundo lugar, Au NCs se sintetizan en las plantillas de proteínas precargado con diferentes fármacos, y el efecto de carga de fármaco en las intensidades de fluorescencia relativas de resultante Au NCs se estudia, que proporcionan la medida de la fuerza de unión relativa. Finalmente, el protocolo de cribado Au NC-fármaco se modifica para medición cuantitativa de la proteína-fármaco constante de unión (K D) mediante la variación del contenido de fármaco precargado en la proteína de una concentración fija.

<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

Se demuestra un nuevo método de detección de drogas para determinar la afinidad de unión de pequeñas moléculas de fármacos a una proteína diana mediante la formación de nanoclusters fluorescentes oro (Au CN) dentro de la proteína cargada con fármaco, basados ​​en la señal diferencial de fluorescencia emitida por el Au NCs. Proteínas tales como albúmina de suero de albúmina humana (HSA) y albúmina de suero bovino (BSA) se seleccionan como las proteínas modelo. Cuatro pequeños fármacos moleculares (por ejemplo, ibuprofeno, warfarina, fenitoína y sulfanilamida) de diferentes afinidades de unión a las proteínas de albúmina se ponen a prueba. Se encontró que la tasa de formación de Au NCs fluorescentes dentro de la proteína albúmina cargadas de fármaco en condiciones desnaturalizantes (es decir, 60 ° C o en presencia de urea) es más lento que el formado en la proteína prístina (sin drogas). Por otra parte, la intensidad fluorescente de la CN como formado se encontró que se correlaciona inversamente con las afinidades de unión de estos fármacos a las proteínas de albúmina. Particularmente,cuanto mayor es la afinidad de unión a proteína de drogas, más lenta será la velocidad de formación de Au CN, y por lo tanto se observa una intensidad de fluorescencia inferior de la resultante Au NCs. La intensidad de fluorescencia de la resultante Au NCs por lo tanto, proporciona una medida sencilla de la fuerza de unión relativa de los diferentes fármacos ensayados. Este método también es extensible para medir la constante de unión específica de proteína-fármaco (K D) simplemente variando el contenido de fármaco precargado en la proteína a una concentración de proteína fija. Los resultados medidos coinciden bien con los valores obtenidos utilizando otros métodos de prestigio pero más complicados.

Desplegamiento de la proteína es un procedimiento importante para la formación de proteínas con plantilla Au NCs porque los grupos funcionales más reactivos (por ejemplo, residuos de tirosina) de una proteína pueden estar expuestos a reducir los iones Au encapsulados y por lo tanto acelerar la velocidad de formación de Au NCs. Agentes de calefacción y de desnaturalización externa son dos medios comunes para promover el proceso de desarrollo de proteínas. La Figura 1 muestra el efecto d...

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.

Una rápida y cuantitativa método fluorimétrico para la proteína-Targeting Pequeña Molécula 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 vistas.  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.

Video: 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.

Una mejor visualización y análisis cuantitativo de los efectos de drogas Uso de 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 ...

Investigación

Bioingeniería

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 biofilm Chip (Ca BChip) para la selección de alto rendimiento 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.

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.

Inducido por fármacos Sensibilización de adenilciclasa: Racionalización Ensayo y miniaturización de moléculas pequeñas y siRNA aplicaciones de cribado

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.

Mezcla rápida Preparación de Bioinspirada nanoescala hidroxiapatita para aplicaciones 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.

Análisis automatizado contracción de ingeniería tisular para la Detección del Corazón Humano cardíaca 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 la terapia regenerativa inyectable y drogas de alto rendimiento

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.

Reprimiendo la transcripción genética redirigiendo la maquinaria celular con modificadores químicos de epigenéticas

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.

Purificación y análisis de un anticuerpo monoclonal de células de ovario de hámster chino utilizando un sistema automatizado de microbiorreactores

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;

Aplicaciones para paneles de iluminación compatibles con microplacas de código abierto

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