Reconocemos el apoyo financiero de CPRIT (RP100119) y la Fundación Welch (I-1665).

Protocolo completo tarda aproximadamente 4 días. 1. Preparación de las células para la transfección siRNA y Reportero Vamos a introducir aquí un protocolo para interrogar bibliotecas siRNA utilizando un pequeño conjunto de siRNAs (384 diferentes siRNA piscinas matriz en formato de 96 pocillos con cada grupo que consiste en 4x siRNAs dirigidos a un solo gen) de un genoma de gran biblioteca de siRNA con fines ilustrativos. Para este estudio en particular, vamos a explotar las células HCT116 que exhiben señalización Wnt y Kras desviadas, y la actividad de p53. La línea celular apropiada en estudios destinados a interrogar a otros procesos celulares de interés tendrá que ser identificado y evaluado de una manera similar. <ol><li> Lavar 5 x 10 cm 2 placas de 80-90% confluentes células HCT116 con PBS y luego recoger las células por tripsinización utilizando 1 ml de solución de tripsina / placa seguido de neutralización con 10 ml de medio Eagle modificado de Dulbecco (DMEM) que contenía 2% de FeTal suero bovino (FBS) y 1% de penicilina / estreptomicina / placa. </li><li> Transferir las células en suspensión a un tubo cónico de 50 ml y centrifugar a ~ 130 xg durante 5 min, eliminar el sobrenadante y volver a suspender el sedimento celular en 10 ml de DMEM / FBS al 2% / 1% de penicilina / estreptomicina. </li><li> Contar el número de células con un contador de células y, a continuación, hacer una solución de células con 10 5 células / ml, mantener a 150 ml de suspensión celular para el siguiente paso. Placa 10 4 células (100 l) en cada pocillo de una placa de cultivo celular de 96 pocillos usando un dispensador de líquido automatizado Multidrop (en adelante Multidrop). La suspensión de células debe ser suficiente en este caso para el chapado 12 x placas de 96 pocillos. </li><li> Se incuban las placas con las células a 37 ° C en una incubadora con 5% de CO 2, mientras que la preparación de la mezcla de transfección. </li></ol> 2. Preparación del Construct Stock Solution Periodista <ol><li> Aislar reportero de alta calidad constructo de ADN utilizando midiprep kits estándar (NucleoBond Xtra Midi-producido porClontech) y diluir el ADN hasta una concentración final de 1 mg / ml para cada reportero. </li><li> Preparar 100 l de solución madre reportero construir mediante la combinación de 30 l de p53-FL, 30 l de 8x TCF-RL, 30 l de Elk1-Gal4, 6 l UAS-CL y 4 l de H 2 O para lograr una mezcla de ADN con una final 1:1:1:0.2 relación de los periodistas. Concentración de ADN final de este ADN mezcla solución madre es de 0,3 mg / ml. </li></ol> 3. Preparación de la ARNsi / ADN de la mezcla de transfección En esta parte del protocolo, prepararemos siRNA / mezclas de transfección de ADN que serán suficientes para la transfección de 12 x 96 y placas. Por esta biblioteca de prueba de 4 x 96 y placa de siRNAs, vamos a transfectar cada grupo siRNA por triplicado. El reactivo de transfección que utilizaremos se llama Effectene (Qiagen). En nuestras manos este es el único reactivo que funciona para la entrega simultánea de siRNA y ADN en las células cultivadas. <ol><li> Diluir 80 l de reportero construir Stock sollución en 8 ml de tampón CE (Effectene kit) para lograr una solución de ADN con una concentración final de 3 g / ml. En general, preparar suficiente mezcla de solución de ADN para su uso inmediato. </li><li> Placa de 20 l de esta solución de ADN a cada pocillo de un 96 así / placa. El número de placas de 96 pocillos dependerá de cuántos serán probados piscinas siRNA. Por ejemplo, en este caso tendremos que 4 x placas de 96 pocillos para evaluar 384 piscinas siRNA diferentes. </li><li> Los siRNAs a ensayar deben estar a una concentración madre de 5 mM. Si no, pueden ser réplica chapada y se diluyó a esta concentración utilizando el tampón de dilución recomendada asociada con cada biblioteca. </li><li> Transferir 2 l de cada 5 mM de stock ARNsi en el correspondiente pocillo de la placa de PCR que contiene la población de ADN diluido con un Liquid Handler automatizado Biomek. Dependiendo de la escala del estudio, una pipeta de múltiples canales puede ser suficiente. </li><li> Ahora, vamos a añadir 1 l de solución Enhancer (kit Effectene) a cada pocillo de la DNA / siRNA plate. De nuevo, esto se puede lograr ya sea con un manipulador de líquidos automatizado o pipeta multicanal. </li><li> Permitir que la reacción reforzador que se produzca a temperatura ambiente durante 5 min y después añadir 3 l de reactivo de transfección Effectene a cada pocillo e incubar a TA durante 10 min adicionales. </li><li> Para detener la formación de complejos de transfección, añadir 10 l de DMEM / FBS al 2% / 1% de penicilina / estreptomicina a cada pocillo de la placa de PCR. </li><li> Transferir 10 l de la mezcla de transfección a cada pocillo de la placa de 96 pocillos que contiene las células (recuperados de la incubadora de células). Como se realizará cada transfección por triplicado, la misma mezcla se aplica a dos adicionales placas de 96 pocillos que contienen células. </li><li> Se incuban las células con mezclas de transfección añadido a 37 ° C en un ambiente humidificado con 5% de CO 2 durante 36 horas. </li></ol> 4. Determinar las actividades de luciferasa a partir de muestras de células Después de 36 horas, las actividades luciferasa están listos para la mediciónen las células transfectadas. El punto final se determinará en función de cada experimento. En nuestro estudio, un tiempo de incubación de 36 horas se había producido previamente una señal suficientemente fuerte para decir, la determinación de los resultados. Como este estudio incorpora múltiples reporteros (uno secretada en el medio de cultivo, y los otros dos se expresa en el citoplasma de la célula), obtendremos mediciones tanto de medio de cultivo, así como un lisado celular. <ol><li> Detección de la actividad de CL en el medio de cultivo: <ol><li> 20 l de medio de cultivo es réplica chapada en un blanco opaco de 96 pocillos (ya sea usando el Liquid Handler Biomek o pipeta multicanal). </li><li> Añadir 20 l de tampón de ensayo CL (Targeting Systems) a cada pocillo. </li><li> Añadir 10 l de sustrato luciferina Cypridina (Targeting Systems) a cada pocillo y detectar la actividad de CL utilizando un luminómetro (el lector de placas Pherastar producido por BMG por ejemplo). </li></ol></li><li> La detección de FL y RL actividad: <ol><li> Lyse la cells quitando primero el medio de cultivo. Esto se puede lograr rápidamente girando plato boca abajo y lanzando medio en un fregadero. Medio restante puede eliminarse golpeando suavemente la placa boca abajo sobre toallas de papel. Añadir 30 l de 1X tampón de lisis pasiva (Promega) a cada pocillo de células utilizando el multipunto y colocar en una plataforma oscilante (Bellco es una empresa que fabrica estos) establecen una velocidad media durante 5 min a temperatura ambiente. </li><li> Añadir 20 l de reactivo de ensayo de luciferasa II (parte del Kit de luciferasa Dual Promega) utilizando la actividad FL multipunto y medir inmediatamente usando el luminómetro. A continuación, agregue Stop &amp; Glo reactivo (Promega) que calmar la actividad FL y permitir la medición de la actividad de RL. Nota: a menos que utilice mezclas de sustratos que permiten duración de la señal extendida (como Kits de luciferasa Dual-Glo de Promega), el uso de kits de flash requiere la medición rápida después de la adición de sustrato (a menos de 5 minutos). </li></ol></li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+systems+in+Drosophila:+a+synthesis+of+time+and+space.">Gene expression systems in Drosophila: a synthesis of time and space.</a> Trends in Genetics: TIG. 20, 384-391 (2004).</li><li>Smirnova, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+Neh2-luciferase+reporter+and+its+application+for+high+throughput+screening+and+real-time+monitoring+of+Nrf2+activators.">Development of Neh2-luciferase reporter and its application for high throughput screening and real-time monitoring of Nrf2 activators.</a> Chem. 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Genet. 19, 379-383 (1998).</li><li>Korinek, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+members+of+the+Tcf+family+implicated+in+Wnt/beta-catenin+signaling+during+embryogenesis+in+the+mouse.">Two members of the Tcf family implicated in Wnt/beta-catenin signaling during embryogenesis in the mouse.</a> Mol. Cell Biol. 18, 1248-1256 (1998).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+molecular+characterization+of+human+colon+and+rectal+cancer.">Comprehensive molecular characterization of human colon and rectal cancer.</a> Nature. 487, 330-337 (2012).</li><li>Koboldt, D. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+analysis+of+genome-wide+RNA+interference+screens.">Integrative analysis of genome-wide RNA interference screens.</a> Science Signaling. 2, pt4 (2009).</li><li>Falschlehner, C., Steinbrink, S., Erdmann, G., Boutros, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+RNAi+screening+to+dissect+cellular+pathways:+a+how-to+guide.">High-throughput RNAi screening to dissect cellular pathways: a how-to guide.</a> Biotechnology Journal. 5, 368-376 (2010).</li><li>Boutros, M., Bras, L. 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Aceptar y LL son autores de una patente pendiente en relación con la tecnología de multiplexado luciferasa.

Hay varios proveedores de sistemas de reportero luciferasa basados ​​(tales como Promega, sistemas de focalización, New England Biolabs, y Thermo Fisher) que proporcionan útiles recursos en línea para aquellos que entretener una pantalla de alto rendimiento para la primera vez. Además, un número de excelentes críticas con respecto a la optimización de la utilización de pantallas de alto rendimiento para el descubrimiento de genes y cómo RNAi basada en los resultados de detección pueden estar integrados con...

Una variedad de los reporteros de luciferasa basados ​​para el seguimiento de una gran variedad de procesos biológicos celulares están disponibles comercialmente. La mayoría de estas construcciones de ADN que codifican proteínas de luciferasa que son inducidas a expresar por estímulos o perturbaciones celulares específicos. Los reporteros transcripcionalmente basados ​​en la mayoría de los sólidos colocan la expresión de una enzima luciferasa bajo el control de elementos potenciadores sintéticos o regiones promotoras de genes bien establecidos para su fiabilidad en informar de un evento de 1,2 transcripcional fisiológicamente relevante. También hay sistemas de trans-reportero de luciferasa que utilizan GAL4-UAS para conducir expresión de la proteína luciferasa. Gal4 es un activador transcripcional de levadura y la UAS es un potenciador de Gal4 que se une específicamente a activar la transcripción de secuencias de genes situados corriente abajo de la misma 3. Estos tipos de sistemas de luciferasa están típicamente soportadas por dos plásmidos de ADN - uno codifica la proteína GAL4 fusionado a la REGULATORY porción de una proteína de interés, y el segundo codifica una proteína luciferasa colocado bajo el control de una o más secuencias UAS. Por lo tanto, la señal de luciferasa celular refleja el nivel de actividad de la proteína de interés. Otro uso común de los reporteros de luciferasa basados ​​es para el seguimiento de la estabilidad de proteínas mediante el cual una proteína de interés se fusiona con una enzima luciferasa 4. Sin importar el tipo de reportero, un caveat emptor se observa aquí no se puede asumir la especificidad de cualquier periodista que ha sido así interrogado. Por lo tanto, se requiere la debida diligencia en la incorporación de nuevas construcciones reportero en cualquier estrategia de investigación. La selección de la enzima luciferasa de lectura puede ser tan importante como la fiabilidad de los elementos de ADN que controlan su expresión. Considerando que la luciferasa de luciérnaga (FL) es la enzima más comúnmente utilizado en construcciones disponibles comercialmente, la llegada de nuevos sistemas de informadores de luciferasa que no requieren ATP (como en el casode reacciones FL-based) o que incorporan enzimas más estables que emiten señales más fuertes promesa de mejorar la eficiencia y la fiabilidad de esta plataforma de investigación. Una nota importante sobre la selección de los reporteros de luciferasa en los estudios químicos es la susceptibilidad de FL a la inhibición química que podría dar lugar a informes falsos. En nuestra experiencia, otras enzimas tales como la luciferasa de Renilla o Gaussia (RL y GL, respectivamente) que utilizan coelenterazina como sustrato son menos fácilmente inhibida por moléculas pequeñas 5. El formato más común para la multiplexación de luciferasa lecturas del implica el uso de FL y RL en gran medida dada la disponibilidad de fácil de utilizar con los kits de la capacidad de medir secuencialmente las actividades enzimáticas a partir de una sola muestra. En la mayoría de los kits, las muestras se expusieron primero a luciferina para revelar los niveles de actividad FL seguido de un reactivo de extinción que se depositan simultáneamente con coelenterazina para producir una segundaseñal RL dary. Con la adición de otras luciferasas que pueden ser secretadas tales como GL o que el uso de otro sustrato tal como Cypridina luciferasa (CL), un mayor número de posibilidades para la generación de datos de alta contenido utilizando luciferasas han surgido. Un ejemplo de un genoma de gran pantalla de RNAi mediante esta técnica se puede encontrar aquí 6. Estos avances tecnológicos tienen a su vez estimulado el desarrollo de mecanismos específicos para saciar cada tipo de enzima luciferasa para limitar la diafonía 7. En nuestras manos, observamos una mayor frecuencia de &quot;efectos de borde&quot; inexplicables (tendencias en la actividad celular asociada con el borde de las placas de cultivo de alto título), con luciferasas secretadas tales como GL o CL. Por otra parte, tales luciferasas secretadas son útiles para ensayos cinéticos, ya que permiten muestreo de la señal sin adulterar la viabilidad celular. Para nuestro estudio presentado aquí, vamos a monitorear simultáneamente la actividad de tres cáncer relevanteprocesos celulares: el p53, Kras y Wnt vías de transducción de señales. Los periodistas incorporados en nuestro estudio son el FL reportero pp53-TA-Luc de Clontech (en adelante, el periodista p53-FL) 8, un sistema reportero Elk1-GAL4/UAS-CL (en adelante, Elk-1 reportero; Agilent), y el reportero de 8X TCF RL que incorpora múltiples elementos potenciadores sintéticos reconocidos por la vía Wnt reguladores transcripcionales conocidos como factores de células T (o TCF) 9-11.

<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>PathDetect Elk1 Trans-Reporting System</td>
			<td>Agilent Technologies Inc.</td>
			<td>219005</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pathway Profiling Luciferase System 4 pp53-TA-Luc Vector</td>
			<td>Clontech Lab Inc.</td>
			<td>631914</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Effectene Transfection Reagent</td>
			<td>Qiagen Inc.</td>
			<td>301427</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dual-Luciferase Reporter Assay System</td>
			<td>Promega Corp.</td>
			<td>E1960</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cypridina Luciferase Assay reagent</td>
			<td>Targeting Systems</td>
			<td>VLAR-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HCT116 cells</td>
			<td>ATCC</td>
			<td>CCL-247</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CytoTox-Fluo Cytotoxicity Assay</td>
			<td>Promega Corp.</td>
			<td>G9260</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>MultiFlo Microplate Dispenser</td>
			<td>Labsystems Inc.</td>
			<td>Model 832</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biomek FXP Laboratory Automation Workstation</td>
			<td>Beckman Coulter Inc.</td>
			<td>A31842</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PHERAstar FS-multi-mode HTS microplate reader</td>
			<td>BMG Labtech Inc.</td>
			<td>0471-101A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bright-Line Reichert Hemacytometers</td>
			<td>Hausser Scientific Company</td>
			<td>1490</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a multiplexed luciferase-based screening platform for interrogating cancer-associated signal transduction in cultured cells

Interrogatorio escala del genoma de la función génica mediante RNA de interferencia (RNAi) es muy prometedora para la rápida identificación de las vulnerabilidades de células de cáncer químicamente manejables. Limitar el potencial de esta tecnología es la incapacidad para delinear rápidamente la base mecánica de los resultados fenotípicos y por lo tanto informar sobre el desarrollo de estrategias terapéuticas dirigidos molecularmente. Resumimos aquí los métodos de deconstruir fenotipos celulares inducidos por genes RNAi mediada por la orientación utilizando sistemas indicadores multiplexados que permiten el seguimiento del cáncer de los procesos clave asociados a células. Esta metodología de cribado de alto contenido es versátil y se puede adaptar fácilmente para la detección de otros tipos de bibliotecas moleculares grandes.

A pesar de los avances en la cartografía del paisaje mutacional de varios tipos de cáncer que utilizan enormes esfuerzos de secuenciación del genoma 12-14, seguimos sin contar con un enfoque sistemático para la traducción de las observaciones en las estrategias de intervención. En el cáncer colorrectal (CRC), las mutaciones que se prevé que afectará a los componentes de los tres procesos celulares - Wnt / β-catenina, p53, Kras y señalización - se encuentran en el 99% de todos los tumores que sugie...

Watch this Scientific Journal Video about A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells at JoVE.com

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

El logro de un nivel de comprensión de los sistemas de procesos celulares es un objetivo de la biología celular moderna. Se describe aquí estrategias para luciferase reporteros multiplexación de varias funciones celulares puntos finales para interrogar la función de genes utilizando RNAi bibliotecas escala del genoma.

Una plataforma de detección de luciferasa basado multiplexado para interrogar cáncer asociado transducción de señales en las células cultivadas

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

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11.0K vistas.  UT Southwestern Medical Center. El logro de un nivel de comprensión de los sistemas de procesos celulares es un objetivo de la biología celular moderna. Se describe aquí estrategias para luciferase reporteros multiplexación de varias funciones celulares puntos finales para interrogar la función de genes utilizando RNAi bibliotecas escala del genoma.

Video: A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will be identifiable at any age. As recently reviewed by Mangin and colleagues2, several methods exist to quantify different characteristics of gyrification. For instance, sulcal morphometry can be used to measure shape descriptors such as the depth, length or indices of inter-hemispheric asymmetry3. These geometrical properties have the advantage of being easy to interpret. However, sulcal morphometry tightly relies on the accurate identification of a given set of sulci and hence provides a fragmented description of gyrification. A more fine-grained quantification of gyrification can be achieved with curvature-based measurements, where smoothed absolute mean curvature is typically computed at thousands of points over the cortical surface4. The curvature is however not straightforward to comprehend, as it remains unclear if there is any direct relationship between the curvedness and a biologically meaningful correlate such as cortical volume or surface. To address the diverse issues raised by the measurement of cortical folding, we previously developed an algorithm to quantify local gyrification with an exquisite spatial resolution and of simple interpretation. Our method is inspired of the Gyrification Index5, a method originally used in comparative neuroanatomy to evaluate the cortical folding differences across species. In our implementation, which we name local Gyrification Index (lGI1), we measure the amount of cortex buried within the sulcal folds as compared with the amount of visible cortex in circular regions of interest. Given that the cortex grows primarily through radial expansion6, our method was specifically designed to identify early defects of cortical development.
In this article, we detail the computation of local Gyrification Index, which is now freely distributed as a part of the FreeSurfer Software (<a href="http://surfer.nmr.mgh.harvard.edu/">http://surfer.nmr.mgh.harvard.edu/</a>, Martinos Center for Biomedical Imaging, Massachusetts General Hospital). FreeSurfer provides a set of automated reconstruction tools of the brain's cortical surface from structural MRI data. The cortical surface extracted in the native space of the images with sub-millimeter accuracy is then further used for the creation of an outer surface, which will serve as a basis for the lGI calculation. A circular region of interest is then delineated on the outer surface, and its corresponding region of interest on the cortical surface is identified using a matching algorithm as described in our validation study1. This process is repeatedly iterated with largely overlapping regions of interest, resulting in cortical maps of gyrification for subsequent statistical comparisons (Fig. 1). Of note, another measurement of local gyrification with a similar inspiration was proposed by Toro and colleagues7, where the folding index at each point is computed as the ratio of the cortical area contained in a sphere divided by the area of a disc with the same radius. The two implementations differ in that the one by Toro et al. is based on Euclidian distances and thus considers discontinuous patches of cortical area, whereas ours uses a strict geodesic algorithm and include only the continuous patch of cortical area opening at the brain surface in a circular region of interest.

Measuring gyrification (cortical folding) at any age represents a window into early brain development. Hence, we previously developed an algorithm to measure local gyrification at thousands of points over the hemisphere1. In this paper, we detail the computation of this local gyrification index.

Cómo medir plegable cortical a partir de imágenes MR: un tutorial paso a paso para Calcular Índice Gyrification Local

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will ...

Investigación

Medicina

High Content Screening in Neurodegenerative Diseases

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be addressed as a matter of great urgency1-4. Multiple complementary 'omics' approaches have provided clues as to the genetic risk factors and pathogenic mechanisms underlying numerous neurodegenerative diseases, but most findings still require functional validation5. For example, a recent genome wide association study for Parkinson's Disease (PD), identified many new loci as risk factors for the disease, but the underlying causative variant(s) or pathogenic mechanism is not known6, 7. As each associated region can contain several genes, the functional evaluation of each of the genes on phenotypes associated with the disease, using traditional cell biology techniques would take too long. 
	There is also a need to understand the molecular networks that link genetic mutations to the phenotypes they cause. It is expected that disease phenotypes are the result of multiple interactions that have been disrupted. Reconstruction of these networks using traditional molecular methods would be time consuming. Moreover, network predictions from independent studies of individual components, the reductionism approach, will probably underestimate the network complexity8. This underestimation could, in part, explain the low success rate of drug approval due to undesirable or toxic side effects. Gaining a network perspective of disease related pathways using HT/HC cellular screening approaches, and identifying key nodes within these pathways, could lead to the identification of targets that are more suited for therapeutic intervention.
	High-throughput screening (HTS) is an ideal methodology to address these issues9-12. but traditional methods were one dimensional whole-well cell assays, that used simplistic readouts for complex biological processes. They were unable to simultaneously quantify the many phenotypes observed in neurodegenerative diseases such as axonal transport deficits or alterations in morphology properties13, 14. This approach could not be used to investigate the dynamic nature of cellular processes or pathogenic events that occur in a subset of cells. To quantify such features one has to move to multi-dimensional phenotypes termed high-content screening (HCS)4, 15-17. HCS is the cell-based quantification of several processes simultaneously, which provides a more detailed representation of the cellular response to various perturbations compared to HTS. 
	HCS has many advantages over HTS18, 19, but conducting a high-throughput (HT)-high-content (HC) screen in neuronal models is problematic due to high cost, environmental variation and human error. In order to detect cellular responses on a 'phenomics' scale using HC imaging one has to reduce variation and error, while increasing sensitivity and reproducibility. 
	Herein we describe a method to accurately and reliably conduct shRNA screens using automated cell culturing20 and HC imaging in neuronal cellular models. We describe how we have used this methodology to identify modulators for one particular protein, DJ1, which when mutated causes autosomal recessive parkinsonism21. 
	Combining the versatility of HC imaging with HT methods, it is possible to accurately quantify a plethora of phenotypes. This could subsequently be utilized to advance our understanding of the genome, the pathways involved in disease pathogenesis as well as identify potential therapeutic targets.

We describe a methodology combining automated cell culturing with high-content imaging to visualize and quantify multiple cellular processes and structures, in a high-throughput manner. Such methods can aid in the further functional annotation of genomes as well as identify disease gene networks and potential drug targets.

Filtrado de contenido de alta en las enfermedades neurodegenerativas

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be ...

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
	After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
	Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

El uso de la cistometría en pequeños roedores: un estudio de la vejiga chemosensation

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

La detección de Modificadores melanoma utilizando un modelo de pez cebra Tumor autóctona

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

El estudio de cáncer de páncreas Tallo Características de las células para el desarrollo de nuevas estrategias de tratamiento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &amp;#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

Un alto contenido<em&gt; In Vitro</em&gt; Islotes pancreáticos de células β plataforma de descubrimiento de replicación

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have been limited compounds available so far to attenuate TLR signaling and there have been no clinically approved TLR inhibitors (except the anti-malarial drug hydroxychloroquine) in clinical use. In light of rapid advances in nanotechnology, manipulation of immune responsiveness using nano-devices may provide a new strategy to treat these diseases. Herein, we present a high throughput screening method for quickly identifying novel bioactive nanoparticles that inhibit TLR signaling in phagocytic immune cells. This screening platform is built on THP-1 cell-based reporter cells with colorimetric and luciferase assays. The reporter cells are engineered from the human THP-1 monocytic cell line by stable integration of two inducible reporter constructs. One expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-&#954;B and AP-1, and the other expresses a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs).Upon TLR stimulation, the reporter cells activate transcription factors and subsequently produce SEAP and/or luciferase, which can be detected using their corresponding substrate reagents. Using a library of peptide-gold nanoparticle (GNP) hybrids established in our previous studies as an example, we identified one peptide-GNP hybrid that could effectively inhibit the two arms of TLR4 signaling cascade triggered by its prototypical ligand, lipopolysaccharide (LPS). The findings were validated by standard biochemical techniques including immunoblotting. Further analysis established that this lead hybrid had a broad inhibitory spectrum, acting on multiple TLR pathways, including TLR2, 3, 4, and 5. This experimental approach allows a rapid assessment of whether a nanoparticle (or other therapeutic compounds) can modulate specific TLR signaling in phagocytic immune cells.

Toll-like receptor (TLR) signaling plays an important role in the pathophysiology of many human inflammatory diseases, and regulating TLR responses by bioactive nanoparticles is anticipated to be beneficial in many inflammatory conditions. THP-1 cell-based reporter cells provide a versatile and robust screening platform for identifying novel inhibitors of TLR signaling.

Proyección de nanopartículas bioactivas en células inmunológicas fagocíticas para inhibidores de la señalización de receptores de tipo Toll

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have ...

Routine Screening Method for Microparticles in Platelet Transfusions

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood banks. Cells fragment off microparticles (MP) when they are stressed. Blood and blood components may contain cellular fragments from a variety of cells, most notably from activated platelets. When performing their roles as innate immune cells and major players in coagulation and hemostasis, platelets change shape and generate microparticles. With dynamic light scattering (DLS)-based microparticle detection, it is possible to differentiate activated (high microparticle) from non-activated (low microparticle) platelets in transfusions, and optimize the use of this scarce blood product. Previous research suggests that providing non-activated platelets for prophylactic use in hematology-oncology patients could reduce their risk of becoming refractory and improve patient care. The goal of this screening method is to routinely differentiate activated from non-activated platelets. The method described here outlines the steps to be performed for routine platelet inventory management in a hospital blood bank: obtaining a sample from a platelet transfusion, loading the sample into the capillary for DLS measurement, performing the DLS test to identify microparticles, and using the reported microparticle content to identify activated platelets.

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative in hospital blood banks. The goal is to differentiate activated from non-activated platelets to optimize the use of platelets. Providing non-activated platelets to hematology-oncology patients might reduce their high risk to become refractory.

Rutina de detección del método de micropartículas en las transfusiones de plaquetas

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood ...

Hydra, a Computer-Based Platform for Aiding Clinicians in Cardiovascular Analysis and Diagnosis

Cardiovascular diseases (CVDs) are the leading cause of death throughout the world. The total risk of developing CVD is determined by the combined effect of different cardiovascular risk factors (e.g., diabetes, raised blood pressure, unhealthy diet, tobacco use, stress, etc.) that commonly coexist and act multiplicatively. Most CVDs can be prevented by an early identification of the highest risk factors and an appropriate treatment. The stratification of cardiovascular risk factors involves a wide range of parameters and tests that specialists use in their clinical practice. In addition to cardiovascular (CV) risk stratification, ambulatory blood pressure monitoring (ABPM) also provides relevant information for diagnostic and treatment purposes. This work presents a list of protocols based on the Hydra platform, a web-based system for clinical decision support which incorporates a set of functionalities and services that are required for complete cardiovascular analysis, risk assessment, early diagnosis, treatment and monitoring of patients over time. The program includes tools for inputting and managing comprehensive patient data, organized into different checkups to track the evolution over time. It also has a risk stratification tool to compute a CV risk factor based upon several risk stratification tables of reference. Additionally, the program includes a tool that incorporates ABPM analysis and allows the extraction of valuable information by monitoring blood pressure over a specific period of time. Finally, the reporting service summarizes the most relevant information in a set of reports that aid clinicians in their clinical decision-making process.

This article presents a protocol based on Hydra &#8212; a web-based system for clinical decision support that integrates a full and detailed set of functionalities and services required by physicians for complete cardiovascular analysis, risk assessment, early diagnosis, treatment, and monitoring over time.

Hydra, una plataforma basada en la computadora para ayudar a los clínicos en el diagnóstico y análisis Cardiovascular

Cardiovascular diseases (CVDs) are the leading cause of death throughout the world. The total risk of developing CVD is determined by the combined effect ...

In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells

Acute respiratory distress syndrome (ARDS) is a syndrome of diffuse alveolar injury with impaired alveolar fluid clearance and severe inflammation. The use of halogenated agents, such as sevoflurane or isoflurane, for the sedation of intensive care unit (ICU) patients can improve gas exchange, reduce alveolar edema, and attenuate inflammation during ARDS. However, data on the use of inhaled agents for continuous sedation in the ICU to treat or prevent lung damage is lacking. To study the effects of halogenated agents on alveolar epithelial cells under "physiologic" conditions, we describe an easy system to culture cells at the air-liquid interface and expose them to halogenated agents to provide precise controlled "air" fractions and "medium" concentrations for these agents. We developed a sealed air-tight chamber in which plates with human alveolar epithelial immortalized cells could be exposed to a precise, controlled fraction of sevoflurane or isoflurane using a continuous gas flow provided by an anesthetic machine circuit. Cells were exposed to 4% of sevoflurane and 1% of isoflurane for 24 hours. Gas mass spectrometry was performed to determine the concentration of halogenated agents dissolved in the medium. After the first hour, the concentrations of sevoflurane and isoflurane in the medium were 251 mg/L and 25 mg/L, respectively. The curves representing the concentrations of both sevoflurane and isoflurane dissolved in the medium showed similar courses over time, with a plateau reached at one hour after exposure. 
This protocol was specifically designed to reach precise and controlled concentrations of sevoflurane or isoflurane in vitro to improve our understanding of mechanisms involved in epithelial lung injury during ARDS and to test novel therapies for the syndrome.

In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells

We describe an easy protocol specifically designed to reach precise and controlled concentrations of sevoflurane or isoflurane in vitro in order to improve our understanding of mechanisms involved in the epithelial lung injury and to test novel therapies for acute respiratory distress syndrome.

In Vitro Método de Control de concentraciones de Gases halogenados en cultivos de células epiteliales alveolares

Acute respiratory distress syndrome (ARDS) is a syndrome of diffuse alveolar injury with impaired alveolar fluid clearance and severe inflammation. The ...