Name: investigation of beige fat biology and metabolism using the crispr suntag-p65-hsf1 activation system
Uploaded: 2023-01-06T14:20:00
Description: Watch this Scientific Journal Video about Investigation of Beige Fat Biology and Metabolism Using the CRISPR SunTag-p65-HSF1 Activation System at JoVE.com

Los autores agradecen el apoyo recibido del Centro Multidisciplinar para Investigação Biológica na Área da Ciência em Animais de Laboratório (Cemib), Unicamp, para la generación de ratones AdipoSPH, el Laboratorio de Inmmunometabolismo y Señalización Celular, y el Instituto Nacional de Ciencia y Tecnología sobre Fotónica Aplicada a la Biología Celular (INFABIC) por todo el apoyo experimental. Agradecemos el apoyo financiero de la Fundación de Investigación de São Paulo (FAPESP): 2019/15025-5; 2020/09308-1; 2020/14725-0; 2021/11841-2.

Los estudios en animales se realizaron de acuerdo con la Guía para el Cuidado y Uso de Animales de Laboratorio de la Universidad de Campinas (protocolo CEUA #5810-1/2021).
1. Clonación molecular
<ol><li>Diseño de ARN guía única (sgRNAs)<ol><li>Diseñar sgRNAs para la activación de CRISPR utilizando CHOPCHOP, disponibles en https://chopchop.cbu.uib.no/, o cualquier otra herramienta adecuada. Utilice los siguientes parámetros para diseñar sgRNA dirigidos al gen Prdm16:...

<ol>
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Una de las aplicaciones no editativas más útiles de la tecnología CRISPR es la interrogación de la función génica mediante la activación de genes endógenos utilizando sistemas CRISPRa6. SPH es un potente CRISPRa que fue descrito originalmente para inducir la conversión de astrocitos en neuronas activas al dirigirse a varios genes neurogénicos14. En este estudio, AdipoSPH demostró ser una herramienta adecuada para investigar la biología de la grasa beige mediante...

Los adipocitos beige (o brite) son adipocitos ricos en mitocondrias que expresan la proteína 1 (UCP1) y que residen dentro de los depósitos de tejido adiposo blanco (WAT). La grasa beige emerge de un subconjunto de progenitores adipocitos o adipocitos blancos maduros en respuesta a la exposición al frío y otros estímulos 1,2. Los adipocitos beige pueden convertir la energía en calor de una manera dependiente de UCP1 o independiente3. Independientemente de su función termogénica, la grasa beige también puede mejorar la salud metabólica por otros medios, como la secreción de adipoquinas y actividades antiinflamatorias y antifibróticas. Los estudios en ratones y humanos han demostrado que la inducción de grasa beige mejora la homeostasis de la glucosa y los lípidos de todo el cuerpo3. Sin embargo, aunque nuestro conocimiento de la biología de la grasa beige ha evolucionado rápidamente en los últimos años, la mayoría de sus beneficios metabólicos y mecanismos relacionados aún no se comprenden completamente.
Las repeticiones palindrómicas cortas agrupadas regularmente interespaciadas (CRISPR) se describieron por primera vez en células eucariotas como una herramienta capaz de generar una ruptura de doble cadena (DSB) en un sitio específico del genoma a través de la actividad nucleasa de la proteína Cas9 4,5. Cas9 es guiado por un ARN sintético de guía única (sgRNA) para dirigirse a una región genómica específica, lo que lleva a un ADN DSB. Además de utilizar la nucleasa Cas9 con fines de edición, la tecnología CRISPR-Cas9 ha evolucionado para ser utilizada como una herramienta de regulación génica específica de secuencia6. El desarrollo de una proteína Cas9 catalíticamente inactiva (dCas9) y la asociación de módulos transcripcionales capaces de mejorar la expresión génica ha dado lugar a herramientas de activación CRISPR (CRISPRa). Han surgido varios sistemas CRISPRa, como VP647,8, mediador de activación sinérgica (SAM)9, SunTag10,11, VPR12,13 y SunTag-p65-HSF1 (SPH)14, que combina los componentes de los activadores SAM y SunTag. Recientemente se ha demostrado que la expresión inducida de genes neurogénicos en neuroblastos N2a y astrocitos primarios es mayor utilizando SPH en comparación con otros sistemas CRISPRa14, demostrando SPH como una herramienta CRISPRa prometedora.
Aquí, aprovechamos un ratón SPH14 previamente desarrollado para generar un modelo de ratón condicional que expresa SPH específicamente en adipocitos utilizando el linaje de adiponectina Cre (AdipoSPH). Usando un virus adenoasociado (AAV) portador del ARNg dirigido al gen endógeno Prdm16, se indujo el pardeamiento (conversión de blanco a beige) de WAT inguinal (iWAT) para aumentar la expresión del programa de genes termogénicos. Además, la sobreexpresión in vitro de Prdm16 mejoró el consumo de oxígeno. Por lo tanto, este protocolo proporciona un modelo versátil de ratón SPH para explorar los mecanismos del desarrollo de grasa beige dentro del tejido adiposo.

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		<tr>
			<td>3,3&#39;,5-Triiodo-L-thyronine</td>
			<td>Sigma-Aldrich</td>
			<td>T2877</td>
			<td></td>
		</tr>
		<tr>
			<td>3-Isobutyl-1-methylxanthine</td>
			<td>Sigma-Aldrich</td>
			<td>I5879</td>
			<td></td>
		</tr>
		<tr>
			<td>AAVpro 293T Cell Line</td>
			<td>Takarabio</td>
			<td>632273</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra Centrifugal Filter</td>
			<td>Merckmillipore</td>
			<td>UFC510008</td>
			<td>100 KDa</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma-Aldrich</td>
			<td>D1756</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modification of Eagles Medium (DMEM)</td>
			<td>Corning</td>
			<td>10-017-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM) F-12, GlutaMAX&#8482; supplement</td>
			<td>Gibco</td>
			<td>10565-018</td>
			<td>high concentrations of glucose, amino acids, and vitamins</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline (DPBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8662</td>
			<td></td>
		</tr>
		<tr>
			<td>Excelta Self-Opening Micro Scissors</td>
			<td>Fisher Scientific</td>
			<td>17-467-496</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Cell Scrapers (100 pk)</td>
			<td>Fisher Scientific</td>
			<td>08-100-241</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand High Precision Straight Tapered Ultra Fine Point Tweezers/Forceps</td>
			<td>Fisher Scientific</td>
			<td>12-000-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Sharp-Pointed Dissecting Scissors</td>
			<td>Fisher Scientific</td>
			<td>08-940</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexadimethrine bromide (Polybrene)</td>
			<td>Sigma-Aldrich</td>
			<td>H9268</td>
			<td></td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Sigma-Aldrich</td>
			<td>I7378</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I9278</td>
			<td></td>
		</tr>
		<tr>
			<td>LigaFast Rapid DNA Ligation System</td>
			<td>Promega</td>
			<td>M8225</td>
			<td></td>
		</tr>
		<tr>
			<td>Maxiprep purification kit&#160;</td>
			<td>Qiagen</td>
			<td>12162</td>
			<td></td>
		</tr>
		<tr>
			<td>Microliter syringe</td>
			<td>Hamilton</td>
			<td>80308</td>
			<td>Model 701</td>
		</tr>
		<tr>
			<td>NEB 10-beta/Stable&#160;</td>
			<td>New England Biolabs</td>
			<td>C3019H</td>
			<td>E. coli competent cells</td>
		</tr>
		<tr>
			<td>pAAV2/8&#160;</td>
			<td>Addgene&#160;</td>
			<td>112864</td>
			<td></td>
		</tr>
		<tr>
			<td>pAAV-U6-gRNA-CBh-mCherry</td>
			<td>Addgene&#160;</td>
			<td>91947</td>
			<td></td>
		</tr>
		<tr>
			<td>pAdDeltaF6&#160;</td>
			<td>Addgene&#160;</td>
			<td>112867</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG 8000</td>
			<td>Sigma-Aldrich</td>
			<td>89510</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine</td>
			<td>Sigma-Aldrich</td>
			<td>23966</td>
			<td>Linear, MW 25000</td>
		</tr>
		<tr>
			<td>Povidone-iodine</td>
			<td>Rioqu&#237;mica</td>
			<td>510101303</td>
			<td>Antiseptic</td>
		</tr>
		<tr>
			<td>Rosiglitazone</td>
			<td>Sigma-Aldrich</td>
			<td>R2408</td>
			<td></td>
		</tr>
		<tr>
			<td>SacI enzyme</td>
			<td>New England Biolabs</td>
			<td>R0156</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Design Premier Adson Forceps</td>
			<td>Fisher Scientific</td>
			<td>22-079-741</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Hamilton</td>
			<td>475-40417</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Promega</td>
			<td>M180B</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA ligase buffer&#160;</td>
			<td>New England Biolabs</td>
			<td>B0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 PNK enzyme kit</td>
			<td>New England Biolabs</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr>
			<td>Tramadol Hydrochloride</td>
			<td>SEM</td>
			<td>43930</td>
			<td></td>
		</tr>
		<tr>
			<td>Vidisic Gel&#160;</td>
			<td>Bausch + Lomb&#160;</td>
			<td>99620</td>
			<td></td>
		</tr>
	</tbody>
</table>

investigation of beige fat biology and metabolism using the crispr suntag-p65-hsf1 activation system

La tecnología de repeticiones palindrómicas cortas agrupadas regularmente interespaciadas (CRISPR) ha provocado una revolución en la biología, y las herramientas recientes se han aplicado mucho más allá de la edición de genes descrita originalmente. El sistema de activación CRISPR (CRISPRa) combina la proteína Cas9 catalíticamente inactiva (dCas9) con distintos módulos de transcripción para inducir la expresión génica endógena. SunTag-p65-HSF1 (SPH) es una tecnología CRISPRa recientemente desarrollada que combina componentes de mediadores de activación sinérgicos (SAM) con los activadores SunTag. Este sistema permite la sobreexpresión de genes únicos o múltiples mediante el diseño de un ARN guía única personalizado (sgRNA). En este estudio, se utilizó un ratón SPH previamente desarrollado para generar un ratón condicional que expresa SPH en adipocitos (linaje adiponectina Cre), llamado AdipoSPH. Para inducir un fenotipo de grasa blanca a beige (pardeamiento), se inyectó un virus adenoasociado (AAV) portador de sgRNA dirigido al gen endógeno Prdm16 (un factor de transcripción bien establecido relacionado con el desarrollo de grasa marrón y beige) en el tejido adiposo blanco inguinal (iWAT). Este modelo de ratón indujo la expresión endógena de Prdm16 y activó el programa del gen termogénico. Además, la sobreexpresión de Prdm16 inducida por SPH in vitro aumentó el consumo de oxígeno de los adipocitos beige, fenocopiando los resultados de un modelo previo de ratón transgénico Prdm16. Por lo tanto, este protocolo describe un modelo de ratón versátil, rentable y rentable para investigar la biología del tejido adiposo.

Los ratones AdipoSPH se desarrollaron mediante la cría de cepas de ratones SPH y Adipoq-Cre. Ambas cepas de ratón tenían un fondo híbrido C57BL6J-DBA/2J (según el proveedor comercial; ver Tabla de materiales). El linaje del ratón SPH fue descrito originalmente por Zhou et al.14.
Desarrollo de adipocitos beige in vivo a través de la sobreexpresión de Prdm16 mediada por AdipoSPH Para evaluar la capaci...

Watch this Scientific Journal Video about Investigation of Beige Fat Biology and Metabolism Using the CRISPR SunTag-p65-HSF1 Activation System at JoVE.com

Investigation of Beige Fat Biology and Metabolism Using the CRISPR SunTag-p65-HSF1 Activation System

Este protocolo presenta el uso de CRISPR SunTag-p65-HSF1 (SPH) en adipocitos (AdipoSPH) como una estrategia alternativa al virus adenoasociado (AAV) para investigar la biología de la grasa beige. La inyección in vivo de ARNg portador de AAV dirigida al gen endógeno Prdm16 es suficiente para inducir el desarrollo de grasa beige y mejorar el programa de genes termogénicos.

Investigación de la biología y el metabolismo de la grasa beige utilizando el sistema de activación CRISPR SunTag-p65-HSF1

Clustered regularly interspaced short palindromic repeats (CRISPR) technology has prompted a revolution in biology, and recent tools have been applied far ...

Este protocolo presenta el Sistema de Activación SPH CRISPR como una estrategia alternativa a los vectores virales convencionales para realizar ensayos de función de ganancia en adipocitos. Permite la sobreexpresión de genes endógenos de adipocitos únicos o múltiples dentro del complejo entorno celular del tejido adiposo mediante la entrega de un ARN guía único personalizado utilizando un virus adenoasociado. Los pasos desafiantes de este protocolo están relacionados con la clonación del ARN guía único y la producción e inyección de virus adenoasociados en el tejido adiposo.Para comenzar, diseñe ARN guía única o ARNg para la activación de CRISPR utilizando chopchop o cualquier otra herramienta adecuada. Diseñar el sgRNA dirigido al gen PRDM16 utilizando los siguientes parámetros:Target como PRDM16 en un musculus musculoso usando un CRISPR/Cas9 y para como activación. Agregue voladizos al sgRNA para que coincida con el sitio de restricción Sac1 en la columna vertebral vectorial PAAVU6GRNACBHM cereza.Incluye 5'AGCT 3' donde n corresponde a nucleótidos. Obtener la secuencia inversa del complemento 3'sgRNA utilizando la herramienta indicada. A continuación, para el recocido de oligonucleótidos complementarios monocatenarios, agregue un microlitro de cada oligonucleótido monocatenario de 5' y 3', un microlitro de tampón ligasa T4, 0.5 microlitros de polinucleótido quinasa T4 y 6.5 microlitros de agua para un volumen de reacción final de 10 microlitros.Analice los oligonucleótidos monocatenarios complementarios usando un termociclador a 37 grados centígrados durante 30 minutos y 95 grados centígrados durante cinco minutos, seguido de una tasa de reducción de cinco grados centígrados por minuto. Luego, para la ligadura de oligonucleótidos de ARNg recocidos, agregue 25 nanogramos de cereza PAAVU6GRNACBHM plásmido a dos microlitros de oligonucleótidos sgRNA recocidos, un microlitro de enzima Sac1, dos microlitros de tampón de ligasa de ADN T4 10X, un microlitro de ligasa de ADN T4 y agua a un volumen de reacción final de 10 microlitros. Usando un termociclador, realice la ligadura incubando la mezcla de reacción durante 15 ciclos a 37 grados centígrados durante cinco minutos y 25 grados centígrados durante cinco minutos, seguido de una retención a cuatro grados centígrados.A continuación, transforme las células competentes de Escherichia coli DH10B con cuatro microlitros del producto de ligadura mediante choque térmico a 42 grados centígrados durante 45 segundos y extiéndalas sobre una placa de agar que contiene 10 microgramos por mililitro de ampicilina. Confirme las colonias transformadas por reacción en cadena de la polimerasa de colonias o PCR utilizando la mezcla maestra de PCR, elija la colonia y mézclela con cinco microlitros de mezcla maestra, 0.1 microlitros de cebador universal, 0.1 microlitros de cebador inverso sgRNA y cinco microlitros de agua. Ejecute la PCR utilizando la desnaturalización inicial a 94 grados centígrados durante dos minutos, seguida de 35 ciclos de desnaturalización a 94 grados centígrados durante 20 segundos, recocido durante 30 segundos a 60 grados centígrados, extensión a 72 grados centígrados durante 30 segundos y un paso final de alargamiento a 72 grados centígrados durante cinco minutos.Después de la PCR, resuelva el ADN usando electroforesis en gel de agarosa en tampón TAE 0.5X a 90 voltios durante 30 minutos. Envíe muestras positivas para la secuenciación de Sanger utilizando imprimación universal. A continuación, purifica el plásmido de un clon positivo usando un kit de purificación de plásmidos siguiendo las instrucciones del fabricante.Coloque el ratón anestesiado en posición supina y afeite un área pequeña en los flancos proximales a las articulaciones de la cadera para el tejido adiposo blanco inguinal o inyecciones IWAT con una afeitadora. Añadir crema depilatoria durante cinco minutos. Retire la crema residual con agua para evitar quemaduras en la piel.Desinfecte la piel usando tres rondas alternas de aplicación de la solución de povidona yodada sobre la piel con gasa limpia y alcohol al 70%. Luego, haga una incisión de uno o dos centímetros con tijeras esterilizadas en el área proximal de las articulaciones y mantenga la piel abierta con fórceps para exponer el depósito de grasa. El WAT se puede unir a la piel en ambos lados extendiéndola desde el principio desde la espalda y hacia los testículos.Con fórceps, tire suavemente del depósito de grasa hacia arriba a través de la incisión para asegurarse de que la inyección esté en la ubicación y profundidad correctas. Llene la jeringa de microlitros con 2,5 microlitros del virus adenoasociado o un AAV que contenga el sgRNA dirigido al gen PRDM16 endógeno. Inserte cuidadosamente la aguja en un ángulo de 30-45 grados en el IWAT.Repita la inyección cinco veces en diferentes ubicaciones del tejido para infectar homogéneamente toda la almohadilla de grasa IWAT. Coloque el ratón sobre la almohadilla térmica hasta que recupere la conciencia. Monitoree al animal cada 10 a 15 minutos hasta que se recupere por completo.Después de que el animal recupere la conciencia, observe el perfil de la locomotora, que debe ser lineal y no tener signos de angustia o dolor. Vea las células vasculares estromales o SVF derivadas de adipo SPH ratón IWAT en una placa de 6 pocillos que contiene medio DMEM completo durante una o dos horas, aspire el medio, lave el pocillo dos veces usando PBS y reemplácelo con un medio fresco y completo. Incubar las células a 37 grados centígrados, 5% de dióxido de carbono y 95% de humedad hasta que las células alcancen 70-80% de confluencia.En el día 0, inducir la diferenciación tratando las células con el medio de inducción y después de dos días reemplazar el medio de inducción con el medio de mantenimiento. Nuevamente después de dos días, en el cuarto día, reemplace el medio de mantenimiento con un medio de mantenimiento nuevo durante 2-3 días. Cambiar el medio de mantenimiento cada 48 horas hasta que los preadipocitos estén completamente diferenciados en adipocitos.Observe los adipocitos maduros usando microscopía óptica ya que las células diferenciadas parecen estar cargadas de gotas de lípidos. Después de cultivar las células en una placa de cultivo de 6 pocillos con el medio completo hasta que las células alcancen el 70-80% de confluencia, mezcle 5.6 * 10 ^ 10 genoma viral por microlitro de ARNg portador de AAV PRDM16 con dos mililitros de medio completo y bromuro de hexadimetrina. Transducir las células reemplazando el medio completo y agregando el medio completo que contiene AAV.Incubar las células transducidas durante 12 horas a 37 grados centígrados, 95% de humedad y 5% de dióxido de carbono. Divida y vea las células como se describe para la proliferación celular y la diferenciación en adipocitos beige. Las imágenes de inmunofluorescencia de IWAT de ratones adipo SPH demostraron la expresión de dCas9 tanto en el grupo control como en el PRDM16.La expresión de PRDM16 inducida por SPH indujo claramente una acumulación generalizada de adipocitos beige multioculares en IWAT. Además, la PCR cuantitativa reveló una mayor expresión de PRDM16 y el programa de genes termogénicos en el grupo PRDM16 en comparación con el control. Del mismo modo, el análisis de expresión génica in vitro confirmó el aumento de la expresión del gen PRDM16 endógeno y los genes termogénicos en el grupo de sobreexpresión PRDM16 en comparación con el control.La expresión de PRDM16 inducida por SPH resultó en un mayor consumo de oxígeno basal y máximo que en las células de control. Es importante destacar que los datos indicaron una mejor respiración desacoplada en el grupo PRDM16 en comparación con el grupo de control. El modelo Adipo SPH es adecuado para investigar múltiples genes y elementos reguladores dentro de los adipocitos.Este método abre la posibilidad de una comprensión más profunda de la biología de la grasa beige.

investigation-beige-fat-biology-metabolism-using-crispr-suntag-p65

Research

JoVE Journal

Bioengineering

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Bacterial Detección e Identificación Usando sensores electroquímicos

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Investigación

Bioingeniería

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

A detailed understanding of the complexity and relative variability within the human cardiac venous system is crucial for the development of cardiac devices that require access to these vessels. For example, cardiac venous anatomy is known to be one of the key limitations for the proper delivery of cardiac resynchronization therapy (CRT)1 Therefore, the development of a database of anatomical parameters for human cardiac venous systems can aid in the design of CRT delivery devices to overcome such a limitation. In this research project, the anatomical parameters were obtained from 3D reconstructions of the venous system using contrast-computed tomography (CT) imaging and modeling software (Materialise, Leuven, Belgium). The following parameters were assessed for each vein: arc length, tortuousity, branching angle, distance to the coronary sinus ostium, and vessel diameter.
	CRT is a potential treatment for patients with electromechanical dyssynchrony. Approximately 10-20% of heart failure patients may benefit from CRT2. Electromechanical dyssynchrony implies that parts of the myocardium activate and contract earlier or later than the normal conduction pathway of the heart. In CRT, dyssynchronous areas of the myocardium are treated with electrical stimulation. CRT pacing typically involves pacing leads that stimulate the right atrium (RA), right ventricle (RV), and left ventricle (LV) to produce more resynchronized rhythms. The LV lead is typically implanted within a cardiac vein, with the aim to overlay it within the site of latest myocardial activation. 
	We believe that the models obtained and the analyses thereof will promote the anatomical education for patients, students, clinicians, and medical device designers. The methodologies employed here can also be utilized to study other anatomical features of our human heart specimens, such as the coronary arteries. To further encourage the educational value of this research, we have shared the venous models on our free access website: <a href="http://www.vhlab.umn.edu/atlas" target="_blank">www.vhlab.umn.edu/atlas</a>.

The objective of this research is to recreate and then access the anatomy of the human cardiac venous system using 3D reconstructions generated from contrast-computed tomography scans.

Las reconstrucciones anatómicas del sistema venoso cardíaco humana usando tomografía computarizada con contraste de las muestras de perfusión fijos

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180&#176; Curved Artery Test Section

The arterial network in the human vasculature comprises of ubiquitously present blood vessels with complex geometries (branches, curvatures and tortuosity). Secondary flow structures are vortical flow patterns that occur in curved arteries due to the combined action of centrifugal forces, adverse pressure gradients and inflow characteristics. Such flow morphologies are greatly affected by pulsatility and multiple harmonics of physiological inflow conditions and vary greatly in size-strength-shape characteristics compared to non-physiological (steady and oscillatory) flows 1 - 7.
Secondary flow structures may ultimately influence the wall shear stress and exposure time of blood-borne particles toward progression of atherosclerosis, restenosis, sensitization of platelets and thrombosis 4 - 6, 8 - 13. Therefore, the ability to detect and characterize these structures under laboratory-controlled conditions is precursor to further clinical investigations.
A common surgical treatment to atherosclerosis is stent implantation, to open up stenosed arteries for unobstructed blood flow. But the concomitant flow perturbations due to stent installations result in multi-scale secondary flow morphologies 4 - 6. Progressively higher order complexities such as asymmetry and loss in coherence can be induced by ensuing stent failures vis-&#224;-vis those under unperturbed flows 5. These stent failures have been classified as &#34;Types I-to-IV&#34; based on failure considerations and clinical severity 14.
This study presents a protocol for the experimental investigation of the complex secondary flow structures due to complete transverse stent fracture and linear displacement of fractured parts (&#34;Type IV&#34;) in a curved artery model. The experimental method involves the implementation of particle image velocimetry (2C-2D PIV) techniques with an archetypal carotid artery inflow waveform, a refractive index matched blood-analog working fluid for phase-averaged measurements 15 - 18. Quantitative identification of secondary flow structures was achieved using concepts of flow physics, critical point theory and a novel wavelet transform algorithm applied to experimental PIV data 5, 6, 19 - 26.

Stent implants in stenosed arterial curvatures are prone to "Type IV" failures involving the complete transverse fracture of stents and linear displacement of the fractured parts. We present a protocol for detection of secondary flow (vortical) structures in a curved artery model, downstream of clinically relevant "Type IV" stent failures.

Investigación experimental de estructuras secundarias de flujo aguas abajo de una Falla Modelo Tipo IV stent en una sección de prueba 180 ° de la arteria curvada

The arterial network in the human vasculature comprises of ubiquitously present blood vessels with complex geometries (branches, curvatures and ...

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

Transplante de islotes en el cojín de grasa epididimal de ratones diabéticos

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy ...

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is made by creating two modules: a synthetically engineered strain of E. coli cells and a functionalized material interface. Within this paper, we detail a protocol for genetically engineering selected behaviors within a strain of E. coli using molecular cloning strategies. Once developed, this strain produces elevated levels of biotin when exposed to a chemical inducer. Additionally, we detail protocols for creating two different functionalized surfaces, each of which is able to respond to cell-synthesized biotin. Taken together, we present a methodology for creating a linked, abiotic-biotic system that allows engineered cells to control material composition and assembly on nonliving substrates.

This paper presents a series of protocols for developing engineered cells and functionalized surfaces that enable synthetically engineered E. coli to control and manipulate programmable material surfaces.

El uso de la biología sintética para Ingeniero células vivas que interactúan con materiales programables

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide &#34;NGG&#34; protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

Eficiente generación y edición de IPSCs alimentador-libre de las células pancreáticas humanas utilizando el sistema CRISPR Cas9

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

Catéter de luz intracardiaca para monitorear el metabolismo celular utilizando espectroscopia de absorbancia transmural de corazones de mamíferos perfundidos

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has facilitated advanced genetic modification of Saccharomyces cerevisiae, a model organism and workhorse in industrial biotechnology. CRISPR-associated protein 12a (Cas12a), an RNA-guided endonuclease with features distinguishable from Cas9 is applied in this work, further extending the molecular toolbox for genome editing purposes. A benefit of the CRISPR/Cas12a system is that it can be used in multiplex genome editing with multiple guide RNAs expressed from a single transcriptional unit (single CRISPR RNA (crRNA) array). We present a protocol for multiplex integration of multiple heterologous genes into independent loci of the S. cerevisiae genome using the CRISPR/Cas12a system with multiple crRNAs expressed from a single crRNA array construct. The proposed method exploits the ability of S. cerevisiae to perform in vivo recombination of DNA fragments to assemble the single crRNA array into a plasmid that can be used for transformant selection, as well as the assembly of donor DNA sequences that integrate into the genome at intended positions. Cas12a is pre-expressed constitutively, facilitating cleavage of the S.&#160;cerevisiae genome at the intended positions upon expression of the single crRNA array. The protocol includes the design and construction of a single crRNA array and donor DNA expression cassettes, and exploits an integration approach making use of unique 50-bp DNA connectors sequences and separate integration flank DNA sequences, which simplifies experimental design through standardization and modularization and extends the range of applications. Finally, we demonstrate a straightforward technique for creating yeast pixel art with an acoustic liquid handler using differently colored carotenoid producing yeast strains that were constructed.

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

The CRISPR/Cas12a system in combination with a single crRNA array enables efficient multiplex editing of the S.&#160;cerevisiae genome at multiple loci simultaneously. This is demonstrated by constructing carotenoid producing yeast strains which are subsequently used to create yeast pixel art.

CRISPR/Cas12a Multiplex Genoma Edición de Saccharomyces cerevisiae y la creación de Arte de píxeles de levadura

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) ...

Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System

Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. This work describes the key steps leading to the construction of efficient sgRNA vectors using a strategy that allows the efficient detection of the positive colonies by PCR prior to DNA sequencing. Since efficient genome editing using the CRISPR system requires a highly efficient sgRNA, a preselection of candidate sgRNA targets is necessary to save time and effort. A dual luciferase reporter system has been developed to evaluate knockout efficiency by examining double-strand break repair via single strand annealing. Here, we use this reporter system to pick up the preferred xCas9/sgRNA target from candidate sgRNA vectors for specific gene editing. The protocol outlined will provide a preferred sgRNA/CRISPR enzyme vector in 10 days (starting with appropriately designed oligonucleotides).

Here, we present a protocol describing a streamlined method for the efficient generation of plasmids expressing both the CRISPR enzyme and associated single guide RNA (sgRNAs). Co-transfection of mammalian cells with this sgRNA/CRISPR vector and a dual luciferase reporter vector that examines double-strand break repair allows evaluation of knockout efficiency.

Construcción de Plásmidos CRISPR y Detección de Eficiencia Knockout en Células de Mamíferos a través de un Sistema Dual de Reporteros Luciferase

Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. ...

Measuring 3D In-vivo Shoulder Kinematics using Biplanar Videoradiography

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, multiple ligaments, and approximately 20 muscles. Unfortunately, shoulder pathologies (e.g., rotator cuff tears, joint dislocations, arthritis) are common, resulting in substantial pain, disability, and decreased quality of life. The specific etiology for many of these pathologic conditions is not fully understood, but it is generally accepted that shoulder pathology is often associated with altered joint motion. Unfortunately, measuring shoulder motion with the necessary level of accuracy to investigate motion-based hypotheses is not trivial. However, radiographic-based motion measurement techniques have provided the advancement necessary to investigate motion-based hypotheses and provide a mechanistic understanding of shoulder function. Thus, the purpose of this article is to describe the approaches for measuring shoulder motion using a custom biplanar videoradiography system. The specific objectives of this article are to describe the protocols to acquire biplanar videoradiographic images of the shoulder complex, acquire CT scans, develop 3D bone models, locate anatomical landmarks, track the position and orientation of the humerus, scapula, and torso from the biplanar radiographic images, and calculate the kinematic outcome measures. In addition, the article will describe special considerations unique to the shoulder when measuring joint kinematics using this approach.

Biplane videoradiography can quantify shoulder kinematics with a high degree of accuracy. The protocol described herein was specifically designed to track the scapula, humerus, and the ribs during planar humeral elevation, and outlines the procedures for data collection, processing, and analysis. Unique considerations for data collection are also described.

Medición de la cinemática del hombro 3D in vivo mediante videorradiografía biplanar

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, ...