Agradecemos sinceramente a la Fundación Whitehall por su apoyo financiero y fondos de inicio del Scripps Research Institute por llevar a cabo este trabajo.

1. Preparación De Ganglios Abdominales, Mediciones Electrofisiológicas, Y El Aislamiento De Las Neuronas Individuales Identificadas Del Ganglio Abdominal De Aplysia californica
<ol><li>Mantenga Aplysia en el acuario de laboratorio con agua de mar artificial circulante (ASW) a 16 ° C bajo condiciones de luz: oscuras de 12:12.</li>
	<li>Aislamiento del ganglio abdominal.<ol>
<li>Anestesiar a los animales inyectando 380 mM mgCl2 solución durante 5-10 min (equivalente al 30-35% del peso corporal del animal).</li>
		<li>Identificar el ganglio abdominal en función de su posición en el sistema nervioso central24,28.</li>
		<li>Retirar los ganglios mediante operación quirúrgica y almacenarlos inmediatamente en agua de mar artificial consistente en 450 mM de NaCl, 10 mM de KCl, 10 mM de CaCl2,55 mM de MgCl2,2,5 mM de NaHCO3,y 10 mM de HEPES (pH 7,4).</li>
	</ol>
</li>
	<li>Tratamiento con proteasas.<ol>
<li>Incubar ganglio abdominal durante 30 min a 34 °C±0,5 en una placa de Petri con proteasa (dispase) al 0,1% diluida en ASW descrita anteriormente. NOTA: La cantidad de la proteasa utilizada debe estandarizarse con la edad y el peso de los animales. En general, los animales más viejos y más grandes requerirían más proteasa.</li>
	</ol>
</li>
	<li>Retiro de la envaesda de los ganglios.<ol>
<li>Después del tratamiento enzimático, fije los ganglios a la base de silicona de Sylgard de una cámara celular (Ø = 1 cm; vol. = 0,3 ml) y perfunda con ASW (caudal: 150 μl/min) a temperatura ambiente (18 °C±2). NOTA: Con el fin de aumentar la visibilidad de las neuronas identificadas, coloque el ganglio en la parte superior de un cilindro de vidrio de policarbonato(Figura 1)(diámetro Ø = 2 mm de cámara celular modificada) que se puede iluminar fácilmente desde la parte inferior utilizando un estereomicroscopio binocular con aumento de 20X y 40X.</li>
		<li>Retire cuidadosamente la vainquia del ganglio mediante el uso de tórceps y microscisores.</li>
	</ol>
</li>
	<li>Grabación electrofisiológica simultánea de dos neuronas.<ol>
<li>Identificar la neurona de interés.</li>
		<li>Empalar la neurona con un solo microelectrodo agudo intracelular con resistencia 10-15 MΩ lleno de 3 M KCl solución.</li>
		<li>Registre la actividad neuronal (paso de banda de 10 kHz) utilizando un sistema de grabación intracelular.</li>
		<li>Procese los datos de registro utilizando software de electrofisiología como Axograph. NOTA: Uno puede llevar a cabo fácilmente la grabación de múltiples neuronas. Las neuronas L7, L11 o R15 y R2 se identifican fácilmente en función de su posición. Se requieren dos amplificadores y dos micromanipuladores para grabar simultáneamente desde dos neuronas L7 y R15 (Figura 2).</li>
	</ol>
</li>
	<li>Aplicación de fármacos.<ol>
<li>Aplicar el fármaco de elección en la cámara celular por pipeteo suave o inyectar directamente en las neuronas.</li>
		<li>Realizar mediciones electrofisiológicas. NOTA: Dependiendo del objetivo experimental, se podrían medir diferentes parámetros electrofisiológicos de las neuronas como los cambios en el potencial de membrana (MP) o el potencial de acción (AP) antes, durante y después de la aplicación del fármaco.</li>
	</ol>
</li>
	<li>Aislamiento de neuronas individuales.<ol>
<li>Al concluir los experimentos electrofisiológicos, detenga la perfusión de ASW y lave el ganglio con etanol al 100%.  La exposición al etanol petrificará las neuronas y, usando los pórceps finos, hará que sea fácil quitar las neuronas individuales sin dañar otras neuronas del ganglio.</li>
		<li>Retire las neuronas individuales debajo de un estereomicroscopio y transfiérelo a un pequeño tubo de Eppendorf que contenga un reactivo de extracción de ARN de 500 μl helado. Las neuronas individuales aisladas se pueden almacenar en reactivos de extracción de ARN a -80 °C para futuros análisis.</li>
	</ol>
</li>
</ol>2. Aislamiento de ARN de neuronas individuales y análisis de expresión génica por PCR cuantitativa en tiempo real (qPCR)
<ol><li>Precauciones para minimizar la degradación del ARN: Las ribonucleasas (RNasas) degradan el ARN y son enzimas muy estables y activas. Siga estos pasos durante el manejo de los ENR.<ol>
<li>Limpie el área de trabajo y los instrumentos con agentes desactivadores de RNase.</li>
		<li>Use guantes en todo momento mientras manipula el ARN y cámbiese los guantes con frecuencia.</li>
		<li>Use solo plásticos libres de ARNsa para manejar los ARNs.</li>
	</ol></li>
	<li>Aislar el ARN total de las neuronas individuales:<ol>
<li>El protocolo trizol estándar para el aislamiento de ARN se puede utilizar para aislar LOS ARN de neuronas individuales. Añadir brevemente 20% v/v de cloroformo a Trizol, mezclar bien por breve vórtice.</li>
		<li>Coloque los tubos en el rotador durante 10 min a 4 °C. Haga girar la mezcla Trizol-Cloroformo a 12.000 x g durante 15 min a 4 °C.</li>
		<li>Recoger la fase acuosa y transferir a un tubo fresco.</li>
		<li>Añadir solución de acetato de sodio (pH 5,5) a una concentración final de 0,3 M, 100 ng/ml del coprecipitante GlycoBlue, y 2,5 volúmenes de etanol al 100% para precipitar el ARN.</li>
		<li>Mezcle bien las muestras e incube a -80 °C durante la noche.</li>
		<li>Gire las muestras a 12.000 x g durante 20 min a 4 °C y un pequeño pellet azul será visible en la parte inferior del tubo.</li>
		<li>Retire el sobrenadante y lave el pellet con 850 μl de etanol helado al 75% a 12.000 x g durante 10 min a 4 °C.</li>
		<li>Retire el sobrenadante con cuidado y seque al aire el pellet de ARN durante 7-10 min, máximo. NOTA: Asegúrese de que el ARN no se deja secar durante mucho tiempo como encima-secar el pellet de ARN hace que la solubilización sea muy difícil.
</li>
		<li>Una vez secado, resuspend el pellet de ARN en 10 μl de agua libre de RNAse y determinar la concentración de ARN. NOTA: Determine la concentración de ARN utilizando un espectrofotómetro. Cuando no se use de inmediato, guarde el ARN a -80 °C.</li>
	</ol></li>
	<li>Síntesis de aRNA a partir de neuronas individuales:<ol>
<li>Amplificar el ARN (una o dos rondas dependiendo del objetivo experimental) utilizando el sistema de amplificación de ARN lineal disponible en el comercio. NOTA: El ARN total de neuronas individuales varió de ~10-60 ng. El rendimiento esperado de la primera ronda de amplificación es de ~ 100-300 ng y la segunda ronda de amplificación es de ~ 0.8-1.5 μg de ARN.</li>
	</ol></li>
	<li>Transcripción inversa del ARN:<ol>
<li>Para generar ADNc a partir de aRNA, utilice 1,0 μg de ARN en 20 μl de una reacción de transcripción inversa. Varios kits están disponibles comercialmente para este propósito. NOTA: El ADNc se sintetizó de acuerdo con el protocolo del fabricante utilizando los siguientes ajustes en el ciclador térmico. 5 min a 25 °C 30 min a 42 °C 5 min a 85 °C Mantener a 4 °C</li>
		<li>Después del paso de transcripción inversa, almacene el ADNc a -20 °C para el almacenamiento a largo plazo.</li>
	</ol></li>
	<li>Diseño y estandarización de cebadores: Varios programas de software excelentes están disponibles, de forma gratuita, para el diseño de cebadores para amplificaciones en tiempo real.<ol>
<li>Seleccione 18-24 oligonucleótidos de mer que produzcan amplicones de 70-110 pb y sintetice los cebadores utilizando fuentes comerciales. NOTA: Se deben diseñar de dos a tres pares de cebadores para cada gen de interés. Cada conjunto de cebadores debe ser estandarizado por qPCR. Los pares de cebadores delanteros e inversos que se utilizaron para el gen CREB1(Figura 4)son los siguientes: Juego de cebadores 1 Adelante: 5'-TGATTCTGACGCAAAGAAAAGA-3' Reverso: 5'-ACCGTGAGCAGTCAGTTGTAGA-3' Juego de cebadores 2 Delantero: 5'-AGGGAATGTCGATGAAAGAAGA-3' Reverso: 5'-GACACACAGGAAGTATGCCAAA-3'</li>
		<li>Para seleccionar el mejor cebador que se utilizará para los análisis aguas abajo, monitoree el valor de Ct y la forma de la curva de amplificación en qPCR. NOTA: Los cebadores que dan valores de Ct (Umbral de ciclo) entre 10-35 en la amplificación de 40 rondas y curvas suaves durante la fase exponencial de la PCR seguida de una meseta suave son óptimos para los análisis de expresión génica.</li>
	</ol></li>
	<li>PCR cuantitativa en tiempo real (qPCR): Nota: Utilice tubos o placas apropiados que sean compatibles con la máquina qPCR.<ol>
<li>Diluya el ADNc a 5x con agua libre de nucleasa.</li>
		<li>A 2 μl de ADNc diluido, agregue 8 μl de una mezcla maestra qPCR que contenga 2 μl deH2O, 5 μl de 2x mezcla maestra verde SYBR y 1,0 μl de imprimación directa e inversa de 10 μM (cada una).</li>
		<li>Cierre los tubos y selle la placa después de pipetear el ADNc y la mezcla maestra. Mezcle el contenido tocando suavemente y girando hacia abajo a 1.500 rpm durante 30 segundos.</li>
		<li>Proceda a configurar la máquina qPCR.</li>
	</ol></li>
</ol>

Los autores no tienen ningún interés financiero en competencia.

La neurona R15 participa en la regulación de los sistemas cardiovascular, digestivo, respiratorio y reproductivo30. Una actividad que estalla regularmente rítmica del AP es una característica de R15. Como se muestra en la sección de resultados, el registro emparejado de R15 y L7 muestran que la preparación de los ganglios ha preservado la actividad de las neuronas R15. Las neuronas R15 y L7 respondieron apropiadamente a Ach. Esta preparación de los ganglios se podía mantener hasta 8-10 horas y la activi...

El cerebro humano es extraordinariamente complejo con casi 100 mil millones de neuronas y billones de conexiones sinápticas. Hay casi un número igual de células no neuronales que interactúan con las neuronas y regulan su función en el cerebro. Las neuronas se organizan en circuitos que regulan comportamientos específicos. A pesar de los avances en nuestra comprensión de las funciones cerebrales y los circuitos neuronales, poco se sabe sobre la identidad de los componentes de los circuitos que controlan un comportamiento específico. El conocimiento de las identidades de varios componentes de un circuito facilitará grandemente nuestra comprensión de la base celular y molecular del comportamiento y ayuda en el desarrollo de las estrategias terapéuticas nuevas para los desordenes neuropsiquiátricos.
El caracol marino Aplysia californica ha sido un caballo de batalla para determinar los circuitos neuronales subyacentes a comportamientos específicos1-14. El sistema nervioso de Aplysia contiene aproximadamente 20.000 neuronas que se organizan en 9 ganglios diferentes. Las neuronas de Aplysia son grandes y se pueden identificar fácilmente en función de su tamaño, propiedades eléctricas y posición en los ganglios. Aplysia tiene un rico repertorio de comportamientos que pueden ser estudiados. Uno de los comportamientos bien estudiados es el reflejo branquial-abstinencia (GWR). Los componentes centrales de este reflejo están situados en los ganglios abdominales. Se han mapeado componentes de los circuitos GWR y se han determinado las contribuciones de varios componentes. Es importante destacar que los circuitos GWR se someten a aprendizaje asociativo y no asociativo5,6,15-19. Décadas de estudio sobre este reflejo también han identificado varias vías de señalización que tienen un papel clave en el aprendizaje y la memoria20-24.
Varias preparaciones diferentes de Aplysia se utilizaron para estudiar las bases celulares y moleculares del almacenamiento de la memoria. Estos incluyen el animal intacto2,3,la preparación semi-intacta1,7,13,14,16 y la reconstitución de los principales componentes de los circuitos neuronales25-29. Una preparación reducida para explorar los ganglios de Aplysia para los análisis electrofisiológicos y moleculares de circuitos neuronales identificados se describe aquí. Las cuatro neuronas identificadas siguientes fueron estudiadas. R15, una neurona que estallaba, L7 y L11, dos diversas neuronas de motor y R2, una neurona colinérgica fueron estudiados. R2 es la neurona más grande descrita en el sistema nervioso de los invertebrados. Brevemente, esta metodología implica el tratamiento proteasa de ganglios, mediciones electrofisiológicas antes y después de los tratamientos farmacológicos, y el aislamiento de neuronas individuales para el análisis cuantitativo de la expresión génica. Esta metodología nos permite combinar análisis moleculares con el registro simultáneo de múltiples neuronas. Esta metodología se utilizó con éxito para estudiar las respuestas de las neuronas R15 y L7 a la acetilcolina (Ach) por grabaciones intracelulares emparejadas. Después de mediciones electrofisiológicas R15 y L7 y otras neuronas identificadas como L11 y R2 se aislaron para el análisis cuantitativo de reacción en cadena de la polimerasa (qPCR) de la expresión de CREB1, un factor de transcripción importante para el almacenamiento de memoria.

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			<td height="101" style="height:101px;width:120px;">Aplysia</td>
			<td style="width:123px;">National Aplysia Resource Facility, University of Miami</td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">NaCl</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:129px;">S 3014-1KG</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:120px;">KCl</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:129px;">P 9333-500G</td>
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		<tr height="27">
			<td height="27" style="height:27px;width:120px;">CaCl2&#8226;2H2O</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:129px;">C5080- 500G</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:120px;">MgCl2&#8226;6H2O</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:129px;">BP 214-501</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:120px;">NaHCO4</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:129px;">S 6297-250G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">HEPES</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:129px;">H 3375-500G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">Protease</td>
			<td style="width:123px;">GIBCO</td>
			<td style="width:129px;">17105-042</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">Trizol</td>
			<td style="width:123px;">Ambion</td>
			<td style="width:129px;">15596-026</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Chloroform</td>
			<td style="width:123px;">MP Biomedicals</td>
			<td style="width:129px;">2194002</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">100% Ethanol</td>
			<td style="width:123px;">ACROS</td>
			<td style="width:129px;">64-17-5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">GlycoBlue</td>
			<td style="width:123px;">Ambion</td>
			<td style="width:129px;">AM9515</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">3 M NaOAc, pH 5.5</td>
			<td style="width:123px;">Ambion</td>
			<td style="width:129px;">AM9740</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Nuclease free water</td>
			<td style="width:123px;">Ambion</td>
			<td style="width:129px;">AM9737</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:120px;">MessageAmp II aRNA Amplification Kit</td>
			<td style="width:123px;">Ambion</td>
			<td style="width:129px;">AM1751</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">qScript cDNA SuperMix</td>
			<td style="width:123px;">Quanta Biosciences</td>
			<td style="width:129px;">95048-100</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:120px;">Power SYBR Green PCR Master Mix</td>
			<td style="width:123px;">Applied Biosystems</td>
			<td style="width:129px;">4367659</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Forceps</td>
			<td style="width:123px;">Fine Science Tools</td>
			<td style="width:129px;">11252-20</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Scissors</td>
			<td style="width:123px;">Fine Science Tools</td>
			<td style="width:129px;">15000-08</td>
		</tr>
		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:120px;">Stainless Steel Minutien Pins&#160;</td>
			<td rowspan="2" style="width:123px;">Fine Science Tools</td>
			<td style="width:129px;">26002-10 or</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:129px;">26002-20</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Veriti Thermal Cycler</td>
			<td style="width:123px;">Applied Biosystems</td>
			<td style="width:129px;">Veriti Thermal Cycler</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">5430R Centrifuge</td>
			<td style="width:123px;">Eppendorf</td>
			<td style="width:129px;">5430R Centrifuge</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">7900HT Fast Real-Time PCR</td>
			<td style="width:123px;">Applied Biosystems</td>
			<td style="width:129px;">7900HT Fast Real-Time PCR</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:120px;">Amplifier</td>
			<td>BRAMP-01R</td>
			<td style="width:129px;">NPI Electronics</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:120px;">Digidata Converter</td>
			<td style="width:123px;">Instrutech ITC-18</td>
			<td style="width:129px;">HEKA ELEKTRONIK</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:120px;">Micro Manipulator</td>
			<td style="width:123px;">Patch Star</td>
			<td style="width:129px;">Scientifica</td>
		</tr>
	</tbody>
</table>

aplysia ganglia preparation for electrophysiological and molecular analyses of single neurons

Un desafío importante en neurobiología es entender los fundamentos moleculares de los circuitos neuronales que gobiernan un comportamiento específico. Una vez identificados los mecanismos moleculares específicos, se pueden desarrollar nuevas estrategias terapéuticas para tratar anomalías en comportamientos específicos causadas por enfermedades degenerativas o envejecimiento del sistema nervioso. El caracol marino Aplysia californica es muy adecuado para las investigaciones de las bases celulares y moleculares del comportamiento porque los circuitos neuronales subyacentes a un comportamiento específico podrían determinarse fácilmente y los componentes individuales de los circuitos podrían ser fácilmente manipulados. Estas ventajas de Aplysia han llevado a varios descubrimientos fundamentales de la neurobiología del aprendizaje y de la memoria. Aquí se describe una preparación del sistema nervioso Aplysia para los análisis electrofisiológicos y moleculares de las neuronas individuales. Brevemente, el ganglio diseccionado del sistema nervioso se expone a la proteasa para eliminar la vaina ganglionar de tal manera que las neuronas están expuestas pero conservan la actividad neuronal como en el animal intacto. Esta preparación se utiliza para llevar a cabo mediciones electrofisiológicas de neuronas únicas o múltiples. Es importante destacar que, después de la grabación utilizando una metodología simple, las neuronas podrían aislarse directamente de los ganglios para el análisis de expresión génica. Estos protocolos se utilizaron para llevar a cabo grabaciones electrofisiológicas simultáneas de las neuronas L7 y R15, estudiar su respuesta a la acetilcolina y la cuantificación de la expresión del gen CREB1 en aislados de las neuronas L7, L11, R15 y R2 de Aplysia.

Los pesos de los animales que fueron utilizados en este estudio se extendieron a partir del 100-200 g. Siguiendo los protocolos descritos, se realizaron mediciones electrofisiológicas y análisis moleculares de neuronas de ganglios abdominales aislados de animales que oscilaban entre 2-5 g y 200-300 g.
La estandarización del tratamiento con proteasa es importante para el éxito de las mediciones electrofisiológicas de las neuronas en los ganglios. Inicialmente, se utilizaron concentraciones...

Watch this Scientific Journal Video about Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons at JoVE.com

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

El caracol marino Aplysia californica ha sido ampliamente utilizado como modelo neurobiológico para los estudios sobre bases celulares y moleculares del comportamiento. Aquí se describe una metodología para explorar el sistema nervioso de Aplysia para los análisis electrofisiológicos y moleculares de neuronas individuales de circuitos neuronales identificados.

Aplysia Preparación De Ganglios Para Análisis Electrofisiológicos Y Moleculares De Neuronas Individuales

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific ...

aplysia-ganglia-preparation-for-electrophysiological-molecular

Research

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Bioengineering

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

9.0K vistas.  The Scripps Research Institute, Florida. El caracol marino Aplysia californica ha sido ampliamente utilizado como modelo neurobiológico para los estudios sobre bases celulares y moleculares del comportamiento. Aquí se describe una metodología para explorar el sistema nervioso de Aplysia para los análisis electrofisiológicos y moleculares de neuronas individuales de circuitos neuronales identificados.

Video: Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc) 7-9. This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments 10. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one 12. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage12.
Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks)11,12, fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO4 blunt-ended break (DNase I-type breaks)11,12, if T4 DNA ligase is present in the solution 13,14 . When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO4 blunt-ended breaks, the ligase reacts with 5'PO4 DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO4 blunt-ended DNA breaks.
This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

En la Asamblea in vitro de la semi-artificial de la máquina molecular y su utilización para la detección del daño del ADN

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

Investigación

Bioingeniería

Molecular Entanglement and Electrospinnability of Biopolymers

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular entanglement of the constituent polymers in the spinning dope was found to be an essential prerequisite for successful electrospinning. Rheology is a powerful tool to probe the molecular conformation and interaction of biopolymers. In this report, we demonstrate the protocol for utilizing rheology to evaluate the electrospinnability of two biopolymers, starch and pullulan, from their dimethyl sulfoxide (DMSO)/water dispersions. Well-formed starch and pullulan fibers with average diameters in the submicron to micron range were obtained. Electrospinnability was evaluated by visual and microscopic observation of the fibers formed. By correlating the rheological properties of the dispersions to their electrospinnability, we demonstrate that molecular conformation, molecular entanglement, and shear viscosity all affect electrospinning. Rheology is not only useful in solvent system selection and process optimization, but also in understanding the mechanism of fiber formation on a molecular level.

Electrospinning is a fascinating technique used to fabricate micro- to nano-scale fibers from a wide variety of materials. Molecular entanglement of the constituent polymers in the spinning dope is essential for successful electrospinning. We present a protocol for utilizing rheology to evaluate the electrospinnability of two biopolymers, starch and pullulan.

Molecular Enredo y Electrospinnability de Biopolímeros

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

Tejido picado en colágeno comprimido: Un Bio Transplant que contiene células para la reparación reconstructiva-puesta en escena individual

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

Una técnica-Label libre para el Imaging espacio-temporal de las secreciones sola célula

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Preparation of Silicon Nanowire Field-effect Transistor for Chemical and Biosensing Applications

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we describe the preparation of polycrystalline silicon nanowire field-effect transistors (pSNWFETs) that serve as biosensing devices for biomarker detection. A protocol for chemical and biomolecular sensing by using pSNWFETs is presented. The pSNWFET device was demonstrated to be a promising transducer for real-time, label-free, and ultra-high-sensitivity biosensing applications. The source/drain channel conductivity of a pSNWFET is sensitive to changes in the environment around its silicon nanowire (SNW) surface. Thus, by immobilizing probes on the SNW surface, the pSNWFET can be used to detect various biotargets ranging from small molecules (dopamine) to macromolecules (DNA and proteins). Immobilizing a bioprobe on the SNW surface, which is a multistep procedure, is vital for determining the specificity of the biosensor. It is essential that every step of the immobilization procedure is correctly performed. We verified surface modifications by directly observing the shift in the electric properties of the pSNWFET following each modification step. Additionally, X-ray photoelectron spectroscopy was used to examine the surface composition following each modification. Finally, we demonstrated DNA sensing on the pSNWFET. This protocol provides step-by-step procedures for verifying bioprobe immobilization and subsequent DNA biosensing application.

We describe key steps for biosensing by using polysilicon nanowire field-effect transistors, including the preparation of the device and the immobilization and confirmation of a DNA molecular probe on the nanowire surface, as well as conditions for DNA sensing.

Preparación de silicio de nanocables de transistor de efecto de campo de químicos y biosensores Aplicaciones

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

Ingeniería Molecular Recognition con polímeros biomiméticos en una sola pared nanotubos de carbono

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; however such coordinates must represent homologous structures across specimens. While many biological objects consist of easily identified landmarks to satisfy the assumption of homology, many lack such structures. One potential solution is to mathematically place semi-landmarks on an object that represent the same morphological region across specimens. Here, we illustrate a recently developed pipeline to mathematically define semi-landmarks from the mouse baculum (penis bone). Our methods should be applicable to a wide range of objects.

Many biological structures lack easily definable landmarks, making it difficult to apply modern morphometric methods. Here we illustrate methods to study the mouse baculum (a bone in the penis), including dissection and microCT scanning, followed by computational methods to define semi-landmarks that are used to quantify size and shape variation.

Disección, de exploración y morfométricos microTC Los análisis de la Baculum

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

Preparación, purificación y uso de liposomas que contienen ácidos grasos

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Absorción de nuevas nanopartículas recubiertas de lípidos que contienen Falcarindiol por células madre mesenquimales humanas

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Evaluation of Photosynthetic Behaviors by Simultaneous Measurements of Leaf Reflectance and Chlorophyll Fluorescence Analyses

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many parameters that efficiently measure photosynthesis. Leaf reflectance analysis provides several vegetation indices in ecology and agriculture, including the photochemical reflectance index (PRI), which can be used as an indicator of thermal energy dissipation during photosynthesis because it correlates with non-photochemical quenching (NPQ). However, since NPQ is a composite parameter, its validation is required to understand the nature of the PRI parameter. To obtain physiological evidence for evaluation of the PRI parameter, we simultaneously measured chlorophyll fluorescence and leaf reflectance in xanthophyll cycle defective mutant (npq1) and wild-type Arabidopsis plants. Additionally, the qZ parameter, which likely reflects the xanthophyll cycle, was extracted from the results of chlorophyll fluorescence analysis by monitoring relaxation kinetics of NPQ after switching the light off. These simultaneous measurements were carried out using a pulse-amplitude modulation (PAM) chlorophyll fluorometer and a spectral radiometer. The fiber probes from both instruments were positioned close to each other to detect signals from the same leaf position. An external light source was used to activate photosynthesis, and the measuring lights and saturated light were provided from the PAM instrument. This experimental system enabled us to monitor light-dependent PRI in the intact plant and revealed that light-dependent changes in PRI differ significantly between the wild type and npq1 mutant. Furthermore, PRI was strongly correlated with qZ, meaning that qZ reflects the xanthophyll cycle. Together, these measurements demonstrated that simultaneous measurement of leaf reflectance and chlorophyll fluorescence is a valid approach for parameter evaluation.

We describe a new technical approach to study photosynthetic responses in higher plants involving simultaneous measurements of chlorophyll a fluorescence and leaf reflectance using a PAM and a spectral radiometer for the detection of signals from the same leaf area in Arabidopsis.

Evaluación de comportamientos fotosintéticos mediante mediciones simultáneas de la reflectancia de la hoja y análisis de fluorescencia de clorofila

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many ...