Los autores desean agradecer al Dr. Paul Witting y Genevieve Fong por su ayuda con la extracción de ARNm y PCR, y la Garnett Passe y Rodney Williams Memorial Fundación para el apoyo.

Declaración de Ética: Todos los procedimientos descritos a continuación fueron aprobados por la Universidad de Comité de Ética Animal Sydney. 1. Los animales NOTA: 1 y 8 - Un 9 meses de edad ratones (C57 / BL6) se obtuvieron del Centro de Recursos Animales (Perth, Australia). Los animales fueron alojados en el Fondo para Bosch de roedores en la Universidad de Sydney. <ol><li> Ratones Casa en jaulas estándar del ratón en un 12/12 horas de luz / oscuridad ciclo con el acceso a alimentos y agua ad libitum. </li><li> Divida a los ratones en cada grupo de edad en el infrarrojo cercano (NIR) tratados o tratamiento simulado (control) grupos de comparación. </li></ol> 2. Infrarrojo Cercano (NIR) Irradiación y Sham Tratamiento <ol><li> Afeitarse la piel en la región de la cabeza y el cuello del ratón con una afeitadora eléctrica tan estrechamente como sea posible para prevenir el rebrote rápida del cabello antes de la finalización del régimen de tratamiento. A fin de mantener el estado libre de patógenos de los ratones, utilice etanol al 70%para limpiar instrumentos entre los animales afeitado y desinfectante veterinaria F10 entre los animales de jaulas separadas. Haga esto 2-3 días antes del comienzo del tratamiento para que los animales no son excesivamente manipulado. </li><li> Restringir ratón manteniendo el extremo proximal de la cola y dejar que se relaje en la palma de una mano o de banco con el fin de minimizar el estrés que podría conducir a resultados falsos. </li><li> Sostenga el NIR (670 nm) LED (diodo emisor de luz) dispositivo 1-2 cm de distancia de la zona expuesta (rebajado) y encienda el dispositivo durante 90 segundos (Figura 1). NOTA: El cambio de temperatura como resultado de 90 segundos de exposición se midió como &lt;0,2 ° C en 100 ml de agua. </li><li> Repita los pasos 2.2 a 2.3 para el grupo de tratamiento simulado de los animales, pero deje el dispositivo apagado (Figura 1B). </li><li> Repita los pasos 2.2 a 2.3 para el grupo de tratamiento bloqueado-NIR de los animales, pero cubre el dispositivo con papel de aluminio. </li><li> Repita los pasos 2.2 a 2.5 días a las approximatintervalos hr e 24 durante 5 días consecutivos. </li><li> Extraer el epitelio vestibular sensorial (3 crestas y 1 mácula utricular) de ambos oídos en el post-tratamiento quinto día (véase la sección 3). </li></ol> 3. Extracción de tejido 20 <ol><li> Preparar 300 ml de un fluido cefalorraquídeo artificial (ACSF) basado en glicerol que consiste en (en mM): 26 NaHCO3, 11 glucosa, 250 glicerol, 2,5 KCl, 1,2 NaH 2 PO 4, 1,2 MgCl2, y 2.5 CaCl 2. Antes de la adición de CaCl2, gas la solución con carbógeno (95% de O2 y 5% de CO 2) para establecer un pH de 7,4 y evitar la precipitación de calcio (nubosidad). Enfriar la solución en un congelador a -80ºC durante 45 min de manera que se forma una suspensión de hielo. </li><li> Preparar el tampón de lisis aislamiento de ARN en microtubos de tapa de rosca marcados (se detiene el estallido de las tapas cuando los cambios de presión del tubo entre la congelación y descongelación en nitrógeno líquido) de acuerdoa las instrucciones del fabricante o las prácticas habituales de laboratorio. Tener nitrógeno líquido listo en un frasco Dewar de aluminio. NOTA: Utilice ropa protectora y eyeware al manejar nitrógeno líquido. Asegúrese de que se utiliza nitrógeno líquido en una habitación bien ventilada, ya que el volumen de su forma de gas es aproximadamente 700 veces mayor que la de su forma líquida y puede causar asfixia. </li><li> Profundamente anestesiar a los ratones con ketamina (400 mg / kg) a través de una inyección intra-peritoneal. Deje que el reflejo de las extremidades posteriores se desplome por completo como indicación de que los ratones están completamente anestesiados. </li><li> Decapitar a los ratones con tijeras de acero inoxidable afiladas y hacer una incisión a lo largo de la piel sagital del cráneo usando una cuchilla de afeitar (redondeado # 22). En este punto y los pasos a lo largo de 3.5 a 3.9, mantener la bóveda del cráneo, el cerebro y aparato vestibular subyacente lo más fresco posible por la aplicación regular de ACSF helada sobre el tejido. </li><li> Utilizando el brazo con punta de tijeras patrón estándar hacer una Small incisión en el cráneo en Lambda y cortar a lo largo de la sutura sagital. </li><li> Deslice suavemente un brazo de una gubia poco profundas debajo del hueso parietal de mantenimiento de la cuchilla lo más cerca posible a la superficie inferior del hueso sin arrastrar el cerebro. Seguros y alejarse del hueso parietal lateral y el hueso occipital posterior hasta que se vea el cerebro. </li><li> Use una pequeña espátula de acero inoxidable y levante el cerebro lejos de la anterior y la fosa craneal media para exponer el nervio vestíbulo coclear (CN VIII). Seccionar el nervio para evitar tensiones innecesarias en los axones aferentes primarias que inervan directamente las células ciliadas vestibulares. </li><li> Retire el cerebro en su totalidad después de la sección del CN VIII. </li><li> Observe el laberinto óseo contiene la cóclea y los órganos vestibulares periféricas en la fosa craneal media. Haga dos incisiones pequeñas al lado de cada laberinto óseo y extirpar toda la estructura sosteniendo el canal semicircular anterior y tirando despuésaliado. </li><li> Inmediatamente sumerja laberintos extirpados en un plato de la disección de la solución que contiene ACSF enfriado en hielo (como se describe en el paso 3.1), mientras continua perfusión con carbógeno. </li><li> Bajo un microscopio estereoscópico, mantenga pulsado el laberinto de la cóclea y fijarlo a la parte inferior del plato con fórceps. <ol><li> El uso de fórceps recto finas rayar una pequeña abertura en el hueso por encima del canal semicircular (SSC) ampolla anterior. </li><li> Ampliar suavemente esta apertura al llegar inmediatamente debajo del hueso y chasqueando hacia el exterior lejos de la ampolla. Tenga precaución para no empujar fórceps en la abertura y dañar el laberinto membranoso frágil continuación. Continuar de esta manera hasta el utrículo, anterior y lateral ampollas están expuestos. Si es posible eliminar la ampolla posterior también. </li></ol></li><li> Con unas pinzas finas, levante suavemente el utrículo y ampollas lejos del laberinto óseo hasta que estén completamente separados. Siempre que sea posible, mantenerlospor sus canales semicirculares asociados para evitar dañar el epitelio sensorial. En algunos casos, puede ser necesario cortar con tijeras iris para liberar las ampollas de la médula la parte proximal del conducto membranoso semicircular. </li><li> Agarrar firmemente los órganos vestibulares entre la punta del fórceps y el lugar en el tampón de lisis preparado anteriormente en el paso 3.2. Agite suavemente la pinza alrededor en el búfer para asegurar órganos vestibulares han desprendido de los fórceps. Compruebe este es el caso de llevar a los fórceps bajo el microscopio estereoscópico. <ol><li> Tornillo en la tapa de los microtúbulos y congelar la muestra en nitrógeno líquido inmediatamente. </li></ol></li></ol> 4. Extracción de ARN y RT-PCR <ol><li> Siga métodos estándar de ARN mensajero (ARNm) de extracción de acuerdo con las instrucciones del fabricante o protocolos de laboratorio preferidos. NOTA: Un kit comercial que utilizó ARN portador para ayudar a aislar pequeños rendimientos de ARNm se utilizó en esteprotocolo. Volúmenes de elución más bajas pueden ser usados ​​para aumentar las concentraciones finales de mRNA. NOTA: Negativo controles &quot;sin enzima&quot; (CNE) y controles &quot;sin plantilla de ADN&quot; (NTC) también deben completarse para asegurar la validez de los efectos observados. </li><li> Aplicar métodos estándar de la transcripción inversa de ARNm en ADN complementario (ADNc) y la amplificación de los genes diana 21-23. </li></ol>

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Los autores declaran que no tienen intereses financieros en competencia.

Los resultados representativos descritos aquí muestran que breve entrega transcraneal de la luz NIR (90 seg / día durante 5 días) es suficiente para elevar los niveles de expresión de antioxidantes en ratones de edad avanzada en comparación con los ratones tratados con placebo. Mientras que el calor emitido podría representar una fuente de mitocondrial y / o activación neuronal, según lo informado por vestibular rata aferentes 24 - nuestra medición del calor emitido por el NIR LED dispositivo fue &lt...

Disminución en los resultados equilibrio y caídas posteriores son comunes, y por desgracia a menudo características definitorias de envejecimiento natural 1. El impacto de esta disminución puede ser tanto físico y social, y reduce significativamente la calidad de vida de las personas mayores. En respuesta, las terapias físicas y de rehabilitación han sido el foco de la investigación de cataratas, pero no se han asociado con la reducción constante en la prevalencia de caídas repetidas. Al mismo tiempo, el trabajo de investigación de los cambios en el sistema vestibular periférico o central (el sistema responsable de mantener el equilibrio) es escasa, y posibles estrategias terapéuticas dirigidas a estos sistemas y las causas subyacentes del desequilibrio limitado. Trabajos recientes sobre los trastornos neurodegenerativos asociados con la edad, incluyendo la degeneración macular relacionada con la edad 2-4, modelos de enfermedad de Alzheimer 5-8, y la enfermedad de Parkinson 9-12 han demostrado efectos neuroprotectores de simple aplicación no invasiva de infrarrojo cercano (NIR) la luz. Además, en el sistema vestibular, NIR se ha utilizado para aumentar la actividad de las neuronas aferentes primarias vestibulares in vitro 13. Aunque no se entiende bien el mecanismo de la luz NIR, la mayoría de los estudios que utilizan NIR han sugerido que NIR estimula las mitocondrias complejo IV (citocromo c oxidasa) 14-17 para facilitar el metabolismo celular. En el epitelio sensorial vestibular la placa subcuticular de células que el cabello de tipo es denso en las mitocondrias de 18 años y, como tal, puede representar un punto de acción para el tratamiento terapéutico NIR. Aquí, una breve régimen, el tratamiento no invasivo de transcraneal aplica NIR que se puede utilizar para medir el metabolismo celular (y por implicación, la función mitocondrial) en el ratón epitelio sensorial vestibular se describe. También se discutió es una preparación del epitelio sensorial vestibular y se muestra que NIR aumenta la expresión de un ubiquitonos anti-oxidante (superóxido dismutasa 1) en el epitelio sensorial - previamente demostrado ser importante para la supervivencia de las células ciliadas cóclea 19.

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			<td height="21" style="height:21px;width:216px;">Isolate II RNA Micro Kit</td>
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near infrared (nir) light increases expression of a marker of mitochondrial function in the mouse vestibular sensory epithelium

Las estrategias para atenuar la disminución en la función del equilibrio con la edad son predominantemente centrada en terapias físicas que incluyen tareas de equilibrio y ejercicios. Sin embargo, estos enfoques no abordan las causas subyacentes del declive de equilibrio. Utilizando ratones, se evaluó el impacto de la luz del infrarrojo cercano (NIR) en el metabolismo de las células en el epitelio sensorial vestibular. Los datos recogidos muestran que esta intervención simple y seguro puede proteger estas células vulnerables de los efectos perjudiciales del envejecimiento natural. ARNm se extrajo a partir del epitelio sensorial vestibular periférica aislada (ampullaris crista y la mácula del utrículo) y posteriormente se transcribe en una biblioteca de ADNc. Después, esta biblioteca se probaron para la expresión de antioxidante ubicua (SOD-1). Entonces Antioxidante expresión génica se utilizó para cuantificar el metabolismo celular. Utilizando la entrega transcraneal de Nir en jóvenes (4 semanas) y mayores (8-9 meses) los ratones, y un régimen de tratamiento breve (90 seg / día durante 5 díasays), este trabajo sugiere Nir solo puede ser suficiente para mejorar la función mitocondrial en el epitelio sensorial vestibular. Dado que actualmente no hay métodos disponibles, asequibles, no invasivos de la terapia para mejorar la función de las células ciliadas vestibulares, la aplicación de radiación NIR externa proporciona una posible estrategia para contrarrestar el impacto del envejecimiento en el metabolismo celular enel epitelio sensorial vestibular.

Para comparar el impacto del tratamiento en Nir joven (4 semanas) y mayores (8-9 meses) los ratones que mide la expresión de antioxidante superóxido dismutasa 1 (SOD-1) en jóvenes (n = 16) y mayores (n = 20) Los ratones que fueron NIR-tratada, tratada simulada, o bloqueado-NIR. La Figura 2 muestra un aumento significativo en normalizado SOD-1 la expresión de β-actina de más de 2 veces en los animales jóvenes tratados con NIR jóvenes en comparación con los animales tratados con placebo (p &lt;0,...

Watch this Scientific Journal Video about Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium at JoVE.com

Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

La disfunción mitocondrial es una característica de la senescencia celular. En este trabajo se utiliza el tratamiento no invasivo de infrarrojo cercano (NIR) para mejorar la función mitocondrial en el ratón epitelio sensorial vestibular envejecimiento.

Infrarrojo Cercano (NIR) Luz aumenta la expresión de un marcador de la función mitocondrial en el ratón vestibular sensorial Epitelio

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and ...

near-infrared-nir-light-increases-expression-marker-mitochondrial

Research

JoVE Journal

Biology

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

9.8K vistas.  University of Sydney.  La disfunción mitocondrial es una característica de la senescencia celular. En este trabajo se utiliza el tratamiento no invasivo de infrarrojo cercano (NIR) para mejorar la función mitocondrial en el ratón epitelio sensorial vestibular envejecimiento. 

Video: Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Exploring Cognitive Functions in Babies, Children &amp; Adults with Near Infrared Spectroscopy

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as language1,2,3,4,5,6,7,8,9,10, memory11, and attention12 is underway worldwide involving adults, children and infants 3,4,13,14,15,16,17,18,19 with typical and atypical cognition20,21,22. The contemporary challenge of using fNIRS for cognitive neuroscience is to achieve systematic analyses of data such that they are universally interpretable23,24,25,26, and thus may advance important scientific questions about the functional organization and neural systems underlying human higher cognition.
Existing neuroimaging technologies have either less robust temporal or spatial resolution. Event Related Potentials and Magneto Encephalography (ERP and MEG) have excellent temporal resolution, whereas Positron Emission Tomography and functional Magnetic Resonance Imaging (PET and fMRI) have better spatial resolution. Using non-ionizing wavelengths of light in the near-infrared range (700-1000 nm), where oxy-hemoglobin is preferentially absorbed by 680 nm and deoxy-hemoglobin is preferentially absorbed by 830 nm (e.g., indeed, the very wavelengths hardwired into the fNIRS Hitachi ETG-400 system illustrated here), fNIRS is well suited for studies of higher cognition because it has both good temporal resolution (~5s) without the use of radiation and good spatial resolution (~4 cm depth), and does not require participants to be in an enclosed structure27,28. Participants cortical activity can be assessed while comfortably seated in an ordinary chair (adults, children) or even seated in mom s lap (infants). Notably, NIRS is uniquely portable (the size of a desktop computer), virtually silent, and can tolerate a participants subtle movement. This is particularly outstanding for the neural study of human language, which necessarily has as one of its key components the movement of the mouth in speech production or the hands in sign language. 
The way in which the hemodynamic response is localized is by an array of laser emitters and detectors. Emitters emit a known intensity of non-ionizing light while detectors detect the amount reflected back from the cortical surface. The closer together the optodes, the greater the spatial resolution, whereas the further apart the optodes, the greater depth of penetration. For the fNIRS Hitachi ETG-4000 system optimal penetration / resolution the optode array is set to 2cm. 
Our goal is to demonstrate our method of acquiring and analyzing fNIRS data to help standardize the field and enable different fNIRS labs worldwide to have a common background.

Here we describe a data collection and data analysis method for functional Near Infrared Spectroscopy (fNIRS), a novel non-invasive brain imaging system used in cognitive neuroscience, particularly in studying child brain development. This method provides a universal standard of data acquisition and analysis vital to data interpretation and scientific discovery.

Explorar las funciones cognitivas en bebés, niños y adultos con Espectroscopia de Infrarrojo Cercano

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as ...

Investigación

Biología

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Utilizando el sistema de electroporación Gene Emisores MXcell para transfectar células primarias con alta eficiencia

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

Hi-C: Un método para estudiar la arquitectura tridimensional de los genomas.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

El uso de un contador de células automatizado para simplificar estudios de expresión génica: siRNA Derribo de IL-4 la expresión génica en células dependientes Namalwa

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

En la reconstitución in vitro de los activos T. castaneum telomerasa

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

El uso de alta resolución de tomografía computarizada para visualizar la estructura tridimensional y la función de la Planta Vasculatura

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

Perfiles de expresión de microARN de las células iPS humanas, de la retina del epitelio pigmentario Derivado de Ips y Fetal epitelio pigmentario de la retina

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Método para la Evaluación de los efectos de un rango de longitudes de onda e intensidades de / Terapia de luz del infrarrojo cercano Red sobre el estrés oxidativo<em&gt; In Vitro</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

Estudiar mitocondrial Estructura y función en<em&gt; Drosophila</em&gt; Los ovarios

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

Cultivos celulares primarios de células del epitelio pigmentario de la retina del ratón

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...