Este trabajo fue apoyado por becas de LAP (PI): Institutos Nacionales de Salud R21 HD50558, otorgado 2005-07; Nacional Institutos Nacionales de Salud R01 HD045822, otorgado 2004-09; Dana beca de la Fundación, otorgado 2004-06, la Fundación Canadiense para la Innovación (&quot;TPI&quot; de subvención), otorgado 2008-2012, de Ontario Research Fund Grant, otorgado 2008-2012.

Parte 1: Antes de la llegada de los participantes en el laboratorio <ol><li> Asegúrese de que la habitación esté libre de artículos extraños que pueden ser una distracción para el participante. </li><li> Set-up y cargar el protocolo experimental en el fNIRS Hitachi ETG-4000. </li><li> Configure su paradigma experimental. Paradigmas experimentales se pueden programar con el software de presentación diferente, incluyendo Eprime, Presentación, Psyscope o una caja de herramientas Matlab psicología basada. Aquí se utiliza caja de herramientas Matlab psicología basada. </li><li> El momento es clave para el análisis de datos, por lo tanto el paradigma experimental debe estar perfectamente sincronizada con la recopilación de datos. El Hitachi fNIRS ETG-4000 ha provocando capacidades, teniendo en cuenta el paradigma experimental para iniciar la recolección de datos, o viceversa. Prueba de activación del programa de presentación de fNIRS Hitachi ETG-4000. Activación se puede hacer utilizando en paralelo, en serie, o puertos USB. Aquí mostramos activación a través del puerto paralelo. </li><li> Antes de iniciar el estudio fNIRS es importante para llevar a cabo selección de los participantes del fondo. En el laboratorio Petitto, llevamos a cabo la investigación de antecedentes por tener los participantes o sus padres llenar adecuado estudio de los cuestionarios estandarizados 29. </li></ol> Participante llega <ol start="6"><li> Es importante llevar a cabo la sesión y para tratar a los participantes de una manera profesional. El participante o los padres de los participantes / tutores legales deben firmar un formulario de consentimiento antes de que el experimento comienza. Es de vital importancia dar las gracias al participante por su tiempo en estos experimentos importantes y emocionantes. </li><li> El participante está sentado cómodamente cerca de la sala de pruebas fNIRS. Un participante niño puede estar sentado en el regazo de sus padres. </li></ol> Parte 2: Colocación de Optodes y Uso del sistema de 10-20 Otro componente del método de análisis que permite la interpretación de datos consistente es la estandarización del protocolo de grabación fNIRS. Esto implica la colocación de optode, el posicionamiento de los participantes, y la activación de software de presentación de estímulo. Tanto la precisión neuro-anatómico de colocación de sondas y la confirmación de las regiones de interés (ROI) se consiguen mediante el sistema de 10 a 20 3,4,30. Además, la localización estereotáctica de la matriz de la sonda fue confirmada en el cráneo de los participantes mediante la superposición de información de seguimiento en 3D de un sistema de Polhemus Fast Trak en una resonancia magnética anatómica co-registro de escaneo de los participantes realizó con cápsulas de vitamina E colocados en cada ubicación de la sonda 3,4. posicionamiento óptimo de los participantes consistía en colocar a los participantes cómodamente en una silla reclinable, con fibra óptica que cuelgan libremente, sin contacto con el cuerpo o en la silla. <ol><li> Las mediciones se toman después de la cabeza con una cinta métrica y por escrito en la hoja de datos de los participantes: <ul><li> Nasion al inion alrededor </li><li> Nasion hasta Inions sobre la parte superior </li><li> Oreja a oreja sobre la parte superior </li></ul></li><li> Cinta quirúrgica puede ser utilizado para marcar lugares específicos de destino. En este experimento vamos a marcar Fp, T3/T4, F8/F7 </li><li> Matrices Optode se colocan en la cabeza de los participantes con optodes específica anclada en 10-20 puntos según las indicaciones de los propósitos del experimento. </li></ol> Parte 4: Prueba de la matriz Optode <ol><li> Introducción a la Hitachi ETG-4000 Interface GUI y pruebas de la sonda. </li><li> Prueba de la señal: optodes Una vez se colocan en el cuero cabelludo de los participantes, la calidad de la señal se pone a prueba. Si un optode no tiene una señal clara, los investigadores retire suavemente el pelo de la conexión de la optode y el cuero cabelludo. En ocasiones, la optodes posible que tenga que ser limpiado con una gasa con alcohol. </li></ol> Parte 5: Ejecutar el experimento. <ol><li> Al menos dos experimentadores debe estar siempre presente en la sala, una observación de la Hitachi fNIRS ETG-4000 en tiempo real, leer y observar el otro participante. Tener una cámara de vídeo centrado en el participante es muy recomendable para las observaciones post-hoc. Una de las ventajas de la Hitachi fNIRS ETG-4000 es que el vídeo y la señal de fNIRS están sincronizados y co-registrados. Un registro que contiene toda la información pertinente y los archivos generados se mantiene. </li><li> Hay métodos bien establecidos de la construcción de paradigmas experimentales hemodinámica, es decir, el diseño de bloques y diseños relacionados con eventos. Para una descripción más completa, por favor vea el artículo de revisión reciente 31. </li></ol> Parte 6: Análisis Una vez que todos los datos han sido recogidos, el participante se le agradece por su tiempo y voluntad de participar y sale del laboratorio. Como el análisis no se realiza en el Hitachi fNIRS ETG-4000, ya que, en cambio, los datos se exportan a un equipo de análisis. <ol><li> La conversión de mV a las concentraciones de hemoglobina. Como los valores de atenuación de primas se recogen en la atenuación de la fuerza de láser (medida en mV), estos valores deben ser convertidos a la hemoglobina oxigenada y desoxigenada valiosaes. Esto se hace con el archivo de Beer-Lambert ecuación. </li><li> La aplicación de la modificación de Beer-Lambert se lleva a cabo en dos etapas. Bajo el supuesto de que la dispersión es constante a lo largo de la longitud del camino, en primer lugar la atenuación para cada longitud de onda (ΔA λ (t)) se calcula mediante la comparación de la densidad óptica de la intensidad de la luz durante la tarea (tarea I) a la línea de base calculada de la señal ( Yo línea de base). Los valores de ΔA para cada longitud de onda y el punto de muestreo de tiempo (t) para resolver el modificado la ecuación de Beer-Lambert. <table border="0"><tbody><tr><td> La ecuación 1 <img style="float:left;" src="/files/ftp_upload/1268/eq1.jpg" alt="la ecuación 1" /></td><td> La ecuación 2 <img style="float:left;" src="/files/ftp_upload/1268/eq2.jpg" alt="la ecuación 2" /></td></tr></tbody></table></li></ol> λ 1 ∈ desoxi, λ 1 ∈ oxi, λ 2 λ ∈ desoxi y 2 ∈ oxígeno son las constantes de los coeficientes de extinción que medir la fracción de luz perdida a la absorción por unidad de distancia de concentración en el tejido. La desoxi C resultante y los valores de C son las concentraciones de oxígeno de la hemoglobina desoxigenada y oxigenada para cada t. Parte 7: Resultados de Representante Los resultados típicos respuesta hemodinámica en varias características distintivas. En la respuesta de oxihemoglobina, que es la primera característica de un baño. Esta caída se produce como una región de neuronas se activa y se agota el oxígeno disponible. A medida que aumenta el flujo de sangre, transportado por la hemoglobina oxigenada, la respuesta de oxihemoglobina aumenta rápidamente por encima de los niveles de referencia inicial a un nivel de estado estacionario. Cuando la región ya no se activa, la respuesta de la hemoglobina se desintegra oxi-durante los próximos 12 a 15 segundos y lentamente vuelve a caer a los niveles basales. Hay de vez en cuando un undershoot que ocurre antes de la respuesta hemodinámica de regresar a los niveles de referencia inicial. Los malos resultados son por lo general en forma de optodes no bien asentada en el cuero cabelludo o el movimiento excesivo. Este tipo de ruido - llamado &quot;Flatling &#39;- son evidentes en la señal de que los valores de microvoltios saturar y una serie de canales diferentes, se mueven tanto y la respuesta de oxi-desoxi-de una manera coordinada. DEMOSTRACIÓN: Agitar la fibra óptica. Análisis estadístico: El oxígeno extraído y deoxi-hemoglobina valores para cada canal, para cada participante y para cada tarea puede ser sometido a un análisis estadístico convencional, incluyendo pruebas t, ANOVA, correlación, etc

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Neurophysiol. 10, 370-371 (1958).</li><li>Amaro, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Brain Cogn. 60, 220-232 (2006).</li></ol>

The authors have nothing to disclose.

En este estudio, hemos demostrado el uso de una novela, no invasiva, la tecnología de imagen cerebral para investigar fNIRS funcionamiento del cerebro humano en relación con la cognición y la percepción humanas. fNIRS imágenes del cerebro puede representar el futuro de la imagen cerebral no invasiva, especialmente con las poblaciones de lactantes y niños, que algún día podría estar ampliamente disponible en los laboratorios de investigación, consultorios médicos, y en el sistema escolar permite a los médicos...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>ETG-4000</td>
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<td>Matlab</td>
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<td>The Mathworks</td>
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exploring cognitive functions in babies, children &amp; adults with near infrared spectroscopy

Una explosión de funcionales espectroscopia de infrarrojo cercano (fNIRS) estudios de investigación de la activación cortical en relación con los procesos cognitivos superiores, tales como el lenguaje 1,2,3,4,5,6,7,8,9,10, memoria 11, y la atención 12 está en marcha en todo el mundo los adultos involucran, niños y bebés 3,4,13,14,15,16,17,18,19 con la cognición típicos y atípicos 20,21,22. El reto actual de la utilización de fNIRS de la neurociencia cognitiva es lograr un análisis sistemático de estos datos que son universalmente interpretable 23,24,25,26, y por lo tanto puede avanzar importantes cuestiones científicas sobre la organización funcional y los sistemas neurales que subyacen a la cognición humana superior. Las tecnologías de neuroimagen han resolución espacial o temporal sea menos robusto. Eventos relacionados encefalografía Potenciales y Magneto (ERP y MEG) tienen una resolución temporal excelente, mientras que la tomografía por emisión de positrones y resonancia magnética funcional (fMRI y PET) tienen una mejor resolución espacial. El uso no ionizantes longitudes de onda de la luz en el rango del infrarrojo cercano (700-1000 nm), donde es preferentemente oxihemoglobina absorbe 680 nm y deoxi-hemoglobina es preferentemente absorbida por 830 nm (por ejemplo, de hecho, las longitudes de onda muy cableado en la fNIRS Hitachi ETG-400 del sistema se muestra aquí), fNIRS es muy adecuado para el estudio de la cognición superior porque tiene una buena resolución temporal (~ 5 años) sin el uso de la radiación y la buena resolución espacial (~ 4 cm de profundidad), y no exigir a los participantes a estar en una estructura cerrada 27,28. Actividad cortical participantes pueden ser evaluados, cómodamente sentados en una silla ordinaria (adultos, niños) o incluso sentado en el regazo de mamá s (niños). Cabe destacar que NIRS es el único portátil (del tamaño de una computadora de escritorio), prácticamente en silencio, y puede tolerar un movimiento sutil participantes. Esto es particularmente excepcional para el estudio neuronal del lenguaje humano, que necesariamente tiene como uno de sus componentes clave del movimiento de la boca en la producción del habla o de las manos en el lenguaje de señas. La forma en que se localiza en la respuesta hemodinámica es un conjunto de emisores láser y detectores. Emisores emiten una intensidad conocida de no ionizantes luz, mientras que los detectores de detectar la cantidad reflejada desde la superficie cortical. Cuanto más cerca estén los optodes, mayor es la resolución espacial, mientras que los más alejados de la optodes, la mayor profundidad de la penetración. Para el fNIRS Hitachi ETG-4000 la penetración óptima del sistema / resolución de la matriz optode se encuentra a 2 cm. Nuestro objetivo es demostrar nuestro método de adquisición y análisis de datos fNIRS para ayudar a estandarizar el campo y permitir que los laboratorios de todo el mundo fNIRS diferentes para tener un fondo común.

Watch this Scientific Journal Video about Exploring Cognitive Functions in Babies, Children & Adults with Near Infrared Spectroscopy at JoVE.com

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

Aquí se describe un método de recolección de datos y análisis de datos para funcionales espectroscopia de infrarrojo cercano (fNIRS), una novela de sistema no invasivo usa imágenes del cerebro en la neurociencia cognitiva, especialmente en el estudio del desarrollo del cerebro del niño. Este método proporciona un estándar universal de adquisición de datos y el análisis fundamental para la interpretación de datos y descubrimientos científicos.

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 ...

exploring-cognitive-functions-babies-children-adults-with-near

Research

JoVE Journal

Biology

20.4K vistas.  University of Michigan, Ann Arbor. Aquí se describe un método de recolección de datos y análisis de datos para funcionales espectroscopia de infrarrojo cercano (fNIRS), una novela de sistema no invasivo usa imágenes del cerebro en la neurociencia cognitiva, especialmente en el estudio del desarrollo del cerebro del niño. Este método proporciona un estándar universal de adquisición de datos y el análisis fundamental para la interpretación de datos y descubrimientos científicos.

Video: Exploring Cognitive Functions in Babies, Children & Adults with Near Infrared Spectroscopy

Recordings of Neural Circuit Activation in Freely Behaving Animals

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. Traditional non-invasive methods require at least partially restrained research subjects, and they only allow identification of large numbers of simultaneously activated neurons. On the other hand, small ensembles of neurons or individual neurons can only be measured using single-cell recordings obtained from largely reduced preparations. Since the expression of natural behavior is limited in restrained and dissected animals, the underlying neural mechanisms that control such behavior are difficult to identify. 
Here, I present a non-invasive physiological technique that allows measuring neural circuit activation in freely behaving animals. Using a pair of wire electrodes inside a water-filled chamber, the bath electrodes record neural and muscular field potentials generated by juvenile crayfish during natural or experimentally evoked escape responses. The primary escape responses of crayfish are mediated by three different types of tail-flips which move the animals away from the point of stimulation. Each type of tail-flip is controlled by its own neural circuit; the two fastest and most powerful escape responses require activation of different sets of large &#8220;command&#8221; neurons. In combination with behavioral observations, the bath electrode recordings allow unambiguous identification of these neurons and the associated neural circuits. Thus activity of neural circuitry underlying naturally occurring behavior can be measured in unrestrained animals and in different behavioral contexts.

Non-invasive measurements of neural activity patterns in freely behaving animals are obtained by combining neurophysiological recordings with high speed videography.

Grabaciones de la activación de circuitos neuronales en animales comportarse libremente

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. ...

Investigación

Biología

Assembly, Tuning and Use of an Apertureless Near Field Infrared Microscope for Protein Imaging

This paper aims to instruct the reader in the assembly and operation of an infrared near-field microscope for imaging beyond the diffraction limit.  The apertureless near-field microscope is a light scattering-type instrument that provides infrared spectra at circa 20 nm resolution. A complete list of components and a step-by-step protocol for use is provided.  Common errors in assembly and instrument tuning are discussed.  A representative data set that shows the secondary structure of an amyloid fibril is presented.  

The assembly of a nearfield infrared microscope for imaging protein aggregates is described.

Montaje, ajuste y uso de un microscopio de campo Apertureless de Infrarrojo Cercano para la imagen de proteínas

This paper aims to instruct the reader in the assembly and operation of an infrared near-field microscope for imaging beyond the diffraction limit.  The ...

Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG

Magnetoencephalography is a technique that detects magnetic fields associated with cortical activity [1]. The electrophysiological activity of the brain generates electric fields  - that can be recorded using electroencephalography (EEG)- and their concomitant magnetic fields  - detected by MEG. MEG signals are detected by specialized sensors known as superconducting quantum interference devices (SQUIDs). Superconducting sensors require cooling with liquid helium at -270 &deg;C. They are contained inside a vacumm-insulated helmet called a dewar, which is filled with liquid. SQUIDS are placed in fixed positions inside the helmet dewar in the helium coolant, and a subject's head is placed inside the helmet dewar for MEG measurements. The helmet dewar must be sized to satisfy opposing constraints. Clearly, it must be large enough to fit most or all of the heads in the population that will be studied. However, the helmet must also be small enough to keep most of the SQUID sensors within range of the tiny cerebral fields that they are to measure. Conventional whole-head MEG systems are designed to accommodate more than 90% of adult heads. However adult systems are not well suited for measuring brain function in pre-school chidren whose heads have a radius several cm smaller than adults. The KIT-Macquarie Brain Research Laboratory at Macquarie University uses a MEG system custom sized to fit the heads of pre-school children. This child system has 64 first-order axial gradiometers with a 50 mm baseline[2] and is contained inside a magnetically-shielded room (MSR) together with a conventional adult-sized MEG system [3,4]. There are three main advantages of the customized helmet dewar for studying children. First, the smaller radius of the sensor configuration brings the SQUID sensors into range of the neuromagnetic signals of children's heads. Second, the smaller helmet allows full insertion of a child's head into the dewar. Full insertion is prevented in adult dewar helmets because of the smaller crown to shoulder distance in children. These two factors are fundamental in recording brain activity using MEG because neuromagnetic signals attenuate rapidly with distance. Third, the customized child helmet aids in the symmetric positioning of the head and limits the freedom of movement of the child's head within the dewar. When used with a protocol that aligns the requirements of data collection with the motivational and behavioral capacities of children, these features significantly facilitate setup, positioning, and measurement of MEG signals.

The advent of MEG systems sized for young children opens important new opportunities to study brain development. The new system, together with a protocol that aligns experimental requirements with the capacities of children, can be used to study cognitive and language processes in healthy, awake children aged three to six.

La medición de la función cerebral neuromagnetic En Pre-escolar Niños con tamaño personalizado MEG

Magnetoencephalography is a technique that detects magnetic fields associated with cortical activity [1]. The electrophysiological activity of the brain ...

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 ...

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

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and exercise. However, these approaches do not address the underlying causes of balance decline. Using mice, the impact of near infrared light (NIr) on the metabolism of cells in the vestibular sensory epithelium was assessed. Data collected shows that this simple and safe intervention may protect these vulnerable cells from the deleterious effects of natural aging. mRNA was extracted from the isolated peripheral vestibular sensory epithelium (crista ampullaris and utricular macula) and subsequently transcribed into a cDNA library. This library was then probed for the expression of ubiquitous antioxidant (SOD-1). Antioxidant gene expression was then used to quantify cellular metabolism. Using transcranial delivery of NIr in young (4 weeks) and older (8 - 9 months) mice, and a brief treatment regime (90 sec/day for 5 days), this work suggests NIr alone may be sufficient to improve mitochondrial function in the vestibular sensory epithelium. Since there are currently no available, affordable, non-invasive methods of therapy to improve vestibular hair cell function, the application of external NIr radiation provides a potential strategy to counteract the impact of aging on cellular metabolism inthe vestibular sensory epithelium.

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

Mitochondrial dysfunction is a hallmark of cellular senescence. This paper uses non-invasive near-infrared (NIr) treatment to improve mitochondrial function in the aging mouse vestibular sensory epithelium.

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 ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

Reconstitución funcional y la actividad del canal Las mediciones de Purificada Wildtype y mutante CFTR Proteína

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision

Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA interactions. ChIP coupled to high throughput sequencing (ChIP-seq) is commonly used to determine the genomic location of proteins that interact with chromatin. However, ChIP-seq is hampered by relatively low mapping resolution of several hundred base pairs and high background signal. The ChIP-exo method is a refined version of ChIP-seq that substantially improves upon both resolution and noise. The key distinction of the ChIP-exo methodology is the incorporation of lambda exonuclease digestion in the library preparation workflow to effectively footprint the left and right 5&#39; DNA borders of the protein-DNA crosslink site. The ChIP-exo libraries are then subjected to high throughput sequencing. The resulting data can be leveraged to provide unique and ultra-high resolution insights into the functional organization of the genome. Here, we describe the ChIP-exo method that we have optimized and streamlined for mammalian systems and next-generation sequencing-by-synthesis platform.

Here, we present a protocol to achieve near base pair resolution of protein-DNA interactions. This is obtained by exonuclease treatment of DNA fragments selectively enriched by chromatin immunoprecipitation (ChIP-exo) followed by high throughput sequencing.

El Método chip-exo: La identificación de las interacciones proteína-ADN con zona de Base Par de precisión

Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA ...

Production of Near-Infrared Sensitive, Core-Shell Vaccine Delivery Platform

Vaccine delivery strategies that can limit the exposure of cargo to organic solvent while enabling novel release profiles are crucial for improving immunization coverage worldwide. Here, a novel injectable, ultraviolet- curable and delayed burst release- enabling vaccine delivery platform called polybubbles is introduced. Cargo was injected into polyester-based polybubbles that were formed in 10% carboxymethycellulose -based aqueous solution. This paper includes protocols to maintain spherical shape of the polybubbles and optimize cargo placement and retention to maximize the amount of cargo within the polybubbles. To ensure safety, chlorinated solvent content within the polybubbles were analyzed using neutron activation analysis. Release studies were conducted with small molecules as cargo within the polybubble to confirm delayed burst release. To further show the potential for on-demand delivery of the cargo, gold nanorods were mixed within the polymer shell to enable near-infrared laser activation.

This article describes the protocols used to produce a novel vaccine delivery platform, &#34;polybubbles,&#34; to enable delayed burst release. Polyesters including poly(lactic-co-glycolic acid) and polycaprolactone were used to form the polybubbles and small molecules and antigen were used as cargo.

Producción de la plataforma de entrega de vacunas Core-Shell, sensible al infrarrojo cercano

Vaccine delivery strategies that can limit the exposure of cargo to organic solvent while enabling novel release profiles are crucial for improving ...

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity is characterized by an increase in adipose tissue (AT) mass due to adipocyte hyperplasia and/or hypertrophia, leading to profound remodeling of its three-dimensional structure. Indeed, the maximal capacity of AT to expand during obesity is pivotal to the development of obesity-associated pathologies. This AT expansion is an important homeostatic mechanism to enable adaptation to an excess of energy intake and to avoid deleterious lipid spillover to other metabolic organs, such as muscle and liver. Therefore, understanding the structural remodeling that leads to the failure of AT expansion is a fundamental question with high clinical applicability. In this article, we describe a simple and fast clearing method that is routinely used in our laboratory to explore the morphology of mouse and human white adipose tissue by fluorescent imaging. This optimized AT clearing method is easily performed in any standard laboratory equipped with a chemical hood, a temperature-controlled orbital shaker and a fluorescent microscope. Moreover, the chemical compounds used are readily available. Importantly, this method allows one to resolve the 3D AT structure by staining various markers to specifically visualize the adipocytes, the neuronal and vascular networks, and the innate and adaptive immune cells distribution.

Here, we describe a simple, inexpensive and fast clearing method to resolve the 3D structure of both mouse and human white adipose tissue using a combination of markers to visualize vasculature, nuclei, immune cells, neurons, and lipid-droplet coat proteins by fluorescent imaging.

Explorar la estructura del tejido adiposo mediante la limpieza de metilsalicilatos y las imágenes 3D

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity ...

Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of both conventional and unconventional myosins under load with unprecedented time resolution. In particular, the possibility to probe myosin motors under constant force right after the actin-myosin bond formation, together with the high rate of the force feedback (200 kHz), has shown UFFCS to be a valuable tool to study the load dependence of fast dynamics such as the myosin working stroke. Moreover, UFFCS enables the study of how processive and non-processive myosin-actin interactions are influenced by the intensity and direction of the applied force.
By following this protocol, it will be possible to perform ultrafast force-clamp experiments on processive myosin-5 motors and on a variety of unconventional myosins. By some adjustments, the protocol could also be easily extended to the study of other classes of processive motors such as kinesins and dyneins. The protocol includes all the necessary steps, from the setup of the experimental apparatus to sample preparation, calibration procedures, data acquisition and analysis.

Presented here is a comprehensive protocol to perform ultrafast force-clamp experiments on processive myosin-5 motors, which could be easily extended to the study of other classes of processive motors. The protocol details all the necessary steps, from the setup of the experimental apparatus to sample preparation, data acquisition and analysis.

Disección de las propiedades mecanoenzimáticas de miosinas procesivas con espectroscopia ultrarrápida de pinza de fuerza

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of ...