Ce travail a été soutenu par des subventions aux LAP (PI): National Institutes of Health R21 HD50558, décerné 2005-07; national Institutes of Health R01 HD045822, décerné 2004-09; Dana Foundation Grant, décerné 2004-06; Fondation canadienne pour l&#39;innovation («CFI» de subvention), décerné 2008-2012; Le Fonds ontarien de recherche sur Grant, décerné 2008-2012.

Partie 1: Avant d&#39;arriver participant au laboratoire <ol><li> S&#39;assurer que la salle est libre d&#39;articles superflus qui peuvent être dérangeantes pour le participant. </li><li> Mise en place et de la charge sur le protocole expérimental fNIRS Hitachi GTE-4000 du système. </li><li> Configurez votre paradigme expérimental. Paradigmes expérimentaux peuvent être programmés avec le logiciel de présentation différente, incluant ePRIME, Présentation, Psyscope ou une boîte à outils Matlab psychologie basée. Ici, nous utilisons boîte à outils Matlab psychologie basée. </li><li> Le timing est clé pour l&#39;analyse de données, donc le paradigme expérimental doit être parfaitement synchronisé avec la collecte de données. Le Hitachi fNIRS GTE-4000 a déclenchement des capacités, permettant ainsi le paradigme expérimental pour déclencher la collecte de données ou vice versa. Test de déclenchement du programme de présentation de fNIRS Hitachi GTE-4000. Le déclenchement peut être fait en utilisant en parallèle, des feuilletons, ou des ports USB. Ici, nous montrons le déclenchement via le port parallèle. </li><li> Avant de commencer l&#39;étude fNIRS il est important d&#39;effectuer la vérification des antécédents des participants. Dans le laboratoire Petitto, nous procédons à la vérification des antécédents en ayant les participants ou leurs parents de remplir d&#39;étude appropriés à des questionnaires standardisés 29. </li></ol> Participant arrive <ol start="6"><li> Il est important de mener la session et pour traiter les participants d&#39;une manière professionnelle. Le participant ou des parents des participants / tuteurs légaux doivent signer un formulaire de consentement avant le début de l&#39;expérience. Il est vital pour remercier les participants pour leur temps dans ces expériences importantes et passionnantes. </li><li> Le participant est assis confortablement près de la salle de test fNIRS. Un participant du nourrisson peut être assis sur les genoux d&#39;un parent. </li></ol> Partie 2: Placer optodes &amp; Utiliser le système de 10 à 20 Une autre composante de la méthode d&#39;analyse qui permet l&#39;interprétation des données cohérentes est la standardisation de l&#39;enregistrement fNIRS protocole. Cela implique le placement optode, le positionnement des participants, et le déclenchement d&#39;un logiciel de présentation de relance. Tant la précision neuro-anatomiques de placement des sondes et la confirmation des régions d&#39;intérêt (ROI) sont obtenus en utilisant le système de 10 à 20 3,4,30. En outre, la localisation stéréotaxique du tableau de sonde a été confirmé sur le crâne du participant en superposant des informations de tracking 3D à partir d&#39;un système de Polhemus Fast Trak sur une IRM anatomique de co-enregistrement de balayage du participant réalisé avec des capsules de vitamine E placés à chaque emplacement 3,4 sonde. positionnement optimal des participants impliqués placer des participants confortablement dans un fauteuil inclinable, avec la fibre optique ballants, sans contact avec le corps ou une chaise. <ol><li> Les mesures suivantes sont prises la tête avec un ruban à mesurer et écrit sur les données des participants de la feuille: <ul><li> Nasion d&#39;Inion autour </li><li> Nasion Inions sur le dessus </li><li> Une oreille à plus haut </li></ul></li><li> Ruban adhésif chirurgical peut être utilisé pour marquer les endroits cibles spécifiques. Dans cette expérience, nous célébrerons Fp, T3/T4, F8/F7 </li><li> Tableaux Optode sont placés sur la tête des participants avec optodes spécifiques ancrés à 10-20 points comme dirigé par les buts de l&#39;expérience. </li></ol> Partie 4: Test de la baie de Optode <ol><li> Introduction à l&#39;ETG Hitachi-4000 Interface graphique et le test de la sonde. </li><li> Test de signal: optodes fois sont placés sur le cuir chevelu des participants, la qualité du signal est testé. Si une optode n&#39;a pas un signal clair, les chercheurs retirez délicatement les cheveux de la connexion de l&#39;optode et le cuir chevelu. À l&#39;occasion de la optodes peut-être besoin d&#39;être essuyé avec une compresse imbibée d&#39;alcool. </li></ol> Partie 5: Exécution de l&#39;expérience. <ol><li> Au moins deux expérimentateurs doivent être toujours présents dans la salle, l&#39;un observant le Hitachi fNIRS GTE-4000 en temps réel de lire et d&#39;observer l&#39;autre participant. Ayant une caméra vidéo axé sur des participants est fortement recommandé pour le post-hoc des observations. Un avantage de l&#39;Hitachi fNIRS GTE-4000 est que le signal vidéo et fNIRS sont synchronisés et co-enregistré. Un log contenant toutes les informations pertinentes et les fichiers générés est maintenu. </li><li> Il ya bien établi des méthodes de construction expérimentale paradigmes hémodynamique, à savoir bloc de conception et d&#39;événements liés conceptions. Pour une description plus complète s&#39;il vous plaît consulter le document récent examen 31. </li></ol> Partie 6: Analyse Une fois toutes les données ont été recueillies, le participant est remercié pour leur temps et leur volonté de participer et quitte le laboratoire. Comme l&#39;analyse n&#39;est pas faite sur le Hitachi fNIRS GTE-4000, comme, au contraire, les données sont exportées vers un ordinateur d&#39;analyse. <ol><li> Conversion de uV à des concentrations d&#39;hémoglobine. Comme valeurs d&#39;atténuation brutes sont recueillies dans l&#39;atténuation de la force au laser (tel que mesuré dans uV), ces valeurs doivent être converties à l&#39;hémoglobine oxygénée et désoxygénée Values. Ceci est fait selon la méthode modifiée de Beer-Lambert équation. </li><li> L&#39;application de la modification de Beer-Lambert est menée en deux étapes. Sous l&#39;hypothèse que la dispersion est constante sur la longueur du chemin, d&#39;abord l&#39;atténuation pour chaque longueur d&#39;onde (λ Aa (t)) est calculé en comparant la densité optique de l&#39;intensité lumineuse durant la tâche (tâche I) à la ligne de base calculée sur le signal ( J&#39;ai de base). Les valeurs Aa pour chaque longueur d&#39;onde et le point le temps d&#39;échantillonnage (t) pour résoudre le modifiés équation de Beer-Lambert. <table border="0"><tbody><tr><td> L&#39;équation 1 <img style="float:left;" src="/files/ftp_upload/1268/eq1.jpg" alt="l&#39;équation 1" /></td><td> Équation 2 <img style="float:left;" src="/files/ftp_upload/1268/eq2.jpg" alt="l&#39;équation 2" /></td></tr></tbody></table></li></ol> λ ∈ 1 désoxy, λ ∈ 1 oxy, λ ∈ 2 désoxy et λ ∈ 2 oxy sont les constantes pour les coefficients d&#39;extinction qui mesurent la fraction de lumière perdue à l&#39;absorption par distance unité de concentration dans les tissus. Le désoxy C et C résultante des valeurs oxy sont les concentrations d&#39;hémoglobine désoxygénée et oxygénée pour chaque t. Partie 7: Résultats Représentant Les résultats typiques de la réponse hémodynamique à plusieurs caractéristiques distinctes. Dans la réponse oxyhémoglobine, il est d&#39;abord un plongeon caractéristique. Cette trempette se produit comme une région de neurones s&#39;active et épuise l&#39;oxygène disponible. Comme le flux sanguin augmente, portant hémoglobine oxygénée, la réponse oxyhémoglobine augmente rapidement au-dessus du niveau de référence initial à un niveau stable. Lorsque la région n&#39;est plus activée, les désintégrations de réponse oxyhémoglobine au cours des 12-15 prochaines secondes et lentement retombe à des niveaux de référence. Il ya parfois une undershoot qui survient avant la réponse hémodynamique retour aux niveaux de base initial. Mauvais résultats sont généralement sous la forme d&#39;optodes pas correctement assis sur le cuir chevelu ou les mouvements excessifs. Ces types de bruit - appelé «Flatling» - sont évidentes dans le signal que les valeurs microvolt saturer et un certain nombre de différents canaux, déplacer les deux oxy-et désoxy-réponse d&#39;une manière coordonnée. DÉMONSTRATION: Secouer la fibre optique. Analyses statistiques: L&#39;oxy extraites et désoxyhémoglobine valeurs pour chaque canal, pour chaque participant et pour chaque tâche peut alors être soumis à une analyse statistique classiques, y compris les tests t, ANOVA, corrélations etc

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The authors have nothing to disclose.

Dans cette étude, nous avons démontré l&#39;utilisation d&#39;un roman, une technologie non invasive fNIRS imagerie cérébrale pour étudier le fonctionnement du cerveau humain par rapport à la cognition humaine et la perception. l&#39;imagerie cérébrale fNIRS peut représenter l&#39;avenir de l&#39;imagerie cérébrale non invasive, en particulier avec les populations infantile et juvénile, qui pourraient un jour être largement disponibles dans les laboratoires de recherche, les bureaux des médecins, et dans ...

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<td>ETG-4000</td>
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<td>Matlab</td>
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<td>Psychology toolbox</td>
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exploring cognitive functions in babies, children &amp; adults with near infrared spectroscopy

Une explosion de fonctionnelles Proche spectroscopie infrarouge (fNIRS) études portant sur ​​l&#39;activation corticale en relation avec les processus cognitifs supérieurs, tels que la langue 1,2,3,4,5,6,7,8,9,10, mémoire 11, 12 et l&#39;attention est en cours dans le monde entier adultes impliquant les enfants et les nourrissons à la cognition 3,4,13,14,15,16,17,18,19 typiques et atypiques 20,21,22. Le défi contemporain de l&#39;aide pour les neurosciences cognitives fNIRS est de réaliser des analyses systématiques de ces données qu&#39;elles sont universellement interprétable 23,24,25,26, et donc peut avancer des questions scientifiques importantes sur l&#39;organisation fonctionnelle et les systèmes neuronaux sous-jacents humaine cognition supérieure. Technologies existantes ont neuroimagerie soit moins robuste résolution temporelle ou spatiale. Événement Potentiels et magnéto-encéphalographie (ERP et MEG) ont une excellente résolution temporelle, tandis que la tomographie par émission de positons et l&#39;imagerie par résonance magnétique (TEP et IRMf) ont une meilleure résolution spatiale. L&#39;utilisation non ionisants longueurs d&#39;onde de la lumière dans le proche infrarouge (700-1000 nm), où l&#39;oxyhémoglobine est préférentiellement absorbée par 680 nm et désoxy-hémoglobine est préférentiellement absorbée par 830 nm (par exemple, en effet, les longueurs d&#39;onde très câblées dans l&#39;fNIRS Hitachi GTE-400 système illustré ici), fNIRS est bien adapté pour les études de cognition supérieure, car il a à la fois bonne résolution temporelle (~ 5s) sans l&#39;utilisation des rayonnements et une bonne résolution spatiale (profondeur ~ 4 cm), et ne exigent que les participants à être dans une structure fermée 27,28. L&#39;activité corticale participants peuvent être évalués tout confortablement assis dans une chaise ordinaire (adultes, enfants) ou même assis sur les genoux de maman s (bébés). Notamment, la SPIR est uniquement portables (de la taille d&#39;un ordinateur de bureau), pratiquement silencieux, et peut tolérer un mouvement participants subtile. Ceci est particulièrement remarquable pour l&#39;étude neurales du langage humain, qui a nécessairement comme une de ses principales composantes du mouvement de la bouche dans la production de la parole ou les mains dans le langage des signes. La façon dont la réponse hémodynamique est localisée est par un réseau d&#39;émetteurs laser et les détecteurs. Les émetteurs émettent une intensité connue des non-ionisantes lumière tandis que les détecteurs de détecter la quantité réfléchie par la surface corticale. Le rapprochement entre l&#39;optodes, plus la résolution spatiale, tandis que les plus éloignés de la optodes, la plus grande profondeur de pénétration. Pour la fNIRS Hitachi GTE-4000 un système optimal de pénétration / la résolution du tableau optode est réglé sur 2 cm. Notre objectif est de démontrer notre méthode d&#39;acquisition et d&#39;analyse des données fNIRS pour standardiser le terrain et permettent différents laboratoires dans le monde entier fNIRS pour avoir un fond commun.

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

Nous décrivons ici une méthode de collecte de données et d&#39;analyse des données fonctionnelles Proche spectroscopie infrarouge (fNIRS), un roman non-invasive du système d&#39;imagerie cérébrale utilisées en neurosciences cognitives, en particulier dans l&#39;étude du développement du cerveau des enfants. Cette méthode fournit un standard universel d&#39;acquisition de données et d&#39;analyse indispensable à l&#39;interprétation des données et la découverte scientifique.

L&#39;exploration des fonctions cognitives chez les bébés, enfants et adultes avec la spectroscopie proche infrarouge

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

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Biology

20.4K vues.  University of Michigan, Ann Arbor. Nous décrivons ici une méthode de collecte de données et d'analyse des données fonctionnelles Proche spectroscopie infrarouge (fNIRS), un roman non-invasive du système d'imagerie cérébrale utilisées en neurosciences cognitives, en particulier dans l'étude du développement du cerveau des enfants. Cette méthode fournit un standard universel d'acquisition de données et d'analyse indispensable à l'interprétation des données et la découv...

Vidéo: 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.

Enregistrements de l&#39;activation des circuits neuronaux dans les Librement Behaving Animaux

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

Recherche

Biologie

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.

Assemblée, Tuning et utilisation d&#39;un microscope en champ proche infrarouge pour l&#39;imagerie Apertureless protéines

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.

Mesure de la fonction cérébrale Neuromagnetic En pré-scolaire des enfants à la coutume entreprises 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.

Méthode d&#39;évaluation des effets d&#39;une gamme de longueurs d&#39;onde et des intensités de Luminothérapie Rouge / proche infrarouge sur le stress oxydatif<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.

Proche infrarouge (NIR) Lumière augmente l&#39;expression d&#39;un marqueur de mitochondrial Fonction chez la souris vestibulaire sensoriel épithélium

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.

Reconstitution fonctionnelle et Activité du Chan mesures de type sauvage purifiée et Mutant protéine CFTR

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.

La méthode ChIP-exo: Identification de protéine-ADN Interactions avec les environs de Base Paire de précision

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.

Production d’une plate-forme de livraison de vaccins sensibles et proches de la coquille centrale

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.

Exploration de la structure des tissus adipeux par compensation du méthylsalicylate et imagerie 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.

Dissection des propriétés mécanoenzymatiques des myosines processives avec la spectroscopie ultrarapide à pince de force

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