Este trabalho foi financiado por doações de LAP (PI): National Institutes of Health R21 HD50558, concedido 2005-07; Nacional Institutes of Health R01 HD045822, concedido 2004-09; Dana Foundation Grant, concedido 2004-06; Fundação Canadense para Inovação (&quot;TPI&quot; subvenção), concedido 2008-2012; O Ontario Research Fund Grant, concedido 2008-2012.

Parte 1: Antes de participante chegar ao laboratório <ol><li> Assegurar que a sala está livre de artigos estranhos que podem ser uma distração para o participante. </li><li> Set-up e carregar protocolo experimental sobre a Hitachi fNIRS sistema ETG-4000. </li><li> Set-up seu paradigma experimental. Paradigmas experimentais pode ser programado com software de apresentação diferente, incluindo Eprime, Apresentação Psyscope, ou uma caixa de ferramentas Matlab psicologia baseada. Aqui usamos Matlab toolbox psicologia baseada. </li><li> Momento é chave para a análise de dados, assim, o paradigma experimental devem estar perfeitamente sincronizada com a coleta de dados. A Hitachi fNIRS ETG-4000 tem capacidade de disparo, permitindo o paradigma experimental para acionar a coleta de dados ou vice-versa. Teste de disparo do programa de apresentação de fNIRS Hitachi ETG-4000. Disparo pode ser feito usando paralelas, folhetins, ou portas USB. Aqui nós mostramos provocando via porta paralela. </li><li> Antes de iniciar o estudo fNIRS é importante a realização de seleção do fundo participante. No laboratório Petitto, realizamos seleção do fundo, tendo os participantes ou seus pais preencher estudo apropriado questionários padronizados 29. </li></ol> Chega participante <ol start="6"><li> É importante realizar a sessão e para tratar os participantes de uma forma profissional. O participante ou os pais dos participantes / representantes legais devem assinar um termo de consentimento antes do experimento começa. É vital para agradecer o participante para o seu tempo nestas experiências importantes e emocionantes. </li><li> O participante está sentado confortavelmente perto da sala de ensaios fNIRS. Um participante bebê pode estar sentado no colo de um pai. </li></ol> Parte 2: Colocar Optodes &amp; Usando o sistema 10-20 Outro componente do método de análise que permite a interpretação de dados consistente é a padronização de protocolo fNIRS gravação. Isso implica a colocação optode, posicionamento participante e desencadeamento de software apresentação do estímulo. Tanto a colocação de neuro-anatômicas precisas de sondas e da confirmação de regiões de interesse (ROIs) são obtidos utilizando o sistema 20/10 3,4,30. Além disso, a localização estereotáxica da matriz sonda foi confirmado no crânio do participante através da sobreposição de informações de rastreamento 3D a partir de um sistema Polhemus trak rápido para um anatômicas scan co-registro de ressonância magnética do participante realizada com cápsulas de vitamina E colocados em cada local da sonda de 3,4. posicionamento participante ideal consistia em colocar os participantes confortavelmente em uma cadeira reclinável, com fibra ótica soltos, sem contato com o corpo ou cadeira. <ol><li> As medições cabeça seguintes são tomadas com uma fita métrica e escrito na folha de dados participantes: <ul><li> Násio ao redor ínion </li><li> Násio para Inions por cima </li><li> Orelha a orelha por cima </li></ul></li><li> Fita cirúrgica pode ser usado para marcar locais de destino específico. Neste experimento vamos marcar Fp, T3/T4, F8/F7 </li><li> Arrays optode são colocados na cabeça dos participantes com optodes específicas ancorada em 20/10 pontos como dirigido pelos propósitos do experimento. </li></ol> Parte 4: Testando a matriz optode <ol><li> Introdução à interface GUI Hitachi ETG-4000 e testes de sonda. </li><li> Teste do sinal: optodes Uma vez são colocados no couro cabeludo dos participantes, a qualidade do sinal é testado. Se um optode não tem um sinal claro, os pesquisadores remover suavemente o cabelo a partir da conexão do optode e do couro cabeludo. Na ocasião, o optodes pode precisar de ser limpo com um algodão embebido em álcool. </li></ol> Parte 5: A execução do experimento. <ol><li> Pelo menos dois experimentadores deve estar sempre presente na sala, um observando o Hitachi fNIRS ETG-4000 em tempo real lido e os outros observando o participante. Ter uma câmera de vídeo focado em participante é altamente recomendado para post-hoc observações. Uma vantagem da Hitachi fNIRS ETG-4000 é que o sinal de vídeo e fNIRS são sincronizados e co-registrados. Um log contendo todas as informações e arquivos gerados é mantida. </li><li> Há bem estabelecidos métodos de construção de paradigmas experimentais hemodinâmica, ou seja, Bloco de design e eventos relacionados com projetos. Para uma descrição mais completa veja o artigo de revisão recente 31. </li></ol> Parte 6: Análise Depois de todos os dados foram coletados, o participante é agradeceu por seu tempo e vontade de participar e deixa o laboratório. Como a análise não é feita no Hitachi fNIRS ETG-4000, como, em vez disso, os dados são exportados para um computador de análise. <ol><li> Conversão de mV para concentrações de hemoglobina. Como valores de atenuação-primas são coletados na atenuação da força laser (medida em mV), esses valores devem ser convertidos para oxigenado e desoxigenado hemoglobina values. Isso é feito usando a equação modificada Beer-Lambert. </li><li> A aplicação da modificação Beer-Lambert é realizado em duas etapas. Sob a suposição de que a dispersão é constante ao longo do comprimento do caminho, primeiro a atenuação para cada comprimento de onda (ΔA λ (t)) é calculado através da comparação da densidade óptica de intensidade de luz durante a tarefa (tarefa I) para a linha de base calculada do sinal ( Eu linha de base). Os valores de ΔA para cada comprimento de onda e tempo de amostragem (t) para resolver a equação modificada Beer-Lambert. <table border="0"><tbody><tr><td> Equação 1 <img style="float:left;" src="/files/ftp_upload/1268/eq1.jpg" alt="equação 1" /></td><td> Equação 2 <img style="float:left;" src="/files/ftp_upload/1268/eq2.jpg" alt="equação 2" /></td></tr></tbody></table></li></ol> λ ∈ um desoxi, λ ∈ um oxi, λ 2 e λ ∈ desoxi 2 ∈ oxi são as constantes para os coeficientes de extinção que medem a fração da luz perdeu a absorção por unidade de distância de concentração no tecido. O desoxi C e os valores resultantes oxi C são as concentrações de hemoglobina desoxigenada e oxigenado para cada t. Parte 7: Resultados Representante Típico resultados hemodinâmicos de resposta em várias características distintas. Na resposta oxi-hemoglobina, há primeiro um mergulho característica. Este mergulho ocorre como uma região de neurônios ativa e esgota o oxigênio disponível. À medida que aumenta o fluxo sanguíneo, levando hemoglobina oxigenada, a resposta oxi-hemoglobina aumenta rapidamente acima dos níveis basais iniciais para um nível de estado estacionário. Quando a região não está sendo ativado, o decai oxi-hemoglobina resposta nos próximos 12-15 segundos e lentamente cai de volta aos níveis basais. Há ocasionalmente um undershoot que ocorre antes da resposta hemodinâmica retornando aos níveis basais iniciais. Maus resultados são geralmente na forma de optodes não devidamente sentado no couro cabeludo ou movimento excessivo. Estes tipos de ruído - chamado &quot;Flatling &#39;- são evidentes no sinal de como os valores microvolt saturar e um número de canais diferentes, move tanto oxi e desoxi-resposta de forma coordenada. DEMONSTRAÇÃO: Agitando a fibra óptica. Análises estatísticas: Os oxi extraído e desoxi-hemoglobina valores para cada canal, para cada participante e para cada tarefa pode então ser submetidos à análise estatística convencional, incluindo os testes t, ANOVAs, correlações etc

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The authors have nothing to disclose.

Neste estudo, demonstramos o uso de um romance não-invasivo fNIRS, a tecnologia de imagens cerebrais para investigar o funcionamento do cérebro humano em relação à cognição humana e percepção. fNIRS imagens do cérebro pode representar o futuro da não-invasivo de imagens cerebrais, particularmente com bebês e crianças populações, que podem um dia estar amplamente disponível em laboratórios de pesquisa, consultórios médicos, e nos sistemas de ensino permitindo que os médicos para aplicar os conheciment...

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exploring cognitive functions in babies, children &amp; adults with near infrared spectroscopy

Uma explosão de Near Infrared Spectroscopy funcional (fNIRS) estudos que investigam a activação cortical em relação a processos cognitivos superiores, como a linguagem 1,2,3,4,5,6,7,8,9,10, memória 11, 12 e atenção está em andamento em todo o mundo, envolvendo adultos, crianças e bebés 3,4,13,14,15,16,17,18,19 com a cognição típicos e atípicos 20,21,22. O desafio contemporâneo de usar fNIRS para a neurociência cognitiva é a realização de análises sistemáticas de dados de tal forma que eles são universalmente interpretável 23,24,25,26, e, assim, pode avançar importantes questões científicas sobre a organização funcional e sistemas neurais subjacentes maior cognição humana. Tecnologias de neuroimagem existentes ou menos robustos resolução temporal ou espacial. Eventos relacionados Potenciais e Magneto encefalografia (ERP e MEG) têm excelente resolução temporal, enquanto Positron Emission Tomography e ressonância magnética funcional (PET e fMRI) têm melhor resolução espacial. Usando não-ionizante comprimentos de onda da luz na faixa do infravermelho próximo (700-1000 nm), onde oxi-hemoglobina é preferencialmente absorvido por 680 nm e desoxi-hemoglobina é preferencialmente absorvido por 830 nm (por exemplo, de fato, os comprimentos de onda muito hardwired em o fNIRS Hitachi ETG-400 sistema ilustrado aqui), fNIRS é bem adequado para estudos de cognição mais elevados, porque tem tanto boa resolução temporal (~ 5s) sem o uso de radiação e boa resolução espacial (~ 4 cm de profundidade), e não exigem que os participantes estar em uma estrutura fechada 27,28. Participantes atividade cortical pode ser avaliado, enquanto confortavelmente sentado em uma cadeira comum (adultos, crianças) ou até mesmo sentado no colo da mãe s (crianças). Nomeadamente, NIRS é o único portátil (do tamanho de um computador de mesa), praticamente em silêncio, e pode tolerar um movimento sutil participantes. Isto é particularmente notável para o estudo neurais da linguagem humana, que necessariamente tem como um de seus componentes-chave do movimento da boca na produção da fala ou as mãos em linguagem gestual. A maneira em que a resposta hemodinâmica é localizado é por um conjunto de emissores laser e detectores. Emissores emitem uma intensidade conhecida de não-ionizante luz, enquanto detectores de detectar a quantidade refletida de volta a partir da superfície cortical. O aproximar do optodes, maior resolução espacial, enquanto a mais distante do optodes, maior a profundidade da penetração. Para o fNIRS Hitachi ETG-4000 ideal do sistema de penetração / resolução matriz optode está definido para 2 centímetros. Nosso objetivo é demonstrar o nosso método de adquirir e analisar dados fNIRS para ajudar a padronizar o campo e permitir que laboratórios diferentes fNIRS em todo o mundo para ter um fundo comum.

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

Aqui nós descrevemos um método de coleta de dados e análise de dados para funcional espectroscopia no infravermelho próximo (fNIRS), um romance não-invasivo sistema de imagens do cérebro usada em neurociência cognitiva, particularmente no estudo do desenvolvimento do cérebro da criança. Este método fornece um padrão universal de aquisição de dados e análise vital para a interpretação de dados e descoberta científica.

Explorando Funções Cognitivas em Bebés, Crianças e Adultos com Espectroscopia no Infravermelho Próximo

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 visualizações.  University of Michigan, Ann Arbor. Aqui nós descrevemos um método de coleta de dados e análise de dados para funcional espectroscopia no infravermelho próximo (fNIRS), um romance não-invasivo sistema de imagens do cérebro usada em neurociência cognitiva, particularmente no estudo do desenvolvimento do cérebro da criança. Este método fornece um padrão universal de aquisição de dados e análise vital para a interpretação de dados e descoberta científica.

Vídeo: 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.

Gravações da ativação do circuito neural no Livremente Behaving Animais

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

Biologia

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.

Assembléia, Tuning e Uso de um microscópio de campo Apertureless Near Infrared for Imaging Protein

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.

Medição da função cerebral Neuromagnetic Em pré-escolares Com Personalizado MEG Sized

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 de Avaliação de efeitos de uma gama de comprimentos de onda e intensidades de Terapia da Luz Vermelha / near-infrared sobre o estresse 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.

Infravermelho Próximo (NIR) Luz aumenta a expressão de um marcador da função mitocondrial no mouse Vestibular Sensorial Epithelium

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.

Reconstituição funcional e atividade do canal Medidas de purificada tipo selvagem e mutante CFTR Protein

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.

O Método ChIP-exo: Identificando Protein-DNA Interações com perto da base Pair Precision

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.

Produção de plataforma de entrega de vacinas de vacinação de infravermelho próximo e infravermelho

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.

Explorando estrutura tecidual adiposa por limpeza de metilsalicylate e imagem 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.

Dissecando as propriedades mecanoenzimáticas de miosinas processivas com espectroscopia de força-braçadeira ultrarrápida

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