Bu çalışma LAP (PI) hibeleri tarafından desteklenen: Ulusal Sağlık R21 HD50558 Enstitüleri, 2005-07 ödüllendirildi; Ulusal Sağlık R01 HD045822 Enstitüleri, 2004-09 ödüllendirildi; Dana Vakfı Hibe Kanada Yenilikçilik Vakfı&#39;nın (&quot;CFI&quot; hibe) ödüllendirildi; 2004-06 ödüllendirildi 2008-2012; Ontario Araştırma Fonu Hibe, 2008-2012 ödüllendirdi.

Bölüm 1: katılımcıya önce laboratuara gelen <ol><li> Oda katılımcıya rahatsız edici olabilir gereksiz makaleler ücretsiz olduğundan emin olun. </li><li> Set-up ve deneysel protokol fNIRS Hitachi ETG-4000 sistem üzerinde yük. </li><li> Deneysel paradigma set-up. Deneysel paradigmalar Eprime, Tanıtım, Psyscope veya bir Matlab tabanlı psikoloji araç kutusu dahil olmak üzere farklı sunum yazılımı ile programlanmış olabilir. Burada Matlab tabanlı psikoloji araç kutusu kullanın. </li><li> Zamanlama veri analizi için önemli olan, bu nedenle deneysel paradigma veri toplama ile mükemmel zamanlanmış olmalıdır. FNIRS Hitachi ETG-4000, veri toplama ya da tam tersi tetiklemek için deneysel paradigma için izin yetenekleri tetikleme. FNIRS Hitachi ETG-4000 sunum programı tetikleme sınayın. Tetikleme, paralel dizi kullanarak veya USB portları yapılması olabilir. Burada paralel port üzerinden tetikleme göstermektedir. </li><li> FNIRS çalışmaya başlamadan önce katılımcı arka plan tarama yapmak için önemlidir. Petitto laboratuvarda, katılımcı ya da ebeveynleri çalışmaya uygun standart anketlerle 29 doldurunuz sahip arka plan tarama yapıyoruz. </li></ol> Katılımcı Geldi <ol start="6"><li> Oturumu yürütmek ve profesyonel bir şekilde katılımcılara tedavi için önemlidir. Katılımcı veya katılımcıların ebeveynleri / yasal vasiler, deney başlamadan önce bir onay formu imzalamanız gerekir. Bu önemli ve heyecan verici bu deneylerde onların zaman katılımcı teşekkür etmek hayati önem taşımaktadır. </li><li> Katılımcı fNIRS test odasına rahat yakın oturmuştur. Bir bebek katılımcı, bir ebeveynin kucağında oturmuş olabilir. </li></ol> Bölüm 2: Optodes yerleştirilmesi ve 10-20 sistemini kullanarak Analiz yöntemi, tutarlı bir veri yorumlama sağlayan diğer bir bileşeni fNIRS kayıt protokol standardizasyon. Bu optode yerleştirme, katılımcı konumlandırma ve uyaran sunum yazılımı, tetikleme gerektirir. Probların doğru nöro-anatomik yerleşimi ve faiz (ROI) bölgeleri onay Her ikisi de, 10-20 sistemi 3,4,30 kullanılarak elde edilir . Ayrıca, prob dizi stereotaktik yerelleştirme Polhemus Fast Trak sistemi, her prob konumu 3,4 bulunan E vitamini kapsülleri ile yapılan katılımcının bir anatomik MR co-kayıt tarama üzerine 3D izleme bilgilerini çakıştırılarak katılımcının kafatası teyit edilmiştir . Optimal katılımcı konumlandırma gevşek vücut ya da sandalye ile temas etmeden asılı fiber optik, katılımcıların yerleştirerek uzanmış bir sandalyede rahat bir yer. <ol><li> Aşağıdaki baş ölçümleri bir mezura ile çekilen ve katılımcılara bilgi formunda yazılı: <ul><li> Inion için nasion etrafında </li><li> Üstünden Inions için nasion </li><li> Kulak üstünden Kulak için </li></ul></li><li> Cerrahi bant, hedeflenen belirli yerleri işaretlemek için de kullanılabilir. Bu deneyde, Fp, T3/T4 F8/F7 işareti </li><li> Optode diziler, deney amacıyla yönettiği 10-20 puanlık demirli özel optodes katılımcıların kafasına yerleştirilir. </li></ol> Bölüm 4: Optode Array Test <ol><li> Hitachi ETG-4000 GUI Arabirimi ve prob test Giriş. </li><li> Test sinyali: Once optodes katılımcılar &#39;kafa derisi üzerine yerleştirilir, sinyal kalitesi test edilir. Bir optode net bir sinyal yoksa, araştırmacılar optode ve kafa derisi bağlantı hafifçe saç kaldırmak. Optodes vesileyle bir alkollü bez ile silinmelidir gerekebilir. </li></ol> Bölüm 5: Deney. <ol><li> Her zaman en az iki deneyci odasında bulunması gereken bir okuma fNIRS Hitachi ETG-4000 gerçek-zamanlı gözlemleme ve diğer katılımcı gözlemleyerek. Katılımcı odaklanmış bir video kameraya sahip yüksek post-hoc gözlemler için tavsiye edilir. FNIRS Hitachi ETG-4000 bir avantajı, video ve fNIRS sinyal senkronize ve co-kayıtlı olduğunu. Ilgili tüm bilgileri ve dosyaları içeren bir günlük tutulur. </li><li> Deneysel hemodinamik paradigmalar binanın köklü bir yöntem vardır, yani tasarım ve Olay ile ilgili tasarımlar engelleyin. Son derlemede 31 daha eksiksiz bir açıklaması için bakınız. </li></ol> Bölüm 6: Analiz Bir kez tüm veri toplanan katılımcı katılmak için zaman ve istekli teşekkür etti ve laboratuar bırakır. FNIRS Hitachi ETG-4000 tarihinde yapılan analiz değildir, bunun yerine, bir analiz bilgisayara veri ihraç edilmektedir. <ol><li> Hemoglobin konsantrasyonları μV dönüşüm. Lazer gücünün zayıflaması (μV olarak ölçülür) toplanan ham zayıflama değerleri olarak bu değerler, oksijenli ve deoxygenated hemoglobin valu dönüştürülmesi gerekires. Bu değiştirilmiş Beer-Lambert denklemi kullanılarak yapılır. </li><li> Değiştirilmiş Beer-Lambert uygulama iki adımda yapılır. Saçılma, ilk yol uzunluğu, sinyal hesaplanan bazal her dalga boyu (ΔA λ (t)) görev (görev I) sırasında ışık şiddeti optik yoğunluk karşılaştırılarak hesaplanır (zayıflama boyunca sabit olduğu varsayımı altında Ben bazal). Her dalga boyu için ΔA değerleri ve örneklenmiş bir zaman noktasında (t) değiştirilmiş Beer-Lambert denklemi çözmek için. <table border="0"><tbody><tr><td> Denklem 1 <img style="float:left;" src="/files/ftp_upload/1268/eq1.jpg" alt="denklemi 1" /></td><td> Denklem 2 <img style="float:left;" src="/files/ftp_upload/1268/eq2.jpg" alt="denklem 2" /></td></tr></tbody></table></li></ol> λ 1 ∈ deoksi, 1 λ ∈ oksi 2 λ ∈ deoksi ve λ 2 ∈ oksi doku birim konsantrasyon mesafe başına emilimini kaybetti ışık kısmını ölçmek yok katsayıları için sabitlerdir. Çıkan C deoksi ve C oksi değerleri her t deoxygenated ve oksijenli hemoglobin konsantrasyonları. Bölüm 7: Temsilci Sonuçlar Birçok farklı özellikleri tipik hemodinamik yanıtı sonuçları. Oksi-hemoglobin yanıt olarak, karakteristik bir daldırma vardır. Bu dalış, nöronların bir bölge aktive eder ve mevcut oksijeni tüketir oluşur. Kan akımı arttıkça, oksijenli hemoglobin taşıyan, oksi-hemoglobin yanıtı istikrarlı bir devlet düzeyinde ilk başlangıç ​​seviyesinin üstünde hızla yükselir. Bölgede artık aktive edilirken, Oksi-hemoglobin sonraki 12-15 saniye içinde yanıt bozunur ve yavaşça bazal düzeylere geri düşer. Ilk başlangıç ​​seviyelerine geri dönen hemodinamik yanıt öncesinde gerçekleştirilen bir inişte bazen yoktur. Kötü sonuçlar, düzgün bir kafa derisi veya aşırı hareketlilik üzerine oturtulmuş optodes form genellikle. Mikrovolt değerleri doyurabilecek ve farklı kanalların bir numarası, oksi-ve deoksi-yanıt koordineli bir biçimde hareket olarak sinyal gürültü Bu tür &#39;Flatling&#39; denilen belirgindir. GÖSTERİ: fiber optik Çalkalamalı. İstatistiksel Analizler: Her katılımcı için ve her görev için her kanal için çıkarılan oksi ve deoksi-hemoglobin değerleri, sonra t-testleri, ANOVAs, korelasyon vb dahil olmak üzere, geleneksel istatistiksel analiz sunulacaktır.

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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.

Bu çalışmada, insan biliş ve algı ile ilgili olarak, insan beyin fonksiyonu araştırmak için bir roman, non-invaziv fNIRS beyin görüntüleme teknolojisi kullanımı gösterdi. fNIRS beyin görüntüleme, özellikle klinisyenlerin temel bilimsel bulguları uygulamak için izin bebek ve çocuk bir gün araştırma laboratuarlarında yaygın olarak bulunabilir popülasyonları, doktor muayenehanesi ve okul sistemleri, non-invaziv beyin görüntüleme geleceği temsil edebilir klinik uygulamada beyin.

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

Fonksiyonel Infrared Spektroskopisi (fNIRS) gibi dil 1,2,3,4,5,6,7,8,9,10, bellek 11 gibi yüksek bilişsel süreçleri ile ilgili kortikal aktivasyon araştıran çalışmalar ve dikkat 12 civarında bir patlama devam içeren dünya çapında yetişkinler, çocuklar ve bebekler 3,4,13,14,15,16,17,18,19 tipik ve atipik biliş 20,21,22. Bilişsel sinirbilim fNIRS kullanarak çağdaş meydan onlar 23,24,25,26 evrensel olarak yorumlanabilir, ve böylece fonksiyonel organizasyon ve insan yüksek biliş altında yatan sinir sistemleri ile ilgili önemli bilimsel soruları önceden bu tür verilerin sistematik analizler elde etmektir. Mevcut beyin görüntüleme teknolojileri, zamansal ve mekansal çözünürlükte ya da daha az güçlü. , Pozitron Emisyon Tomografi ve fonksiyonel Manyetik Rezonans Görüntüleme (PET ve fMRI) iyi mekansal çözünürlüğe sahip ise Olaylarla İlgili Potansiyeller ve Magneto Ensefalografi (ERP ve MEG), mükemmel zamansal çözünürlüğe sahip. Oksi-hemoglobin tercihen 680 nm ve deoksi-hemoglobin tarafından emilir yakın-kızılötesi aralığı (700-1000 nm), non-iyonize dalga boylarında ışık kullanarak tercihen 830 nm (örneğin, gerçekten, çok dalga boylarını içine kablolu tarafından emilir Burada gösterilen fNIRS Hitachi ETG-400 sistemi) de iyi temporal çözünürlük (~ 5 saniye), radyasyon ve iyi uzaysal çözünürlük (~ 4 cm derinlik) kullanmadan, çünkü, fNIRS yüksek biliş çalışmaları için uygundur katılımcıların kapalı bir yapısı 27,28 olması gerekir. Sıradan bir sandalye (yetişkinler, çocuklar) rahatça oturarak veya hatta anne s tur (bebeklerde) otururken Katılımcılar kortikal aktivite tespit edilebilir. Özellikle, NIRS neredeyse sessiz, (bir masaüstü bilgisayar boyutunda) benzersiz taşınabilir ve katılımcılar ince hareketi tolere edebilir. Bu, mutlaka onun anahtar bileşenleri işaret dili konuşma üretim veya eller ağız hareketi biri olarak insan dili, sinir çalışması için, özellikle göz alıcı. Hemodinamik yanıt lokalize olduğu yolu lazer üreten ve dedektörleri bir dizi. Dedektörleri geri kortikal yüzeyinden yansıyan miktarını tespit ederken Yayıcılar bilinen bir yoğunluk non-iyonize ışık yayarlar. Optodes birbirine yaklaştırır, daha fazla uzaysal çözünürlüğü daha ayrı optodes ise, daha derin penetrasyon. FNIRS Hitachi ETG-4000 sisteminin optimum penetrasyon / çözünürlük optode dizi 2cm. Amacımız alanında standartlaştırmak yardımcı olmak ve ortak bir arka plana sahip, dünya çapında farklı fNIRS laboratuvarları etkinleştirmek için fNIRS veri edinme ve analiz yöntemi ortaya koymaktır.

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

Burada Near Infrared Spektroskopisi (fNIRS), bilişsel sinirbilim kullanılan non-invaziv bir roman beyin görüntüleme sistemi, özellikle çocuğun beyin gelişimi eğitim işlevsel bir veri toplama ve analiz yöntemi açıklar. Bu yöntem, veri toplama ve verilerin yorumlanması ve bilimsel keşif için hayati analiz evrensel bir standart sağlar.

Yakın Kızılötesi Spektroskopisi ile bebekler, Çocuk ve Yetişkin Bilişsel Fonksiyonları keşfetmek

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

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Research

JoVE Journal

Biology

20.4K Görüntüleme.  University of Michigan, Ann Arbor. Burada Near Infrared Spektroskopisi (fNIRS), bilişsel sinirbilim kullanılan non-invaziv bir roman beyin görüntüleme sistemi, özellikle çocuğun beyin gelişimi eğitim işlevsel bir veri toplama ve analiz yöntemi açıklar. Bu yöntem, veri toplama ve verilerin yorumlanması ve bilimsel keşif için hayati analiz evrensel bir standart sağlar.

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.

Serbestçe Hayvanlar Behaving Sinir Devre Etkinleştirme kayıtları

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

Biyoloji

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.

Kurul, Tuning ve Protein Görüntüleme için bir Apertureless Yakın Alan Kızılötesi Mikroskop Kullanımı

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.

Özel Ölçekli MEG ile Okul Öncesi Çocuk Neuromagnetic Beyin Fonksiyonu Ölçümleri

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.

Dalga boyundaki ve Oksidatif Stres Kırmızı / yakın-kızılötesi ışık Terapisi şiddetler bir Range Etkilerinin Değerlendirilmesi için Yöntemi<em&gt; İn 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.

Yakın Kızılötesi (NIR) Işık Fare Vestibüler Duyu Epitelinin Mitokondriyal Fonksiyonun bir Marker Anlatım artırır

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.

Saf Yabanitip ve Mutant CFTR Protein Fonksiyonel Sulandırma ve Kanal Aktivite Ölçümleri

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.

ChIP-ekzo Yöntem: Base Çifti Hassas Protein-DNA Etkileşimleri belirlenmesi

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.

Yakın Kızılötesi Hassas, Çekirdek Kabuğu Aşı Dağıtım Platformu Üretimi

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.

Metiyatisilin Temizleme ve 3D Görüntüleme ile Yağ Dokusu Yapısının Araştırılması

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.

İşlemsel Miyosinlerin Mekanoenzimatik Özelliklerinin Ultra Hızlı Kuvvet-Kelepçe Spektroskopisi ile Diseksiyonu

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