이 작품은 무릎 (PI)에 보조금 지원되었습니다 국민 건강 R21 HD50558의 연구소, 2005-07를 수여; 국립 건강 R01 HD045822의 기관은 2004-09을 수상, 다나 재단 그랜트, 혁신에 대한 캐나다 재단 ( &quot;CFI&quot;부여), 수상, 2004-06를 수여 2008년에서 2012년까지; 온타리오 연구 기금 그랜트, 2008-2012을 수여.

1 부 : 참가자하기 전에 연구실에 도착 <ol><li> 객실 참가자에게 혼란있을 수 있습니다 관계없는 기사를 무료로되었는지 확인하십시오. </li><li> 셋업 및 fNIRS 히타치 ETG - 4000 시스템에서 실험 프로토콜을로드합니다. </li><li> 귀하의 실험 패러다임 - 설정합니다. 실험 패러다임은 Eprime, 프리젠 테이션, Psyscope 또는 Matlab 기반의 심리 도구 상자를 포함한 다양한 프레 젠 테이션 소프트웨어로 프로그래밍할 수 있습니다. 여기 Matlab 기반의 심리 도구 상자를 사용합니다. </li><li> 타이밍 데이터 분석을위한 핵심이며, 따라서 실험 패러다임이 완벽한 데이터 수집과 초과해야합니다. fNIRS 히타치 ETG - 4000은 데이터 수집이나 반대를 유도하는 실험 패러다임에 대한 수 있도록 기능을 실행하고 있습니다. fNIRS 히타치 ETG - 4000에서 프레 젠 테이션 프로그램의 실행 테스트합니다. 트리거링은 병렬 연속을 사용하거나, USB 포트 할 수 있습니다. 여기서 우리는 병렬 포트를 통해 실행 보여줍니다. </li><li> 전에 fNIRS 연구를 시작하기에 그것은 참가자 배경 검사를 실시하는 것이 중요합니다. Petitto 연구실에서 우리는 참가자 또는 부모가 공부에 적합한 표준화 설문에게 29 기입함으로써 백그라운드 검사를 실시합니다. </li></ol> 참가자 도착 <ol start="6"><li> 세션을 실시하고 전문적인 방식으로 참가자를 치료하는 것이 중요합니다. 실험을 시작하기 전에 참가자 또는 참가자들의 부모 / 법적 보호자 동의서에 서명해야합니다. 그것이 중요하고 흥미로운 실험에서 시간을 참가자에게 감사 중요합니다. </li><li> 참가자는 fNIRS 테스트 룸으로 편안하게 가까이 앉아있다. 유아 참가자는 부모의 무릎에 앉아 있습니다. </li></ol> 2 부 : Optodes 배치 및 10-20 시스템을 사용 일관성있는 데이터 해석을 가능하게 분석 방법의 또 다른 구성 요소는 fNIRS 녹음 프로토콜의 표준화이다. 이것은 optode 배치, 참가자의 위치와 자극 프레 젠 테이션 소프트웨어의 실행을 알아볼 수 있습니다. 프로브의 정확한 신경 해부 배치 및이자 (로아)의 영역의 확인 모두 10-20 3,4,30 시스템을 사용하여 얻을 수 있습니다. 또한, 탐침 배열의 stereotactic 지방화는 각 프로브 위치 3,4에 배치 비타민 E의 캡슐 실시 참가자의 해부 MRI 공동 등록 검사에 Polhemus 빠른 트락 시스템에서 3D 추적 정보를 오버레이하여 참가자의 두개골에서 확인되었다. 최적의 참가자 위치 시체 또는 의자와 연락없이 느슨하게 매달려 광섬유와 reclining 의자에 편안하게 참가자를 배치 참여. <ol><li> 다음 헤드 측정은 줄자로 찍은 및 참가자 데이터 시트에 쓰여지는 다음과 같습니다 <ul><li> Inion에 Nasion 주변 </li><li> 맨 위에 Inions에 Nasion </li><li> 맨 위에 이어에 이어 </li></ul></li><li> 외과 테이프는 특정 타겟 위치를 표시하는 데 사용할 수 있습니다. 이 실험에서 우리는 FP, T3/T4, F8/F7를 표시합니다 </li><li> Optode의 배열로 실험의 목적 감독 10-20 지점에서 정박 특정 optodes와 함께 참가자들의 머리에 게재됩니다. </li></ol> 파트 4 : Optode 배열을 테스트 <ol><li> 히타치 ETG - 4000 GUI 인터페이스 및 프로브 테스트 소개. </li><li> 신호를 테스트 : 일단 optodes가 참가자들의 두피에 배치, 신호 품질 테스트입니다. optode가 명확한 신호가없는 경우, 연구자들은 부드럽게 optode과 두피의 연결에서 머리를 제거합니다. 때때로 optodes은 알코올 면봉과 함께 닦아해야 할 수 있습니다. </li></ol> 5 부 : 실험을 실행. <ol><li> 적어도 두 경험은 항상 방에 존재해야하며 하나는 fNIRS 히타치 ETG - 4000 실시간 밖으로 읽고 관찰하고 다른 참가자를 관찰. 참가자에 초점을 맞춘 비디오 카메라를 갖는 것은 매우 사후 특별 관찰하는 것이 좋습니다. fNIRS 히타치 ETG - 4000의 장점은 비디오와 fNIRS 신호가 서로 연관과 공동으로 등록되어있다. 생성된 모든 관련 정보 및 파일을 포함하는 로그는 유지됩니다. </li><li> 실험 hemodynamic 패러다임 구축의 잘 설립 방법이있다, 즉 설계 및 이벤트 관련 디자인을 차단합니다. 보다 자세한 설명을 보려면 최근 검토 종이 31 참조하시기 바랍니다. </li></ol> 6 부 : 분석 일단 모든 데이터가 수집되었고, 참가자들은 시간과 참여 의지에 대한 감사와 실험실을 단풍입니다. 분석 fNIRS 히타치 ETG - 4000에서 수행 아니므로, 같은 대신, 데이터는 분석 컴퓨터에 수출하고 있습니다. <ol><li> 헤모글로빈 농도로 μV에서 전환. 원시 감쇠 값은 레이저 강도의 감쇠합니다 (μV로 측정)에서 수집 있으므로이 값은 산소와 헤모글로빈 deoxygenated valu로 변환해야합니다말이지. 이것은 수정 비어 - 램버트 방정식을 사용하여 수행됩니다. </li><li> 수정된 맥주 - 램버트의 응용 프로그램은 두 단계로 진행됩니다. 산란 처음 경로 길이, 신호의 계산 기준 각 파장 (ΔA λ (t)가) 작업 (I 작업) 중 광도의 광학 밀도를 비교하여 계산 (대한 감쇠를 통해 지속되는 가정하에 I 기준). 각 파장에 대해 ΔA의 가치와 샘플 시점 (T)는 수정 맥주 램버트 방정식을 해결하기 위해. <table border="0"><tbody><tr><td> 방정식 1 <img style="float:left;" src="/files/ftp_upload/1268/eq1.jpg" alt="방정식 1" /></td><td> 방정식 2 <img style="float:left;" src="/files/ftp_upload/1268/eq2.jpg" alt="방정식 2" /></td></tr></tbody></table></li></ol> λ 1 ∈ 데옥시, λ ∈ 일 옥시, λ 2 ∈ 데옥시 및 λ ∈ 옥시이이 조직의 단위 농도 거리마다 흡수하는 손실 빛의 분율을 측정 흡광 계수에 대한 상수입니다. 결과 C의 데옥시와 C 옥시 값은 각 t에 대한 deoxygenated과 산소 헤모글로빈의 농도입니다. 7 부 : 대표 결과 몇 가지 뚜렷한 특징은 전형적인 hemodynamic 응답 결과. 옥시 헤모글로빈 응답에 특성 물놀이가 먼저 있습니다. 뉴런의 지역 활성화와 사용 가능한 산소를 depletes로 수영을 발생합니다. 혈액 흐름이 증가할수록 산소 헤모글로빈를 들고, 옥시 헤모글로빈 응답은 안정된 상태 수준으로 초기 기본 수준 이상 빠르게 상승합니다. 영역이 더 이상 활성화되지중인 경우, 옥시 헤모글로빈 응답 다음 12~15초 이상 자연 붕괴 천천히가 기본 수준하기 위해 뒤로 빠졌습니다. 초기 기본 수준으로 돌아가는 hemodynamic 응답하기 전에 발생 undershoot가 종종있다. 잘못된 결과가 제대로 두피이나 과도한 운동에 자리에 착석하지 optodes의 형태로 일반적입니다. 소음 이러한 유형 - &#39;Flatling&#39;라는 - 백만분의 일볼트 가치가 포화하고 다른 채널의 번호, 조율된 방식으로 옥시과 데옥시 - 응답 모두 이동 신호에 분명 있습니다. 데모 : 광섬유를 흔들어봐. 통계 분석 : 각 참가자에 대해 각 작업에 대한 각 채널에 대해 추출 옥시과 데옥시 - 헤모글로빈이 값은, 다음 T - 테스트, ANOVAs, 상관 관계 등, 기존의 통계 분석에 제출하실 수 있습니다

<ol>
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The authors have nothing to disclose.

본 연구에서는, 우리는 인간의 인식과 지각과 관련하여 인간의 두뇌 기능을 조사하기 위해 소설, 비침습 fNIRS 뇌 이미징 기술의 사용을 시연했다. fNIRS 두뇌 이미징 특히 임상가에 대한 기본적인 과학적 발견을 적용할 수 있도록 유아 및 어린이 일일 연구 실험실에서 널리 사용할 수있는 집단, 의사 &#39;사무실과 학교 시스템과 함께, 비침습 뇌 이미징의 미래를 대표하는 수도 있습니다 그들의 임상...

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

적외선 분광학 (fNIRS) 같은 언어 1,2,3,4,5,6,7,8,9,10, 메모리 11으로 높은인지 과정에 관련하여 대뇌 피질의 활성을 조사 연구하고, 관심 니어 12 기능의 폭발 진행 세계와 관련된 성인, 전형 및 비정형 인식 20,21,22과 함께 어린이 및 유아 3,4,13,14,15,16,17,18,19입니다. 인지 신경 과학에 대한 fNIRS를 사용하는 현대적인 도전들은 23,24,25,26 보편 interpretable 있으며, 따라서 기능적 조직과 인간의 높은 인식을 기본 신경 시스템에 대한 중요한 과학적 질문을 사전에 수도와 같은 데이터의 체계적 분석을 달성하는 것입니다. 기존 neuroimaging 기술 중 덜 강력한 시간적 혹은 공간적 해상도 있습니다. 양전자 방출의 Tomography 및 기능 자기 공명 이미징 (PET와 fMRI)가 더 나은 공간 해상도를 가지고 반면, 이벤트 관련 후보와 매그 니 토는 Encephalography (ERP 및 MEG)는, 우수한 현세의 해상도를했습니다. 옥시 헤모글로빈이 우선적으로 680 nm의 및 데옥시 - 헤모글로빈에 의해 흡수되는 가까운 적외선 범위 (700-1000 NM)에 빛의 비 이온화 파장을 사용하여이 우선적으로 830 nm의 (예, 게다가 매우 파장이에 연결된 의해 흡수 그것이 방사선 좋은 공간 해상도 (~ 4cm 깊이)를 사용하지 않고 모두 좋은 시간적 해상도 (~ 기들)을 가지고 있으며,하지 않기 때문에 여기에 그림 fNIRS 히타치 ETG - 400 시스템), fNIRS 잘 상위인지의 연구에 가장 적합합니다 참가자가 동봉된 구조 27,28에 필요합니다. 편안 일반 의자 (어른, 어린이)에 앉아있는 혹은 엄마의 무릎 (유아)에 앉아있는 동안 참가자 대뇌 피질의 활동이 평가하실 수 있습니다. 특히, NIRS는 거의 침묵 (데스크탑 컴퓨터의 크기) 독특한 휴대용이며, 참가자 미묘한 움직임을 용납하실 수 있습니다. 이것은 반드시 핵심 구성 요소는 수화에서 음성 생산이나 손에 입의 움직임 중 하나로가 인간 언어의 신경 연구에 특히 뛰어난 있습니다. hemodynamic 응답화된되는 방법은 레이저 방출 및 감지기의 배열입니다. 감지기는 금액을 다시 대뇌 피질의 표면에서 반사를 감지하면서 방사체 비 이온화 빛의 알려진 강도를 내보냅니다. 가까이 함께 optodes을 더 큰 공간적 해상도, 더 떨어져 optodes 반면, 침투의 더 깊이. fNIRS 히타치 ETG - 4000 시스템은 최적의 보급 / 해상도 optode 배열은 2cm로 설정됩니다하십시오. 우리의 목표는 현장을 표준화 도움 공통 배경을 가지고 세계 여러 fNIRS 연구소를 활성화 fNIRS 데이터를 수집하고 분석 우리의 방법을 입증하는 것입니다.

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

여기 우리는 특히 어린이 두뇌 개발을 공부에 적외선 분광학 (fNIRS),인지 신경 과학에 사용되는 소설 비침습 두뇌 이미징 시스템, 주변 기능을위한 데이터 수집 및 데이터 분석 방법을 설명합니다. 이 방법은 데이터 수집 및 데이터 해석과 과학적 발견에 필수적인 분석의 보편적인 기준을 제공합니다.

가까운 적외선 분광학과 함께 아기, 어린이 및 성인의인지 기능을 탐색

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 조회수.  University of Michigan, Ann Arbor. 여기 우리는 특히 어린이 두뇌 개발을 공부에 적외선 분광학 (fNIRS),인지 신경 과학에 사용되는 소설 비침습 두뇌 이미징 시스템, 주변 기능을위한 데이터 수집 및 데이터 분석 방법을 설명합니다. 이 방법은 데이터 수집 및 데이터 해석과 과학적 발견에 필수적인 분석의 보편적인 기준을 제공합니다.

비디오: 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.

자유롭게 동물 행동의 신경 회로 활성화의 기록

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

연구

생물학

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.

조립, 조정 및 단백질 이미징을위한 Apertureless 니어 필드 적외선 분광 현미경의 사용

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.

사용자 정의 크기 MEG 사전 - 학교 어린이 Neuromagnetic 뇌 기능의 측정

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.

파장과 산화 스트레스에 레드 / 근적외선 치료의 강렬의 범위의 효과의 평가를위한 방법<em&gt; 체외</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.

근적외선 (NIR) 빛은 마우스 전정 감각 상피 세포에서 미토콘드리아 기능의 마커의 발현을 증가

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.

정제 된 야생형과 돌연변이 체 CFTR 단백질의 기능의 재구성 및 채널 활동 측정

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.

칩 엑소 방법 : 가까운 자료 쌍의 정밀과 단백질 DNA 상호 작용을 확인

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.

근적외선 민감성 코어 쉘 백신 전달 플랫폼 생산

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.

메틸살리실레이트 클리어링 및 3D 이미징에 의한 지방 조직 구조 탐구

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.

초고속 포스 클램프 분광법을 사용한 공정 미오신의 기계적 효소 특성 해부

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