우리는 소중한 의견을위한 그림 3과 교수 요나 Bergquist에 대한 데이터를 기여한 한나 워너 감사드립니다. 스웨덴 연구 협의회 (부여 522-2006-6416 (MA), 521-2007-5407 (MA), 아케 Wiberg의 재단 (MA, JH), 과학 로얄 스웨덴어 아카데미 (MA, JH), 그리고 스웨덴의 화학 사회 (JH)가 기꺼이 재정적인 지원을 인정한다.

<ol>
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저자가 공개하는 게 없다.

neuropeptides의 연구에 든 이미징 질량 분광법을 고용의 몇 가지 장점이 있습니다. MS 데이터의 편견 분석은 특정 뇌 핵을 표시하거나 같은 striatum의 단지 dorsolateral 부분은 특정 pathophysiological 조건과 연결된 곳이 여기 제시된 결과에 있습니다. 공간 정보를 유지함으로써 그것은 전체 뇌 부분의 분석 또는 펩타이드 추출물에서 전통 peptidomics 연구를 사용 비해 높은 감도와 낮은 다양성과 통계적 분석을 수행하기 위해 관심 영역을 재정의하는 데 후 가능합니다. 또한, 그것은 든 IMS는 즉시 이전에 알려지지 않은 사후 translational 수정을 감지할 수 실현하는 것이 중요하지만, 구조적 분석은 수정되어 정확한 아미노산 위치를 결정하기 위해 따라야합니다. 든 IMS 데이터를 시각화의 일반적인 함정은 BL에서 선형 광학 규모로 최대 피크 강도를 매핑 포함대신 100 % 모든 부분의 최대 피크 강도가 어디 흔한 절대 규모에 대한 모든 부분을 매핑의 실험 시리즈의 각각의 섹션에서 (그림 3)에 대한 컬러 (100 %)까지 (0 %) 응답 (그림 5) . 후자의 방법은 그룹 데이터 및 치료 그룹 사이의 차이의 시각화의 비교를 허용합니다. 든 IMS 분석의 주요 장애물은 특정 질량 봉우리에 펩티드의 과제입니다. 온 조직 탠덤 - 질량 분광법 때로는 가능하지만 종종 13,14 꽤 어려움이 증명한다. 우리는 반대 위상 LC-MS/MS 뒤에는 강력한 양이온 교환 크로마 토그래피, 대한 예비 분별 (분리)를 포함하여 더 전통적인 접근 방식이 성공적으로 시퀀스 많은 neuropeptides 특히 opioid 펩티드하는 데 사용할 수있는 것을 발견했습니다. 그것은 같은 마스코트와 같은 일반적인 검색 엔진을 사용하여 모든 데이터베이스 항목이 일치하지 않는 양질의 MS / MS 스펙트럼을 얻기 위해 여전히 흔치 않습니다. 이러한 경우에는 손으로 드 노보-시퀀싱은 onl입니다Y 옵션도 있습니다. 피크 정체성의 궁극적인 증명은 적절한 녹아웃 마우스에서 조직 섹션 든 IMS에 의해 얻을 수있다, 그러나 이것은 항상 사용할거나 불가능합니다. 대안은 서구 immunoblotting이나 immunohistochemistry에 의해 예를 들어, 정반대로 다른 방법으로 결과를 확인하는 것입니다. 이것은 종종 항체 및 신규 항체를 확인하는 작업을 상당히 높이는 포함될 수 있습니다. 이 프로토콜에 제시된 일반적인 전략은 몇 가지 섹션 및 실험 조건을 포함한 대규모 neuropeptide 든 IMS 실험에 최적화되어 있습니다. 메커니즘 밑에있는 고통과 중독의 약물에 대한 내생적인 반응을 포함하여 연구의 다양한 분야에서 취업으로 프로토콜은 특히 opioid 펩티드에 최적화되었으며, 향후 연구에 큰 영향을 미칠 것이다.

1.1 샘플 수집 및 준비 <ol><li> Isoflurane (Apoteksbolaget AB, 스웨덴) </li><li> 수술 도구, 가위의 두 켤레 rongeur, 주걱 </li><li> 잘게 깔린 드라이 아이스 </li><li> 알루미늄 호일 및 저장을위한 비닐 봉지 </li><li> 그라 이오 스탯 (Microm, 하이 델베르크, 독일) </li><li> 인듐 - 주석 산화물 (ITO) 코팅 든 슬라이드 (Bruker Daltonics, 독일) </li><li> 최적의 절삭 온도 매체 (-10 월) (사쿠라 Finetek 유럽, 네덜란드) </li><li> 진공 dessicator </li></ol> 1.2 매트릭스 응용 <ol><li> 의 Acetonitrile (ACN), 메탄올 (MeOH), 에탄올 (EtOH) 프로 분석 학년 (머크, 독일). </li><li> Trifluoroacetic 산성 (TFA) (시그마 알드리치, 세인트 루이스, 미주리). </li><li> 물이 밀리 Q 정화 시스템 (Millipore, 베드 포드, MA)로 정화되었다. </li><li> 칩-1000 (Shimadzu, 일본) 또는 세로 630 망보고 (LabCyte, 서니 베일, 캘리포니아) </li></ol> 1.3 질량 분광법 <ol><li> ULtraflex II (Bruker Daltonics) </li><li> FlexImaging 소프트웨어 (V 2.0, Bruker Daltonics) </li><li> 펩타이드 또는 단백질 보정 표준 (Bruker Daltonics) </li></ol> 1.4 데이터 처리 <ol><li> 기원 v.8.1 (OriginLab 사, Norhampton, MA) </li><li> Matlab 버전 7.9.0.529 (MathWorks, Natic​​k, MA) </li><li> pBin ( <a href="http://www.vicc.org/biostatistics/software.php" target="_blank">http://www.vicc.org/biostatistics/software.php</a> ) </li></ol> 1.5 Neuropeptide 식별 <ol><li> 초음파 sonicator </li><li> 10 kDa 분자량 차단 필터 (Millipore, 베드 포드, MA) </li><li> SP Sephadex C 25 겔 (시그마, 독일) </li><li> Hypersil 칼럼 (ThermoFisher) </li><li> 피리딘, 프로 분석 등급 개미의 산 (FA), Acetonitrile (ACN), 메탄올 (MeOH), 에탄올 (EtOH) (머크, 독일). </li><li> Trifluoroacetic 산성 (TFA) 시그마 알드리치 (세인트 루이스, 미주리, 미국). </li><li> 애질런트 1100 nanoflow LC, microWPS, C18 컬럼 (0.075x150mm) </li><li> 애질런트 1100 마이크로 fractioncollector, Prespotted anchorchip 대상 플레​​이트 (Bruker Daltonics) </li><li> BioTools 소프트웨어 (V 3.1 SR2, Bruker Daltonics) </li><li> 바이오 작품 소프트웨어 (v.3.3, 써모 피셔, <a href="http://www.thermoscientific.com" target="_blank">www.thermoscientific.com</a> ) </li><li> 마스코트 소프트웨어 (V 2.2 MatrixScience; <a href="http://www.matrixscience.com" target="_blank">www.matrixscience.com</a> ) </li><li> Swepep 데이터베이스 ( <a href="http://www.swepep.org" target="_blank">www.swepep.org</a> ) </li></ol>

maldi imaging mass spectrometry of neuropeptides in parkinson's disease

든 이미징 질량 분석법 (IMS)은 생물 학적 조직 샘플 2 (Fig.1)의 분자 종의 공간적 분석을 용이하게하는 강력한 방법이다. 12 μm의 얇은 조직 섹션은 일반적으로 든 TOF / TOF 질량 분석기를 사용하여 질량 분석기로 감지할 수 온전 펩티드와 단백질의 탈착 및 이온화를 촉진 든 행렬로 덮여 있습니다. 일반적으로 피크 수백은 단일 쥐의 뇌 조직 섹션에서 평가 할 수 있습니다. 일반적으로 사용되는 영상 기술 달리, 이러한 방식은 관심 분자의 사전 지식을 필요로하지 않습니다 높은 분자 특이성과 감도 2를 유지하면서 여러 분자 종 혼자 가잖아하고 종합적인 분석을 허용합니다. 여기에서 우리는 동물 모델 파킨슨병 (PD)의 쥐의 두뇌에 neuropeptides의 특정 지역과 관련된 유통 프로필을 elucidating위한 든 IMS 기반의 접근 방식을 설명합니다. PD 나이 3,4 65 명 이상 1 %의 유행과 함께 일반적인 neurodegenerative 질병입니다. 가장 일반적인 증상 치료는 L-DOPA 5를 사용 도파민 교체에 따라 달라집니다. 그러나 이것은 L-DOPA 유발 dyskinesias (뚜껑) 1,3,6 되나 불수의 이상 운동을 포함한 심각한 부작용이 동반된다. 뚜껑에서 가장 두드러진 분자 변화 중 하나는 opioid 전구체 prodynorphin의 mRNA 7 upregulation이다. dynorphin의 펩티드는 본질적으로 운동 제어 7,8에 관련된 뇌 영역에서 neurotransmission 보죠. 그러나, neuropeptide 전구체의 처리에서 발생한 정확한 opioid 펩티드의 연대를 특징되지 않았습니다. 따라서, 우리는 실험 파킨슨병 및 L-DOPA 유도 운동 이상증의 동물 모델에서 든 IMS를 활용. 든 이미징 질량 분광법은 neuropeptide characteriza에 관하여 특히 유리한 것으로 판명기, 일반적으로 사용되는 항체 기반의 접근 방식부터 알려진 펩타이드 시퀀스와 이전에 관찰 포스트 translational 수정을 대상으로하고 있습니다. 반대로 든 IMS는 새로운 펩타이드 가공 제품을 해명하기 때문에의 연결을 전송 neuropeptide 변조의 새로운 분자 메커니즘을 밝힐 수 있습니다. neuropeptides의 절대 액수가 든 IMS에 의해 결정될 수 없지만 펩타이드 이온의 상대적인 풍요로움은 건강과 질병의 레벨 변경에 대한 통찰력을 제공, 대량 스펙트럼에서 delineated 수 있습니다. 여기에 제시된 예제에서는 dynorphin B, 알파 neoendorphin과 물질 P의 정상 농도는 크게 dorsolateral 증가된다는 것을 발견했다지만, 심한 운동 이상증과 동물의 dorsomedial, striatum는 (그림 얼굴, 몸통과 orolingual 근육을 포함하지 5). 또한, 든 IMS는 기능성 inacti의 이전에 알려지지 않은 메커니즘을 대표하는, 운동 이상증의 심각도 및 DES-티로신 알파 neoendorphin 수준 간의 상관 관계를 밝혀N-터미널 티로신의 제거 등 striatum에서 dynorphins의 vation는 dynorphin의 opioid - 수용체 결합 능력 9를 줄여줍니다. 이 든 IMS와 결과가 생물 의학 연구의 모든 분야에 응용 프로그램에 대한 기술의 잠재력을 강조를 사용하여 뚜껑의 neuropeptide 특성에 관한 최초의 연구이다.

Watch this Scientific Journal Video about MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease at JoVE.com

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson&#039;s Disease

L-DOPA를 사용하여 도파민 대체 약물은 파킨슨병에서 가장 일반적으로 사용되는 증상을 치료하지만, 불수의 이상 운동, 되나 운동 이상증을 포함한 부작용이 동반된다<sup&gt; 1</sup&gt;. 여기서 든 이미징 질량 분석법을위한 프로토콜은 운동 이상증과 관련된 쥐의 두뇌 neuropeptide 수준의 변화를 감지하여 그 제공됩니다.

파킨슨병의 Neuropeptides의 든 이미징 질량 분광법

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 ...

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MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

25.8K 조회수.  Uppsala University. L-DOPA를 사용하여 도파민 대체 약물은 파킨슨병에서 가장 일반적으로 사용되는 증상을 치료하지만, 불수의 이상 운동, 되나 운동 이상증을 포함한 부작용이 동반된다 1. 여기서 든 이미징 질량 분석법을위한 프로토콜은 운동 이상증과 관련된 쥐의 두뇌 neuropeptide 수준의 변화를 감지하여 그 제공됩니다.

비디오: MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson&#039;s Disease

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

파킨슨병의 일방적-6-OHDA lesioned 마우스 모델의 개발

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

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Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

인간의 기본 섬유 아세포의 유도를위한 피부 펀치 생검 절편 문화

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

파킨슨 병 분야에서 Mitochondrial Phenotypes을 모니터링하기위한 기본 인간 섬유 아세포의 사용

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson&#039;s Disease

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

파킨슨 병에서 감수성 메커니즘을 마스크를 해제하는 유전자 환경 상호 작용 모델

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Controlling Parkinson's Disease With Adaptive Deep Brain Stimulation

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time according to fluctuating disease and medication state. In the present realization of adaptive DBS we record and stimulate from the DBS electrodes implanted in the subthalamic nucleus of patients with Parkinson&#39;s disease in the early post-operative period. Local field potentials are analogue filtered between 3 and 47 Hz before being passed to a data acquisition unit where they are digitally filtered again around the patient specific beta peak, rectified and smoothed to give an online reading of the beta amplitude. A threshold for beta amplitude is set heuristically, which, if crossed, passes a trigger signal to the stimulator. The stimulator then ramps up stimulation to a pre-determined clinically effective voltage over 250 msec and continues to stimulate until the beta amplitude again falls down below threshold. Stimulation continues in this manner with brief episodes of ramped DBS during periods of heightened beta power.
Clinical efficacy is assessed after a minimum period of stabilization (5 min) through the unblinded and blinded video assessment of motor function using a selection of scores from the Unified Parkinson&#39;s Rating Scale (UPDRS). Recent work has demonstrated a reduction in power consumption with aDBS as well as an improvement in clinical scores compared to conventional DBS. Chronic aDBS could now be trialed in Parkinsonism.

Controlling Parkinson&#039;s Disease With Adaptive Deep Brain Stimulation

Adaptive deep brain stimulation (aDBS) is effective for Parkinson&#8217;s disease, improving symptoms and reducing power consumption compared to conventional deep brain stimulation (cDBS). In aDBS we track a local field potential biomarker (beta oscillatory amplitude) in real time and use this to control the timing of stimulation.

적응 깊은 뇌 자극으로 파킨슨 병을 제어

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

골격근 질환의 정량적 자기 공명 영상

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

액체 및 고체 종양 유래의 암세포 프로테옴 정량적 질량 분석 프로파일

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

Development of an Alpha-synuclein Based Rat Model for Parkinson&#039;s Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

재조합 아데노 - 관련 바이러스 성 벡터의 정위 주사를 통해 파킨슨 병에 대한 알파 - 시누 클레인 기반 쥐 모델 개발

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS)

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a specific anatomical region. Within the brain, metals have a highly compartmentalized distribution, depending on the primary function they play within the central nervous system. Imaging the spatial distribution of metals has provided unique insight into the biochemical architecture of the brain, allowing direct correlation between neuroanatomical regions and their known function with regard to metal-dependent processes. In addition, several age-related neurological disorders feature disrupted metal homeostasis, which is often confined to small regions of the brain that are otherwise difficult to analyze. Here, we describe a comprehensive method for quantitatively imaging metals in the mouse brain, using laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) and specially designed image processing software. Focusing on iron, copper and zinc, which are three of the most abundant and disease-relevant metals within the brain, we describe the essential steps in sample preparation, analysis, quantitative measurements and image processing to produce maps of metal distribution within the low micrometer resolution range. This technique, applicable to any cut tissue section, is capable of demonstrating the highly variable distribution of metals within an organ or system, and can be used to identify changes in metal homeostasis and absolute levels within fine anatomical structures.

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry LA-ICP-MS

Quantitatively mapping metals in tissue by laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) is a sensitive analytical technique that can provide new insight into how metals participate in normal function and disease processes. Here, we describe a protocol for quantitatively imaging metals in thin sections of mouse neurological tissue.

유도 결합 플라즈마 - - 레이저 박리에 의한 뇌 조직의 이미징 금속 질량 분석 (LA-ICP-MS)

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a ...

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI&#8212;Application in Premanifest Huntington's Disease

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy begins many years prior to the motor onset (during the &#34;premanifest&#34; period), but the spatiotemporal pattern of regional atrophy across the brain has not been fully characterized. Here we demonstrate an online cloud-computing platform, &#34;MRICloud&#34;, which provides atlas-based whole-brain segmentation of T1-weighted images at multiple granularity levels, and thereby, enables us to access the regional features of brain anatomy. We then describe a regression model that detects statistically significant inflection points, at which regional brain atrophy starts to be noticeable, i.e. the &#34;change-point&#34;, with respect to a disease progression index. We used the CAG-age product (CAP) score to index the disease progression in HD patients. Change-point analysis of the volumetric measurements from the segmentation pipeline, therefore, provides important information of the order and pattern of structural atrophy across the brain. The paper illustrates the use of these techniques on T1-weighted MRI data of premanifest HD subjects from a large multicenter PREDICT-HD study. This design potentially has wide applications in a range of neurodegenerative diseases to investigate the dynamic changes of brain anatomy.

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI&amp;#8212;Application in Premanifest Huntington&#039;s Disease

This paper describes a statistical model for volumetric MRI data analysis, which identifies the &#34;change-point&#34; when brain atrophy begins in premanifest Huntington&#39;s disease. Whole-brain mapping of the change-points is achieved based on brain volumes obtained using an atlas-based segmentation pipeline of T1-weighted images.

전체 두뇌 세분화 및 해 부 뇌 MRI의 변화 포인트 분석-Premanifest Huntington의 질병에 응용

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy ...