실험 염증의 유도 : <ol><li> 첫 학위 햇볕은 보정, 자외선 B 소스 (Saalmann Multitester SBB LT 499, Saalmann GmbH를, 헤리퍼드, 독일)의 도움을 허벅지의 비 빠져있었단 피부에 유도합니다. 햇볕에 노출이 피부의 완전한 붉어지는 것, 또는 세 번 최소한의 선용 복용량 (MED)을 원인 UVB의 빛의 양을 필요로 세 번 적용하여 microdialysis 실험을 시작하기 전에 24 시간 유도합니다. MED는 연구 개시 전에 각 과목에서 결정됩니다. 에너지의 정확한 전달이 함께 보장되는 UV - m (RM 12, 박사 Grobel UV - 전자 GmbH를, Ettlingen, 독일). </li></ol> 카테 테르 배치 및 샘플 컬렉션 <ol><li> 멸균 장갑, 23 게이지 (G) 바늘, 30G의 바늘로 1cc 주사기, 1 % lidocaine 1 무균 바늘 홀더, 1cc luer 팁 주사기와 준비 영역 준비, 알코올 면봉, 2x2 멸균 거즈 패드, microdialysis의 카테터는 하나 첨부 4cm 23G 바늘 (피부 투석, 에를랑겐, 독일), 그리고 드라이 아이스에 100μl 샘플 당 4μl 테아제 억제제 솔루션 1.5 ML Microcent 수집 튜브 (E &amp; K 과학적, 산타 클라라, CA.) 최종. 프로 테아제 억제제 솔루션은 테아제 억제제 칵테일의 타블렛을 (로체 진단, GmbH를, 만하임, 독일)를 사용하여 제조 업체의 지시마다 이루어집니다. </li><li> lactated 링어의 용액에 1 %의 인간 알부민 솔루션을 준비하고 1cc luer 팁 주사기로 그려, 주사기에 남아 기포가가 없는지 만들기. </li><li> 고정밀 주입 펌프 (펌프 22, 하버드 장치, 사우스 내틱, MA에 장착 크래들 (microdialysis 멀티 주사기 랙, 하버드 장치, 사우스 내틱, MA)에 1% 인간 알부민 - 풍부한 대타의 락트 솔루션 주사기로드 ). 주사기를 운전 주입 펌프의 막대가 주사기의 플런저와 함께 전체 접촉에 있는지 확인하십시오. 주사기의 직경이 정확 펌프에 입력되었는지 확인 후 펌프 속도를 설정합니다. 일반적으로 사용되는 주입 속도는 2 사이 4μl/min까지 다양합니다. </li><li> 당신이에서 샘플을 수집하려는 영역을 정의합니다. 같은 햇볕 위에서 설명한, 햇볕의 가장자리에 카테터를 삽입 계획으로 인위적으로 만들어 염증 지역에서 샘플링 때. </li><li> 당신은 알코올에서 샘플을 수집하고자하는 작은 병변의 반대편 가장자리 영역 또는 사이트를 청소합니다. 이 청소 구역은 카테터를위한 목적으로 삽입하고 종료 지점 것입니다. 충분히 내부 cutaneously lidocaine 주입 (주입에있는 피부의 창백에 의해 입증)이 사이트에서 아주 작은 물집을 (2mm 최대) 만들 수 있습니다. microdialysis의 카테터의 입구와 출구 지점에서 내부 피부 lidocaine 이러한 한계의 주입은 bioassay 결과를 변경하지 말아야하지만, 학습 과목 훨씬 덜 괴롭 카테터의 삽입을 않습니다. </li><li> 무균 기법을 사용하여 탐구 피부 영역의 길이를 통해 첨부 23G 바늘의 도움으로 microdialysis의 카테터를 삽입합니다. 삽입의 깊이가 커티스와 subcutis 사이의 인터페이스에 있습니다. 적절한 깊이에 삽입 광학 확인할 수 있습니다. 바늘과 연결된 카테터가 앞으로 이동으로 바늘은 피부뿐만 아니라 피부의 일부 창백 약간 상향 폴딩을 일으킬 것입니다. 아무도 출혈이 발생해서는 안됩니다. 출혈이 발생하는 경우 이것은 바늘이 깊이 예정보다 있으며 삽입의 깊이가 조정할 수있다는 것을 나타냅니다. 바늘이 피부의 출구 지점을 통해 밀고있다 일단 당신이 카테 테르의 길이를 따라 방울을 볼 때까지, 1 % 알부민 풍부한 대타의 락트 솔루션 카테터를 플러시. 이것은주의 깊게 microdialysis의 카테터의 말초 끝에 주사기 (23G 바늘)의 바늘 끝을 삽입하고, 천천히 솔루션을 주사하여 이루어집니다. </li><li> 플러싱 후 카테터가 피부를 통해 연결된 카테터 먹이를 바늘에 부드럽게 당기십시오. 카테터는 작은 직경, 그리고 결과적으로되기 때문에 젠틀 처리가 필요합니다. 그것이 완전히 피부를 통해 스레드 때까지 카테터를 당겨 즉, 카테터의 말초 끝을 포함 큰 플라스틱 튜브의 기본은 진입 점에서 피부를 접​​촉. 위에서 언급한 바와 같이 프로 시저의 끝 부분에 출혈은 거의 최적의 카테 테르의 위치를​​ 확인합니다. </li><li> 주입 펌프의 선반에 휴식 주사기의 23G 바늘 끝에 카테터의 말초 끝을 연결합니다. 관리는 카테터의 벽을 통해 바늘을 펑쳐링 피하기 위해 필요합니다, 이것은 잠재적인 누수의 원인 것입니다. </li><li> 카테 테르의 근위 끝에서 바늘을 잘라요. </li><li> ¼ 인치 핑크색 테이프를 사용하여 카테터의 출구 사이트에 가까운 피부에있는 준비 1.5ml Microcent 수집 튜브를 연결합니다. microdialysate의 수집을 허용하도록 컬렉션 관에 카테 테르의 끝에 놓습니다. 예정된 R에서 재관류 펌프를 시작합니다먹고 간헐적으로 그 전달과 수집 량이 일치 확인합니다. 경우에는 볼륨은 피부 항목 사이트에서 누설 (때로는 더 단단히 피부 항목 사이트에 플라스틱 튜브가 내장된 카테터의 일부를 당기는 해결할 수 있습니다) 제외되어야를 일치하지 않습니다. 배달 및 수집 볼륨이 일치하지 않는 경우 또는 카테터를 교체해야 할 수도 있습니다. </li><li> 일단 샘플링이 완료되면 샘플은 -80 º C.에 보관까지 드라이 아이스에 표시됩니다 </li></ol>

<ol>
	<li>Angst, M. S., Clark, J. D., Carvalho, B., Tingle, M., Schmelz, M., Yeomans, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+profile+in+human+skin+in+response+to+experimental+inflammation,+noxious+stimulation+and+administration+of+a+COX-inhibitor:+A+microdialysis+study.">Cytokine profile in human skin in response to experimental inflammation, noxious stimulation and administration of a COX-inhibitor: A microdialysis study.</a> Pain. 139, 15-27 (2008).</li></ol>

The authors have nothing to disclose.

microdialysis 기법과 멀티 플렉스 면역 분석법 기술의 결합 사용은 관심의 조직에 생화 학적 사건에 대한 통찰력을 제공하는 귀중한 실시간에 - 생체내 인간 bioassay입니다. 이러한 통증 시험으로 행동 검사와 결합, 방법은 통증과 염증을 중재 biochemicals의 복잡한 상호 작용을 연구하고 있습니다. 방법은 주변 조직 병리학과 염증과 만성 통증 조건의 병리에 기계론의 통찰력을 제공 통증과 염증, 2) 중재 조직 구체적인...

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		<div>
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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Microdialysis catheter</td>
<td>Tool</td>
<td>Dermal Dialysis</td>
<td>NA</td>
<td>&nbsp;</td>
<tr>
<td>Pump 22</td>
<td>Tool</td>
<td>Harvard Apparatus</td>
<td>552219</td>
<td>&nbsp;</td>
<tr>
<td>Saalmann Multitester SBB LT 400</td>
<td>Tool</td>
<td>Saalmann GmbH</td>
<td>NA</td>
<td>&nbsp;</td>
<tr>
<td>RM-12 IP-65 Radiometer</td>
<td>Tool</td>
<td>Dr. Grobel UV Elektronik GmbH</td>
<td>82120000/81112100</td>
<td>&nbsp;</td>
<tr>
<td>Complete Proteinase Inhibitor</td>
<td>Reagent</td>
<td>Roche Bioscience</td>
<td>11836145001</td>
<td>&nbsp;</td>
<tr>
<td>Microcent tube</td>
<td>Tool</td>
<td>E&K Scientific</td>
<td>380150-X</td>
<td>&nbsp;</td>
<tr>
<td>Albumin 5%</td>
<td>Reagent</td>
<td>ZLB Behring</td>
<td>NA</td>
<td>&nbsp;</td>
<tr>
<td>Lactated Ringer</td>
<td>Reagent</td>
<td>Hospira Inc</td>
<td>NA</td>
<td>&nbsp;</td>
<tr>
<td>Xylocaine-MPF</td>
<td>Other</td>
<td>Astra Zeneca</td>
<td>NA</td>
<td>&nbsp;</td>
<tr>
<td>Syringe 1mL</td>
<td>Tool</td>
<td>Becton Dickinson Co</td>
<td>309602</td>
<td>&nbsp;</td>		</tbody>
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human in-vivo bioassay for the tissue-specific measurement of nociceptive and inflammatory mediators

이 - 생체내 인간 bioassay 인간 자원 봉사자와 환자를 공부하는 데 사용할 수 있습니다. 샘플은 같은 무균 삽입 microdialysis의 카테터 (피부 투석, 에를랑겐, 독일)를 통해 피부와 같은 조직 관련 사이트에서 수집하고 있습니다. 이 예제에서 설명된 인간 자원 봉사자에 실험적으로 염증이 피부의 침입형 유체의 컬렉션입니다. 샘플 수집은 다른 실험 테스트와 함께하실 수 있습니다. 예를 들어, 로컬 출시 biochemicals 및 실험적으로 염증이 피부에 아픈 자극에 주관적인 감성의 동시 평가는 통증과 염증의 생체를 식별하는 중요한 생화학 - 행동 링크를 제공합니다. 살아있는 인간 유기체로 표시 분석 고통 및 / 또는 염증을 기본 조직을 특정 프로세스에 기계론의 통찰력을 허용합니다. 이러한 신약 후보로 - - 통증 및 / 또는 염증성 조건의 치료를 타겟팅하는 방법은 또 기존 또는 소설 개입의 효과를 조사하기 위해 적합합니다. 이 문서는 인간의 학습 과목의 실험적 염증 피부 병변에서 중간 액체를 수집하기 위해 microdialysis 기법의 사용에 대한 자세한 설명을 제공합니다. 중간 유체 샘플은 일반적으로 50 microliters으로 볼륨에서와 같이 작은 샘플 100 analytes까지 시금 수 있도록 멀티 플렉스 비드 어레이 immunoassays의 도움으로 처리됩니다.

Watch this Scientific Journal Video about Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators at JoVE.com

Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators

기술은 통증과 염증을 중재 biochemicals의 측정을위한 관련 조직 사이트 (여기서, 실험적으로 염증 피부)에서 중간 액체 샘플에서 - 생체내 수집 제공됩니다.

Nociceptive 및 염증성 중재자의 조직 특정 측정을위한 인간 - VIVO의 Bioassay

This in-vivo human bioassay can be used to study human volunteers and patients. Samples are collected from pertinent tissue sites such as the skin via ...

human-vivo-bioassay-for-tissue-specific-measurement-nociceptive

Research

JoVE Journal

Biology

10.2K 조회수.  Stanford University School of Medicine. 기술은 통증과 염증을 중재 biochemicals의 측정을위한 관련 조직 사이트 (여기서, 실험적으로 염증 피부)에서 중간 액체 샘플에서 - 생체내 수집 제공됩니다.

비디오: Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

내부 핵심 현미경에 의해 골격근의 생체내 미세 순환 측정

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

연구

생물학

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds 

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. Collecting site-specific biochemical markers allows us to evaluate the relationship between surgical wound and serum levels; determine any associations between mediator release, pain and analgesic consumption; and evaluate the effect of systemic and peripheral drug administration on surgical wound biochemistry.
This methodology has been applied to healthy women undergoing elective cesarean delivery with spinal anesthesia. Wound exudate and serum mediators, in conjunction with pain scores and analgesics consumption were measured at 1, 6, 24, and 48 hours post-cesarean delivery. Biochemical mediators that were detected included IL-1&beta;, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNF&alpha;, INF&gamma;, G-CSF, GM-CSF, MCP-1 and MIP-1&beta;, nerve growth factor (NGF), prostaglandin E2 (PG-E2) and substance P. We found no correlations between wound and serum cytokines concentrations or time-release profiles (J Pain. 2008 Jul 9(7):650-7). This article describes and demonstrates the feasibility of collecting and assaying nociceptive and inflammatory mediators in surgical wounds at specific time points. The lack of significant correlations between serum and wound levels shows the importance of determining site-specific release if surgical wounds and localized pathologies are to be studied.

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds

A technique to collect and measure surgical wound biochemical mediators at specific time points.

외과 듀엣으로 Nociceptive 및 염증성 중재자를 수집 및 측정

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. ...

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH are highly regulated in yeast cells and occupy a central role in overall pH homeostasis. We describe protocols for ratiometric measurement of pH in vivo using pH-sensitive fluorophores localized to the vacuole or cytosol. Vacuolar pH is measured using BCECF, which localizes to the vacuole in yeast when introduced into cells in its acetoxymethyl ester form. Cytosolic pH is measured with a pH-sensitive GFP expressed under control of a yeast promoter, yeast pHluorin. Methods for measurement of fluorescence ratios in yeast cell suspensions in a fluorimeter are described. Through these protocols, single time point measurements of pH under different conditions or in different yeast mutants have been compared and changes in pH over time have been monitored. These methods have also been adapted to a fluorescence plate reader format for high-throughput experiments. Advantages of ratiometric pH measurements over other approaches currently in use, potential experimental problems and solutions, and prospects for future use of these techniques are also described.

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).

액포와 세포질의 pH 측정<em&gt; 생체 내</em&gt; 효모 세포 현탁액의

Vacuolar and  cytosolic pH are highly regulated in yeast cells and occupy a central role in  overall pH homeostasis. We describe  protocols for ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

Danio rerio를 사용하여 병적 인 인간 게놈의 생체 모델링에

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

혈관 내피 성장 인자의 평가에 대한 간단한 생물 검정

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

소뇌 대동맥 파열 상해 : 효율적<em&gt; In Vivo</em&gt; 평활근 세포 증식과 내피 기능 모델

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

유세포분석으로 생체내 단백질 자가 조립 검출 및 특성화

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

조직별 샤페론 상호 작용을 위해 검사하기 위해 Caenorhabditis elegans를 사용

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

In vivo Measurement of Knee Extensor Muscle Function in Mice

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the clinic and the research laboratory, maximal muscular strength is widely measured longitudinally in humans, with knee extensor musculature the most reported functional outcome. Pathology of the knee extensor muscle complex is well documented in aging, orthopedic injury, disease, and disuse; knee extensor strength is closely related to functional capacity and injury risk, underscoring the importance of reliable measurement of knee extensor strength. Repeatable, in vivo assessment of knee extensor strength in pre-clinical rodent studies offers valuable functional endpoints for studies exploring osteoarthritis or knee injury. We report an in vivo and non-invasive protocol to repeatedly measure isometric peak tetanic torque of the knee extensors in mice across time. We demonstrate consistency using this novel method to measure knee extensor strength with repeated assessment in multiple mice producing similar results.

Quantification of knee extensor maximal strength is imperative to understand functional adaptations to aging, disease, injury, and rehabilitation. We present a novel method to repeatedly measure in vivo knee extension isometric peak tetanic torque.

마우스에서 무릎 엑스텐서 근육 기능의 생체 측정

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

진핵 세포에서 번역 단지의 신속한 생체 내 고정 및 격리

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...