実験的な炎症の誘導： <ol><li>第一度日焼けはキャリブレーション、紫外線Bのソース（SaalmannマルチテスターSBB LT 499、Saalmann社、ヘレフォード、ドイツ）の助けを借りて、太ももの非日焼けした肌に誘導される。日焼けは、露出した皮膚の完全な発赤、または3回最小限の紅斑量（MED）を引き起こすために必要なUVB光線の3倍を適用することにより、マイクロダイアリシス実験を開始する前に24時間誘導される。 MEDは、研究開始前に各被験者に決定されます。エネルギーの正確な納期は、UV計（RM 12、博士グローベルUV -電子社、Ettlingenの、ドイツ）で保証されています。 </li></ol>カテーテル留置とサンプルの収集： <ol><li>滅菌手袋、23ゲージ（G）針、30Gの針と1cc注射器、1％リドカイン1滅菌ニードルホルダー、1ccルアーチップシリンジを使用してステージング領域を準備し、アルコール綿棒、2x2の滅菌ガーゼパッド、マイクロダイアリシスカテーテルは、一度に添付4センチメートル23G針（皮膚透析、エルランゲン、ドイツ）、そしてドライアイス上に100μlのサンプル当たり4μlプロテアーゼ阻害剤溶液と1.5ミリリットルMicrocentコレクションチューブ（E＆Kサイエンティフィック、サンタクララ、カリフォルニア州）に終了。プロテアーゼ阻害剤溶液は、プロテアーゼインヒビターカクテル錠剤（Roche Diagnostics社、社、マンハイム、ドイツ）を使用して、製造元の指示ごとに行われます。 </li><li>乳酸リンゲル液の1％ヒトアルブミン溶液を調製し、1ccルアーチップシリンジにそれを描く、注射器に残っているに気泡がないことを確認すること。 </li><li>高精度の注入ポンプ（ポンプ22、ハーバード装置、南ネイティック、マサチューセッツ州に取り付けたクレードル（微小透析マルチシリンジラック、ハーバード装置、南ネイティック、マサチューセッツ州）に1％ヒトアルブミンの濃縮乳酸溶液を注射器をロードする）。注射器を駆動する輸液ポンプのバーは、シリンジのプランジャーと完全に接触していることを確認してください。シリンジの直径が正確にポンプに入力されていることを確認した後にポンプ速度を設定します。一般的に使用される注入速度は2〜4μl/minの範囲です。 </li><li>あなたからのサンプルを収集する予定の領域を定義します。このような日​​焼け上記の、日焼けの端にカテーテルを挿入する上での計画として人工的に作られた炎症部位からサンプリングするとき。 </li><li>あなたがアルコールと、からサンプルを収集する予定の小さな病変の反対側の端の領域、またはサイトをクリーニングします。これらのクリーンなエリアでは、意図した挿入とカテーテルのための出口点になります。ちょうど足りるだけの非常に小さな水疱（2mmの最大）を作成するために、これらのサイトで（注射で皮膚の蒼白によって証明される）内皮下リドカイン注入する。マイクロダイアリシスカテーテルの入口と出口でイントラ皮膚リドカインのような限られた量の注射は、バイオアッセイの結果を変更しないでくださいしかし、研究対象者に対して大幅に少ない痛みカテーテルの挿入を行うことはありません。 </li><li>無菌技術を用いて、探索皮膚領域の長さ以上添付23G針の助けを借りて、微小透析カテーテルを挿入する。挿入の深さは、皮膚と皮下組織の間のインタフェースになります。十分な深さで挿入は光学的に検証することができます。針と接続されたカテーテルを前方に移動されると、針は皮膚の少し上の折りたたみだけでなく、皮膚の一部白化を引き起こす必要があります。出血は発生しないはずです。出血が発生した場合、これは針が深く、意図したよりも置かれていることを示し、挿入の深さは調節する必要があります。針が皮膚の出口ポイントを介してプッシュされた後に、カテーテルの長さに沿って液滴を表示されるまで、1％アルブミンの濃縮乳酸リンゲル液ソリューションでカテーテルをフラッシュする。これは、慎重にマイクロダイアリシスカテーテルの遠位端にシリンジ（23G針）の針の端を挿入し、ゆっくりと溶液を注入することにより行われます。 </li><li>カテーテルをフラッシュした後、皮膚を介して接続されたカテーテルを養うために針を軽く引っ張る。カテーテルが小径、そして結果的に壊れやすいので、丁重な扱いが必要です。それは完全に皮膚を介してスレッド化されるまで、カテーテルを引っ張って、すなわち、カテーテルの遠位端を埋め込む大きなプラスチックチューブのベースには、エントリポイントで肌に触れる。前述のように、手順の最後にない出血の少ない、最適なカテーテルの配置を確認する。 </li><li>輸液ポンプのラックで休んで注射器の23G針の先端にカテーテルの遠位端を取り付けます。ケアは、潜在的な漏れの原因となるカテーテルの壁を通して針を穿刺を回避するために必要です。 </li><li>カテーテルの近位端から針をカット。 </li><li> ¼インチのピンクのテープを使用して、カテーテルの出口部に近い皮膚に準備さ1.5ミリリットルMicrocentコレクションチューブを取り付けます。 microdialysateのコレクションを可能にするためにコレクションチューブにカテーテルの先端を置きます。所定Rにおける灌流ポンプを起動します。食べて、断続的に配信され、収集されたボリュームが一致していることを確認。ケース内のボリュームが一致しない、皮膚の侵入部位からの漏れは（時々皮膚の侵入部位に、より緊密にプラスチックチューブにより埋め込まれているカテーテルの部分を引いて解決することができます）除外する必要があります。配信され、収集されたボリュームが一致しない場合あるいは、カテーテルを交換する必要があります。 </li><li>サンプリングが完了すると、サンプルは-80℃で貯蔵するまでドライアイス上に配置されています</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.

微小透析技術と多重イムノアッセイ技術の併用は、関心のある組織の生化学的事象についての洞察を提供する人間のバイオアッセイ、in vivoで、リアルタイムの貴重なものです。このような痛みのテストのような行動テストと組み合わせることで、方法は痛みや炎症を介在する生化学物質の複雑な相互作用を研究することができます。方法は、末梢組織病理で炎症と慢性疼痛の条件の病理にメカニズムの洞察...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Microdialysis catheter</td>
<td>Tool</td>
<td>Dermal Dialysis</td>
<td>NA</td>
<td>&nbsp;</td>
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<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>
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<td>Complete Proteinase Inhibitor</td>
<td>Reagent</td>
<td>Roche Bioscience</td>
<td>11836145001</td>
<td>&nbsp;</td>
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<td>Microcent tube</td>
<td>Tool</td>
<td>E&K Scientific</td>
<td>380150-X</td>
<td>&nbsp;</td>
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<td>Albumin 5%</td>
<td>Reagent</td>
<td>ZLB Behring</td>
<td>NA</td>
<td>&nbsp;</td>
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<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>
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human in-vivo bioassay for the tissue-specific measurement of nociceptive and inflammatory mediators

この生体内のヒトバイオアッセイは、ヒトのボランティアと患者を研究するために使用することができます。サンプルは、無菌的に挿入した微小透析カテーテル（皮膚透析、エルランゲン、ドイツ）を経由して皮膚などの関連する組織のサイトから収集されます。この例に示すようにヒトのボランティアで実験的に炎症を起こしている皮膚から間質液のコレクションです。サンプルのコレクションは、他の実験的なテストと組み合わせることができます。例えば、局所的に放出さ生化学および実験的に炎症を起こしている皮膚の痛みを伴う刺激に対する主観的な感度の同時評価は、痛みや炎症のバイオマーカーを識別するための重要な生化学的、行動的リンクを提供しています。生きている人間の生体に提示アッセイは、疼痛および/または炎症の基礎となる組織特異的なプロセスへの機械的な洞察が可能になります。このような新薬候補として - - 痛みおよび/または炎症性状態の治療をターゲッティング法もよく、既存または新規の介入の有効性を調べるために適しています。この記事では、人間の被験者の実験的炎症を起こしている皮膚の病変から間質液を収集するためのマイクロダイアリシス技術の使用に関する詳細な説明を提供します。間質液のサンプルは、通常、50マイクロリットルのようなボリュームのような小さなサンプルで100検体までアッセイすることが可能マルチプレックスビーズアレイのイムノアッセイの助けを借りて処理されます。

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

技術は、痛みや炎症を介在する生化学物質の測定のための適切な組織のサイト（ここで、実験的に炎症を起こしている皮膚）から間質液サンプルの生体内のコレクションのために提示されます。

侵害受容性および炎症性メディエーターの組織特異的な測定のためのヒトのin vivoバイオアッセイ

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 ビュー.  Stanford University School of Medicine. 技術は、痛みや炎症を介在する生化学物質の測定のための適切な組織のサイト（ここで、実験的に炎症を起こしている皮膚）から間質液サンプルの生体内のコレクションのために提示されます。

ビデオ: 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.

イントラ不可欠な顕微鏡による骨格筋のin vivo微小循環測定における

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

生物学

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.

手術創における侵害受容性および炎症性メディエーターを収集し、測定

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).

液胞と細胞質のpHの測定<emインビボで&gt;</em&gt;酵母細胞懸濁液に

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.

ゼブラフィッシュを用いた病的ヒトゲノムの in vivoモデルで

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.

血管内皮増殖因子の評価のための簡易バイオアッセイ

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

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

ギャップ結合細胞間コミュニケーション：毒物および毒素の有害効果を評価するための機能バイオマーカー、および天然物の健康上の利点

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

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.

ネズミ大動脈挫傷：効率的<em&gt;インビボ</em&gt;平滑筋細胞の増殖と内皮機​​能のモデル

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

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.

カエノハブディティス・エレガンスを使用して組織特異的シャペロン相互作用をスクリーニングする

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.

マウスにおける膝伸張筋機能のインビボ測定

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.

真核細胞からのトランスレーショナル複合体の迅速な インビボ 固定と分離

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