Deneysel inflamasyon İndüksiyon: <ol><li> Birinci derece güneş yanığı, kalibre, ultraviyole B kaynağı (Saalmann Multitester SBB LT 499, Saalmann GmbH, Hereford, Almanya) yardımı ile uyluk olmayan tabaklanmış deri indüklenir. Güneş yanığı, tam maruz kalan deride kızarıklık ya da üç kez minimal eritemal doz (MED) neden UVB ışık miktarını gerekli üç kez uygulayarak mikrodiyaliz deney başlamadan 24 saat önce meydana gelir. MED, çalışmaya başlamadan önce her konuda belirlenir. Doğru enerji teslimat ile güvence altına alınmıştır UV-metre (RM 12, Dr. Grobel UV-Elektronik GmbH, Ettlingen, Almanya). </li></ol> Kateter yerleştirme ve örnek toplama: <ol><li> Steril eldiven, 1 adet 23 gauge (G) iğne, 1 cc şırınga 30G iğne ile,% 1 lidokain steril iğne tutucu, 1cc döner ucu şırınga ile bir hazırlama alanı hazırlayın, alkol çubuklar, 2x2 steril gazlı bez, mikrodiyaliz kateterler ekli 4cm 23G iğne (Dermal Diyaliz, Erlangen, Almanya), ve kuru buz üzerinde 100μl örnek başına 4μl proteaz inhibitörü çözümü ile 1,5 ml Microcent toplama tüpleri (E ve K Bilimsel, Santa Clara, CA.) sonunda. proteaz inhibitörü çözüm üreticinin talimat başına proteaz inhibitörü kokteyl tablet (Roche Diagnostics GmbH, Mannheim, Almanya) kullanılarak yapılır. </li><li> Laktat ringer solüsyonu% 1 human albumin solüsyonu hazırlayın ve 1cc döner ucu şırınganın içine çekmek, şırınga içinde kalan NO hava kabarcıkları vardır emin olun. </li><li> Şırıngaya bir yüksek hassasiyetli infüzyon pompası (Pompa 22, Harvard Apparatus, Güney Natick, MA üzerine monte beşiği (mikrodiyaliz multi-şırınga Rack, Harvard Apparatus, Güney Natick, MA) içine% 1 insan albumin-zenginleştirilmiş ringer laktat çözüm yükleyin .) Şırınga sürüş infüzyon pompası bar şırınga pistonu ile tam temas olduğundan emin olun. Şırınga çapı doğru pompa girmiş olduğunu onayladıktan sonra pompa hızını ayarlayın. Yaygın olarak kullanılan infüzyon oranları 2 4μl/min arasında değişmektedir. </li><li> Numune planı alan tanımlayın. Yukarıda açıklanan güneş yanığı, güneş yanığı kenarında kateter takmadan planı gibi, yapay olarak oluşturulmuş iltihaplı alan, örnekleme. </li><li> Alkol, örnek toplamak niyetinde küçük bir lezyon ters kenarlarında alan, veya site temizleyin. Bu silinen alanları için amaçlanan kateterin yerleştirilmesi ve çıkış noktaları olacaktır. Yeterli intra-kutanöz lidokain enjekte (enjeksiyon cilt beyazlaşma tarafından kanıtlandığı) bu sitelerin çok küçük bir bleb (maksimum 2 mm) oluşturmak için. Mikrodiyaliz kateter giriş ve çıkış noktaları cilt içi lidokain gibi sınırlı miktarda enjeksiyon biyoassay sonuçları değiştirmez, ancak çalışma konuları için önemli ölçüde daha az ağrılı kateterin yerleştirilmesi yapar. </li><li> Aseptik teknikleri kullanarak, keşfedilmeyi cilt alanının uzunluğu boyunca bağlı 23G iğne yardımı ile mikrodiyaliz kateter yerleştirin. Yerleştirme derinliği kutis ve subkutiste arasında arayüz. Yeterli bir derinliğe yerleştirilmesi optik kontrol edilebilir. Iğne, iğne ve ekli kateter ileri taşınır cilt gibi bazı cilt beyazlaşma biraz yukarı katlanır neden olmalıdır. Kanama meydana gelmelidir. Kanama meydana gelirse bu iğne istenenden daha derin bulunan ve yerleştirme derinliği ayarlanabilir olduğunu gösterir. Iğne derinin çıkış noktası üzerinden itilmiş sonra kateter uzunluğu boyunca damlacıkları görene kadar,% 1 albümin zenginleştirilmiş ringer laktat solüsyonu ile kateter yıkayın. Bu dikkatle mikrodiyaliz kateterin distal ucunu bir şırınga (23G iğne) iğne sonuna ekleme ve yavaş yavaş solüsyon enjekte yapılır. </li><li> Yıkamadan sonra kateter deri yoluyla ekli kateter beslemek için iğne hafifçe çekin. Kateterler, küçük çaplı, ve dolayısıyla kırılgan çünkü hassas davranılması gereklidir. Kateter çekin tamamen deri yoluyla dişli kadar, yani, kateterin distal ucu gömme büyük plastik boru tabanına giriş noktasında cilt dokunur. Yukarıda belirtildiği gibi, prosedürün sonunda kanama çok az optimal kateter onaylar. </li><li> Infüzyon pompası rafa dinlenme şırınga 23G iğne ucu kateterin distal ucu takın. Bakım kateter duvarın üzerinden iğne batırmak önlemek için gereklidir, potansiyel kaçak neden olur. </li><li> Kateterin proksimal ucundan iğne kesin. </li><li> ¼-inç pembe bant kullanımı, kateter çıkış yeri yakın cilt hazırlanan 1.5ml Microcent toplama tüpü takın. Microdialysate toplama izin için toplama tüpüne kateterin ucu yerleştirin. Önceden belirlenmiş r perfüzyon pompası Başlatyedik ve aralıklı bu teslim edilir ve toplanan hacmi kadar maçı kontrol. Miktarlar halinde, cilt giriş yeri kaçağı (bazen daha sıkı bir cilt giriş yeri üzerine plastik boru gömülü kateter parçası çekerek çözülebilir) ekarte edilmelidir kadar maç yok. Teslim edilir ve ciltlik eşleşmiyor Alternatif olarak, eğer bir kateter değiştirilmesi gerekebilir. </li><li> Örnekleme tam sonra, numuneler -80 ° C&#39;de depolama kadar kuru buz konur </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.

Mikrodiyaliz teknikleri ve multipleks immunoassay teknoloji kombine kullanımının ilgi doku biyokimyasal olaylar içgörü sağlayan değerli bir gerçek zamanlı, in vivo insan biyoassay. Ağrı testleri gibi davranış testleri ile birlikte, yöntem, ağrı ve iltihaplanma aracılık biyokimyasalın karmaşık etkileşimleri inceleyerek sağlar. Periferik dokularda patoloji ile kronik enflamatuar ve ağrı koşulları patoloji mekanistik bir kavrayış sağlayan ağrı ve iltihaplanma, 2) arabuluculuk doku spesifik biyobelirteçleri bel...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<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>
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<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>
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human in-vivo bioassay for the tissue-specific measurement of nociceptive and inflammatory mediators

Bu in vivo insan biyoassay insan gönüllüler ve hastalar çalışma için kullanılabilir. Örnekler aseptik takılı mikrodiyaliz kateterler (Dermal Diyaliz, Erlangen, Almanya) ile deri gibi yerinde doku sitelerinden toplanır. Bu örnekte gösterildiği insan gönüllüler üzerinde yapılan deneysel iltihaplı cilt interstisyel sıvı toplanması. Numune toplama diğer deneysel testler ile kombine edilebilir. Örneğin, aynı anda değerlendirme yerel piyasaya biyokimyasalın ve deneysel iltihaplı deride ağrılı uyaranlara subjektif hassasiyet, ağrı ve inflamasyon biyobelirteçleri tanımlamak için kritik biyokimyasal-davranışçı bağlantı sağlar. Sunan yaşayan insan organizması testi doku ağrısı ve / veya inflamasyon altında yatan spesifik süreçlere mekanistik anlayış sağlar. Bu yeni ilaç adaylarının - ağrılı ve / veya inflamatuvar koşulları tedavi hedeflemesi Bu yöntem aynı zamanda mevcut veya yeni müdahalelerin etkinliğini incelemek için uygundur. Bu makale, insan deneklerin deneysel iltihaplı cilt lezyonu interstisyel sıvı toplanması için mikrodiyaliz tekniklerinin kullanımı ile ilgili ayrıntılı bir açıklama sağlayacaktır. İnterstisyel sıvı numuneleri, genellikle multipleks boncuk dizisi immunoassay 50 mikrolitre hacmi gibi küçük numunelerin 100 analitler assaying izin yardımı ile işlenir.

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

In vivo ağrı ve inflamasyon aracılık biyokimyasalın ölçümü için ilgili doku siteleri (deneysel iltihaplı cilt) interstisyel sıvı örneklerinin toplanması için bir teknik sunulmuştur.

Nosiseptif ve İnflamatuar Arabulucular Doku Özel Ölçüm İnsan In-Vivo Biyoassay

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 Görüntüleme.  Stanford University School of Medicine. In vivo ağrı ve inflamasyon aracılık biyokimyasalın ölçümü için ilgili doku siteleri (deneysel iltihaplı cilt) interstisyel sıvı örneklerinin toplanması için bir teknik sunulmuştur.

Video: 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.

İskelet kas-içi hayati Mikroskopi in vivo Mikro sirkülasyon Ölçümü

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

Biyoloji

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.

Cerrahi Yaralar Nosiseptif ve İnflamatuar Arabulucular Toplama ve Ölçüm

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).

Vakuolar ve sitosolik pH Ölçümü<em&gt; In Vivo</em&gt; Maya hücre süspansiyonlarında

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.

Br.rerio kullanarak Morbid İnsan Genomu İn Vivo Modelleme

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.

Vasküler Endotelyal Büyüme Faktörlerinin Değerlendirilmesi için Basit Biyoassay

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.

Murin Aort Kırma Yaralanması: Verimli<emIn vivo</em&gt; Düz Kas Hücresi çoğalması ve Endotel Fonksiyon Modeli

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.

Algılama ve ölçme protein Self-montaj In vivo tarafından akış sitometri

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.

Dokuya Özgü Chaperone Etkileşimlerini Taramak için Caenorhabditis elegans'ı Kullanma

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.

Farelerde Diz Ekstensör Kas fonksiyonunun in vivo ölçümü

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.

Ökaryotik Hücrelerden Çeviri Komplekslerinin Hızlı In Vivo Fiksasyonu ve İzolasyonu

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