Gli autori ringraziano la dottoressa Anne Gibbon, il dottor Christine Mackay e tutto lo staff di Monash Servizi per animali e la casa di fisiologia animale. Gli autori ringraziano Jeremy Weinstein e la squadra Mcube per il video e l&#39;animazione production.a

<ol><li> La pecora è a digiuno per 24 ore, ma ha permesso ad libitum acqua </li><li> La pecora è pesato. </li><li> Anaestheisa è indotta mediante iniezione endovenosa di tiopentone (20mg/kg). </li><li> La profilassi con antibiotici per via endovenosa può essere somministrato. </li><li> Intubazione endotracheale viene eseguita utilizzando una grandezza 7-9 tubo endotracheale cuffiato. </li><li> L&#39;anestesia è sostiene, con isoflurano, 1-3%, in 100% di ossigeno ad una portata di 2 litri al minuto. </li><li> Le osservazioni sono fatti e il livello di anestesia monitoraggio continuo. </li><li> Gli strumenti chirurgici necessari per questa operazione sono: pinza dentata, non pinza dentata, forbici Metzenbaum, forbici Mayo, porta-aghi, manico bisturi (lungo e corto), Kerrison Rongeurs, Rongeurs pituitaria, martello pneumatico chirurgico, punte e guardie, ganci dei nervi (ottuso e micro gancio), ascensore (per esempio, Watson Cheyne), isolati mono polari e bipolari elettrobisturi pinze, distrattore corpo vertebrale (cioè pin Caspar 16 millimetri), perno trapano distrazione e manica trapano, 2-0, e 3-0 curette angolato Misura 7 e 10 ventosa francese e tubo di aspirazione, Divaricatori Langenback e di auto-conservazione divaricatori cervicale. </li><li> La regione del collo è rasato e lavato con una soluzione detergente Betadine. </li><li> L&#39;animale viene quindi posto in posizione supina sul tavolo operatorio e il collo leggermente esteso. </li><li> La pelle è segnata con un ago e un x-ray è preso per confermare la C3 / 4 livello. </li><li> Il collo viene poi preparata con clorexidina alcolica, iodio alcolica e il 70% di etanolo in acqua. </li><li> Un drappo fenestrato piazza è utilizzato per il sito chirurgico e un grande drappo quadrato per il tavolo in testa. </li><li> Un microscopio operatorio, o ingrandimento Loupe e fari, sono utilizzati per la procedura chirurgica. </li><li> L&#39;incisione viene effettuata utilizzando un bisturi nel sito precedentemente identificati utilizzando raggi X </li><li> Successivamente, la dissezione attraverso gli strati investendo viene eseguita utilizzando forbici e un piano sviluppato mediale alla guaina carotidea e laterali alla trachea e l&#39;esofago. </li><li> L&#39;emostasi viene mantenuta attraverso la procedura con diatermia </li><li> Una volta che la colonna vertebrale si trova, la linea mediana dovrebbe essere palpato e occorre prestare attenzione per assicurare che sia retrazione mediale all&#39;arteria carotidea e laterale di trachea ed esofago. </li><li> Un ago baionetta viene quindi inserito il disco (questo impedisce l&#39;avanzamento involontario, che potrebbe rischiare di danneggiare il midollo spinale). Un drappo quadrato è usato per coprire il sito chirurgico. Prima di una radiografia intraoperatoria è preso per confermare la C3 / 4 livello del disco. </li><li> Pin distrazione Casper vengono poi inseriti in C3 e C4, rispettivamente. </li><li> Il disco è incisa con un 15-bisturi lama. </li><li> Il distrattore viene quindi inserito e lo spazio disco leggermente distratto a parte. </li><li> La discectomia è poi effettuata utilizzando Rongeurs e Rongeurs Kerrison. Un trapano e curette può anche essere di aiuto. </li><li> Le placche sono poi preparati con il trapano e curette fino all&#39;osso sanguinamento visualizzati. </li><li> Il legamento longitudinale posteriore può essere aperto, a questo punto, a seconda del disegno sperimentale. Un gancio smussato e 1 millimetro Kerrison sono i più utilizzati per questa procedura. </li><li> La ferita è poi irrigata con soluzione fisiologica, prima dell&#39;inserimento della gabbia intersomatica o materiale di prova. </li><li> La distrazione è rilassato annuncio del dispositivo controllato per verificare che sia saldamente in posizione. </li><li> I perni distrazione vengono poi rimossi e cera osseo possono essere inseriti per ottenere l&#39;emostasi nei fori delle ossa. </li><li> Il muscolo longus coli è chiusa con Vicryl. Chiusura a strati viene quindi eseguita, preferibilmente con una sutura intradermica per evitare la necessità per la rimozione sutura. </li><li> Xylocaine è infiltrato in tutto il sito chirurgico </li><li> Il gas anaestheitc è spento e, quando avviene la respirazione spontanea e segni di deglutizione sono evidenti, il tubo endotracheale viene rimosso. </li><li> L&#39;animale è poi permesso di recuperare in una gabbia metabolica sotto costante osservazione. </li><li> E &#39;importante che l&#39;animale è in posizione eretta con gambe ripiegate sotto di essa, per evitare il rigurgito. </li><li> Una volta che l&#39;animale è in piedi (di solito entro 1 ora) cibo e l&#39;acqua può essere reintrodotta. </li><li> Chiudere monitorati viene eseguita per 24 ore e continuato per 1 settimana. </li><li> L&#39;analgesia viene mantenuta con una patch fentanil per 3 giorni dopo l&#39;operazione. </li></ol>

<ol>
	<li>Angevine, P. D., Arons, R. R., McCormick, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=National+and+regional+rates+and+variation+of+cervical+discectomy+with+and+without+anterior+fusion,+1990-1999.">National and regional rates and variation of cervical discectomy with and without anterior fusion, 1990-1999.</a> Spine. 28, 931-940 (2003).</li><li>Cloward, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anterior+approach+for+removal+of+ruptured+cervical+disks.">The anterior approach for removal of ruptured cervical disks.</a> J Neurosurg. 15, 602-617 (1958).</li><li>Jacobs, W. C., Anderson, P. G., Limbeek, J., Willems, P. C., Pavlov, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+or+double-level+anterior+interbody+fusion+techniques+for+cervical+degenerative+disc+disease.">Single or double-level anterior interbody fusion techniques for cervical degenerative disc disease.</a> Cochrane Database Syst Rev. , CD004958-CD004958 (2004).</li><li>Kandziora, F., Pflugmacher, R., Scholz, M., Schnake, K., Lucke, M., Schr&#246;der, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+between+sheep+and+human+cervical+spines:+an+anatomic,+radiographic,+bone+mineral+density,+and+biomechanical+study.">Comparison between sheep and human cervical spines: an anatomic, radiographic, bone mineral density, and biomechanical study.</a> Spine. 26, 1028-1037 (2001).</li><li>Patil, P. G., Turner, D. A., 	Pietrobon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=National+trends+in+surgical+procedures+for+degenerative+cervical+spine+disease:+1990-2000.">National trends in surgical procedures for degenerative cervical spine disease: 1990-2000.</a> Neurosurgery. 57, 753-758 (2005).</li><li>Smith, G. W., Robinson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+treatment+of+certain+cervical-spine+disorders+by+anterior+removal+of+the+intervertebral+disc+and+interbody+fusion.">The treatment of certain cervical-spine disorders by anterior removal of the intervertebral disc and interbody fusion.</a> J Bone Joint Surg Am. 40-A, 607-624 (1958).</li></ol>

Ci sono alcune fonti di variabilità di prendere in considerazione durante la pianificazione e l&#39;esecuzione di discectomia cervicale anteriore e fusione. Pertanto, per coerenza del modello, gli animali devono essere della stessa età e razza. Noi preferiamo due anni, incroci Leicester pecore merino. Se eseguito correttamente, ci saranno complicazioni minime come sanguinamento, infezione, o la mortalità. Emorragia dovrebbe essere controllato durante tutta la procedura e haemosatsis controllato prima della chiusura. ...

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<td>Marcain</td>
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anterior cervical discectomy and fusion in the ovine model

Discectomia cervicale anteriore e fusione (ACDF) è l&#39;operazione chirurgica più comune per radicolopatia cervicale e / o mielopatia nei pazienti che hanno fallito il trattamento conservativo<sup&gt; 1,5</sup&gt;. Dal momento che l&#39;operazione è stata descritta la prima volta da Cloward<sup&gt; 2</sup&gt; E Smith e Robinson<sup&gt; 6</sup&gt; Nel 1958, una serie di raffinatezze tecniche, materiale dell&#39;innesto e gli impianti sono stati fatti<sup&gt; 3</sup&gt;. In particolare, vi è la necessità di sicuro agenti osteoinduttiva che potrebbero beneficiare i pazienti selezionati. Il modello di ovini ha dimostrato di avere anatomica, biomeccanica, la densità ossea e le proprietà radiologiche che sono simili alla controparte umana, il livello più analoghe C3 / 4<sup&gt; 4</sup&gt;. E &#39;quindi un modello ideale in cui gli studi pre-clinici possono essere eseguite. In particolare, questa metodologia può essere utile ai ricercatori interessati a valutare diversi dispositivi e farmaci biologici, comprese le cellule staminali, per le potenziali applicazioni per chirurgia spinale umano.

Watch this Scientific Journal Video about Anterior Cervical Discectomy and Fusion in the Ovine Model at JoVE.com

Anterior Cervical Discectomy and Fusion in the Ovine Model

Questo video mostra la tecnica di discectomia cervicale anteriore e fusione nel modello ovino.

Discectomia cervicale anteriore e fusione nel modello ovina

Anterior cervical discectomy and fusion (ACDF) is the most common surgical operation for cervical radiculopathy and/or myelopathy in patients who have ...

anterior-cervical-discectomy-and-fusion-in-the-ovine-model

Research

JoVE Journal

Biology

12.8K Views.  Monash University. Questo video mostra la tecnica di discectomia cervicale anteriore e fusione nel modello ovino.

Video: Anterior Cervical Discectomy and Fusion in the Ovine Model

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

Metodo per la misurazione di Cinetica fusione virale a livello di singola particella

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

Ricerca

Biologia

Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs) catalyze intracellular vesicle fusion1-4. Reconstitution assays are essential for dissecting the mechanism and regulation of SNARE-mediated membrane fusion5. In a cell fusion assay6,7, SNARE proteins are expressed ectopically at the cell surface. These "flipped" SNARE proteins drive cell-cell fusion, demonstrating that SNAREs are sufficient to fuse cellular membranes. Because the cell fusion assay is based on microscopic analysis, it is less efficient when used to analyze multiple v- and t-SNARE interactions quantitatively.
Here we describe a new assay8 that quantifies SNARE-mediated cell fusion events by activated expression of &beta;-galactosidase. Two components of the Tet-Off gene expression system9 are used as a readout system: the tetracycline-controlled transactivator (tTA) and a reporter plasmid that encodes the LacZ gene under control of the tetracycline-response element (TRE-LacZ). We transfect tTA into COS-7 cells that express flipped v-SNARE proteins at the cell surface (v-cells) and transfect TRE-LacZ into COS-7 cells that express flipped t-SNARE proteins at the cell surface (t-cells). SNARE-dependent fusion of the v- and t-cells results in the binding of tTA to TRE, the transcriptional activation of LacZ and expression of &beta;-galactosidase. The activity of &beta;-galactosidase is quantified using a colorimetric method by absorbance at 420 nm.
The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments10-15. By expressing VAMPs 1, 3, 4, 5, 7 and 8 at the same level, we compare their membrane fusion activities using the enzymatic cell fusion assay. Based on spectrometric measurement, this assay offers a quantitative approach for analyzing SNARE-mediated membrane fusion and for high-throughput studies.

We have developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of &beta;-galactosidase.

Analisi delle SNARE-mediata fusione della membrana L&#39;utilizzo di un test enzimatico fusione cellulare

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)  proteins on vesicles (v-SNAREs) and on target membranes ...

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) 1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.
The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency 3. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction 4. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues 5. This later change increases muscle stiffness 6. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Changes in limb muscle contractile and passive mechanical properties are important biomarkers for muscle diseases. This manuscript describes physiological assays to measure these properties in the murine extensor digitorum longus and tibialis anterior muscles.

Valutazione della funzione muscolare del muscolo estensore lungo delle dita<em&gt; Ex vivo</em&gt; E Muscolo tibiale anteriore<em&gt; In situ</em&gt; In Mouse

Body  movements are mainly provided by mechanical function of skeletal muscle. Skeletal  muscle is composed of numerous bundles of myofibers that are ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

L&#39;Assemblea e l&#39;applicazione di &quot;Shear Anelli &#39;: A Novel endoteliale modello per Orbital, unidirezionale e periodica del fluido di flusso e lo stress di taglio

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Espressione di proteine di fusione fluorescenti in macrofagi e cellule dendritiche derivate da midollo osseo Murine

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult mice for in vivo longitudinal real-time monitoring of thymocytes&#180; dynamics within a vascularized thymus segment. Following the transplantation, laser scanning microscopy (LSM) through the cornea allows in vivo noninvasive repeated imaging at cellular resolution level. Importantly, the approach adds to previous intravital T-cell maturation imaging models the possibility for continuous progenitor cell recruitment and mature T-cell egress recordings in the same animal. Additional advantages of the system are the transparency of the grafted area, permitting macroscopic rapid monitoring of the implanted tissue, and the accessibility to the implant allowing for localized in addition to systemic treatments. The main limitation being the volume of the tissue that fits in the reduced space of the eye chamber which demands for lobe trimming. Organ integrity is maximized by dissecting thymus lobes in patterns previously shown to be functional for mature T-cell production. The technique is potentially suited to interrogate a milieu of medically relevant questions related to thymus function that include autoimmunity, immunodeficiency and central tolerance; processes which remain mechanistically poorly defined. The fine dissection of mechanisms guiding thymocyte migration, differentiation and selection should lead to novel therapeutic strategies targeting developing T cells.

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The goal of the protocol is to show longitudinal intravital real-time tracking of thymocytes by laser scanning microscopy in thymic implants in the anterior chamber of the mouse eye. The transparency of the cornea and vascularization of the graft allows for continuously recording progenitor cell recruitment and mature T-cell egress.

Inseguimento In Vivo dei timociti nell'alloggiamento anteriore dell'occhio di microscopia a scansione Laser in tempo reale

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

Effetto di proteine fluorescenti su Fusion partner utilizzando saggi di tossicità di Polyglutamine in lievito

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Analisi della spettrometria di massa per identificare l'ubiquilazione del CENP-A con tag EYFP (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing need in developing efficient methods for recombinant antibody production. As of 2019, there were more than 70 FDA-approved monoclonal antibodies, and there is exponential growth potential. Despite their promise, limiting factors for widespread use are manufacturing costs and complexity. Potentially, plants offer low-cost, safe, and easily scalable protein manufacturing&#160;strategies. Plants like Nicotiana benthamiana not only can correctly fold and assemble complex mammalian proteins but also can add critical post-translational modifications similar to those offered by mammalian cell cultures. In this work, by using native GFP and an acid-stable variant of green fluorescent protein (GFP) fused to human monoclonal antibodies, we were able to visualize the entire transient antibody expression and purification process from N. benthamiana plants. Depending on the experiment&#39;s purpose, native GFP fusion can ensure easier visualization during the expression phase in the plants, while acid-stable GFP fusion allows for visualization during downstream processing. This scalable and straightforward procedure can be performed by a single researcher to produce milligram quantities of highly pure antibody or antibody fusion proteins in a matter of days using only a few small plants. Such a technique can be extended to the visualization of any type of antibody purification process and potentially many other proteins, both in plant and other expression systems. Moreover, these techniques can benefit virtual instructions and be executed in a teaching laboratory by undergraduate students possessing minimal prior experience with molecular biology techniques, providing a foundation for project-based exploration with real-world applications.

Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana

We describe here a simple method for expression, extraction, and purification of recombinant human IgG fused to GFP in Nicotiana benthamiana. This protocol can be extended to purification and visualization of numerous proteins that utilize column chromatography. Moreover, the protocol is adaptable to the in-person and virtual college teaching laboratory, providing project-based exploration.

Produzione di proteine di fusione IgG espresse transitoriamente in Nicotiana benthamiana

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing ...