Je voudrais proposer un grand merci à David Carlson, professeur d&#39;anglais à Matsumoto dentaire de l&#39;Université, pour son soutien en nature de rédaction du présent rapport. Je tiens également à remercier Tadafumi Adachi, un séminariste dans notre laboratoire, pour son respect en tant que sujet dans cette enquête.

1. Préparation des électrodes EMG <ol><li> Commencez par préparer neuf électrodes. Huit d&#39;entre eux sont utilisés pour l&#39;enregistrement de signaux à partir de sites sur le muscle, et on est une électrode de référence. </li><li> Après avoir connecté les électrodes d&#39;enregistrement et la référence à l&#39;amplificateur, remplir les disques avec la conductance électrique pâte. Chaque type est OK, mais un type fluide à basse est meilleure. </li><li> Avant de fixer les électrodes à la peau, ont fait l&#39;objet fermement, et de déterminer l&#39;emplacement approximatif du muscle masséter sur le visage. </li><li> Le tendon supérieur du muscle masséter s&#39;attache à l&#39;arcade zygomatique, et son tendon inférieur à l&#39;angle de la mandibule. </li><li> Placer les électrodes d&#39;enregistrement huit sur un long morceau de ruban adhésif en égale distance inter-électrodes, et joignez le tableau de la surface de la peau. L&#39;électrode de premier enregistrement est placé sur l&#39;arcade zygomatique, et le dernier sur l&#39;angle de la mandibule. </li><li> Enfin, joignez l&#39;électrode de référence à l&#39;extrémité du nez. </li></ol> 2. L&#39;enregistrement EMG <ol><li> Pour commencer l&#39;enregistrement, tout d&#39;abord définir les paramètres de l&#39;amplificateur analogique multi-canal (MEG6100). Réglez le gain x 500, le filtre passe-bas à 0,5 Hz, et le filtre passe-haut à 10 kHz. </li><li> Connectez les huit sorties de l&#39;amplificateur analogique à un convertisseur analogique-numérique (PCI-MIO-16E-4). Le signal numérique est traitée avec le logiciel LabVIEW avec un taux d&#39;échantillonnage de 20 kHz. </li><li> Lorsque tout est connecté, EMG records au cours rapide des chewing-gums serrant sur le côté ipsilatéral. </li><li> Les traces des huit canaux sont présentés sur l&#39;écran LabView immédiatement après le processus d&#39;enregistrement a été achevé. </li></ol> 3. L&#39;extraction du PPE et de l&#39;observation de ses fonctions <ol><li> Pour extraire la composante PPE, retirez d&#39;abord la composante du potentiel d&#39;action de l&#39;EMG brutes en utilisant une fréquence de coupure haute du filtre numérique (filtre de Butterworth). </li><li> Commencer à appliquer le filtre avec une fréquence de coupure de 10 Hz. Une vague lente qui dévie dans le sens positif ou négatif devrait apparaître. Cette vague correspond à la PPE, et la différence de polarité reflète la relation puits-source du courant plaque d&#39;extrémité. </li><li> Lors de l&#39;enregistrement à partir du muscle masséter, la déviation la plus négative de l&#39;onde est attendu dans la trace du site d&#39;enregistrement les plus inférieurs, et la polarité doit passer autour de 3 sites éloignés de ce site. </li><li> L&#39;ampleur de la composante PPE peut être mesuré comme un point fort de la déviation ou une zone pendant la flexion. </li><li> Le changement de polarité de la composante PPE peut être déterminé en traçant la vague du PPE de chaque canal contre une chaîne de référence qui a montré le fléchissement le plus négatif. </li><li> Maintenant, en augmentant la fréquence de coupure, l&#39;onde lente deviendra oscillatoire. L&#39;oscillation va augmenter avec l&#39;augmentation de la fréquence de coupure et seront généralement devenu évident, à une fréquence de coupure de 30 Hz. </li><li> La phase de l&#39;onde sera inverse à travers la trace dans lequel la polarité de l&#39;onde de 10Hz filtrée PPE a montré une inversion. </li><li> Le déphasage de l&#39;oscillation peut être vérifié avec les mêmes graphiques qui ont été appliqués à la vague de 10Hz-filtrée PPE. </li><li> De l&#39;intervalle entre les voisins pics positifs et / ou négatifs de l&#39;oscillation, nous pouvons obtenir une approximation de la fréquence de l&#39;oscillation </li></ol> 4. L&#39;enregistrement des résultats EMG <img src="/files/ftp_upload/1653/1653Fig1a.jpg" alt="Figure 1 une" /> Figure 1a: Montré ici est une vue schématique des huit sites le long du muscle masséter qui ont été enregistrées à partir. <img src="/files/ftp_upload/1653/1653Fig1b.jpg" alt="Figure 1 b" /> Figure 1b: Nous voyons d&#39;un échantillon de l&#39;EMG monopolaire enregistrées simultanément à partir des sites sur le muscle masséter en réponse à serrer la gomme rapide sur le côté ipsilatéral. La composante PPE a été extrait en utilisant un filtre coupe-haut numérique avec une fréquence de coupure de 10Hz, qui se superpose à chaque crue EMG. Dans cet essai, la déviation la plus négative a été observée dans les traces-5 et -6 et la polarité changé travers oligo-3. <img src="/files/ftp_upload/1653/1653Fig1c.jpg" alt="Figure 1c" /> Figure 1c: Lorsque la fréquence de coupure du filtre coupe-haut numérique a été mis à 30Hz, l&#39;onde lente montré une oscillation. Sa phase aussi déplacé à travers oligo 3, où la polarité de l&#39;onde 10Hz-filtrée PPE changé. L&#39;intervalle entre les voisins crêtes positives de l&#39;oscillation a été mesurée. Dans ce sujet, la moyenne inter-pic de l&#39;intervalle était de 31,7 ± 6,5 ms de plus de 16 enregistrements.

<ol>
	<li>Eccles, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The physiology of synapses. , (1964).</li><li>Mitzdorf, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+source-density+method+and+application+in+cat+cerebral+cortex:+investigation+of+evoked+potentials+and+EEG+phenomena.">Current source-density method and application in cat cerebral cortex: investigation of evoked potentials and EEG phenomena.</a> Physiol. Rev. 65, 37-100 (1985).</li><li>Rall, W., Shepherd, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theoretical+reconstruction+of+field+potentials+and+dendrodendritic+synaptic+interactions+in+olfactory+bulb.">Theoretical reconstruction of field potentials and dendrodendritic synaptic interactions in olfactory bulb.</a> J. Neurophysiol. 31, 884-915 (1968).</li><li>Richardson, T. L., Turner, R. W., Miller, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action-potential+discharge+in+hippocampal+CA1+pyramidal+neurons:+current+source+density+analysis.">Action-potential discharge in hippocampal CA1 pyramidal neurons: current source density analysis.</a> J. Neurophysiol. 58, 981-996 (1987).</li><li>Kumai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Location+of+the+neuromuscular+junction+of+the+human+masseter+muscle+estimated+from+the+low+frequency+component+of+the+surface+electromyogram.">Location of the neuromuscular junction of the human masseter muscle estimated from the low frequency component of the surface electromyogram.</a> J. Jpn. Physiol. 55, 61-68 (2005).</li><li>Mito, K., Sakamoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+muscle+fiber+conduction+velocity+of+m.+masseter+during+voluntary+isometric+contraction.">Distribution of muscle fiber conduction velocity of m. masseter during voluntary isometric contraction.</a> Electroencephalogr. Clin. Neurophysiol. 40, 275-285 (2000).</li><li>Tokunaga, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+configuration+of+the+myoneural+junctions+of+human+masticatory+muscle+detected+with+matrix+electrode.">Two-dimensional configuration of the myoneural junctions of human masticatory muscle detected with matrix electrode.</a> J. Oral Rehabili. 25, 329-334 (1998).</li><li>Rohen, J. H., Yokochi, C., L&#x0171;tjen-Drecoll, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Color atlas of anatomy. , (2006).</li><li>Wichmann, T., DeLong, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillation+in+the+basal+ganglia.">Oscillation in the basal ganglia.</a> Nature. 400, 621-622 (1999).</li></ol>

<ol><li> Comme le potentiel négatif est considéré comme formé par un écoulement dans les cours du flasque, et la déviation positive, par son flux de sortie [1,2,3,4], la trace montrant la déviation la plus négative doit correspondre à la site de localisation de la jonction neuromusculaire [5]. La figure 1b indique la jonction neuromusculaire du muscle masséter localiser dans son étroite partie inférieure à l&#39;angle de la mandibule, qui est approximativement le même que les résultats obtenus par une autre méthode utilisant la ...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Analogue filter</td>
<td>&nbsp;</td>
<td>Nihon Kohden</td>
<td>MEG6100</td>
<td>pre-,main-amplifier</td>
</tr>
<tr>
<td>A/D converter</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>PCI-MIO-16E-4</td>
<td>PCI board for computer</td>
</tr>
<tr>
<td>Connection interface</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>BNC-2090</td>
<td>8ch BNC adaptor</td>
</tr>
<tr>
<td>Disc electrodes</td>
<td>&nbsp;</td>
<td>Nihon Kohden</td>
<td>NS-11</td>
<td>Ag/AgCl (&Phi;8mm)</td>
</tr>
<tr>
<td>Electrode past</td>
<td>&nbsp;</td>
<td>Sanshin</td>
<td>SA-5</td>
<td>Semi-fluid carbon past</td>
</tr>
<tr>
<td>LabVIEW (Digital filter)</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>V. 8.5</td>
<td>Programming language</td>
</tr>
<tr>
<td>Chewing gum</td>
<td>&nbsp;</td>
<td>Lotte</td>
<td>Green gum</td>
<td>Test food in clenching</td>
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extraction of the epp component from the surface emg

Un électromyogramme de surface (EMG), surtout lorsqu&#39;il est enregistré près de la jonction neuromusculaire, est censé contenir le potentiel des plateaux vertébraux (PPE) composant qui peut être extraite par un filtre de signal approprié. Deux facteurs sont importants: l&#39;EMG doit être enregistrée dans la mode monopolaire, et l&#39;enregistrement doit être fait pour que le signal basse fréquence correspondant au PPE n&#39;est pas éliminée. Ce rapport explique comment extraire la composante PPE de l&#39;EMG du muscle masséter chez un sujet humain. L&#39;EMG de surface est enregistré dans huit sites en utilisant des électrodes à disque traditionnel alignés le long du muscle, avec la distance inter-électrodes égale de l&#39;arcade zygomatique à l&#39;angle de la mandibule en réponse à serrer la gomme rapide. Une électrode de référence est placé sur le bout du nez. La composante PPE est extraite de l&#39;EMG premières en appliquant un coupe-haut filtre numérique (2ème dimension Butterworth filtre) avec une gamme de 10 à 35 Hz. Quand le filtre est fixé à 10 Hz, l&#39;extrait du PPE ondes dévie soit positif ou négatif selon le site d&#39;enregistrement. La différence dans la polarité reflète la relation puits-source du courant plaque d&#39;extrémité, avec le site montrant la déviation la plus négative correspondant à la jonction neuromusculaire. Dans le cas du muscle masséter, la jonction neuromusculaire est estimé à être situé dans la partie inférieure proche de l&#39;angle de la mandibule. La composante PPE présente une oscillation intéressante lorsque la fréquence de coupure du filtre coupe-haut numérique est réglé à 30 Hz. L&#39;oscillation du PPE indique que la contraction musculaire est ajustée de façon intermittente. Tremblements anormal qui accompagne toutes sortes de maladies peuvent être substantiellement en raison de cette oscillation du PPE, qui devient plus lent et il est difficile de cesser.

Watch this Scientific Journal Video about Extraction of the EPP Component from the Surface EMG at JoVE.com

Extraction of the EPP Component from the Surface EMG

Le potentiel de plaque motrice (PPE) composant peut être extraite de l&#39;EMG de surface en utilisant un filtre numérique. L&#39;extrait montre PPE oscillation avec une fréquence d&#39;environ 30 Hz.

Extraction de la composante PPE de l&#39;EMG de surface

A surface electromyogram (EMG), especially when recorded near the neuromuscular junction, is expected to contain the endplate potential (EPP) component ...

extraction-of-the-epp-component-from-the-surface-emg

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JoVE Journal

Biology

12.5K vues.  Matsumoto Dental University. Le potentiel de plaque motrice (PPE) composant peut être extraite de l'EMG de surface en utilisant un filtre numérique. L'extrait montre PPE oscillation avec une fréquence d'environ 30 Hz.

Vidéo: Extraction of the EPP Component from the Surface EMG

Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-dissection du cerveau de rat pour l&#39;ARN ou de l&#39;extraction de protéines à partir région spécifique du cerveau

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates ...

Recherche

Biologie

RNA Extraction from Neuroprecursor Cells Using the Bio-Rad Total RNA Kit

Extraction d&#39;ARN à partir de cellules Neuroprecursor Utiliser le Bio-Rad ARN total Kit

Retrograde Labeling of Retinal Ganglion Cells by Application of Fluoro-Gold on the Surface of Superior Colliculus

Retinal ganglion cell (RGC) counting is essential to evaluate retinal degeneration especially in glaucoma. Reliable RGC labeling is fundamental for evaluating the effects of any treatment. In rat, about 98% of RGCs is known to project to the contralateral superior colliculus (SC) (Forrester and Peters, 1967). Applying fluoro-gold (FG) on the surface of SC can label almost all the RGCs, so that we can focus on this most vulnerable retinal neuron in glaucoma. FG is taken up by the axon terminals of retinal ganglion cells and bilaterally transported retrogradely to its somas in the retina. Compare with retrograde labeling of RGC by putting FG at stump of transected optic nerve for 2 days, the interference of RGC survival is minimized. Compare with cresyl violet staining that stains RGCs, amacrine cells and endothelium of the blood vessel in the retinal ganglion cell layer, this labeling method is more specific to the RGC. This video describes the method of retrograde labeling of RGC by applying FG on the surface of SC. The surgical procedures include drilling the skull; aspirating the cortex to expose the SC and applying gelatin sponge over entire dorsal surface of SC are shown. Useful tips for avoiding massive intracranial bleeding and aspiration of the SC have been given.

This video describes the method of retrograde labeling of RGC by applying fluoro-gold (FG) on the surface of superior colliculus (SC). Technique involves drilling the skull, aspirating the cortex, and applying gelatin sponge over entire dorsal surface of SC.

Marquage rétrograde de cellules ganglionnaires de la rétine par l&#39;application de fluoro-or sur la surface du colliculus supérieur

Retinal ganglion cell (RGC) counting is essential to evaluate retinal degeneration especially in glaucoma. Reliable RGC labeling is fundamental for ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

L&#39;avidité d&#39;un dépistage interaction extracellulaire (AVEXIS) pour la détection évolutive de faible affinité extracellulaires interactions récepteur-ligand

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Bourgeonnement de levure extraction et purification des protéines pour l&#39;étude de la fonction, Interactions, et des modifications post-traductionnelles

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. 
SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

Surface Plasmon Resonance (SPR) is a label-free method for detecting bio-molecular interactions in real time. Herein, a protocol for a membrane protein:receptor interaction experiment is described, while discussing the pros and cons of the technique.

Temps réel Mesures de Membrane Protein Receptor Interactions: Utilisation de résonance plasmonique de surface (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

La technique d&#39;infiltration-centrifugation pour l&#39;extraction de apoplastique Fluid feuilles des plantes Utilisation<em&gt; Phaseolus vulgaris</em&gt;, Un exemple

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

The goal of this protocol is to demonstrate how to monitor fluorescently-tagged protein dynamics on plant cell surfaces with variable-angle epifluorescence microscopy, showing blinking dots of GFP-tagged PATROL1, a membrane trafficking protein, in the cell cortex of the stomatal complex in Arabidopsis thaliana.

Imagerie en temps réel des plantes Dynamics de surface cellulaire avec angle variable épifluorescence Microscopie

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to study the functions and evolution of bacterial symbionts in their host. As most symbionts cannot be cultured in vitro, methods to isolate an adequate quantity of bacteria for genome sequencing are very important. In the whitefly Bemisia tabaci, a number of endosymbionts have been identified and are predicted to be of importance in the development and reproduction of the pests through multiple approaches. However, the mechanism underpinning the associations remains largely unknown. The obstacle partially comes from the fact that the endosymbionts in whitefly, mostly restrained in bacteriocytes, are hard to separate from the host cells. Here we report a step-by-step protocol for the identification, extraction and purification of endosymbionts from the whitefly B. tabaci mainly by dissection and filtration. Endosymbiont samples prepared by this method, although still a mixture of different endosymbiont species, are suitable for subsequent genome sequencing and analysis of the possible roles of endosymbionts in B. tabaci. This method may also be used to isolate endosymbionts from other insects.

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Here, we present a protocol to isolate endosymbionts from the whitefly Bemisia tabaci through dissection and filtration. After amplification, the DNA samples are suitable for subsequent sequencing and study of the mutualism between endosymbionts and whitefly.

Méthodes d&#39;extraction des endosymbionts de la mouche blanche<em&gt; Bemisia tabaci</em

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...

In Vivo Surface Electrocardiography for Adult Zebrafish

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of zebrafish not only as a research model for human cardiac electrophysiology and myopathies but also as a surrogate model in high throughput pharmaceutical screening for potential cardiotoxicities to humans, such as QT prolongation. As such, in vivo electrocardiography for adult zebrafish is an electrical phenotyping tool that is necessary, if not indispensable, for cross-sectional or longitudinal in vivo electrophysiological characterizations. However, too often, the lack of a reliable, practical, and cost-effective recording method remains a major challenge preventing this in vivo diagnostic tool from becoming more readily accessible. Here, we describe a practical, straightforward approach to in vivo electrocardiography for adult zebrafish using a low-maintenance, cost-effective, and comprehensive system that yields consistent, reliable recordings. We illustrate our protocol using healthy adult male zebrafish of 12-18 months of age. We also introduce a rapid real-time interpretation strategy for quality validation to ensure data accuracy and robustness early in the electrocardiogram recording process.

Here, we present a reliable, minimally invasive, and cost-effective method to record and interpret electrocardiograms in live anesthetized adult zebrafish.

Électrocardiographie de surface in Vivo pour le poisson zèbre adulte

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of ...