私はこのレポートを書くのに彼の親切なサポートのために、デビッドカールソン、松本歯科大学の英語の教授に大きな感謝を提供したいと思います。私もこの調査の対象としての彼のコンプライアンスのため忠史足立、私たちの研究室でseminaristを、感謝したいと思います。

1。 EMG電極の調製<ol><li> nine電極を準備することから始めます。それらの8つは筋肉以上のサイトからの信号を記録するために使用、もう一方は参照電極であるされています。 </li><li>アンプへの記録と参照電極を接続した後、電気伝導度のペーストでディスクを埋めます。すべてのタイプはOKですが、低い流体のタイプが優れている。 </li><li>皮膚に電極を取り付ける前に、しっかりと被写体を持っている、と顔咬筋のおおよその位置を決定する。 </li><li>咬筋の優れた腱は、下顎の角度に頬骨弓、およびその下位の腱に付着。 </li><li>同じ電極間距離の粘着テープの長い部分にeight記録電極を置き、そして皮膚の表面に配列を添付。最初の記録電極は、頬骨弓、下顎の角度で最後に置かれます。 </li><li>最後に、鼻の先端に参照電極を取り付けます。 </li></ol> 2。 EMG記録<ol><li>録音を開始するには、初のマルチチャンネルアナログアンプ（MEG6100）のパラメータを設定します。 × 500、0.5 Hzまでの低域通過フィルタ、および10 kHzのハイパスフィルタにゲインを設定します。 </li><li>アナログ - デジタルコンバータ（PCI - MIO - 16E - 4）にアナログアンプの8つの出力を接続します。デジタル信号は20kHzのサンプリングレートでLabVIEWソフトウェアで処理されます。 </li><li>すべてが接続されている場合は、同側で迅速にチューインガム、ガム噛みしめ時の記録的な筋電図。 </li><li> 8つのチャネルからのトレースが記録プロセスが完了した直後に、LabViewのディスプレイ上に表示されます。 </li></ol> 3。その特徴のEPPの抽出と観察<ol><li> EPPの成分を抽出するには、最初の高カットデジタルフィルタ（バターワースフィルタ）を使用して生の筋電図から活動電位のコンポーネントを削除します。 </li><li> 10 Hzのカットオフ周波数とフィルタの適用を開始。正または負の方向に偏向する徐波が表示されます。この波は、EPPに対応し、極性の違いは、エンドプレートの現在のシンクソースの関係を反映している。 </li><li>咬筋から録音する場合、波の最も負のたわみが最も劣るレコーディングのサイトからのトレースが期待されている、と極性は、そのサイトから離れて3つのサイトの周りにシフトすべきである。 </li><li> EPPの成分の大きさは、たわみやたわみ時の面積のピーク点として測定することができる。 </li><li> EPPの成分の極性の変化はほとんど陰性波を示した基準チャネルに対して、各チャンネルからEPPの波をプロットすることにより決定することができます。 </li><li>今のカットオフ周波数を増やすことによって、徐波は振動になります。振動は増加するカットオフ周波数を増加すると一般的には30Hzのカットオフ周波数で明らかになるであろう。 </li><li>波の位相は、10Hzのフィルタ処理されたEPP波の極性が反転を見せたするトレース間の逆になります。 </li><li>振動の位相シフトは、10HzのフィルタリングされたEPPの波に適用された同一のグラフで確認することができます。 </li><li>発振の近隣の正及び/または負のピーク間の間隔から、我々は、発振周波数の近似値を得ることができます</li></ol> 4。 EMGの記録結果<img src="/files/ftp_upload/1653/1653Fig1a.jpg" alt="図1" /> 図1a：ここに示されてから記録された咬筋に沿って8つのサイトの概略図である。 <img src="/files/ftp_upload/1653/1653Fig1b.jpg" alt="図1 B" /> 図1b：我々は同側で迅速なガムの噛みしめに対応して咬筋以上のサイトから同時に記録したモノポーラの筋電図のサンプルを参照してください。 EPPのコンポーネントは、それぞれの生のEMGに重畳される10Hzのカットオフ周波数、高カットデジタルフィルタを用いて抽出した。この試験では、ほとんどの負偏差は、トレース- 5、-6およびトレース- 3の間に変更極性で観察された。 <img src="/files/ftp_upload/1653/1653Fig1c.jpg" alt="図1c" /> 図1cは：ハイカットデジタルフィルタのカットオフ周波数が30Hzをするように設定された場合、徐波が発振を示した。その段階でも10HzのフィルタリングされたEPP波の極性が変更されたトレース3、全体にシフト。発振の隣接する正のピーク間の間隔を測定した。このテーマでは、平均間のピーク間隔は31.7でした​​± 16の録音は6.5ミリ秒。

<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>負の電位がによって、フローの現在のエンドプレートの、と肯定的な偏向によって形成されると考えられているとして、そのフローアウト[1,2,3,4]、ほとんどの陰性波を示すトレースはに対応している必要があります神経筋接合部を配置するサイト[5]。図1bは、咬筋の神経筋接合部がおよそ運動単位活動電位[6,7]の伝導の方法を利用する別の方法で得られた結果と同じである下顎の角度にその劣る部分の近?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<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>
</tr>		</tbody>
		</table>
		</div>

extraction of the epp component from the surface emg

神経筋接合部の近くで記録された特に表面筋電図（EMG）は、、適切な信号をフィルタで抽出することができます終板電位（EPP）コンポーネントを含むことが期待されている。二つの要素が重要になります。EMGはモノポーラ方式で記録されている必要があります、とEPPを対応する低周波信号が除去されていないので、録音が行われている必要があります。このレポートでは、被験者に咬筋の筋電図からEPPの成分を抽出する方法を説明します。表面筋電図は、迅速なガムを噛みしめへの応答における下顎角に頬骨弓から等しい電極間距離で、筋肉を介して一緒に整列従来のディスク電極を用いて8つのサイトから記録されます。参照電極は、鼻の先端に配置されます。 EPPのコンポーネントは、10から35 Hzの範囲で高カットデジタルフィルタ（2次元バターワースフィルタ）を適用することにより、生の筋電図から抽出されます。フィルターは10Hzに設定されている場合、抽出されたEPP波は記録部位に応じて、負または正のそらします。極性の違いは、神経筋接合部に対応するほとんどの陰性波を示すサイトで、エンドプレートの現在のシンクソースの関係を反映している。咬筋の場合には、神経筋接合部が下顎角に近い下部に位置すると推定されている。 EPPの成分は高カットデジタルフィルタのカットオフ周波数を30 Hzに設定されている面白い発振を示す。 EPP振動は筋肉の収縮が断続的に調整されていることを示します。疾患の様々な種類に伴う異常な震えが遅くなり、停止することが困難なこのEPPの振動、と実質的に原因である可能性があります。

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

終板電位（EPP）コンポーネントは、デジタルフィルタを用いて表面筋電図から抽出することができます。抽出されたEPPは約30 Hzの周波数で振動を示しています。

表面筋電からEPPコンポーネントの抽出

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

Research

JoVE Journal

Biology

12.5K ビュー.  Matsumoto Dental University. 終板電位（EPP）コンポーネントは、デジタルフィルタを用いて表面筋電図から抽出することができます。抽出されたEPPは約30 Hzの周波数で振動を示しています。

ビデオ: 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.

RNAまたは特定の脳領域からのタンパク質抽出用のラット脳の微小解剖

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

生物学

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

Bio - Rad社のTotal RNA Kitを使​​用してNeuroprecursor細胞からのRNA抽出

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.

上丘の表面上にフッ素金の適用により網膜神経節細胞の逆行性標識法

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.

低親和性細胞外受容体 - リガンド相互作用の検出のためのスケーラブルな親和性ベースの細胞外相互作用のスクリーニング（AVEXIS）

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.

機能の研究、相互作用、および翻訳後修飾のため出芽酵母タンパク質の抽出と精製

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.

膜タンパク質のリアルタイム測定：受容体相互作用は、表面プラズモン共鳴を使用した（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.

植物の葉からのアポプラスト液の抽出のための浸透·遠心分離技術の使用<em&gt;インゲンマメ</em&gt;例として

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.

可変角度落射蛍光顕微鏡を用いて植物細胞表面ダイナミクスのリアルタイムイメージング

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.

白蝶貝からの内生菌の抽出方法<em&gt;ベミシア・タバチ</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.

成体ゼブラフィッシュの生体内心電図検査

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