이 보고서를 서면으로 자신의 종류의 지원을위한 데이비드 칼슨, 마츠모토 치과 대학의 영어 교수에게 큰 감사를 드리고자합니다. 나는 또한이 수사 대상으로 자신의 준수 Tadafumi 아다치 우리 연구실에서 seminarist을 감사드립니다.

1. EMG 전극의 준비 <ol><li> 구 전극을 준비하여 시작합니다. 그 중 여덟은 근육 이상의 사이트에서 신호를 녹음에 사용하고, 하나는 기준 전극집니다. </li><li> 앰프로 녹음과 참조 전극을 연결하면, 전기 전도성 페이스트로 디스크를 작성하십시오. 모든 종류의 확인을하지만, 낮은 유체의 종류가 더 낫죠. </li><li> 피부에 전극을 연결하기 전에 단단히 주제를 가지고 있고, 얼굴에 턱 근육의 대략적인 위치를 결정합니다. </li><li> 턱 근육의 힘줄이 뛰어난 mandible의 각도로 광대뼈 아치, 그 열악한 힘줄로 연결됩니다. </li><li> 같은 상호 전극 거리에 접착 테이프의 긴 조각에 8 기록 전극을 놓고, 피부 표면에 배열을 연결합니다. 첫번째 기록 전극은 광대뼈 아치와 mandible의 각도에 마지막에 위치합니다. </li><li> 마지막으로, 코 끝에 대한 참조 전극을 첨부합니다. </li></ol> 2. EMG 녹음 <ol><li> 녹음을 시작하려면 먼저 멀티 채널 아날로그 앰프 (MEG6100)의 매개 변수를 설정합니다. X 500, 0.5 Hz로 저역 통과 필터 및 10 kHz에서 수있는 하이 패스 필터 게인을 설정합니다. </li><li> 아날로그 - 디지털 컨버터 (PCI - 미오 - 16E - 4)에 아날로그 앰프의 8 출력을 연결합니다. 디지털 신호는 20kHz의 샘플링 속도와 LabView 소프트웨어와 함께 처리됩니다. </li><li> 모든 연결된 경우, ipsilateral 측면에 빠른 씹는 껌 - clenching 동안 기록 EMGs. </li><li> 레코딩 프로세스가 완료되면 8 채널의 흔적은 즉시 LabView 디스플레이에 표시됩니다. </li></ol> 3. 그 기능의 EPP 추출 및 관찰 <ol><li> EPP 구성 요소를 추출하려면, 먼저 높은 절단 디지털 필터 (버터 워스 필터)를 사용하여 원료 EMGs에서 작업 잠재적인 구성 요소를 제거합니다. </li><li> 10 Hz의 컷 - 오프 주파수와 필터를 적용 시작합니다. 긍정적이거나 부정적인 방향으로 굴절 느린 파도가 나타납니다. 이 파도는 EPP에 해당하고, 극성의 차이는 최종 플레이트 전류의 싱크 소스 관계를 반영합니다. </li><li> 턱 근육에서 녹화 때, 파도의 가장 부정적인 편향은 가장 열악한 녹음 사이트에서 추적 예상되며, 극성 해당 사이트에서 떨어져 3 사이트의 주위에 이동해야합니다. </li><li> EPP 구성 요소의 크기가 편향 동안 편향이나 지역의 피크 포인트로 측정할 수 있습니다. </li><li> EPP 구성 요소의 극성 변화는 대부분 부정적인 편향을 보여주 참조 채널에 대해 각 채널에서 EPP 파를 계획에 의해 결정하실 수 있습니다. </li><li> 이제 차단 주파수를 증가하여 속도가 느린 파도 oscillatory 될 것입니다. 진동이 증가 컷 - 오프 주파수와 함께 증가하고, 일반적으로 30 Hz의 컷 - 오프 주파수에서 분명하게 될 것입니다. </li><li> 물결의 단계는 10Hz 필터 EPP 파도의 극성이 반전을 보여주있는 추적에 걸쳐 반대합니다. </li><li> 진동의 위상 변화는 10Hz - 필터링 EPP 파에 적용했던 동일한 그래프로 확인하실 수 있습니다. </li><li> 진동의 인근 긍정적 및 / 또는 부정적인 봉우리 사이의 간격에서, 우리는 진동의 주파수의 근사치를 얻을 수 </li></ol> 4. EMG 레코딩 결과 <img src="/files/ftp_upload/1653/1653Fig1a.jpg" alt="그림 1" /> 그림 1A : 여기 표시에서 기록된 턱 근육을 따라 8 사이트의 구조입니다. <img src="/files/ftp_upload/1653/1653Fig1b.jpg" alt="그림 1 B" /> 그림 1B : 우리는 ipsilateral 측면에 빠른 잇몸 clenching에 대한 응답으로 턱 근육 이상의 사이트에서 동시에 기록된 monopolar의 EMGs 샘플을 참조하십시오. EPP 구성 요소는 각 원료 EMG에 겹쳐있다 10Hz의 컷 - 오프 주파수와 높은 절단 디지털 필터를 사용하여 추출되었다. 이 재판에서 가장 부정적인 편향은 흔적 - 5 -6에서 관찰되었으며 극성은 추적 - 3에 걸쳐 변경되었습니다. <img src="/files/ftp_upload/1653/1653Fig1c.jpg" alt="그림 1C" /> 그림 1C :이 높은 절단 디지털 필터의 컷 - 오프 주파수가 30Hz로 설정되었을 때, 느린 파도 진동을 전시. 그 단계는 또한 10Hz - 필터링 EPP 파의 극성이 변경 추적 3에 걸쳐 바뀌었다. 진동의 인근 긍정적인 봉우리 사이의 간격은 측정되었다. 이 주제에서는, 평균 남북 정상 간격은 31.7되었습니다 ± 16 녹음을 통해 6.5 밀리초.

<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> 부정적인 가능성에 의해, 흐름의 현재 endplate, 그리고 긍정적인 편향에 의해 형성되는 것으로 간주됩니다로서 흐름 아웃 [1,2,3,4] 가장 부정적인 편향을 보여주는 추적이 일치해야합니다 신경근육학 접합을 찾기 사이트 [5]. 그림 1B는 턱 근육의 신경근육학 교차점 약 모터 단위 작업의 가능성 [6,7]의 전도 방식을 활용 다른 방법에 의해 얻은 결과와 동일합니다 mandible의 각도는 열등 부분 가까이에서 찾을 수 있?...

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<td>Analogue filter</td>
<td>&nbsp;</td>
<td>Nihon Kohden</td>
<td>MEG6100</td>
<td>pre-,main-amplifier</td>
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<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>
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<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

신경근육학 접합 근처에 기록된 특히 표면 electromyogram (EMG)는, 적절한 신호 필터 추출 수있는 endplate 잠재력 (EPP) 구성 요소를 포함할 것으로 예상된다. 두 가지 요소가 중요하다 : EMG는 monopolar 패션에 기록해야하며, EPP를 해당하는 낮은 주파수 신호가 제거되지 않도록 기록 할 수 있어야합니다. 이 보고서는 인간 주제에 턱 근육의 EMG에서 EPP 구성 요소를 추출하는 방법을 설명합니다. 표면 EMG는 빠른 잇몸 clenching에 대한 응답으로 mandible의 각도로 광대뼈 아치에서 같은 상호 전극 거리로, 근육을 통해 따라 정렬된 전통적인 디스크 전극을 사용하여 여덟 사이트에서 기록됩니다. 참조 전극은 코의 끝에 배치됩니다. EPP 구성 요소는 10-35 Hz의 범위 높은 절단 디지털 필터 (2 차원 버터 워스 필터)를 적용하여 원료 EMGs에서 추출됩니다. 필터가 10 Hz로 설정되면, 압축을 푼 EPP 웨이브 녹음이 사이트에 따라 중 부정적 또는 긍정적인 굴절. 극성의 차이는 신경근육학 접합에 해당하는 대부분의 부정적인 편향을 보여주는 사이트로, 최종 플레이트 전류의 싱크 소스 관계를 반영합니다. 턱 근육의 경우, 신경근육학 교차점은 mandible의 각도에 가까운 하부 부분에있는 것으로 추정된다. EPP 구성 요소는 높은 절단 디지털 필터의 컷 - 오프 주파수 30 Hz로 설정됩니다 흥미로운 진동을 전시하고 있습니다. EPP의 진동은 근육 수축이 간헐적으로 방식으로 조정을 나타냅니다. 질병의 다양한 종류와 함께 비정상적인 흔들림은 느린되고 중단이 어려운이 EPP의 진동에 크게 때문일 수 있습니다.

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

endplate 가능성 (EPP) 구성 요소는 디지털 필터를 사용하여 표면 EMG에서 추출하실 수 있습니다. 추출 EPP는 약 30 Hz의 주파수로 진동을 보여줍니다.

표면 EMG에서 EPP 구성 요소의 추출

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 조회수.  Matsumoto Dental University. endplate 가능성 (EPP) 구성 요소는 디지털 필터를 사용하여 표면 EMG에서 추출하실 수 있습니다. 추출 EPP는 약 30 Hz의 주파수로 진동을 보여줍니다.

비디오: 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 또는 특정 뇌 영역에서 단백질 추출에 대한 쥐 두뇌의 마이크로 해부

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

연구

생물학

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

바이오 - 래드 총 RNA 키트를 사용 Neuroprecursor 셀에서 RNA 추출

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.

우수한 Colliculus의 표면에 플루오로 - 골드의 응용 프로그램에 의해 망막 신경절 세포의 퇴보 라벨링

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.

낮은 친화력 세포 수용체 - 리간드 상호 작용의 확장 가능한 감지를위한 욕망 기반의 세포 상호 작용 검사 (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.

함수의 연구, 상호 작용, 그리고 번역 후 수정을위한 신진 효모 단백질 추출 및 정제

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.

막 단백질의 실시간 측정 : 수용체 상호 작용은 표면 플라즈몬 공명 (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.

공장에서 유체 Apoplastic의 추출을위한 침투-원심 분리 기술을 사용하여 잎<em&gt; 위 Phaseolus 심상</em&gt; 예를 들어

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.

가변 각도 표면 형광 현미경으로 식물 세포 표면 역학의 실시간 영상

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.

Whitefly에서 Endosymbionts의 추출을위한 방법<em&gt; 베미 시아 타바시</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.

성인 얼룩말 피시를위한 생체 내 표면 심전도

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