这种生产和试点工作提出了本反映雷纳尔帕雷哈的投入和热情帮助，请丽娜阿吉雷，吉尔伯托科尔多瓦，威廉梅拉，尼利亚Ximena，和达里奥Viveros支持。我们也感谢约翰迈尔斯和3个匿名审稿意见，帮助我们提高了视频和手稿。

1。植物<ol><li>单从成熟的植物扦插作为试点单位。修剪到10厘米的插条，以确保种植材料的统一性。 </li><li>为了防止植物病原体污染，扦插钠2分钟3％的次氯酸钠溶液洗涤，然后用自来水冲洗thoughroughly。 </li><li> CA是种植在每个切削。 36克，在一个圆柱形的PVC管（外直径6.5厘米的高度，5.3厘米）的无菌土密封，下端用发泡胶杯和皑皑的PVC套管。切割置于套管中央开放的泡沫环举行。 </li><li>需要保持土壤墒情充足，植物施肥和浇水。 </li><li>植物会成长为一个月前被昆虫的挑战，使表层根系的发展，这根木质部喂养spittlebug若虫喂网站服务。 </li><li>植根于扦插的PVC管倒立8天前为患进一步刺激表层根系的生长，部分调整为根构型的内在的差异。这个步骤需要人工源的光，这是我们提供24公顷的一天。为了避免与草食性草食动物的混杂逃脱，个别工厂复制表层根系很少或根本没有，这反演处理后从实验所淘汰。这消除应是罕见的，从来没有领先，以消除他们所代表的基因型。 </li></ol> 2。昆虫<ol><li>成人spittlebugs从外地收集，鉴定到种，并介绍产卵的地方，他们提供的易感brachiariagrasses枝叶喂笼。雌虫在土壤中的鸡蛋放在笼子的底部在一个浅可拆卸托盘中。 </li><li>几天后，土壤悬浮于水中，并通过一系列的筛子（42，60和150目），收集鸡蛋。暂停在30％的盐溶液的过筛材料，使成熟的卵子浮动，留下未成熟的卵子和任何残留的有机物。 </li><li>为了防止昆虫病原体污染，蛋是在5分钟钠0.5％次氯酸钠溶液消毒，然后用蒸馏水漂洗。 </li><li>每个测试厂（试点单位），是出没在土壤表面与6个成熟卵子。只有成熟的卵子孵化选择的侵扰。他们可以识别存在的两个红点在其前部，相应的昆虫的眼睛，粉红色标记，在他们的后部分，相应的昆虫的腹部和一个全面扩大鳃盖。 </li><li>成功孵化4 d后证实，在这一点任何未孵化的蛋是从我们的殖民地新生儿spittlebug取代。 </li><li>被安排在一个随机百分之六基因型复制完整的块设计测试工厂。 </li></ol> 3。评价<ol><li>这项实验是评价CA。 30 d后为患（确切的时间有所不同，取决于研究的昆虫物种）。 </li><li>损害是取得了一个可视化规模1至5，其中1对应没有明显可见的损坏，到了山穷水尽的植物（图1）和5对应。评分植物的损害提供了一个宽容的估计。 </li></ol><img src="/files/ftp_upload/3047/3047fig1.jpg" alt="图1" /> 图1。损害的规模，用于评估spittlebug臂形菌的草食动物的耐受性。 1 =没有检测到损坏; 2 = 0-25％的坏死枝叶; 3 = 25-50％的坏死枝叶; 4 = 50-75％的坏死枝叶; 5 = 75-100％的坏死枝叶。 <ol start="3"><li>评估计数达到其最终龄幼虫或成虫期（图2）的昆虫，昆虫的生存。昆虫生存的得分，提供了一种抗菌的估计。 </li></ol><img src="/files/ftp_upload/3047/3047fig2.jpg" alt="图2" /> 图2。Spittlebug的发展，从第一龄（左）成人（右）。只有达到评价之日起至少他们最终龄（箭头）的昆虫被算作"幸存者"。 4。代表性的成果从有代表性的实验筛选抗spittlebug Aeneolamia reducta 臂形杂交的结果列于图3 。 <img src="/files/ftp_upload/3047/3047fig3.jpg" alt="图3" /> 图3:从筛选抗spittlebug Aeneolamia reducta臂形杂交的结果。我们认为如果他们的反应，以草食动物在此图的下半部分属于植物耐。抗菌增加由右至左和从上到下的耐受性增加。箭头指向耐检查。

<ol>
	<li>Painter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Insect resistance in crop plants. , (1951).</li><li>Kogan, M., Ortman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antixenosis-a+new+term+proposed+to+define+Painter's+'Nonpreference'+modality+of+resistance.">Antixenosis-a new term proposed to define Painter's 'Nonpreference' modality of resistance.</a> Bull. Entomol. Soc. Am. 24, 175-176 (1978).</li><li>Kennedy, G., Gould, F., Deponti, O., Stinner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecological,+agricultural,+genetic,+and+commercial+considerations+in+the+deployment+of+insect-resistant+germplasm.">Ecological, agricultural, genetic, and commercial considerations in the deployment of insect-resistant germplasm.</a> Environ. Entomol. 16, 327-338 (1987).</li><li>Holmann, F., Peck, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Economic+damage+of+grassland+spittlebugs+(Homoptera+Cercopidae)+in+Colombia:+a+first+approximation+of+impact+on+animal+production+in+Brachiaria+decumbens.">Economic damage of grassland spittlebugs (Homoptera Cercopidae) in Colombia: a first approximation of impact on animal production in Brachiaria decumbens.</a> Neotrop. Entomol. 31, 275-284 (2002).</li><li>Miles, J. W., Cardona, C., Sotelo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+selection+in+a+synthetic+brachiariagrass+population+improves+resistance+to+three+spittlebug+species.">Recurrent selection in a synthetic brachiariagrass population improves resistance to three spittlebug species.</a> Crop Sci. 46, 1088-1093 (2006).</li><li>Ferrufino, A., Lapointe, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+plant+resistance+in+Brachiaria+grasses+to+the+spittlebug+Zulia+colombiana.">Host plant resistance in Brachiaria grasses to the spittlebug Zulia colombiana.</a> Entomol. Exp. Appl. 51, 155-162 (1989).</li><li>Lapointe, S. L., Serrano, M. S., Arango, G. L., Sotelo, G., Cordoba, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiosis+to+spittlebugs+(Homoptera:+Cercopidae)+in+accessions+of+Brachiaria+spp.">Antibiosis to spittlebugs (Homoptera: Cercopidae) in accessions of Brachiaria spp.</a> J. Econ. Entomol. 82, 1764-1766 (1992).</li><li>Cardona, C., Miles, J. W., Sotelo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+methodology+for+massive+screening+of+Brachiaria+spp.+genotypes+for+resistance+to+Aeneolamia+varia+(Homoptera:+Cercopidae).">An improved methodology for massive screening of Brachiaria spp. genotypes for resistance to Aeneolamia varia (Homoptera: Cercopidae).</a> J. Econ. Entomol. 92, 490-496 (1999).</li><li>Cardona, C., Fory, P., Sotelo, G., Pabon, A., Diaz, G., Miles, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiosis+and+tolerance+to+five+species+of+spittlebug+(Homoptera:+Cercopidae)+in+Brachiaria+spp.:+implications+for+breeding+for+resistance.">Antibiosis and tolerance to five species of spittlebug (Homoptera: Cercopidae) in Brachiaria spp.: implications for breeding for resistance.</a> J. Econ. Entomol. 97, 635-645 (2004).</li><li>Smith, C., Khan, Z., Pathak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Techniques for evaluating insect resistance in crop plants. , (1994).</li><li>Lapointe, S., Sotelo, G., Arango, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+technique+for+rearing+spittlebugs+(Homoptera:+Cercopidae).">Improved technique for rearing spittlebugs (Homoptera: Cercopidae).</a> J. Econ. Entomol. 82, 1764-1766 (1989).</li><li>Lopez, F., Cardona, C., Miles, J. W., Sotelo, G., Montoya, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+resistance+to+adult+spittlebugs+(Hemiptera:+Cercopidae)+in+Brachiaria+spp.:+methods+and+categories+of+resistance.">Screening for resistance to adult spittlebugs (Hemiptera: Cercopidae) in Brachiaria spp.: methods and categories of resistance.</a> J. Econ. Entomol. 102, 1309-1316 (2009).</li></ol>

草食动物的耐药机制之间的歧视，可以照亮作物育种工作3。基于强大的抗菌性可能会影响，在某些情况下，建立更积极的害虫生物型的选择压力。另一方面，容忍的阻力可能会允许害虫种群增加，直到宽容最终被淹没。持久抗性育种，因此，需要慎重考虑什么样的耐药机制将赋予一个特定的作物/害虫系统最稳定[见肯尼迪等。 （1987年）为一个全面的讨论主题] 3。 此视频演示建立在几个以前的研究，估计抗菌和宽容brachiariagrasses 6-9 spittlebug阻力相对贡献。在选择试验或田间试验对比，没有选择的测试，确保所有工厂收到相同的草食动物的压力，从而为控制草食动物的行为（例如，主机的偏好，聚集）所产生的分歧。出于这个原因，但是，没有选择的测试，是不合适的，以评估antixenosis阻力。 人工饲养昆虫的殖民地，是首选的方法，获得抗性筛选实验测试10昆虫。殖民地提供了一个可靠和统一的一个著名实验进行的任何时间在今年11岁的spittlebugs源。然而，它可能是必要的定期注入殖民地的野生个体，以确保殖民地不偏离相关领域的人口 10基因。为了评估这些人工饲养的草食动物的健身的，在他们的攻击，随着时间的推移和监测可能发生的变化，重要的是，所有场次包括已知的阻力水平适当的制衡。 多个参数，必须仔细定义，设计一个合适的抗性筛选试验。我们认为设计我们的测试的几个关键参数包括寄主植物的年龄，品种和发育阶段的spittlebugs，为害的水平，以及植物昆虫接触8,9,12期。可能需要几个回合的实验和协议的细化，达到快速，成本效益，可靠，试​​验充分的预测，在该领域的草食动物阻力。

characterizing herbivore resistance mechanisms: spittlebugs on brachiaria spp. as an example

植物能抵御通过三大机制的草食动物损害:antixenosis，抗菌和宽容 1 。 Antixenosis是该厂房时，避免草食动物是可以选择其他植物的程度。抗菌植物影响的程度，1喂养草食健身。宽容程度的植物能够承受或修复的草食动物所造成的损害，不损害草食动物的生长和繁殖1。草食动物抵抗农产品设置的耐久性取决于上很大程度的耐药机制，在作物育种工作3青睐。 我们展示一个没有选择的实验设计估计抗菌和宽容的阻力臂形SPP spittlebug的相对贡献。非洲属臂形草的几个品种，是宝贵的牧草和牧草，在新热带，但它们可以由几个本地物种spittlebugs（半翅目:Cercopidae）严重挑战4，以评估其耐spittlebugs的，植物无性繁殖扦插和允许增长约1个月，使表层根系的生长spittlebugs可以养活。在这一点上，每个测试厂是单独附近spittlebug鸡蛋孵化的挑战。虫害是允许的进展评估植物的损害和昆虫的生存之前一个月。评分植物的损害提供了一个宽容的估计，而得分昆虫的生存提供了一种抗菌的估计。该协议提供了便利的植物育种目标，以提高商业brachiariagrases 5 spittlebug阻力。

Watch this Scientific Journal Video about Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example at JoVE.com

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

此视频介绍寄主植物抗性机制，以草食动物，并演示了一个没有选择的测试，估计抗菌和宽容spittlebug阻力相对贡献<em&gt;臂形</em&gt; SPP。

表征草食动物的耐药机制:Spittlebugs臂形 SPP。为例

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is ...

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Research

JoVE Journal

Biology

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

13.7K 次观看.  CIAT. 此视频介绍寄主植物抗性机制，以草食动物，并演示了一个没有选择的测试，估计抗菌和宽容spittlebug阻力相对贡献臂形 SPP。

视频: Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

人类DNA修复蛋白的遗传研究利用酵母作为模型系统

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

科研

生物学

Herbivore-induced Blueberry Volatiles and Intra-plant Signaling

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2. These HIPVs are mainly regulated by the defensive plant hormone jasmonic acid (JA) and its volatile derivative methyl jasmonate (MeJA)3,4,5. Over the past 3 decades researchers have documented that HIPVs can repel or attract herbivores, attract the natural enemies of herbivores, and in some cases they can induce or prime plant defenses prior to herbivore attack. In a recent paper6, I reported that feeding by gypsy moth caterpillars, exogenous MeJA application, and mechanical damage induce the emissions of volatiles from blueberry plants, albeit differently. In addition, blueberry branches respond to HIPVs emitted from neighboring branches of the same plant by increasing the levels of JA and resistance to herbivores (i.e., direct plant defenses), and by priming volatile emissions (i.e., indirect plant defenses). Similar findings have been reported recently for sagebrush7, poplar8, and lima beans9..
Here, I describe a push-pull method for collecting blueberry volatiles induced by herbivore (gypsy moth) feeding, exogenous MeJA application, and mechanical damage. The volatile collection unit consists of a 4 L volatile collection chamber, a 2-piece guillotine, an air delivery system that purifies incoming air, and a vacuum system connected to a trap filled with Super-Q adsorbent to collect volatiles5,6,10. Volatiles collected in Super-Q traps are eluted with dichloromethane and then separated and quantified using Gas Chromatography (GC). This volatile collection method was used n my study6 to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants. These methods are described here. Briefly, undamaged blueberry branches are exposed to HIPVs from neighboring branches within the same plant. Using the same techniques described above, volatiles emitted from branches after exposure to HIPVs are collected and analyzed.

A push-pull method for collecting plant volatiles is described. The method allows for a comparison of volatiles induced by herbivore feeding, exogenous methyl jasmonate, and mechanical damage. This technique is also used to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants.

蓝莓虫害诱导挥发物和植物内信令

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2.  These HIPVs are mainly regulated by the defensive ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

测量渗透水渗透系数（P<sub&gt; F</sub&gt;）球形细胞:隔离植物原生质体为例。

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

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.

渗透离心技术质外体中提取植物液使用叶<em&gt;菜豆</em&gt;为例

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

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

成像 FITC-葡聚糖作为胞吐作用调节的记者

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological samples, designed for high-resolution magnetic resonance imaging (MRI), also referred to as MR microscopy (MRM). It may be used at pre-clinical MRI spectrometers, demonstrated on Medicago truncatula root samples. Microcoils increase sensitivity by matching the size of the RF resonator to the size of the sample of interest, thereby enabling higher image resolutions in a given data acquisition time. Due to the relatively simple design, solenoidal microcoils are straightforward and cheap to construct and can be easily adapted to the sample requirements. Systematically, we explain how to calibrate new or home-built microcoils, using a reference solution. The calibration steps include: pulse power determination using a nutation curve; estimation of RF-field homogeneity; and calculating a volume-normalized signal-to-noise ratio (SNR) using standard pulse sequences. Important steps in sample preparation for small biological samples are discussed, as well as possible mitigating factors such as magnetic susceptibility differences. The applications of an optimized solenoid coil are demonstrated by high-resolution (13 x 13 x 13 &#956;m3, 2.2 pL) 3D imaging of a root sample.

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

A protocol to study biological tissue at high spatial resolution using ultra-high field magnetic resonance microscopy (MRM) using microcoils is presented. Step-by-step instructions are provided for characterizing the microcoils. Finally, optimization of imaging is demonstrated on plant roots.

MRM 微可可性能校准和使用在 22 T 的 Medicago 特伦卡图拉根上演示

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological ...

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV

The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as epitranscriptomics. Among others, N6-methyladenosine (m6A) and 5-methylcytosine (m5C) RNA modifications have been described on mRNA molecules and may have a role in modulating cellular processes. Epitranscriptomics is thus a new layer of regulation that must be considered in addition to transcriptomic analyses, as it can also be altered or modulated by exposure to any chemical or biological agent, including viral infections.
Here, we present a workflow that allows analysis of the joint cellular and viral epitranscriptomic landscape of the m6A and m5C marks simultaneously, in cells infected or not with the human immunodeficiency virus (HIV). Upon mRNA isolation and fragmentation from HIV- infected and non-infected cells, we used two different procedures: MeRIP-Seq, an RNA immunoprecipitation-based technique, to enrich for RNA fragments containing the m6A mark and BS-Seq, a bisulfite conversion-based technique, to identify the m5C mark at a single nucleotide resolution. Upon methylation-specific capture, RNA libraries are prepared for high-throughput sequencing. We also developed a dedicated bioinformatics pipeline to identify differentially methylated (DM) transcripts independently from their basal expression profile.
Overall, the methodology allows exploration of multiple epitranscriptomic marks simultaneously and provides an atlas of DM transcripts upon viral infection or any other cell perturbation. This approach offers new opportunities to identify novel players and novel mechanisms of cell response, such as cellular factors promoting or restricting viral replication.

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV

The role of RNA modifications in viral infections is just starting to be explored and could highlight new viral-host interaction mechanisms. In this work, we provide a pipeline to investigate m6A and m5C RNA modifications in the context of viral infections.

探索病毒感染时的m6A和m5C表继转录组:HIV示例

The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as ...

Characterizing Mechanical Properties of Primary Cell Wall in Living Plant Organs Using Atomic Force Microscopy

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the plant. Many sophisticated techniques have been developed to measure these properties; however, atomic force microscopy (AFM) remains the most convenient for studying cell wall elasticity at the cellular level. One of the most important limitations of this technique has been that only superficial or isolated living cells can be studied. Here, the use of atomic force microscopy to investigate the mechanical properties of primary cell walls belonging to the internal tissues of a plant body is presented. This protocol describes measurements of the apparent Young&#39;s modulus of cell walls in roots, but the method can also be applied to other plant organs. The measurements are performed on vibratome-derived sections of plant material in a liquid cell, which allows (i) avoiding the use of plasmolyzing solutions or sample impregnation with wax or resin, (ii) making the experiments fast, and (iii) preventing dehydration of the sample. Both anticlinal and periclinal cell walls can be studied, depending on how the specimen was sectioned. Differences in the mechanical properties of different tissues can be investigated in a single section. The protocol describes the principles of study planning, issues with specimen preparation and measurements, as well as the method of selecting force-deformation curves to avoid the influence of topography on the obtained values of elastic modulus. The method is not limited by sample size but is sensitive to cell size (i.e., cells with a large lumen are difficult to examine).

Studies of cell wall biomechanics are essential for understanding plant growth and morphogenesis. The following protocol is proposed to investigate thin primary cell walls in the internal tissues of young plant organs using atomic force microscopy.

使用原子力显微镜表征活植物器官中原代细胞壁的机械性能

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the ...

An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines

Rickettsioses are caused by a broad range of obligate intracellular bacteria belonging to the genus Rickettsia that can be transmitted to vertebrate hosts through the bite of infected arthropod vectors. To date, emerging or re-emerging epidemic rickettsioses remain a public health risk due to the difficulty in diagnosis, as diagnostic methods are limited and not standardized or universally accessible. Misdiagnosis resulting from a lack of recognition of the signs and symptoms may result in delayed antibiotic treatment and poor health outcomes. A comprehensive understanding of Rickettsia characteristics would ultimately improve clinical diagnosis, assessment, and treatment with improved control and prevention of the disease. 
Functional studies of rickettsial genes are crucial for understanding their role in pathogenesis. This paper describes a procedure for the electroporation of the Rickettsia parkeri strain Tate's Hell with the shuttle vector pRAM18dSFA and the selection of transformed R. parkeri in tick cell culture with antibiotics (spectinomycin and streptomycin). A method is also described for the localization of transformed R. parkeri in tick cells using confocal immunofluorescence microscopy, a useful technique for checking transformation in vector cell lines. Similar approaches are also suitable for the transformation of other rickettsiae.

An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines

Electroporation is a rapid, broadly adopted method for introducing exogenous DNA into the genus Rickettsia. This protocol provides a useful electroporation method for the transformation of obligate intracellular bacteria in the genus Rickettsia.

一种在蜱细胞系中用荧光蛋白表达穿梭载体转化立 克次 体属的电穿孔方法

Rickettsioses are caused by a broad range of obligate intracellular bacteria belonging to the genus Rickettsia that can be transmitted to vertebrate hosts ...

使用 FluoZin-3 测定研究哺乳动物细胞中的锌转运机制

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn2+. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn2+ levels. With the development of intracellular Zn2+ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn2+, detected using fluorescent probes, with the expression of the Zn2+ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn2+ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn2+ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn2+ by utilizing the Zn2+ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn2+. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.

作者聚焦:通过 ZnT1 功能探索细胞锌调节 

Zinc transport has proven challenging to measure due to the weak causal links to protein function and the low temporal resolution. This protocol describes a method for monitoring, with high temporal resolution, Zn2+ extrusion from living cells by utilizing a Zn2+ sensitive fluorescent dye, thus providing a direct measure of Zn2+ efflux.

使用 体外 锌转运测定表征哺乳动物锌转运蛋白

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured ...