作者感谢COPES条例草案“（哈里斯的莫兰种子公司，戴维斯）为这种分析提供了甜瓜果实。特色作物研究倡议计划的竞争性拨款支持这个项目是由授予。 2009-51181-05783从美国农业部国家食品和农业研究所。

1。样品制备<ol><li>在所需的成熟阶段收获果实。用自来水冲洗，以去除污垢和灰尘。 </li><li>选择进行分析的基础上，如果没有外部和内部缺陷，大小均匀性的水果。 </li><li>纵向切开的水果到挥发性抽样楔子。如果适用，去除皮肤，种子，种子腔组织，或坑。水果组织的选择必须是一致的，整个实验过程中，考虑变异在一个单一的水果（即获得同样来自赤道，开花和果蒂部分样品）。 </li><li>结合选定的水果组织，它混合以随机，然后放入搅拌机掂量出200克。 </li><li>加入200毫升饱和氯化钙溶液（372.5 g在20°C的500毫升去离子水）和50μL的2 -甲基异戊酸甲醇100毫米的解决方案。 氯化钙是为了作为AC酶抑制剂活性，这可能发生的经过切割和均质果肉。 2 - 甲基异戊酸增加一条，作为内部标准，监测同质化过程中的任何挥发性化合物可能造成的损失。 </li><li>在实验室搅拌机（华林，美国），30秒18,000 rpm的均质混合物，然后立即倒入玻璃瓶和特氟隆盖密封。保持在室温下匀浆瓶，直到所有的样品制备。 </li><li>瓶倒入匀浆后，等待10分钟允许从液体分离的泡沫，然后吸取5毫升的液体分装，无泡沫，到20毫升玻璃瓶中，密封小瓶装有用聚四氟乙烯与钢螺钉帽/硅橡胶隔垫。这个过程是适合甜瓜和梨匀浆准备。如果其他水果都用于分析，离心步骤可能需要。因此，消除泡沫，然后离心液体沉淀颗粒可能阻碍移液器。准备至少3个样品瓶作为技术复制。 </li><li>在这一点上，样品可立即分析，或在液氮中闪光冻结，并存储在超低温（-80℃）以供日后分析。 </li><li>冷冻样本，分析当天从冰箱取出的样品，并让他们一小时在室温下解冻。解冻后，在分析之前，更换一个新的，有一个干净，干燥的隔瓶帽。如果没有更换，隔隔凝结水解冻被卷入仪器损坏。 </li></ol> 2。气相色谱 - 表面声波（GC-SAW）设置和数据采集<ol><li>上zNose加载适当的分析方法。 对于酯丰富的甜瓜挥发性个人资料的分析，我们在MicroSense版本5.44.22软件（纽伯里公园，加利福尼亚，美国）的参数如下：顶吸进进口为20秒30毫升分钟-1通过泵入口温度在200°C，TENAX阱温度在225℃，载气（氦气纯度99.999％）2.9毫升min-1的流速;柱（DB-5列，1米×0.25毫米ID×0.25微米薄膜厚度）温度从45方案°C至180°C时，速度10°C间秒-1;传感器的温度在40°C，阀门在165°C。每个样品总分析时间为1分钟。 </li><li>非取芯尖的zNose入口连接不锈钢针，并多次与周围空气净化系统，直至基线稳定，无峰大于200计数（CT）检测。 </li><li>调整仪器使用的是直链烷烃（C6 - C14）的解决方案。调的结果是使用仪器软件科瓦茨指数（KI）为单位时间单位转换成洗脱峰的保留时间。因此，系统调整后，保留时间报道碘化钾单位。 </li&gt; <li>在分析之前，让样品平衡30分钟。为了分析的样品瓶之一，插入样品瓶隔一针，以缓解压力。然后插入针连接到小瓶隔仪器入口，并开始顶空进样。分析至少有三个技术复制每个样品。 </li><li>手动启动仪器，通过点击“播放”按钮;泵的启动和退出的蒸气以上的样本呈现。在分析结束时，屏幕上会出现一个色谱，传感器会自动加热到150°C 10秒清理。当系统的状态框“按钮再次变绿，该仪器是准备另一样本进行分析。 </li><li>为了确保一个稳定的基准和适当的系统清洗，运行每个样品之​​间的空气至少有一个空白。可能从样品瓶和瓶盖的挥发性污染监测，分析2小瓶的空白（空帽瓶），在一天的开始和结束。/ LI&gt; </ol> 3。数据导出和分析<ol><li>数据导出到Microsoft Excel文件后使用的“巅峰记录”功能MicroSense软件收购。一旦数据被导出，添加列包含变量和复制的标签。 </li><li>脚本中，我们的发展，名为“reform_data.py”（见图1的数据格式的一个例子之前和之后使用脚本“reform_data的数据格式转化为更容易操作（使用Python 2.6版本;免费提供上线）。 PY“）。源文件（xls格式）和表的名称为输入数据，以及输出所需的文件名（xls格式）的名称，直接在脚本编辑。 </li><li>启动“kim_interface.py”（也写在Python 2.6; 见图2），并在上一步生成的文件导入数据。具体来说，分析的基础上观察和分析每个KI值检测的数量倍（“碘化钾命中”）。因此，该程序显示了每个KI值碘化钾点击条形图。 </li><li>样品的特定子集的评价的碘化钾命中，分析各组的技术复制一起。要做到这一点，分析每一个治疗或变量分别通过检查/取消选中相应的框中。图形用户界面（GUI）功能的详细描述，请参阅图2标题。 </li><li>每个KI使用GUI窗口的宽度确定后，随机选择一些相应的色谱Microsense软件和评估技术之间重叠峰复制。例如两个技术复制的重叠色谱图3。 </li><li>一旦KI窗口个体化，使用“合并”功能在GUI合并属于窗口中，人口最多的碘化钾，KIS。通过使用此功能，标有一系列的KI值的峰合并在一个单一的的碘化钾标签，allowi吴视为一个单一的变量这样的高峰。 要做到这一点，首先点击“合并”按钮来激活该功能，并选择中心窗口作为人口最多的左键点击相应栏碘化钾。一旦酒吧已被选中，它改变颜色变绿。合并KIS落入窗口内选定碘化钾，用鼠标右键单击相应的酒吧，这会导致条变成红色，而中央碘化钾顶部添加了相应的长度的蓝色横条（ 见图4 ）。一旦选定KIS已合并到相应的中央碘化钾，点击“合并”按钮，再次接受更改，这将导致“合并”按钮变黄。 “取消合并”按钮，在出现失误的情况下，也可用。要取消合并，在GUI中点击“取消合并”按钮，然后右键单击红色的酒吧，你要取消合并。由红，酒吧变成蓝色。再次点击“取消合并”按钮，以接受更改。 </li><li>如果一个尝试到incorrectly在一个单一的样本合并到一个单一的KI值的两个峰，一个错误消息被打印出来。在这种情况下，密切检查色谱和重新在该地区的碘化钾窗口。 </li><li>一旦合并行动已全部完成，保存该文件。 </li><li>进行统计分析之前，空气和小瓶空白的色谱分析，以监测可能的污染物。一旦在空白的峰碘化钾已经确定，减去面积峰面积峰目前在样品中检测到的空气和/或小瓶空白。 然后进行统计分析。 </li></ol> 4。代表结果电子鼻是能够探测到在不同的成熟阶段（ 图5）收获的瓜果之间的差异，在动荡的型材。第二十碘化钾窗户被确定所有样品。方差分析表明，14座山峰DETE反恐执行局由成熟阶段之间的显着变化的电子鼻。在图6中，记录了这14个组件的平均峰面积绘制，显示在繁忙的丰度的差异，两个成熟阶段，提早成熟和完全成熟的果实。 <img alt="图1" src="/files/ftp_upload/3821/3821fig1.jpg" /> 图1从仪器软件（A）和改造后，使用的“reform_data.py”脚本（二）执行。导出的数据格式。为了方便数据处理和分析，确定所有的独特的KIS所有样品，然后将数据重新排序的行和列，相应的独特的KIS峰面积与样品的信息。如果不是KI值在样本中检测到一个高峰，相应的单元格是空的。 <img alt="图2" src="/files/ftp_upload/3821/3821fig2.jpg" /> 图2。以股代息的抓屏T文件kim_interface.py“。在中心小区显示每KI与碘化钾的命中数。 “命中每碘化钾”是在一个特定的KI峰值检测样品的数量。在左侧，有三个控制所选数据的黄色盒子。他们显示参数来划分数据集（处理，复制，定性变量等）。在这个数字中，他们是（从上到下）：品种，播种日期和收获时的成熟阶段。底部：按一下3个酒吧和向左或向右移动蓝色酒吧，人们可以选择的的碘化钾范围，和最低的峰面积（&#39;门槛&#39;）最低和最高值。上右：“合并”按钮，允许合并选定的KIS通过手动点击情节酒吧。 “取消合并”按钮，允许一对选定的情况下扭转过程。 <img alt="图3" src="/files/ftp_upload/3821/3821fig3.jpg" /> 图3叠加色谱（在<html>黑色和红色），两个技术复制从甜瓜挥发性顶空，说明在保留时间漂移。 <img alt="图4" src="/files/ftp_upload/3821/3821fig4.jpg" /> 图4。合并过程碘化钾的例子。在中央的阴谋，绿色的酒吧（中央碘化钾）代表的人口最稠密的碘化钾，这已被选择为中心的KI窗口。碘化钾X和碘化钾ŸKIS在窗口的兴趣下降，需要将它们合并到中央的KI。由碘化钾X的酒吧右键点击，它会变成红色，蓝色的相同长度的碘化钾X的酒吧，酒吧在同一时间，出现在顶部的绿色。通过碘化钾Y的蓝色条的长度（合并KIS）重复相同的过程将增加相应的长度。一旦所有的KIS点击绿色的“合并”按钮，合并过程结束，已添加，更改保存，按钮的颜色变成黄色。 / files/ftp_upload/3821/3821fig5.jpg“/&gt; 图5两种不同成熟阶段收获的甜瓜样品色谱图，早熟（上）和完全成熟（下），来说明电子鼻的能力，以检测挥发性丰度的差异。 <img alt="图6" src="/files/ftp_upload/3821/3821fig6.jpg" /> 图6。雷达图显示了14个部分组成，两个样品在两个不同的成熟阶段，提早成熟和完全成熟的瓜峰面积。据报道，在对数刻度的峰面积，帮助可视化的比较。在每个射线年底的数字代表相应科瓦茨指数。

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我们什么都没有透露。

电子鼻代表水果香味或挥发性丰富的样品剖面迅速，客观地评价一个很有前途的方法。然而，移动代表峰识别是一个挑战，并可能导致误解的数据时，两个色谱不完全一致，保留时间。色谱目视检查表明，样品间的保留时间的变化经常导致相同的峰值略有不同的Ki值（约±10）标记。这翻译成夸张独特的KIS检测。以优势的事实（一）不同的化合物，目前在不同的成熟阶段，（b）技术复制的大致相同，两台计算机为基础的脚本（的“kim_merge.py”，其中包含用于处理数据的例程系统地开发集，“kim_interface.py”，它提供了一个图形用户界面（GUI））在一个半自动化的方式比较样本，色谱分析大型数据集所需的时间大大减少。这些程序允许合并，在适当情况下，峰下单碘化钾标签与标记的Ki值的范围。这有两个重要目的：（一）它使统计分析视为一个单一的变量如峰，（二）有利于峰识别和比较，与其他系统和出版值。本文结果表明，冬瓜样本zNose系统结合使用，有足够的KI识别的成熟度与香气分析的基础上，可以歧视。这是一个有前途的新技术，质量控制程序，可用于挥发物的分析。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>氯化钙</td><td> MP的生物医学</td><td> 195088 </td><td></td></tr><tr><td> 2 - 甲基丁基异戊酸</td><td> SAFC全球</td><td> W350613 </td><td> ≥98％，自然，FCC </td></tr><tr><td>甲醇</td><td> Fisher Scientific则</td><td> A411-4 </td><td></td></tr><tr><td>小瓶</td><td> SIGMA / Supelco公司</td><td> SU860098 </td><td></td></tr><tr><td>帽</td><td> SIGMA / Supelco公司</td><td> SU860101 </td><td></td></tr><tr><td>实验室搅拌机</td><td>华林实验室科学</td><td> 7009G </td><td>搅拌器; 1 - 2速升的玻璃容器</td></tr><tr><td>瓶</td><td> Fisher Scientific则</td><td> 06-414-1C </td><td>耐热，500毫升;聚丙烯插件密封</td></tr><tr><td>针</td><td>电子传感器技术</td><td> TLC101046 </td><td>侧孔露儿</td></tr><tr><td>烷烃的解决方案</td><td>电子传感器技术</td><td></td><td> C6 - C14烷烃溶液中甲醇</td></tr><tr><td> zNose </td><td>电子传感器技术</td><td> 4500型</td><td></td></tr><tr><td> DB-5气相色谱柱</td><td>电子传感器技术</td><td> SYS4500C5 </td><td></td></tr><tr><td> MicroSense </td><td>电子传感器技术</td><td>版本5.44.22 </td><td></td></tr><tr><td> Python 2.6中</td><td></td><td></td><td>免费提供上线</td></tr><tr><td> “reform_data.py”和“kim_interface.py的”脚本</td><td></td><td></td><td>脚本作为补充材料上的朱庇特</td></tr></tbody></table>

fruit volatile analysis using an electronic nose

多种多样的生理变化发生在果实成熟，包括发展的一个特定的挥发性混合特征的水果香气。在采收成熟度是影响水果和蔬菜的风味品质的关键因素之一。稳健快速评估的方法，果实成熟，将使改善香气质量管理先进的育种计划，生产实践和采后处理验证。 在过去的三十年中，已进行了大量的研究，发展所谓的电子鼻，这些设备能够快速检测气味和口味2-4。目前，有几种市售电子鼻能够执行挥发性的分析，根据不同的技术。电子鼻（zNose，东部，纽伯里公园，加利福尼亚，美国）在我们的工作，包括（超快速气相色谱耦合表面声波传感器UFGCSAW）。这项技术已经被测试，它能够监控各种商品的质量，包括检测苹果5恶化;芒果6成熟和腐烂的评价; 胸腺种香气分析7 6 8个葡萄浆果中的挥发性化合物;表征植物油和椰子油10搀杂检测。 该系统可以进行香气分析的三个主要步骤：顶采样，挥发性化合物的分离和检测。在一分钟左右，生产和输出，色谱，后清除周期，该仪器是准备作进一步的分析。与zNose所取得的成果，可以比其他气体色谱系统由计算的科瓦茨指数（碘化钾）。一旦仪器已调整烷烃的标准溶液的保留时间被自动转换成KIS。然而，在温度和流量的细微变化，预计随着时间的推移发生，导致保留时间漂移。此外，根据列固定相的极性，重复性的KI计算可以改变几个指标单位11。因此制定了一系列方案和图形界面比较样品在一个半自动化的方式计算的KIS。这些方案降低了色谱分析大型数据集所需的时间和尽量减少误解时，色谱图不完全一致的数据的潜力。 我们提出了一个快速水果中的挥发性化合物分析方法。样品的准备，数据采集和处理程序进行了讨论。

Watch this Scientific Journal Video about Fruit Volatile Analysis Using an Electronic Nose at JoVE.com

Fruit Volatile Analysis Using an Electronic Nose

水果中的挥发性化合物分析的一种快速的方法描述。顶空样品匀浆中的挥发性有机化合物迅速分离，超快速气相色谱耦合（GC）的表面声波（SAW）传感器检测。还讨论了数据处理和分析过程。

果实挥发分析，利用电子鼻

Numerous and diverse physiological changes occur during fruit ripening, including the development of a specific volatile blend that characterizes fruit ...

fruit-volatile-analysis-using-an-electronic-nose

Research

JoVE Journal

Biology

21.1K 次观看.  University of California, Davis. 水果中的挥发性化合物分析的一种快速的方法描述。顶空样品匀浆中的挥发性有机化合物迅速分离，超快速气相色谱耦合（GC）的表面声波（SAW）传感器检测。还讨论了数据处理和分析过程。

视频: Fruit Volatile Analysis Using an Electronic Nose

Studying Aggression in Drosophila (fruit flies)

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources. This complex social behavior is influenced by genetic, hormonal and environmental factors. In many organisms, aggression is critical to survival but controlling and suppressing aggression in distinct contexts also has become increasingly important. In recent years, invertebrates have become increasingly useful as model systems for investigating the genetic and systems biological basis of complex social behavior. This is in part due to the diverse repertoire of behaviors exhibited by these organisms. In the accompanying video, we outline a method for analyzing aggression in Drosophila whose design encompasses important eco-ethological constraints. Details include steps for: making a fighting chamber; isolating and painting flies; adding flies to the fight chamber; and video taping fights. This approach is currently being used to identify candidate genes important in aggression and in elaborating the neuronal circuitry that underlies the output of aggression and other social behaviors.

Studying Aggression in Drosophila fruit flies

学习在果蝇的侵略（果蝇）

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources.  This complex social behavior is influenced by ...

科研

生物学

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

自制的网站定向诱变整个质粒

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine parasitoids known to infect the host during the egg stage1. Second, it has a wide range of potential fruit fly hosts. Perhaps due to its life history, F. arisanus has been a successfully used for biological control of fruit flies in multiple tropical regions2-4. One impediment to the wide use of F. arisanus for fruit fly control is that it is difficult to establish a stable laboratory colony5-9. Despite this difficulty, in the 1990s USDA researchers developed a reliable method to maintain laboratory populations of F. arisanus10-12. There is significant interest in F. arisanus biology13,14, especially regarding its ability to colonize a wide variety of Tephritid hosts14-17; interest is especially driven by the alarming spread of Bactrocera fruit fly pests to new continents in the last decade18. Further research on F. arisanus and additional deployments of this species as a biological control agent will benefit from optimizations and improvements of rearing methods. In this protocol and associated video article we describe an optimized method for rearing F. arisanus based on a previously described approach12. The method we describe here allows rearing of F. arisanus in a small scale without the use of fruit, using materials available in tropical regions around the world and with relatively low manual labor requirements.

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus is an egg-larval parasitoid of Tephritid fruit flies that is successfully used in biological control of these important tropical pests. We describe here an optimized protocol for rearing F. arisanus on a small scale using readily available materials.

一个优化的协议饲养 Fopius arisanus Tephritid果寄生蝇

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine ...

Isolation and Biophysical Study of Fruit Cuticles

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and abiotic stresses and also regulates water flow from the external environment.1 A biopolyester (cutin) and long-chain fatty acids (waxes) form the principal structural framework of the cuticle; the functional integrity of the cuticular layer depends on the outer 'epicuticular' layer as well as the blend consisting of the cutin biopolymer and 'intracuticular' waxes.2 Herein, we describe a comprehensive protocol to extract waxes exhaustively from commercial tomato (Solanum lycopersicum) fruit cuticles or to remove epicuticular and intracuticular waxes sequentially and selectively from the cuticle composite. The method of Jetter and Sch&auml;ffer (2001) was adapted for the stepwise extraction of epicuticular and intracuticular waxes from the fruit cuticle.3,4 To monitor the process of sequential wax removal, solid-state cross-polarization magic-angle-spinning (CPMAS) 13C NMR spectroscopy was used in parallel with atomic force microscopy (AFM), providing molecular-level structural profiles of the bulk materials complemented by information on the microscale topography and roughness of the cuticular surfaces. To evaluate the cross-linking capabilities of dewaxed cuticles from cultivated wild-type and single-gene mutant tomato fruits, MAS 13C NMR was used to compare the relative proportions of oxygenated aliphatic (CHO and CH2O) chemical moieties.
Exhaustive dewaxing by stepwise Soxhlet extraction with a panel of solvents of varying polarity provides an effective means to isolate wax moieties based on the hydrophobic characteristics of their aliphatic and aromatic constituents, while preserving the chemical structure of the cutin biopolyester. The mechanical extraction of epicuticular waxes and selective removal of intracuticular waxes, when monitored by complementary physical methodologies, provides an unprecedented means to investigate the cuticle assembly: this approach reveals the supramolecular organization and structural integration of various types of waxes, the architecture of the cutin-wax matrix, and the chemical composition of each constituent. In addition, solid-state 13C NMR reveals differences in the relative numbers of CHO and CH2O chemical moieties for wild-type and mutant red ripe tomato fruits. The NMR techniques offer exceptional tools to fingerprint the molecular structure of cuticular materials that are insoluble, amorphous, and chemically heterogeneous. As a noninvasive surface-selective imaging technique, AFM furnishes an effective and direct means to probe the structural organization of the cuticular assembly on the nm-&mu;m length scale.

Aerial plant organs are protected by the cuticle, a supramolecular biopolyester-wax assembly. We present protocols to monitor selective removal of epi- and intracuticular waxes from tomato fruit cuticles on molecular and micro scales by solid-state NMR and atomic force microscopy, respectively, and to assess the cross-linking capacity of engineered cuticular biopolyesters.

果实角质层分离和生物物理研究

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and ...

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

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

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

SNARE蛋白介导的膜融合酶的细胞融合含量的分析

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

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

用基团特异 rDNA 引物对水生无脊椎动物进行遗传肠道含量分析的实验室规程

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

Simple Homemade Tools to Handle Fruit Flies&#8212;Drosophila melanogaster

The fruit fly, Drosophila melanogaster, is widely used both in biological research and biology education. Handling adult flies is common but difficult in practice, as adult flies fly. Demonstrated here is how to make some simple and cost-effective tools to address difficult issues in the handling of Drosophila. Holes in foam stoppers are made and pipette tips or funnels are inserted into the holes. Flies then move only in one direction into the pipette tip/funnel assemblage, allowing efficient control of the transfer of adult Drosophila into or out of a vial. Existing protocols have been modified for cool-anesthetizing flies by chilling in crushed ice and transferring them onto a cold, hard icepack surface. The icepack is covered with a piece of medical gauze that keeps immobilized flies from the condensed water when examined under a stereomicroscope. The flies are finally euthanized for counting and sorting or discarded by microwaving. A bottle-shaped cage has also been developed for collecting eggs, as well as a labor-saving device and accompanying protocol for cleaning glass culture vials.

Described here is the use of several homemade tools to transfer, chill, and kill adult Drosophila, as well as to clean glass culture vials and collect eggs. These tools are easy to make and are rather efficient in handling Drosophila.

简单的自制工具处理果蝇*果蝇黑色素

The fruit fly, Drosophila melanogaster, is widely used both in biological research and biology education. Handling adult flies is common but difficult in ...

Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. Insecticide resistance surveillance of mosquito populations is typically done through Centers for Disease Control and Prevention (CDC) bottle bioassays or World Health Organization (WHO) tube tests. However, these methods can result in a high degree of variability in mortality data due to variable insecticide contact with&#160;the insect, the relatively small numbers of organisms tested, extensive variation in mass between populations, and constantly changing environmental conditions, leading to variable outcomes. This paper presents the topical application bioassay, adapted as a high-throughput phenotypic bioassay for both mosquitoes and fruit flies, to test large numbers of insects along&#160;a range of insecticide concentrations.
This assay 1) ensures consistent treatment and insecticide contact with every organism, 2) produces highly specific dose-response curves that account for differences in average mass between strains and sexes (which is particularly important for field-collected organisms), and 3) allows for the calculation of statistically rigorous median lethal doses (LD50), which are necessary for resistance ratio comparisons-an alternative surveillance approach from diagnostic dose mortality, which is also used for larvicide resistance surveillance. This assay will be a complementary tool for accurately phenotyping mosquito populations and, as illustrated using fruit flies, is easily adaptable for use with other insects. We argue that this assay will help fill the gap between genotypic and phenotypic insecticide resistance in multiple insect species.

We describe the methodology and importance of the topical application bioassay to measure insecticide susceptibility in mosquitoes and fruit flies. The presented assay is high-throughput, utilizes insect mass-thus allowing for calculating a mass-relativized lethal dose instead of concentration-and likely has lower variability than other similar methods.

局部应用生物测定以量化蚊子和果蝇的杀虫剂毒性

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. ...

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis

The Oriental fruit fly, Bactrocera dorsalis, is a highly invasive and adaptive pest species that causes damage to citrus and over 150 other fruit crops worldwide. Since adult fruit flies have great flight capacity and females lay their eggs under the skins of fruit, insecticides requiring direct contact with the pest usually perform poorly in the field. With the development of molecular biological tools and high-throughput sequencing technology, many scientists are attempting to develop environmentally friendly pest management strategies. These include RNAi or gene editing-based pesticides that downregulate or silence genes (molecular targets), such as olfactory genes involved in searching behavior, in various insect pests. To adapt these strategies for Oriental fruit fly control, effective methods for functional gene research are needed. Genes with critical functions in the survival and reproduction of B. dorsalis serve as good molecular targets for gene knockdown and/or silencing. The CRISPR/Cas9 system is a reliable technique used for gene editing, especially in insects. This paper presents a systematic method for CRISPR/Cas9 mutagenesis of B. dorsalis, including the design and synthesis of guide RNAs, collecting embryos, embryo injection, insect rearing, and mutant screening. These protocols will serve as a useful guide for generating mutant flies for researchers interested in functional gene studies in B. dorsalis.

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis

This paper presents the step-by-step protocols for CRISPR/Cas9 mutagenesis of the Oriental fruit fly Bactrocera dorsalis. Detailed steps provided by this standardized protocol will serve as a useful guide for generating mutant flies for functional gene studies in B. dorsalis.

东方果蝇背侧芽孢杆菌CRISPR/Cas9诱变实验方案

The Oriental fruit fly, Bactrocera dorsalis, is a highly invasive and adaptive pest species that causes damage to citrus and over 150 other fruit crops ...

Methods Article

果实挥发分析，利用电子鼻