The authors would like to thank Sensors Inc. for providing a PEMS for conducting an inter-laboratory exercise.

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这里所表达的观点是作者的，不得被视为欧盟委员会的官方立场。 商品名称或商品的提及，并不由作者或欧盟委员会构成认可或推荐。

在本文中，RDE过程被描述。几个点值得特别注意，将在这里更详细地讨论。 用于定型的目的，它是强制性确定使用的设备的废气流，如不与车辆的ECU的任何连接一个EFM运作。关于车辆的准备，对EFM和尾管之间的连接是重要的。该材料应温度和废气耐组合物-虽然这不是对NO x的如此重要，这将是对粒子数采样，其中沉积的材料的解吸可导致人为的高排放显著。此外，应避免可累积缩合点。加速过程中形成的冷凝液可以进入到测量系统和破坏或阻止他们。的分析仪的采样点被连接，以便对EFM的下游，以确保整个佛罗里达州流穿过的EFM。在情况下，这是不可能的，它们对EFM的上游连接，对所提取的流的校正，必须作出。分析器应连接的EFM的下游，而没有任何修改的采样线的长度。如果这是不可能的，在额外管道中的停留时间具有以确保正确排放计算要考虑到该软件。该分析仪可以安装内部或车辆外部的，只要安全要求。此外，该分析仪的校准需要注意。它必须在车辆的排放量的预期范围内进行的。否则，的排放测试的有效部分的测量值的99％〜90％的覆盖率的要求可能无法实现。 跳闸验证和排放量的计算通常是由PEMS软件进行。对于正常驾驶，所有的条件可以很容易地满足17 &lt;/ SUP&gt;。例如，根据我们的测量结果，一个正常驱动行程以及动态边界的范围内（ 图4）。然而，攻击性驾驶可以通区域内，特别是在市区或高速公路的部分。另一方面，在荷兰的城市的数据显示，通常驱动也可以超出这些限制18。在未来，经历随着时间的推移，试验进行了更接近，显示的"50％的差异将评估过程11,19的适用性的边界条件，以及评价方法。 不确定性的来源道路载荷的测定二氧化碳排放量与WLTC测量起源;这些测量用于评估与RDE数据评估行驶条件的正常性。理想的情况下，所选择的道路负载类似于那些用在道路上PEMS测试的卸载车辆。由WLTP授予（ 例如，到D的灵活性etermine基于保守的通用参数或者与一个家庭内的最高测试质量的车辆的道路负载）可以在由WLTC确定和以后的道路上测得的CO 2的排放造成实质性的偏差。由此，该方法可以产生实际驾驶严重程度的偏颇的评价。用于设定道路负载的WLTP规定可能潜在需要RDE目的被指定。 应当指出的是，相较于在服务符合规欧洲重负荷，存在一定的差异（ 例如，漂移校正是允许的，OBD连接是必要的，以计算以g /千瓦时排放）由于不同类型对于重型汽车（发动机）6批准程序。的差异超出了本文的范围。随着美国在使用符合规定，有在评价方法的更多不同之处。 在世界范围内，RDE标志着首先监管在道路上测试轻型车辆。在条例427分之2016定义的RDE规定标志着轻型汽车在欧洲型式认可，在RDE补充实验室受控条件下的标准车辆测试的第一个相关的实例。的RDE测试过程允许进行测试，并因此控制，在宽范围的操作条件，并以更强大的和全面的方式比与预定的驱动周期的当前应用的实验室测试车辆污染物排放。 尽管如此，RDE也受到限制。首先，在路上长时间周期模态辐射测量仪嗣继承漂移的风险（ 例如，由于环境温度的变化）。在道路上排放测量因此受到较大不确定性的利润（估计在适用的排放限制的 NO x最多20-30％）比21排放测量■在实验室，即使PEMS分析器履行有关准确度和精密度的实验室分析仪类似的要求。二，PEMS设备的处理需要培训;在道路上进行发射试验还没有插件和播放，它需要的专家。由于在道路上使用PEMS测试仍然相当新颖，培训，使汽车制造商和技术服务来获取和分享最佳实践是必要的。本文章是传播PEMS的处理和车辆排放的道路上测试知识的一种尝试。与RDE规定较大规模的经验，可以通过实验室之间的练习或通过与现有的国际立法基准获得，至今下落不明。作为RDE构成为全球轻型汽车首次在公路上测试程序，欧盟委员会预计的符合性因素进行年度审查，并在中期整个RDE程序的更全面的审查。

车辆在受控的实验室条件下进行测试，以确定他们的官方排放值和燃油消耗（ 例如，联合国欧洲经济委员会（UNECE）第83条）1。对于轻型汽车，法规2007分之7152定义了欧5和6的排放限值，到在各类车辆M1，M2（乘用车），N1和N2（车辆货物运输），必须遵守。合规是由实验室1标准化测试中冷启动后，测量尾气排放的所谓的"I型"试验验证。虽然实验室测试确保重现性和结果的可比性，它仅覆盖一个小范围的的道路上通常发生的环境，驱动和发动机的运行条件。作为事实上，官方实验室检测结果反映越来越少的实际油耗司机的道路3经历。另外，邻正公路车辆排放，尤其是柴油轿车的 NO x排放量也比型式认证值越高4-5。条例二千零七分之七百十五2包含的规定，以确保排放限值是根据车辆的正常操作和使用推崇。各种新的管理组件的管道，以减少观察到的差异，如世界协调轻型程序（WLTP），主要为二氧化碳和燃油消耗，而真正驱动排放（RDE）的测试程序，主要用于污染物。 不可否认，新的监管包常规污染物中最重要的组件是符合排放限值需要遵循RDE过程，证明了真实世界的车辆运行。新的程序将补充排放的测量在底盘测功机，使管制污染物的彻底控制在laborator既实现Y和在道路上。在RDE是根据在公路上与便携式排放测量系统（PEMS）排放测试。 PEMS并不新鲜，尤其是对重型车辆测试。美国环境保护局（US-EPA）已经加入到实验室认证测试基于与PEMS车辆测试不被超过（NTE）的概念更多的排放要求。在欧洲，PEMS基于在职合格的欧VI标准（ISC）的规定适用于欧V发动机6,7。 PEMS度量排放与测量性能（ 例如，线性，准确度）是比得上的实验室级的设备8的发动机排气。最新一代PEMS的权衡30公斤，结构紧凑，并能方便地安装在小型客车，从而对车辆的影响很小。 应付的检测条件，特定的测试和数据评估亲真实世界变异cedures必须执行。在宽范围的高度，温度和驱动条件，可能会发生的测试。然而，关于（我）之旅成分要求（ 例如，大致城市，农村，和高速公路驾驶平摊）和（ii）驾驶动态（ 例如，在允许的范围加速度）旨在确保车辆在一个公平的测试，代表性的，且可靠的方式。尽管如此，由于一些因素（ 例如，交通，驱动程序和风），任何在道路测试仍然存在，在一定程度上随机和非复制的。因此，主要的挑战是，评估事后的测试条件正常，使车辆排放的可靠的评估数据评估方法的开发。为此目的，两种方法均在RDE内通过:移动平均窗口（MAW）和功率分级方法。所述MAW方法把测试成子部分（窗口），并使用特定的距离平均二氧化碳（CO 2 </sUB&gt;）各窗口的排放，以评估操作条件的正常性。功率分级方法归类瞬时公路上排放到基于在车轮上的相应的电源功率箱。所得配电的正常通过与标准化的轮动力频率分布的比较成立。这两种方法都包括标准，以确保实现考试涵盖驾驶的RDE测试过程9-10动态性允许的范围内。这两种方法通常给予10％以内的结果;然而，在50％的量级上的差异已经报道11,12。这两个数据的评价方法进行了深入的评估仍下落不明。欧盟委员会承认这一缺陷在RDE法规13,14的演奏14和预见在不久的将来这两个方法与留住他们或开发一个统一的方法为气态污染物和班驳的评价客观的审查第一百号排放。 到现在为止，两人RDE包已经通过了欧盟成员国的技术委员会汽车（TCMV）及其官方公报欧盟13-15出版后成为法律。第一RDE封装覆盖的边界条件，实际测试过程中，PEMS规格和数据评估方法（MAW和/或功率分级），但不排放限值（包被在18 日的投票由TCMV 2015年5月）。第二RDE包添加适用于RDE测试不被超过（NTE）的排放限值。此外，还引入了互补的边界条件检查过剩或缺乏驾驶动态。每个有效的个人RDE测试的排放量必须低于各自NTE排放限值，在调控的整合因素提及。目前，只有NO排放都包括在内。结合整合因素将出台在两个步骤:一因素的欧6 的 NO x限制（80毫克/公里）将适用于从2017年至2019年新的型式认证，所有新车注册量2.1。整合因素将随后2020年至2021年降低到1.5。 1.5最终欧6一致性因子提供的0.5相比实验室设备和测试条件的可能范围内的测试对测试排放变异性（ 例如，温度PEMS的附加测量不确定性的余量（ 即，50％） ，动力学和高度）。关于CO。，虽然结合符合因素目前没有讨论，在道路上CO排放必须测量和记录，以获得式的批准。第二包是由TCMV于2015年10月的28 日付诸表决。 两个额外的包开球会议举行了1月25 日 2016年第三RDE包将解决粒子数PEMS测试，冷启动E使命和混合动力汽车的测试。板载测量粒子数排放汽车是具有挑战性的，因为没有经过验证的技术尚未确立。新的概念和方法均在有效期内制定2013年和2014年间，包括实时与恒流量采样16气溶胶组合电器检测。这个包是在下半年的2016年第四RDE包将处理有关在业务整合和市场监督测试的需求定义进行表决。这个包的完成是由2017年年初的RDE规定预见427分之201613和14646分之2016目前正与全球统一的轻型车辆测试程序（WLTP）整合成为一个更大的欧盟型式认证法规，这将补充条例二千〇七分之七百一十五2。 本文的目的是提出通过新近通过RDE单组所需的实验程序特征研。的RDE测试程序定义的容许测试条件的界限，也可以用于分析车辆操作和相关的污染物排放量（ 表1）进行测试车辆，仪器的要求，以及评价方法的协议。 1）车辆选择，2）车辆的准备，3）的行程设计，4）行程执行，5）行程验证，和6）的排放量的计算:该程序可以在六个步骤进行总结。如果任何的在任何这些六个步骤的要求没有得到满足时，测试被认为失败。对于RDE测试过程的更详细的描述，读者可参考的调节自身13-14。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>欧共体条例二千零八分之六百九十二附件IIIA </td></tr><tr><td> 1.引言，定义和缩写</td></tr><tr><td> 2.符合性因素一般要求</td></tr><tr><td>要执行3 RDE测试</td></tr><tr><td> 4.一般要求</td></tr><tr><td> 5.边界条件</td></tr><tr><td> 6.旅行要求</td></tr><tr><td> 7.操作要求</td></tr><tr><td> 8.润滑油，燃料油和试剂</td></tr><tr><td> 9.排放和评价之旅</td></tr><tr><td>附录</td></tr><tr><td>附录1:与PEMS车辆排放检测试验方法</td></tr><tr><td>附录2:规格和PEMS元件和信号校准</td></tr><tr><td>附录3:PEMS和非可追踪排气质量流量的验证</td></tr><tr><td>附录4:排放量的测定。 </td></tr><tr><td>附录5:与方法1行程动态条件验证（移动平均窗） </td></tr><tr><td>附录6:与方法2行程动态条件验证（电源BinniNG） </td></tr><tr><td>附录7:车辆的选择初始型号核准检测PEMS </td></tr><tr><td>附录7A:整个行程动态验证</td></tr><tr><td>附录7B:过程，以确定行程累计正收益抬高</td></tr><tr><td>附录8:数据交换和报告要求</td></tr><tr><td>附录9:遵守生产商的证书</td></tr></tbody></table> 表1:快速发展经济体的监管结构的调控被认为是欧盟委员会法规2008分之69210的附件IIIA。所有部件和附件的委员会法规427分之2016（第一包）8进行了说明。附录7a和7b，以及整合的因素，在委员会条例646分之2016（第二包装）9进行说明。

<table border="0" cellpadding="0" cellspacing="0" width="776">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PEMS analyzer</td>
			<td style="width:103px;">Sensors Inc.</td>
			<td style="width:163px;">SEMTECH ECOSTAR</td>
			<td style="width:264px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PEMS analyzer</td>
			<td style="width:103px;">AVL</td>
			<td style="width:163px;">MOVE</td>
			<td>Figure 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PEMS analyzer</td>
			<td style="width:103px;">Horiba</td>
			<td style="width:163px;">OBS</td>
			<td>Figure 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PEMS analyzer</td>
			<td style="width:103px;">MAHA</td>
			<td style="width:163px;">PEMS-GAS</td>
			<td>Figure 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Exhaust Flow meter</td>
			<td style="width:103px;">Sensors Inc.</td>
			<td style="width:163px;">SEMTECH EFM-HS</td>
			<td>EFM-HS specifications of Table 4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">GPS</td>
			<td style="width:103px;">Garmin</td>
			<td style="width:163px;">Drive 50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Weather station</td>
			<td style="width:103px;">Waisala</td>
			<td style="width:163px;">AWS310</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Zero gas</td>
			<td style="width:103px;">Air Liquide</td>
			<td style="width:163px;">AL089</td>
			<td>Alphagaz 1 (N2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Span gas</td>
			<td style="width:103px;">Air Liquide</td>
			<td style="width:163px;">SM190022710IT</td>
			<td>1800 ppm NO in N2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Span gas</td>
			<td style="width:103px;">Air Liquide</td>
			<td style="width:163px;">SM190022710IT</td>
			<td>13% CO2 in N2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Batteries</td>
			<td style="width:103px;">Discover</td>
			<td style="width:163px;">EV12A-A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;"></td>
			<td style="width:103px;"></td>
			<td style="width:163px;"></td>
			<td></td>
		</tr>
		<tr height="43">
			<td colspan="4" height="43" style="height:43px;width:776px;">Mention of trade names or commercial products does not constitute endorsement or recommendation by the authors or the European Commission</td>
		</tr>
	</tbody>
</table>

implementation of portable emissions measurement systems (pems) for the real-driving emissions (rde) regulation in europe

Vehicles are tested in controlled and relatively narrow laboratory conditions to determine their official emission values and reference fuel consumption. However, on the road, ambient and driving conditions can vary over a wide range, sometimes causing emissions to be higher than those measured in the laboratory. For this reason, the European Commission has developed a complementary Real-Driving Emissions (RDE) test procedure using the Portable Emissions Measurement Systems (PEMS) to verify gaseous pollutant and particle number emissions during a wide range of normal operating conditions on the road. This paper presents the newly-adopted RDE test procedure, differentiating six steps: 1) vehicle selection, 2) vehicle preparation, 3) trip design, 4) trip execution, 5) trip verification, and 6) calculation of emissions. Of these steps, vehicle preparation and trip execution are described in greater detail. Examples of trip verification and the calculations of emissions are given.

<html>的RDE要求功能的一个例子进行说明。 选择并准备车辆和设计，并进行了行程:这不是一个型号核准测试，但的RDE程序的应用程序。因此，所选择的车辆，欧元5B轻型涡轮增压汽油直喷汽车（1.2升发动机排量），已经可以在实验室JRC。兼容RDE行程选择（ 图3）。安装和准备PEMS后，行程中进行的。 <img alt="图3" src="/files/ftp_upload/54753/54753fig3.jpg" /> 图3:旅行设计 ，包括城市（≤60公里/小时）之旅，乡村，和高速公路（&gt; 90公里/小时），平摊部分所示。的设计是基于所选择的道路的速度限制。rce.jove.com/files/ftp_upload/54753/54753fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 验证行程:跳闸通过检查（i）该边界和操作条件和（ii）驱动的正常性进行验证。边界和运行条件和行程要求得到满足（ 表5）。环境温度和最大高度分别为0-30℃，≤700微米，适中的范围内均。此行包括市区行驶其次是农村和高速公路驾驶。它持续96分钟，并覆盖至少16公里的距离为每个U / R / M的部分。距离股价29-44％的市区部分，为农村和高速公路部分23-43％的范围内。跳闸表明停止时段，定义为不到1公里/小时的车速的时期，在市区操作持续6-30％的规定范围。据一<html> S中的车辆速度分布而言，试验表明，适当地覆盖的高速公路操作（ⅰ）90和110公里/小时和（ii）速度高于100公里/小时，至少5分钟之间的范围内。最大车速为好低于145公里/小时的阈值，而跳闸，包括停止的市区驱动部的平均速度，是15-40公里/小时的容许范围内。累计正收益海拔在整个行程是低于每百公里1200米极限。的开始和结束点之间的高度差为&lt;100微米。相对正加速度和速度乘以正加速度的第 95 个百分位数为界限（ 见图4）内。使用相同的汽车，以及文献中报道的其它试验更积极驱动的实验数据，示出用于比较17,18。 /ftp_upload/54753/54753fig4.jpg"/&gt; 图4:指标检查过剩或缺乏驾驶动态 。 （ 一 ）城市，农村，和高速公路行驶时的瞬时速度和正加速度的产品的95百分。 （ 二 ）城市，农村，和高速公路行驶过程中的相对正加速度。的空心方块是实验结果。开放三角形是与同车野蛮驾驶的结果。星号在德国城市咄咄逼人之旅。连续线显示了允许的范围。的及格或不及格的区域也显示。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54753/54753fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>条件</td><td>单位</td><td>范围</td><td>旅</td><td>城市的</td><td>乡村</td><td>公路</td><td>注释</td></tr><tr><td>速度</td><td> [公里/小时] </td><td></td><td></td><td> ≤60 </td><td> 60 &lt;V≤90 </td><td> V&gt; 90 </td><td></td></tr><tr><td>有效载荷</td><td> [％] </td><td> 90 </td><td> 75 </td><td></td><td></td><td></td><td>好</td></tr><tr><td>环境温度</td><td> [C] </td><td> -7 ... + 35 </td><td> 19 </td><td></td><td></td><td></td><td> OK（中度） </td></tr><tr><td>最大。高度</td><td> [M]。 </td><td> ≤1,300 </td><td> 302 </td><td></td><td></td><td></td><td> OK（中度） </td></tr><tr><td>开始/结束高差</td><td> [M]。 </td><td> &lt;100 </td><td> 40 </td><td></td><td></td><td></td><td>好</td></tr><tr><td>累积正收益抬高</td><td> [米/ 100公里] </td><td> &lt;1200 </td><td> 636 </td><td></td><td></td><td></td><td>好</td></tr><tr><td>相对正加速</td><td> [米/秒2] </td><td> 图4 </td><td></td><td> 0.215 </td><td> 0.134 </td><td> 0.100 </td><td>好</td></tr><tr><td>速度x正加速度</td><td> [米2 /秒3] </td><td> 图4 </td><td></td><td> 15.5 </td><td> 22.7 </td><td> 21.4 </td><td>好</td></tr><tr><td>旅行时间</td><td> [秒] </td><td> 90-120 </td><td> 96 </td><td></td><td></td><td></td><td>好</td></tr><tr><td>覆盖距离</td><td> [公里] </td><td> &gt; 16 </td><td></td><td> 29 </td><td> 27 </td><td> 23 </td><td>好</td></tr><tr><td>分享</td><td> [％] </td><td> 23（29）-43 </td><td></td><td> 36.7 </td><td> 34.2 </td><td> 29.1 </td><td>好</td></tr><tr><td>停止时间（城市持续时间） </td><td> [％] </td><td> ＆＃160; 6-30 </td><td></td><td> 28.8 </td><td></td><td></td><td>好</td></tr><tr><td> V&gt; 100公里/小时</td><td> [分钟] </td><td> ≥5 </td><td></td><td></td><td></td><td> 9.7 </td><td>好</td></tr><tr><td> V&gt;145公里/小时（高速公路的时间） </td><td> [％] </td><td> &lt;3 </td><td></td><td></td><td></td><td> 0 </td><td>好</td></tr><tr><td>平均速度（市区部分） </td><td> [公里/小时] </td><td> 15-40 </td><td></td><td> 28 </td><td> 75 </td><td> 114 </td><td>好</td></tr></tbody></table> 表5:行程评测总结的边界条件。试验要求;和得到的结果之前和/或跳闸的城市，农村，和高速公路部分期间，分别被列出。 开车的常态是用鱼鳔evaluat进行离子的方法，但不包括冷启动和空转和称重的 NO x排放量的 CO 2排放的偏差根据MAW方法的类型批准周期的大于25％（见附录5）8。使用游离EMROAD软件。 计算RDE排放:结果的分析也与EMROAD软件进行。结果可以在图5中可以看出，城镇的 NO x排放量在同一水平或比相应WLTC相排放（0.02克/公里）下。农村和高速公路的排放均低于各自的WLTC阶段高&gt; 0.05克/公里。平均来说，公路上排放量为0.056克/千米，这是比NTE限制（这种情况下，0.06毫克/公里×2.1合格因子）低。因此，这个特定的车辆会通过RDE试验（即使RDE过程并不适用于欧5车辆）。更多的例子可以在别处找到17-18。 <img alt="图5" src="/files/ftp_upload/54753/54753fig5.jpg" /> 图5:MAW路上行作为MAW速度的函数的NO x的排放量的蓝色正方形显示每个移动平均窗口的各个窗口的平均车辆速度的函数的平均NO x的排放量。实心菱形描绘平均在道路上代表城市，农村，和高速公路驾驶的所有窗口的 NO x排放量。白色圆圈描绘实验室在WLTP的四个阶段的 NO x排放量。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54753/54753fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Implementation of Portable Emissions Measurement Systems PEMS for the Real-driving Emissions RDE Regulation in Europe at JoVE.com

Implementation of Portable Emissions Measurement Systems PEMS for the Real-driving Emissions RDE Regulation in Europe

The European Commission has developed a Real-Driving Emissions (RDE) test procedure to verify pollutant emissions during real-world vehicle operation using the Portable Emissions Measurement Systems (PEMS). This paper presents the experimental procedures required by the newly-adopted RDE test.

便携式排放测量系统（PEMS）在欧洲的真实驾驶排放（RDE）规例的执行情况

Vehicles are tested in controlled and relatively narrow laboratory conditions to determine their official emission values and reference fuel consumption. ...

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Implementation of Portable Emissions Measurement Systems (PEMS) for the Real-driving Emissions (RDE) Regulation in Europe

27.9K 次观看.  Joint Research Centre. The European Commission has developed a Real-Driving Emissions (RDE) test procedure to verify pollutant emissions during real-world vehicle operation using the Portable Emissions Measurement Systems (PEMS). This paper presents the experimental procedures required by the newly-adopted RDE test.

视频: Implementation of Portable Emissions Measurement Systems PEMS for the Real-driving Emissions RDE Regulation in Europe

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

使用吉布森大会和基因枪烟草中瞬时基因表达

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

科研

环境科学

Measurement of Greenhouse Gas Flux from Agricultural Soils Using Static Chambers

Measurement of greenhouse gas (GHG) fluxes between the soil and the atmosphere, in both managed and unmanaged ecosystems, is critical to understanding the biogeochemical drivers of climate change and to the development and evaluation of GHG mitigation strategies based on modulation of landscape management practices. The static chamber-based method described here is based on trapping gases emitted from the soil surface within a chamber and collecting samples from the chamber headspace at regular intervals for analysis by gas chromatography. Change in gas concentration over time is used to calculate flux. This method can be utilized to measure landscape-based flux of carbon dioxide, nitrous oxide, and methane, and to estimate differences between treatments or explore system dynamics over seasons or years. Infrastructure requirements are modest, but a comprehensive experimental design is essential. This method is easily deployed in the field, conforms to established guidelines, and produces data suitable to large-scale GHG emissions studies.

This article showcases the static chamber-based method for measurement of greenhouse gas flux from soil systems. With relatively modest infrastructure investments, measurements may be obtained from multiple treatments/locations and over timeframes ranging from hours to years.

从农业土壤使用静态钱伯斯温室气体流量测量

Measurement of greenhouse gas (GHG) fluxes between the soil and the atmosphere, in both managed and unmanaged ecosystems, is critical to understanding the ...

A New Application of the Electrical Penetration Graph (EPG) for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Electrophysiological properties of cells are often studied in vitro, after dissociating them from their native environments. However, the study of electrical transmission between distant cells in an organism requires in vivo, artifact-free recordings of cells embedded within their native environment. The transmission of electrical signals from wounded to unwounded areas in a plant has since long piqued the interest of botanists. The phloem, the living part of the plant vasculature that is spread throughout the plant, has been postulated as a major tissue in electrical transmission in plants. The lack of suitable electrophysiological methods poses many challenges for the study of the electrical properties of the phloem cells in vivo. Here we present a novel approach for intracellular electrophysiology of sieve elements (SEs) that uses living aphids, or other phloem-feeding hemipteran insects, integrated in the electrical penetration graph (EPG) circuit. The versatility, robustness, and accuracy of this method made it possible to record and study in detail the wound-induced electrical signals in SEs of central veins of the model plant Arabidopsis thaliana1. Here we show that EPG-electrodes can be easily implemented for intracellular electrophysiological recordings of SEs in marginal veins, as well as to study the capacity of SEs to respond with electrical signals to several external stimuli. The EPG approach applied to intracellular electrophysiology of SEs can be implemented to a wide variety of plant species, in a large number of plant/insect combinations, and for many research aims.

A New Application of the Electrical Penetration Graph EPG for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Electrical Penetration Graph (EPG) is a well-established technique for studying the feeding behavior of stylet-bearing insects. Here we show a new application of EPG as a non-invasive tool for the acquisition of intracellular electrophysiology recordings of sieve elements (SEs), the cells that form the phloem vasculature in plants.

在电气贯穿图（EPG）的韧皮部筛分子获取和测量电信号中的新应用

Electrophysiological properties of cells are often studied in vitro, after dissociating them from their native environments. However, the study of ...

The Use of an Automated System (GreenFeed) to Monitor Enteric Methane and Carbon Dioxide Emissions from Ruminant Animals

Ruminant animals (domesticated or wild) emit methane (CH4) through enteric fermentation in their digestive tract and from decomposition of manure during storage. These processes are the major sources of greenhouse gas (GHG) emissions from animal production systems. Techniques for measuring enteric CH4 vary from direct measurements (respiration chambers, which are highly accurate, but with limited applicability) to various indirect methods (sniffers, laser technology, which are practical, but with variable accuracy). The sulfur hexafluoride (SF6)&#160;tracer gas method is commonly used to measure enteric CH4 production by animal scientists and more recently, application of an Automated Head-Chamber System (AHCS) (GreenFeed, C-Lock, Inc., Rapid City, SD), which is the focus of this experiment, has been growing. AHCS is an automated system to monitor CH4 and carbon dioxide (CO2) mass fluxes from the breath of ruminant animals. In a typical AHCS operation, small quantities of baiting feed are dispensed to individual animals to lure them to AHCS multiple times daily. As the animal visits AHCS, a fan system pulls air past the animal&#8217;s muzzle into an intake manifold, and through an air collection pipe where continuous airflow rates are measured. A sub-sample of air is pumped out of the pipe into non-dispersive infra-red sensors for continuous measurement of CH4 and CO2 concentrations. Field comparisons of AHCS to respiration chambers or SF6 have demonstrated that AHCS produces repeatable and accurate CH4 emission results, provided that animal visits to AHCS are sufficient so emission estimates are representative of the diurnal rhythm of rumen gas production. Here, we demonstrate the use of AHCS to measure CO2 and CH4 fluxes from dairy cows given a control diet or a diet supplemented with technical-grade cashew nut shell liquid.

The Use of an Automated System GreenFeed to Monitor Enteric Methane and Carbon Dioxide Emissions from Ruminant Animals

Accuracy and precision of the techniques used to measure methane emissions from ruminant animals are critically important for the success of greenhouse gas mitigation efforts. This manuscript describes the principles and operation of an automated system to monitor methane and carbon dioxide mass fluxes from the breath of ruminant animals.

自动系统（绿色饲料）的用于监视肠道的甲烷和二氧化碳从反刍动物排放

Ruminant animals (domesticated or wild) emit methane (CH4) through enteric fermentation in their digestive tract and from decomposition of manure during ...

Calibrated Passive Sampling - Multi-plot Field Measurements of NH3 Emissions with a Combination of Dynamic Tube Method and Passive Samplers

Agricultural ammonia (NH3) emissions (90% of total EU emissions) are responsible for about 45% airborne eutrophication, 31% soil acidification and 12% fine dust formation within the EU15. But NH3 emissions also mean a considerable loss of nutrients. Many studies on NH3 emission from organic and mineral fertilizer application have been performed in recent decades. Nevertheless, research related to NH3 emissions after application fertilizers is still limited in particular with respect to relationships to emissions, fertilizer type, site conditions and crop growth. Due to the variable response of crops to treatments, effects can only be validated in experimental designs including field replication for statistical testing. The dominating ammonia loss methods yielding quantitative emissions require large field areas, expensive equipment or current supply, which restricts their application in replicated field trials. This protocol describes a new methodology for the measurement of NH3 emissions on many plots linking a simple semi-quantitative measuring method used in all plots, with a quantitative method by simultaneous measurements using both methods on selected plots. As a semi-quantitative measurement method passive samplers are used. The second method is a dynamic chamber method (Dynamic Tube Method) to obtain a transfer quotient, which converts the semi-quantitative losses of the passive sampler to quantitative losses (kg nitrogen ha-1). The principle underlying this approach is that passive samplers placed in a homogeneous experimental field have the same NH3 absorption behavior under identical environmental conditions. Therefore, a transfer co-efficient obtained from single passive samplers can be used to scale the values of all passive samplers used in the same field trial. The method proved valid under a wide range of experimental conditions and is recommended to be used under conditions with bare soil or small canopies (&#60;0.3 m). Results obtained from experiments with taller plants should be treated more carefully.

Calibrated Passive Sampling - Multi-plot Field Measurements of NH3 Emissions with a Combination of Dynamic Tube Method and Passive Samplers

Ammonia emissions are a major threat to the environment by eutrophication, soil acidification and fine particle formation and stem mainly from agricultural sources. This method allows ammonia loss measurements in replicated field trials enabling statistical analysis of emissions and of relationships between crop development and emissions.

校准被动采样 - NH多情节场测量<sub&gt; 3</sub&gt;与动态管法和被动采样的组合排放

Agricultural ammonia (NH3) emissions (90% of total EU emissions) are responsible for about 45% airborne eutrophication, 31% soil acidification and 12% ...

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and detritus, and excreting particulate and dissolved compounds, they serve as important agents for benthic-pelagic coupling. Accurately measuring the compounds removed and excreted by suspension feeders (such as sponges, ascidians, polychaetes, bivalves) is crucial for the study of their physiology, metabolism, and feeding ecology, and is fundamental to determine the ecological relevance of the nutrient fluxes mediated by these organisms. However, the assessment of the rate by which suspension feeders process particulate and dissolved compounds in nature is restricted by the limitations of the currently available methodologies. Our goal was to develop a simple, reliable, and non-intrusive method that would allow clean and controlled water sampling from a specific point, such as the excurrent aperture of benthic suspension feeders, in situ. Our method allows simultaneous sampling of inhaled and exhaled water of the studied organism by using minute tubes installed on a custom-built manipulator device and carefully positioned inside the exhalant orifice of the sampled organism. Piercing a septum on the collecting vessel with a syringe needle attached to the distal end of each tube allows the external pressure to slowly force the sampled water into the vessel through the sampling tube. The slow and controlled sampling rate allows integrating the inherent patchiness in the water while ensuring contamination free sampling. We provide recommendations for the most suitable filtering devices, collection vessel, and storing procedures for the analyses of different particulate and dissolved compounds. The VacuSIP system offers a reliable method for the quantification of undisturbed suspension feeder metabolism in natural conditions that is cheap and easy to learn and apply to assess the physiology and functional role of filter feeders in different ecosystems.

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

We introduce the VacuSIP, a simple, non-intrusive, and reliable method for clean and accurate point sampling of water. The system was developed and evaluated for the simultaneous collection of the water inhaled and exhaled by benthic suspension feeders in situ, to cleanly measure removal and excretion of particulate and dissolved compounds.

VacuSIP，对改进的方法INEX<em&gt;原位</em&gt;颗粒物与测量溶解的化合物加工后主动悬挂给料机

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and ...

Protocols for Quantifying Transferable Pesticide Residues in Turfgrass Systems

Plant canopies in established turfgrass systems can intercept an appreciable amount of sprayed pesticides, which can be transferred through various routes onto humans. For this reason, transferable pesticide residue experiments are required for registration and re-registration by the United States Environmental Protection Agency (USEPA). Although such experiments are required, limited specificity is required pertaining to experimental approach. Experimental approaches used to assess pesticide transfer to humans including hand wiping with cotton gloves, modified California roller (moving a roller of known mass over cotton cloth) and soccer ball roll (ball wrapped with sorbent strip) over three treated turfgrass species (creeping bentgrass, hybrid bermudagrass and tall fescue maintained at 0.4, 5 and 9 cm, respectively) are presented. The modified California roller is the most extensively utilized approach to date, and is best suited for use at low mowing heights due to its reproducibility and large sampling area. The soccer ball roll is a less aggressive transfer approach; however, it mimics a very common occurrence in the most popular international sport, and has many implications for nondietary pesticide exposure from hand-to-mouth contact. Additionally, this approach may be adjusted for other athletic activities with limited modification. Hand wiping is the best approach to transfer pesticides at higher mowing heights, as roller-based approaches can lay blades over; however, it is more subjective due to more variable sampling pressure. Utility of these methods across turfgrass species is presented, and additional considerations to conduct transferable pesticide residue research in turfgrass systems are discussed.

Transferable pesticide residue experiments in turfgrass systems are integral components of human risk exposure assessments. Experimental approaches to measure transferable residues should be adjusted to the human interaction of interest and turfgrass system dynamics. Three transferable pesticide residue protocols are presented and the suitability across three turfgrass systems is discussed.

协议在草坪系统量化转换农药残留

Plant canopies in established turfgrass systems can intercept an appreciable amount of sprayed pesticides, which can be transferred through various routes ...

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

检测幼虫行为反应剖面的实验协议: 在神经刺激咖啡因中的应用

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) in vitro exposure systems are often large and bulky, making transport to the field and operation at the source of emission or within the breathing zone difficult. Through miniaturization of these systems, the lab can be brought to the field, expediting processing time and providing a more appropriate exposure method that does not alter the aerosol prior to contacting the cells. The Portable In vitro Exposure Cassette (PIVEC) adapts a 37 mm filter cassette to allow for in vitro toxicity testing outside of a traditional laboratory setting. The PIVEC was characterized using three sizes of copper nanoparticles to determine deposition efficiency based on gravimetric and particle number concentration analysis. Initial cytotoxicity experiments were performed with exposed lung cells to determine the ability of the system to deposit particles while maintaining cell viability. The PIVEC provides a similar or increased deposition efficiency when comparing to available perpendicular flow in vitro exposure devices. Despite the lower sample throughput, the small size gives some advantages to the current in vitro ALI exposure systems. These include the ability to be worn for personal monitoring, mobility from the laboratory to the source of emission, and the option to set-up multiple systems for spatial resolution while maintaining a lower user cost. The PIVEC is a system capable of collecting aerosols in the field and within the breathing zone onto an air-interfaced, in vitro model.

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

Here, we present a protocol to perform portable cellular aerosol exposures and measure cellular response. The method uses cells, grown at the air-liquid interface, mimicking in vivo physiology. Cellular response to copper nanoparticle aerosols was observed as oxidative stress through reactive oxygen species generation and cytotoxicity as lactate dehydrogenase release.

一种用于气溶胶取样的新型便携式体外曝光盒

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) ...

Split Point Analysis and Uncertainty Quantification of Thermal-Optical Organic/Elemental Carbon Measurements

Researchers from myriad fields frequently seek to quantify and classify concentrations of carbonaceous aerosols as organic carbon (OC) or elemental carbon (EC). This is commonly accomplished using thermal-optical OC/EC analyzers (TOAs), which enable measurement via controlled thermal pyrolysis and oxidation under specific temperature protocols and within constrained atmospheres. Several commercial TOAs exist, including a semi-continuous instrument that enables on-line analyses in the field. This instrument employs an in-test calibration procedure that requires relatively frequent calibration. This article details a calibration protocol for this semi-continuous TOA and presents an open-source software tool for data analysis and rigorous Monte Carlo quantification of uncertainties. Notably, the software tool includes novel means to correct for instrument drift and identify and quantify the uncertainty in the OC/EC split point. This is a significant improvement on the uncertainty estimation in the manufacturer&#8217;s software, which ignores split point uncertainty and otherwise uses fixed equations for relative and absolute errors (generally leading to under-estimated uncertainties and often yielding non-physical results as demonstrated in several example data sets). The demonstrated calibration protocol and new software tool enabling accurate quantification of combined uncertainties from calibration, repeatability, and OC/EC split point are shared with the intent of assisting other researchers in achieving better measurements of OC, EC, and total carbon mass in aerosol samples.

This article presents a protocol and software tool for the quantification of uncertainties in the calibration and data analysis of a semi-continuous thermal-optical organic/elemental carbon analyzer.

热光学有机/元素碳测量的分割点分析与不确定性定量

Researchers from myriad fields frequently seek to quantify and classify concentrations of carbonaceous aerosols as organic carbon (OC) or elemental carbon ...