We thank Victor Marquez for providing DZNep.HCl and Scotty Bolling for manufacturing the bloodfeeders. The Mopti and SUA2La strains of An. gambiae were obtained from the Malaria Research and Reference Reagent Resource Center (MR4). This work was supported by the Fralin Life Science Institute and the grant from National Institutes of Health 1R21AI094289 to Igor V. Sharakhov.

注：该协议的三个部分描述DZNep药物的水溶液暴露于幼虫蚊子，DZNep的基于血液的暴露于成年女性进行研究繁殖力其效果，并且使用简单的比色技术SAH水解酶抑制DZNep测量。这些测定法的示意图示于图1。 1.未成熟蚊子发展和存活率测定注：本节介绍使用水溶性药物，影响蚊子幼虫的发展和生存。 <ol><li>哈奇A.冈比亚鸡蛋在28°C的蒸馏水（DH 2 O）和后他们第二龄幼虫。 </li><li>取一个六孔DNA酶，RNA酶游离细胞培养板和倾10毫升的dh 2 O至每个孔中。 </li><li>选择蚊子的幼虫在2 次龄每孔加入15幼虫。然后加入第二龄幼虫直接制版，PUT斯达康它们在纸巾上为2-3秒，以除去多余的水。 </li><li>标签井用颜色编码的磁带随机实验。标签上的每个板（ 图2），每个测试浓度2孔中。 </li><li>通过在1ml的dh 2 O中溶解为0.3mg DZNep制备1mM的DZNep盐酸盐（MW = 298.73）原液添加DZNep.HCl的原液到标孔中，以达到0.5μM和5.0μM的终浓度。 注：原液可以储存在4 °下进行短期（1天至一个月）或-20 ℃下长期（超过一个月）。 注：加入的储备溶液为上述的浓度不会改变水的体积在孔中显著。然而，在情况下要高得多的浓度，最好是溶解化合物在幼虫介质，以避免任何不必要的稀释的化合物。 </li><li>加的片状鱼食等量至每个孔中。覆盖板s和培养他们在28 °下，在孵化器。 </li><li>记录的死亡率为24小时暴露时间后DZNep处理的和未处理的幼虫蚊子。取下并丢弃任何死幼虫。每天重复这个记录直到所有的幼虫模或化蛹并脱颖而出，成为成年人。记录幼虫的大小，每隔一天， 即，第0天，2，4，6，8，直到它们化蛹。 </li><li>分析使用适当的统计分析软件的幼体发育和死亡数据。 注：条形图，可以用于表示死亡率数据在Microsoft Excel中。多元方差分析（MANOVA）进行了软件如JMP或SPSS就会发现，如果不同剂量的药物导致显著不同的生存蚊子幼虫。 </li></ol> 2.繁殖力测定通过给予药品通过人工进纸器雌蚊注：本部分详细介绍了另外的药物，以血次使用人工馈线系统它馈送到成年雌性蚊子。 <ol><li>设置成蚊笼同等数量的男性和女性的蚊子控制和测试笼。提供10％的糖溶液中浸泡棉球两个笼子里，直到雌性准备喂养（3-5天是最佳的冈比亚按蚊 ）。 </li><li> 2小时前的血液供给，取出棉球，以确保女性是饿了。 </li><li>装配两个人工血液供料器，它由一个倒宽底部玻璃漏斗（直径= 50毫米，长度= 70毫米）和周围的塑料工业胶密封。附加塑料管（直径= 7毫米）至2馈线“网点（直径= 10mm）的通过连接器，用于流入和流出的水​​。标记两个馈线为“控制”和“5μMDZNep”。 </li><li>使用市售的加热元件，设置程序觅食。在“菜单”进入“设置”并选择“设定点”;预装的程序将会出现。选择“SP1”，并设置该温度37 ℃。 注：加热元件确保了血液施用维持在一个恒定的温度。可用于不同的蚊子或昆虫物种附加程序设置。 </li><li>填充有水的水桶和浸入加热元件到水中。 </li><li>切一块方形封口膜（50毫米×50毫米），并伸展它，使薄的膜状的膜。涵盖人工饲养的封口膜的底部。切另一片封口膜（50毫米×10mm的）的，它伸展并密封该馈线的边缘。 </li><li>组装系统中，通过管连接馈线和加热元件。一旦完成，切换系统，选择“SP1”。按“Enter”来启动系统。 注：水会加热到37 °C和保持在该温度。 </li><li>监测日水用水桶温度计é温度。 注：连接器和管道有助于通过该系统保持恒定的水循环，使血液的温度保持在37 ℃。 </li><li>填平底托盘与将用于净化任何血液泄漏的10％漂白溶液。 </li><li>加入2毫升除颤羊的血液离心管。这个标签为“控制”。重复该过程为实验管标记为“5μMDZNep”。新增6微升DZNep原液（见步骤1.5）的实验管标有“5μMDZNep”轻轻颠倒几次混合血液和药品。在室温下孵育该管10分钟。 </li><li>使用移液管，加2ml对照和测试血液到相应标记的馈线的顶部。丢弃在漂白溶液中的吸移管。盖上笼用深色袋子，呼吸的空气笼定期鼓励˚Females喂养。 </li><li>让蚊子喂约30分钟。一旦加料完毕，关掉加热元件，把馈线出来，剥离封口膜。浸泡在漂白剂溶液馈线约5分钟，并在去离子水冲洗干净。 </li><li>取出馈线，把棉球用白糖水的笼子里48小时。将含有水和滤纸在笼子里过夜，以方便产卵一个鸡蛋的菜。标记每个蛋盘与相应的测试或对照的浓度。 </li><li>取出蛋菜第二天检查蛋的数量和结构的立体显微镜下。获得使用Q-colors5软件映像。计数的蛋的数量。分析在从测试和对照笼获得蛋的数量差异。 注意：一个非配对t检验可以用于确定是否蛋的数量是显著差异（p≤0.05）。 </li></ol>蚊子酶抑制3.生化测定通过药物注意：本节介绍使用DTNB作为指标来确定DZNep对成年雄性和雌性蚊子SAH水解酶的抑制效果的酶活性测定。 <ol><li>通过加入Na 214.19克HPO 4到1升的dh 2 O的制备的0.1M的Na 2 HPO 4（磷酸氢二钠）溶液接着，通过添加13.79克的NaH 2 PO 4至1升的dh 2 O的制备的0.1M的NaH 2 PO 4（磷酸二氢钠）溶液</li><li>滴定的0.1M的Na 2 HPO 4溶液与所述的0.1M的NaH 2 PO 4的溶液至pH 8.5。 注意：这将是为0.1M的Na 2 HPO 4（pH为8.5）的工作原液。 </li><li>通过加入0.3％的Triton X-100的制备的的0.1M的Na 2 HPO 4（pH为8.5）均质化的溶液。 注：同质化的解决方案可以保存在4℃。 </li><li>公关epare 1.6毫SAH（MW = 384.4）通过在10毫升的0.1M的Na 2 HPO 4（pH为8.5）中溶解6.15毫克SAH的溶液。 </li><li>通过在10毫升的0.1M的Na 2 HPO 4（pH为8.5）中溶解6.3毫克DTNB的制备1.6毫DTNB（MW = 396.4）溶液。置于冰上新鲜DTNB液备用。 </li><li>通过在1毫升的dh 2 O中溶解为0.3mg DZNep的制备1mM的DZNep.HCl（MW = 298.73）的溶液接下来，稀释1mM的DZNep溶液成一系列浓度从1000微米至0.002微米的dh 2 O. </li><li>为了测量SAH水解酶抑制DZNep，准备蚊子粗酶提取：均质10个非血液喂养成蚊1毫升冰冷的0.1M的NaH 2 PO 4（pH值8.5），含0.3％的Triton X- 100，使用玻璃组织匀浆器。匀浆液转移至1.5ml离心管中。 </li><li>离心匀浆5分钟以10,000 xg离心在4℃下。上清转移到一个干净的1.5 ml离心管。使用上清液作为酶源SAH生物化学测定。 </li><li>对于空白处理（ 即无DZNep，无酶）中，添加50微升1.6毫SAH，50微升1.6毫DTNB，并加入100μl的0.1M的Na 2 HPO 4（pH为8.5），以的96孔的各孔，平底微孔板。 </li><li>对于控制（ 即没有DZNep），加入50微升1.6毫米SAH，50微升1.6毫米DTNB，50微升的0.1M的Na 2 HPO 4（pH值8.5）和50微升酶（从原油中提取SAH水解获得）个别井。 </li><li>对于DZNep治疗，加50微升1.6毫米SAH，50微升1.6毫米DTNB，50微升酶和50微升选择DZNep集中到个别井。准备每次治疗和控制4个重复。 </li><li>使用96孔酶标仪读取SAH酶样品的​​光密度（OD）在405nm进行5分钟，在20秒的间隔。 </li><li>减去控制空白OD次治疗OD从每个获得良好。计算使用下面的等式其余SAH水解酶活性的百分比：％残留活性=（处理的OD /对照OD值）×100。 </li></ol>

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The authors have nothing to disclose.

有几个步骤，这个协议的成功应用至关重要。对于幼虫检测，应注意正确的标签和复制每个测试浓度。随机化的测试样品和添加药物的指定量的各试验孔中是本实验装置的一个重要部分。之前加入第二龄蚊子幼虫到96孔微量，每个幼虫可以放在纸巾2-3秒，以除去多余的水。为了防止干燥，幼虫必须立即转移到用钝钳子的微孔板。这使水在各微孔板的体积以及与药物的浓度保持为意。当用于...

疟疾是负责的最高数目昆虫有关的死亡，在世界。每年发生大约2.19亿箱子全球，造成约66万死亡病例，主要是在非洲1。尽管共同努力，疟疾项目面临着一些挑战。而杀虫处理的蚊帐和程序室内滞留喷洒形式的关键部件，阻力在当地居民杀虫剂阻碍这些努力2。在杀虫剂抗性蚊子种群的快速增长在很大程度上归因于疟蚊的快速适应变化的环境，并利用不同的壁龛3,4,5的能力。为了克服抗药性的现有机制，新型杀虫剂目标和下一代化合物的探索是必要的。一个简单的，一步一步的协议确定的疟疾米不同人生阶段的实验杀虫剂的功效osquitoes将显著加强这些努力。 对细胞系和动物模型中药物作用的药理学研究已经建立的使用后生药物作为用于调节细胞和生物体的遗传学和生理学的有用工具。 DNA甲基化和组蛋白修饰是影响在多细胞生物体的基因表达不改变基本的DNA序列6 2后生机制。翻译后修饰，例如甲基化在维持细胞完整性和基因表达至关重要的作用，并且可影响几个基本过程7,8,9。研究在一些昆虫物种突出表观遗传学的重要性在涉及卵子发生过程和干细胞维持10，以及剂量补偿11。然而，这些方面在病媒尚待开发。用化合物调节该系统可蚊子为我们提供了在景点融入新颖杀虫剂目标的途径。 3- Deazaneplanocin A（DZNep）是一种已知的组蛋白甲基化抑制剂，其在不同类型的癌症的影响进行了研究12,13,14,15,16。 DZNep是一种稳定的水溶性后生药物间接抑制组蛋白赖氨酸 N- -methyltransferase（EZH2），在哺乳动物细胞中的Polycomb压制性复杂2（PRC2）的一个组件。 PRC2在调节干细胞的多细胞生物的生长中起重要作用，并且组蛋白甲基化是将PRC2介导的基因沉默的一个关键方面。在免疫缺陷小鼠中，细胞预先用DZNep已经显示出少致瘤17。这种药物正在成为用于研究其他疾病，如非酒精性脂肪肝疾病，其中EZH2牵连18。 DZNep是一个既定的S -adenosylhomocysteine（SAH）水解酶抑制剂19，20。的SAH水解导致一个抑制SAH的累积和，反过来，通过限制可用的甲基供体基团导致甲基转移酶活性的抑制。 SAH是由许多生物，包括昆虫，在他们的代谢途径利用的氨基酸衍生物。最近的一项研究表明，DZNep低剂量可能会影响滞育和延缓发展的昆虫21。 这里，一个强大的协议来调查对蚊子的各种生命阶段的水溶性化合物的效果十分发达。这个协议的三个部分，包括：用于检查在未成熟的蚊子，成人吸血雌性和雄性和雌性蚊子的酶活性的水溶性化合物的效果的说明。首先，DZNep溶解在水中以研究未成熟蚊发育和存活。这被执行以两种浓度进行比较，从在药物暴露10倍的增加所引起的任何差异。探索成人的雌蚊，DZNep药物的效果被添加到除颤羊血和供给血液中人为地女性。接着，对生殖力的药物的结果进行检测。最后，使用5,5&#39;- dithiobis-（2-硝基苯甲酸）（DTNB）作为指标来确定DZNep对成年雄性和雌性蚊子SAH水解酶抑制效果进行的酶活性测定。虽然该协议被显影用疟蚊， 冈比亚按蚊 ，它可以容易地适应于学习的任何种类的蚊子或其他昆虫感兴趣化合物的作用。在这个协议中详述的技术可能不被有效地应用到具有在水中或含水介质有限或没有溶解性的药物。

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			<td height="21" style="height:21px;width:219px;">Name of the Reagent/Equipment&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:192px;">Company</td>
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			<td style="width:125px;">Comments</td>
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			<td height="21" style="height:21px;">96-well microplate</td>
			<td>Fisher Scientific</td>
			<td>12565561</td>
			<td></td>
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			<td height="21" style="height:21px;">Cell culture plate</td>
			<td>CytoOne</td>
			<td>CC7682-7506</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Sorvall Fresco</td>
			<td>76003758</td>
			<td>A different centrifuge can be used</td>
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			<td height="21" style="height:21px;">Colored tape rolls</td>
			<td>Fisher</td>
			<td>S68134</td>
			<td></td>
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			<td height="21" style="height:21px;">Dissection microscope</td>
			<td>Olympus</td>
			<td>SZ</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">DTNB</td>
			<td>Sigma Aldrich</td>
			<td>D8130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DZNep.HCl</td>
			<td>Sigma Aldrich</td>
			<td>SMLO305</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Egg dish cups</td>
			<td></td>
			<td></td>
			<td></td>
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			<td height="21" style="height:21px;">Filter papers</td>
			<td>Fisher</td>
			<td>09-795E</td>
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			<td height="21" style="height:21px;">Glass feeders</td>
			<td>Virginia Tech</td>
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			<td></td>
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			<td height="21" style="height:21px;">Glass tissue homogenizer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating element</td>
			<td>Fisher Scientific</td>
			<td style="width:133px;">NC0520091</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td>Percival scientific</td>
			<td>I36VLC8</td>
			<td>A different incubator can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge tube, &#160;2 ml</td>
			<td>Axygen</td>
			<td>22-283</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge tube, 1.5 ml</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td></td>
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			<td height="21" style="height:21px;">Micropipette</td>
			<td>Eppendorf</td>
			<td>4910 000.069</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4</td>
			<td>Fisher Scientific</td>
			<td>M-3154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaH2PO4</td>
			<td>Fisher Scientific</td>
			<td>M-8643</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pH meter&#160;</td>
			<td>Mettler Toledo 7easy</td>
			<td>S20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plate reader</td>
			<td>Spectramax</td>
			<td>M2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SAH</td>
			<td>Sigma Aldrich</td>
			<td>A9384</td>
			<td>store at -20C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
	</tbody>
</table>

toxicological assays for testing effects of an epigenetic drug on development, fecundity and survivorship of malaria mosquitoes

杀虫性带来的疟疾控制规划的一大难题。蚊子适应范围广的变化迅速的环境中，使得疟疾控制一个无所不在的问题，在热带国家。杀虫剂抗性种群的出现值得新型药物靶点的途径和化合物矢量控制蚊子的探索。后生药物确立在癌症研究，但没有多少人知道他们对昆虫的影响。这项研究提供了一个简单的协议，用于检查3 Deazaneplanocin A（DZNep），实验后生药物用于癌症治疗的毒性作用，对疟蚊， 冈比亚按蚊 。在死亡率和降低尺寸的浓度依赖性增加，观察暴露于DZNep未成熟蚊子，而化合物还原成蚊相对于对照治疗的繁殖力。此外，还有S中的药物依赖性降低-腺苷高半胱氨酸（SAH）水解酶的蚊子暴露于DZNep相对于对照治疗活动。这些协议提供研究者用一个简单的，一步一步的程序，以评估多个毒理学终点的实验性药物，反过来，表现出探索的水溶性后生药物或化合物的毒性效应独特的多管齐下的方法利率对蚊子和其他昆虫。

图1是测定的示意图;它描述了各个步骤本文中列出的程序。作为该协议基于蚊不同的生命阶段，没有特定的顺序应遵循此处详述的实验。用户可以选择要进行的一个或多个测定中的同时，这取决于样品的可用性。 图2演示了该板设置为未成熟蚊子发展和存活测定法。测试浓度和重复的数量可以根据用户的要求和药物的可用性而改变。对于未成熟?...

Watch this Scientific Journal Video about Toxicological Assays for Testing Effects of an Epigenetic Drug on Development, Fecundity and Survivorship of Malaria Mosquitoes at JoVE.com

Toxicological Assays for Testing Effects of an Epigenetic Drug on Development, Fecundity and Survivorship of Malaria Mosquitoes

A protocol is developed to examine the effects of an epigenetic drug DZNep on the development, fecundity and survivorship of mosquitoes. Here we describe procedures for the aqueous exposure of DZNep to immature mosquitoes and a blood-based exposure of DZNep to adult mosquitoes in addition to measuring SAH hydrolase inhibition.

毒理学化验一个后生药物对疟蚊的发育，繁殖和存活率测试效果

Insecticidal resistance poses a major problem for malaria control programs. Mosquitoes adapt to a wide range of changes in the environment quickly, making ...

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Research

JoVE Journal

Biology

8.4K 次观看.  Virginia Tech. A protocol is developed to examine the effects of an epigenetic drug DZNep on the development, fecundity and survivorship of mosquitoes. Here we describe procedures for the aqueous exposure of DZNep to immature mosquitoes and a blood-based exposure of DZNep to adult mosquitoes in addition to measuring SAH hydrolase inhibition. 

视频: Toxicological Assays for Testing Effects of an Epigenetic Drug on Development, Fecundity and Survivorship of Malaria Mosquitoes

Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This video protocol demonstrates a method used by the James laboratory to microinject DNA constructs into Anopheles stephensi embryos for the generation of transformed mosquitoes. Techniques for preparing microinjection needles, collecting and preparing embryos and performing the microinjection are illustrated.

Anopheles stephensi mosquitoes are vectors for malaria inhabiting India and throughout Asia. This video demonstrates the technique for performing microinjections of this species with transgenes that will confer resistance to the malaria to the mosquito. Much of the methodology demonstrated in this video is applicable to microinjection techniques of other mosquito species.

注射的。 stephensi胚胎生成抗药性疟疾的蚊子

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This ...

科研

生物学

Protocol for Dengue Infections in Mosquitoes (A. aegypti) and Infection Phenotype Determination

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of the virus in the mosquito tissues. This protocol is routinely used for studying mosquito-virus interactions, especially for identification of novel host factors that are able to determine vector competence. The entire experiment must be conducted in a BSL2 laboratory. Similar to Plasmodium falciparum infections, proper attire including gloves and lab coat must be worn at all times. After the experiment, all the materials that came in contact with the virus need to be treated with 75% ethanol and bleached before proceeding with normal washing. All other materials need to be autoclaved before discarding them.

Protocol for Dengue Infections in Mosquitoes A. aegypti and Infection Phenotype Determination

Once a gene is identified as potentially refractory for the dengue virus, it must be evaluated for it's role in preventing viral infections within the mosquito. This protocol illustrates how the extent of dengue infections of mosquitoes can be assayed. The techniques for growing up the virus in culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

在蚊子（埃及斑蚊）感染引起的表型测定为登革热感染病例的议定书“

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of ...

Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito.    This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed.  The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito. This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed. The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

恶性疟原虫在蚊子和病毒感染的表型测定感染的议定书

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the ...

Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite.  The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

成蚊（冈比亚按蚊的RNAi实验议定书）

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

Preventing the Spread of Malaria and Dengue Fever Using Genetically Modified Mosquitoes

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced, as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission. Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

防止疟疾的传播和使用基因改造的蚊子登革热

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population ...

Building a Better Mosquito: Identifying the Genes Enabling Malaria and Dengue Fever Resistance in A. gambiae and A. aegypti Mosquitoes

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections.   Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections. Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.

建设一个更美好的蚊子：确定基因在冈比亚按蚊和埃及斑蚊蚊子启用疟疾和登革热的电阻

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus ...

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

使用细胞色素线粒体呼吸功能的可视化 C氧化酶/琥珀酸脱氢酶（COX / SDH），双标组织化学

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Myospheres的产生从人类胚胎干细胞通过表观遗传修饰

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

A Multi-detection Assay for Malaria Transmitting Mosquitoes

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

一种多检测试验为疟疾蚊子发射

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

新型杀虫化学对蚊子毒性测试规程

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...