作者感谢酋长，村长和马查为他们的unwaveing​​合作，在蚊子的现场样品采集的社区。道格·诺里斯博士和利百加肯特的盐析协议提供了宝贵的经验。这项工作是由约翰霍普金斯大学疟疾研究所试点补助金制度。蚊子和寄生虫DNA实验室标准MR4，美国的文化类型和收集捐赠。

1。筹备的蚊子标本解剖<ol><li>将切割封口膜，并安装在解剖显微镜下的小蚊子。 </li><li>量的去离子水软化组织在下拉。 </li><li>切开的蚊子尸体正是在胸部和腹部之间的联合，从而刻痕的头部和胸部，腹部。 </li><li>每个解剖蚊子节转移到一个干净的高压灭菌1.5毫升离心管中prelabeled蚊子ID号码的细节和适当的标记来表示的主体部分（ 例如，"m"为中肠部分，头部和胸部部分的"HT"）。 </li><li>丢弃封口膜处理下一个蚊子之前，仔细地擦拭显微镜载物台与70％酒精蘸Kimwipe。 </li></ol> 2。蚊子标本的DNA提取<ol><li>吸取20微升的样品管和去离子水，使用移液器吸头研磨成联合国淹没蚊子节iform暂停。 </li><li>中加入100μl，蒸压1X PBS / 1％皂素的解决方案样品匀浆和组合，温柔的瞬间涡旋。 </li><li>在室温下孵育20分钟。 </li><li>离心20000×g离心2分钟，弃去上清液。 </li><li>在100μL1X PBS重悬浮颗粒。 </li><li>在20,000×g离心2分钟，再次离心，弃去上清液。 </li><li>通过轻轻涡旋（5秒），20％w / v的Chelex-100树脂的去离子水的悬浮液中重悬沉淀，将75μl无菌去离子水和25μl。 </li><li>皮尔斯孔盖样品管，用无菌23G注射器针头在本生灯火焰。 </li><li>煮沸样品悬浮液的浮动机架在水浴中10分钟（或在加热块）。 </li><li>在20,000 xg离心离心，1分钟和转让随​​后的DNA溶液作为PCR应用的模板用于成prelabeled储存小瓶。 </li></ol> 3。恶性疟原虫基因分型和按蚊SPP。分子生物学鉴定<ol><li> Chelex DNA提取25微升的PCR反应中加入2.5μl检查DNA，确定质量11的 按蚊种10和基因型P.恶性疟原虫 12月中旬肠道和唾液腺感染。 </li><li>为了最大限度地提高扩增产物的产率，特别是对体育恶性疟原虫的检测，包括1.5X牛血清白蛋白（BSA）的PCR检测。下面的反应组合物的建议的:（2.5微升模板，0.2510μM的引物，1.5μM的氯化镁，200μM的dNTPs，1X PCR缓冲液，1.5X BSA和1.0U Taq DNA聚合酶，在25微升的体积）。 </li><li>要检查的DNA提取物质量在节肢动物线粒体烟酰胺腺嘌呤二核苷酸脱氢酶（NADH）亚基4（ND4）基因（引物ND4FW [5&#39;-GTD一TTA TGA TTR建AA-3&#39;]和ND4RV [5&#39;，放大区域CTT CGD CTT CCW ADW CGT TC-3&#39;];期望产品400bp的）11,13。 </li><li>为了区分按蚊冈比亚E SL近缘 ​​种（An.冈比亚ss和阿拉伯按蚊只），扩增侧翼区域SNP位点的基因间隔区（IGS），（引物UN [5&#39;-GTGTGCCCCTTCCTCGATGT 3]，GA [5&#39;-CTGGTTTGGTCGGCACGTTT 3&#39;]，AR [5&#39;-AAGTGTCCTTCTCCATCCTA-3&#39;]期望产品的390bp 的冈比亚β，315bp的阿拉伯按蚊 ）10,11。 </li><li>对于打字P.恶性疟原虫抗叶酸剂的耐药性基因多态性，进行巢式PCR扩增侧翼区域的氨基酸的密码子108，51，59，16和164中的寄生虫的二氢叶酸还原酶（DHFR）基因: 主轮引物 :M1 [5&#39;-TTTATGATGGAACAAGTCTGC-3&#39;]和M5 [5&#39;-AGTATATACATCGCTAACAGA-3&#39;] 中学轮引物:M3 [5&#39;-TTTATGATGGAACAAGTCTGCGACGTT-3&#39;]与F / [5&#39;-AAATTCTTGATAAACAACGGAAACCTTTTA-3&#39;] 或 F [5&#39;-GAAATGTAATTCCCTAGATATGGAATATT-3] M4 [5&#39;-TTAATTTCCCAAGTAAAACTATTAGAGCTTC-3与&#39;]）。 含氨基酸的密码子436，437，540，581一个，也放大区域ND 613的P.恶性疟原虫二氢喋呤合成酶（，DHPS），基因: 主轮引物 R2 [5&#39;-AACCTAAACGTGCTGTTCAA-3&#39;]与R / [5&#39;-AATTGTGTGATTTGTCCACAA3&#39;]; 二级轮引物 J:[5&#39;-TGCTAGTGTTATAGATATAGGTGGAGAAAGC-3&#39;]与K / [5&#39;-CTATAACGAGGTATTGCATTTATTGCAAGAA-3&#39;] 或 K:与K / [5&#39;-TGCTAGTGTTATAGATATAGGATGAGCATC-3&#39;]， 或 L [5&#39;-ATAGGATACTATTTGATATTGATATTGGACCAGGATTCG-3&#39;]与L / [5&#39;-5TATTACAACATTTTGATCATTCGCGCAACCGG-3&#39;] 12。 </li><li>主题4微升的扩增子检测抗叶酸药耐药相关多态性等位基因特异的限制性内切酶消化，按照制造商的说明，在30μl的反应12。 </li><li>主题5μl的PCR扩增子（或15微升限制性消化）每车道溴化乙锭染色的2％琼脂糖凝胶电泳（100 - 120V）和可视化条带在紫外透照。 </li></ol>

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没有利益冲突的声明。

简化Chelex这里介绍的方法，使提取质量按蚊属和P.恶性疟原虫的蚊子标本，适合不同的PCR应用DNA。这种技术可以用于传播疟疾的蚊子的分子鉴定和耐药监测P.恶性疟原虫基因蚊子为国家疟疾控制计划。简化Chelex技术的优点包括简单，试剂少，因此成本，安全性和较短的处理时间比标准协议，如盐析方法10（2小时47分钟，过夜的步骤， 表1）（37分钟）。上述优势和最小的试剂要求（试剂）相比，目前的标准协议（10个试剂， 表1），11,15，简化Chelex协议，特别是友好流行国家实验室，那里的恒定试剂供应链往往是难以维护。该方法的一个限制是一样的标准协议，它也发生在蚊虫珠被16 PCR抑制剂。然而，这是容易地解除通过包含在检测的BSA。 BSA也被成功地采用作为一个放大增强剂针对在其他PCR应用程序17,18的抑制剂。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> Chelex-100 ,50-100干爽网面</td><td> BioRad公司</td><td> ＃143-2832 </td><td></td></tr><tr><td>皂甙</td><td> SIGMA </td><td> 142-7425 </td><td></td></tr><tr><td> BSA </td><td> New England Biolabs公司</td><td> B9001S </td><td></td></tr></tbody></table>

a simple chelex protocol for dna extraction from anopheles spp.

疾病流行的国家越来越多地采用高效的打字，识别和监视对疟疾寄生虫和媒介蚊虫的分子工具，作为一个组成部分，其控制方案1,2,3,4,5。可持续发展建立在操作这些准确的方法研究，以加强控制和消除疟疾的努力，简单又经济实惠的方法，吝啬的试剂和设备的要求是必不可少的6,7,8。在这里，我们提出了一个简单的Chelex从现场采集的蚊虫标本中提取疟疾寄生虫与载体DNA为基础的技术。 形态确定了72， 冈比亚按蚊SL。从156，由除虫菊喷雾捕获卧室的家庭在2,000 平方公里 ，附近的马查疟疾研究所在捕获的蚊子。经过解剖，分离的头部和胸部，腹部所有72个按蚊GAM两个部分BIAE sl的蚊子，单独放置在1.5毫升微量离心管中，并浸没在20微升的去离子水。使用无菌移液管尖，每个蚊子部分分别均匀的匀化的去离子水的悬浮液中。 10微升的随后从每个蚊子部匀浆中，被保留，而其他的10微升转移到一个单独的蒸压1.5 ml管。独立的等分试样进行DNA提取，不论是由简化的Chelex或标准盐析萃取协议9,10。盐析协议是所谓的和广泛使用的，因为它采用高盐浓度代替有害的有机溶剂（如苯酚和氯仿），用于DNA提取过程中的步骤9的蛋白质沉淀。 提取物被用作PCR扩增的模板，使用引物针对节肢动物线粒体烟酰胺腺嘌呤二核苷酸脱氢酶检查DNA的质量激酶（NADH）亚单位基因（ND4）11，PCR鉴定冈比亚按蚊亲缘种10和巢式PCR分型恶性疟原虫感染12。比较使用DNA质量（ND4），PCR结果显示93％的敏感性和特异性82％Chelex方法相对于既定的盐析协议。相应的值的敏感性和特异性分别为100％和78％，分别使用近缘种鉴定PCR和92％和80％，分别为P.恶性疟原虫检测PCR。有给扩增子的的Chelex或定期盐析协议在所有三个PCR应用与信号的样本比例无显着差异。 Chelex方法需要三个简单的试剂和37分钟完成，而盐析协议entailed 10个不同的试剂和2小时47分钟的处理时间，包括一晚一步。我们的研究结果表明Chelex法是与现有的盐析提取，并可以取代一个简单的和可持续的方法在资源有限的环境中恒定试剂的供应链往往是难以维持的。

结果PCR检测蚊子的DNA提取物质量（ 图1）， 阿拉伯按蚊的分子鉴定（ 图2），P. 的例子恶性疟原虫的检测（ 图3）表明，简化的的Chelex方法产生相似的结果的标准盐析协议10，尽管少得多的步骤（ 表1）。在各自的提取物具有可比性的DNA的质量，这是不足为奇的，样品阳性率相对于至An。冈比亚按蚊亲缘种以及寄生虫感染率差异无统计学意义的基础上麦克尼马尔的卡方检验（ 图3）。 灵敏度（％）被计算为TP /（TP + FN）* 100，其中TP表示真阳性和FN表示假阴性。特异性（％）被确定为TN /（TN + FP）* 100，TN表示真阴性和FP为假阳性。 DNA的质量（ND4）PCR结果显示93％的敏感性和82％的特异性的Chelex方法相比既定的盐析协议为金标准。相应的值的敏感性和特异性分别为100％和78％，分别使用近缘种鉴定PCR和92％和80％，分别为P.恶性疟原虫检测PCR。 到反应混合物中，加入BSA导致在一般的PCR阳性增加（ 图3）由于对救济PCR抑制剂14，为简化的Chelex，定期盐析协议。然而，这增加没有统计学意义，除了DNA的质量（ND4 PCR）Chelex提取物（P = 0.039）。等位基因特异的限制性内切酶消化对P.恶性疟原虫 DHFR（或其它目标基因）扩增子可以为药物抗性等位基因（ 图4，，唾液腺数据未示出 ）中肠和唾液腺疟疾感染的基因分型。 <table bo的乐趣="1"&gt; <tbody><tr><td colspan="2"> 简单Chelex程序 </td><td colspan="2"> 标准的盐析过程 </td></tr><tr><td> 步骤 </td><td> 试剂 </td><td> 步骤 </td><td> 试剂 </td></tr><tr><td><ol><li>将试样在100微升的1X PBS / 1％皂苷溶液（2分钟）1.5毫升微量离心管中，并匀化。 </li><li>发布，在室温下20分钟。 </li><li>转速为20000×g离心2分钟，弃去上清液。 </li><li>加入100μl1X PBS，轻轻混匀，短暂涡旋式（3秒）。 </li><li>转速为20000×g离心2分钟，弃去上清液。 </li><li>中加入25μl的20％w / v的Chelex在去离子水和75微升无菌去离子水。 </li><li>皮尔斯孔在盖体的样品管使用热，无菌23G皮下注射针和沸腾管中内容物的10分钟。 </li><li>旋转的离心管20000×g离心1分钟。 </li><li>将上清转移到无菌prelabeled的样品瓶和存储的DNA溶液在-20℃下直至使用（2.5微升在25μl的PCR反应）。 </li></ol></td><td> PBS 皂甙 Chelex-100珠</td><td><ol><li>样品在100微升本德尔缓冲区* 1％的DEPC（2分钟）至1.5 ml离心管中，均质。 </li><li>在65℃下孵育1小时。 </li><li>加入15微升冷的8 M的醋酸钾。轻轻混匀，冰上孵育45分钟。 </li><li>附带样品在微量离心管中，以20,000 xg离心10分钟，并转移上清至新的1.5毫升的试管。 </li><li>颠倒离心管，加入250μl的100％乙醇，混合均匀。 </li><li>样品在室温下孵育5分钟。 </li><li>附带样品在20,000 xg离心15分钟。 </li><li>吸液，弃去上清液，留下沉淀完全干燥，在管（30米在）。 </li><li>重悬在20微升RNA酶和0.1X SSC +（10微克/毫升），4℃过夜沉淀℃。 </li><li>加入80微升DEPC水和存储使用在-20°C至。 </li></ol></td><td>焦碳酸二乙酯（DEPC） *本德尔缓冲液: 0.1M NaCl的 0.2 M蔗糖 的0.1M Tris-盐酸 的0.05M EDTA，pH值为9.1 0.5％SDS在DEPC水 8 M乙酸钾乙醇 0.1X盐渍柠檬酸钠（SSC）缓冲液 RNA酶（10微克/毫升） </td></tr><tr><td colspan="2">总时间:37分钟</td><td colspan="2">总时间:2小时47分钟，再加上过夜</td></tr></tbody></table> 表1中。步骤一步所用试剂和时间要求为简单Chelex协议比较站在ARD蚊子标本的DNA提取盐析法。 <img alt="图1" src="/files/ftp_upload/3281/3281fig1.jpg" /> 图1。ND4线粒体PCR扩增的DNA质量比较从简化的Chelex的"C"和盐析"M"的提取物对阿拉伯按蚊现场样品的标准。 NC，阴性对照; M1和C1，M2和C2，M3和C3，和M4和C4是成对的盐析法和Chelex提取物相同的一个。阿拉伯按蚊的蚊子标本。 L，100 bp的DNA阶梯。 <img alt="图2" src="/files/ftp_upload/3281/3281fig2.jpg" /> 图2。C2和C1，M1，M2，C3和M3，C4和M4分别表示的扩增子从盐析"M"简化的Chelex"C"相同的蚊子标本的DNA提取物，AR， 按蚊阿拉伯按蚊的分子鉴定PCR。阿拉伯按蚊阳性对照组，L，100 bp的DNA梯状条带。 <img alt="图3" src="/files/ftp_upload/3281/3281fig3.jpg" /> 图3 Chelex和，盐析蚊子现场样品的DNA提取物， 阿拉伯按蚊 （AR）10，节肢动物NADH脱氢酶基因（ND4）DNA的质量测试11，与抗叶酸药物性 P分子鉴定的PCR检测的阳性检测。 。恶性疟原虫 DHFR基因分型12（F-M4）运行，或在反应混合物中不含BSA的测定结果所示。使用麦克尼马尔的卡方检验的，从DNA％的正的PCR之间的差异，以确定是否从简化Chelex萃取和盐析法协议的标准有统计学显着性。 <img alt="图4" src="/files/ftp_upload/3281/3281fig4.jpg" /> <stroNG&gt; Chelex协议简化应用基因分型P.图4。 疟原虫感染的蚊子（中肠资料显示）的多药耐药基因。该BSTN我消化扩增子P. 108位密码子的两侧恶性疟原虫 DHFR基因12显示cycloguanil耐S108T突变体的的蚊子样本（M1-M5，中肠的数据显示）。 U，未消化的522 bp的扩增产物; FCR3，实验室标准P.恶性疟原虫阳性对照克隆的S108T; K1，P.恶性疟原虫实验室标准克隆阴性对照携带cycloguanil敏感S108N L，100 bp的DNA梯状条带。

Watch this Scientific Journal Video about A Simple Chelex Protocol for DNA Extraction from Anopheles spp. at JoVE.com

用于从按蚊属中提取 DNA 的简单 Chelex 方案。 |免疫学和感染|视频

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

一种快速，经济实惠的方式来提取质量疟疾寄生虫的蚊子标本与载体DNA的方法。利用Chelex树脂螯合性能，简单的方法使基因分型疟疾寄生虫的蚊子中肠和唾液腺阶段，以及分子鉴定<em&gt;按蚊</em近缘种PCR。

一个简单的的Chelex协议对DNA的提取<em&gt;按蚊</em&gt;属。

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

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A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

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54.7K 次观看.  Malaria Institute at Macha. 一种快速，经济实惠的方式来提取质量疟疾寄生虫的蚊子标本与载体DNA的方法。利用Chelex树脂螯合性能，简单的方法使基因分型疟疾寄生虫的蚊子中肠和唾液腺阶段，以及分子鉴定按蚊

视频: A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens1, 2. The innate immune system of mosquitoes consists of several lines of defense 3. Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut 4 enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens 5, 6. Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages 7. The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells. 
Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis 8, and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen 9, 10. These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively 11. More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues 11. While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A simplified yet accurate method to collect and stain mosquito hemocytes is described. Our method combines the simplicity of perfusion with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes. This method facilitates studies requiring knowledge of the types of hemocytes and their abundance.

从黄热病的蚊子的血细胞的收集和染色的议定书埃及伊蚊</em

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

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免疫与感染

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation

Hens can be immunized by means of i.m. vaccination (Musculus pectoralis, left and right, injection volume 0.5-1.0 ml) or by means of Gene-Gun plasmid-immunization. Dependent on the immunogenicity of the antigen, high antibody-titres (up to 1:100,000 - 1:1,000,000) can be achieved after only one or 3 - 4 boost immunizations. Normally, a hen lays eggs continuously for about 72 weeks, thereafter the laying capacity decreases. This protocol describes the extraction of total IgY from egg yolk by means of a precipitation procedure (PEG. Polson et al. 1980). The method involves two important steps. The first one is the removal of lipids and the second is the precipitation of total IgY from the supernatant of step one. After dialysis against a buffer (normally PBS) the IgY-extract can be stored at -20&deg;C for more than a year. The purity of the extract is around 80 %, the total IgY per egg varies from 40-80 mg, dependent on the age of the laying hen. The total IgY content increases with the age of the hen from around 40 mg/egg up to 80 mg/egg (concerning PEG precipitation). The laying capacity of a hen per year is around 325 eggs. That means a total potential harvest of 20 g total IgY/year based on a mean IgY content of 60 mg total IgY/egg (see Table 1).

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation

This protocol describes in particular the extraction of total IgY from egg yolk by means of polyethylene glycol precipitation and gives general information about IgY technology.

IgY的技术:从蛋黄鸡抗体提取聚乙二醇（PEG）降水量

Hens can be immunized by means of  i.m. vaccination (Musculus pectoralis, left and right, injection volume  0.5-1.0 ml) or by means of Gene-Gun ...

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important plant parasitic nematodes. The microscopic second-stage juvenile (J2), molted once in the egg, is the infective stage. The J2s hatch from the eggs, move freely in the soil within a film of water, and locate root tips of suitable plant species. After penetrating the plant root, they migrate towards the vascular cylinder where they establish a feeding site and initiate feeding using their stylets. The multicellular feeding site is comprised of several enlarged multinuclear cells called 'giant cells' which are formed from cells that underwent karyokinesis (repeated mitosis) without cytokinesis. Neighboring pericycle cells divide and enlarge in size giving rise to a typical gall or root knot, the characteristic symptom of root-knot nematode infection. Once feeding is initiated, J2s become sedentary and undergo three additional molts to become adults. The adult female lays 150-250 eggs in a gelatinous matrix on or below the surface of the root. From the eggs new infective J2s hatch and start a new cycle. The root-knot nematode life cycle is completed in 4-6 weeks at 26-28&deg;C.
Here we present the traditional protocol to infect plants, grown in pots, with root-knot nematodes and two methods for high-throughput assays. The first high-throughput method is used for plants with small seeds such as tomato while the second is for plants with large seeds such as cowpea and common bean. Large seeds support extended seedling growth with minimal nutrient supplement. The first high throughput assay utilizes seedlings grown in sand in trays while in the second assay plants are grown in pouches in the absence of soil. The seedling growth pouch is made of a 15.5 x 12.5cm paper wick, folded at the top to form a 2-cm-deep trough in which the seed or seedling is placed. The paper wick is contained inside a transparent plastic pouch. These growth pouches allow direct observation of nematode infection symptoms, galling of roots and egg mass production, under the surface of a transparent pouch. Both methods allow the use of the screened plants, after phenotyping, for crossing or seed production. An additional advantage of the use of growth pouches is the small space requirement because pouches are stored in plastic hanging folders arranged in racks.

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Two distinct methods to screen plants with root-knot nematodes are described. The described approaches include high-throughput screens with nematodes in a nondestructive manner facilitating the use of these plants in breeding programs.

根结线虫的高和低通量筛选根结线虫。</em

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important ...

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules 1. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes 2 and restrict their environment to a more favorable low oxygen pressure 3. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.
Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria 4,5. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor 6. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)7,8 has also been associated with severe disease 9.
One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps 10. Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM 11. In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian 12 and Mozambican patients 13, (although not in Malian 14).
With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay 15. Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

一个简单的协议介导的血小板聚集<em&gt;恶性疟原虫</em&gt;受感染的红细胞在一个资源匮乏的设置

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

总蛋白提取和二维凝胶电泳方法<em&gt;鼻疽</em&gt;物种

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

一个协议，用于分析丙型肝炎病毒复制

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

一种简单快速议定书非酶离解新鲜人组织的浸润淋巴细胞的分析

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

从人类腭扁桃体B和T淋巴细胞有效隔离协议

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

一种简单有效的方法构建突变牛痘病毒载体

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.