This research was funded by Doris Duke Charitable Foundation, Burroughs Wellcome Fund, NIH R21DK080994, Duke Chancellor's pilot project fund, the Roche Foundation for Anemia Research and The Bill and Melinda Gates Foundation. G.L. is supported by Duke's UPGG and the NIH (Grant # 5R01AI090141-03) and K.A.W. by the NSF Graduate Research Fellowship Program.

1：隔离从P.小型的RNA 疟原虫在IDC <ol><li>获得疟原虫无性文化29。 注：所需要的培养规模将有所不同根据所需的应用;然而，10毫升培养在3-5％寄生虫血症和5％的血细胞比容提供了充足的RNA进行实时PCR（RT-PCR）。该技术最初是为异步培养物进行了优化，但是当感染周期中的特定时间点需要的话，环阶段寄生虫可以通过山梨醇同步不迟超过10-12小时入侵后同步。同步应该一个周期（约48小时）29后重复。 </li><li>池感染细胞一起（〜5×10 9的RBCs在3-5％的寄生虫血症）和填充冷PBS到管和离心机的顶部，在800×g离心5分钟，无制动以沉淀红细胞。 </li><li>离心后，小心地用吸吸管除去上清液，附到真空中，由于红细胞​​粒料很容易去除。用冷的1×PBS洗涤沉淀一次。 </li><li>在寒冷的0.15％皂苷悬浮细胞沉淀（以1×PBS中），并置于冰上30分钟。 </li><li>离心机的细胞在1500星光12分钟在4°C，除去冷PBS上清（这将是暗红色），并重新悬浮颗粒。再一次重复此步骤。 注意：为了确保存在的微RNA是反射寄生虫内微RNA，而不是红细胞污染，一些寄生虫分离条件进行了测试：1）皂苷裂解，2）皂苷裂解结合RNA酶A处理的宿主细胞的miRNA和3 ）甲基-β-环糊精处理，除去从主机红细胞的寄生虫。所有的处理，得到类似的结果。 </li><li>裂解使用推荐600微升裂解缓冲液分离的寄生虫沉淀。的裂解缓冲液体积这是足够的培养物高达约30 ml的在2％的血细胞比容和5％寄生虫血症。 注：对于较大的寄生虫培养物，该体积能够增加。以确保寄生虫粒料的完全裂解通过彻底涡旋至少1分钟是重要的。此外，为便于提取，裂解的样品可以被转移到一个1.7 ml离心管。 </li><li>添加匀浆添加剂（醋酸酯2钠，pH 4）的60微升，或1/10体积在步骤6中使用的裂解缓冲液，。 </li><li>离开样品在冰上10分钟。 </li><li>添加600微升，或一个体积等于裂解缓冲液，在步骤6中加入酸苯酚choloroform，向每个样品并涡旋1分钟的量。 </li><li>通过离心样品以10,000×g离心5分钟分离水相和有机相。此自旋长于由该试剂盒建议，以确保所有的血红蛋白/疟原虫色素是在有机相中。 </li><li>收集上层（水溶液层），并将其传输至新管。重复步骤9和10，如果水相为白色或（更可能）褐色，这表明亲蛋白质污染。 </li><li>通过移液管确定得到的上清液的体积，然后添加1.25体积的100％乙醇（〜800微升），并通过反相1.7 ml离心管5次混合。 </li><li>穿过一个设置RNA结合滤筒各样品（若干不同，其中是市售的）通过离心在14,000rpm xg离心1分钟或通过使用真空歧管中。 </li><li>洗滤筒700微升所述miRNA的洗涤缓冲液1使用微量，以14000×g离心纺丝1分钟。 </li><li>通过在14000 XG离心每1分钟两次清洗过滤器滤芯500微升洗涤液的2/3。 </li><li>洗脱的RNA用100μlDEPC水，这已被预热到95°C下，在50μl的洗涤两次，以14000×g离心离心每1分钟。测量的RNA通过UV吸光度在260nm处的浓度。 注：一种RNA凝胶（或TBE - 尿素丙烯酰胺或变性甲醛琼脂糖凝胶）可以bë用于评估所述分离的RNA的尺寸分布。 注：这种RNA是适合于通过微阵列，RNA测序，Northern印迹和核糖核酸酶保护测定分析。 </li></ol> 2：定量从P.小型的RNA 疟原虫在IDC <ol><li>确定分离在步骤1使用紫外分光光度法在260nm处个别样本的浓度。 </li><li>生成的实时PCR引物为所需的RNA种类。对于单独的miRNA，我们使用了预制miRNA的qPCR实验，而mRNA的或融合的miRNA表达我们设计引物进行SYBR绿色。聚变的RNA，正向引物是miRNA序列本身（的miR-451：AAACCGTTACCATTACTGAGTT），而反向是基因特异性引物〜100bp的所述miRNA的下游中的mRNA序列（PKA-R：ATCGAACATGCTGTGAACAT）。 </li><li>使cDNA的使用RNA样品或者发夹结构引物的miRNA（使用20-40纳克的RNA）单独或随机六聚体为mRNA和/或miRNA-mRNA融合物种（使用1微克的RNA）。混合RNA和引物5分钟，在65℃，然后冷却至室温。之后，添加逆转录酶混合物（0.5微升反转录酶，0.5μl的RNase抑制剂，1微升的dNTPs（各2.5mM），4μl的5×缓冲液，2微升0.1M DTT，1微升H 2 O）。 <ol><li>运行样品上能够检测或者SYBR绿色或TaqMan探针热循环。 SYBR绿定量PCR 30，用于特定的基因被运行作为3步PCR，95℃15秒，最低引物退火温度零下5℃，30秒，和68℃，30秒，40个循环，使用商业SYBR Green母液。 miRNA的实时PCR被运行作为二步PCR，95℃15秒和60℃1分钟，进行40个循环。 </li><li>量化使用ΔΔCT方法分析和规范他们对任何饶GTP酶（PF08_0110）或18S rRNA的31。 </li></ol></li></ol> 3：前言小RNA进入疟原虫<ol><li>沉淀300微升每转红血细胞（250微升红细胞，约1.5×10 9细胞，最终将被使用）在完整的疟疾介质离心，在800×g离心5分钟，制动器1。 </li><li>用RPMI重悬在完全cytomix（120毫米氯化钾洗两次红细胞; 0.15毫米氯化钙2; 10mM的K 2 HPO 4 / KH 2 PO 4，pH为7.6; 25毫米的HEPES，pH值7.6; 2mM的EGTA，pH值7.6; 5mM的氯化镁 ; pH值用KOH在800 xg离心在制动器1离心5分钟后调整），在50％的血细胞比容。 </li><li>转移细胞到0.2厘米电穿孔杯中。 </li><li>添加的定制合成的寡核苷酸到反应杯10微克，或适量，并悬浮培养物。 </li><li>调整电穿孔至310 V / 950μF，并提供一个脉冲，每个试管中。 </li><li>重悬在300微升红细胞的比色皿内预热完成MAL咏叹调 ​​媒体和板块它们在24孔板和在密闭容器与疟疾血液混合气体在37℃（3％O 2，5％的CO 2）。 </li><li> 4小时后，感染转染的红细胞与同步晚期滋养体至1％的近似最终的寄生虫血症。 </li><li>加入新鲜红细胞转每4-6天，感染的培养，并确定使用的YoYo-1染色％原虫通过流式细胞仪，如第4所示。 </li></ol> 4：测定红细胞的microRNA效应在寄生虫感染率<ol><li>取100微升重悬的文化通过微量移液器和地点在1.7 ml离心管。用1ml 1×PBS中并离心在台式微量离心机离心800×g离心5分钟洗涤两次。 </li><li>重新悬浮颗粒在1毫升0.025％戊二醛。孵育样品20分钟，在室温。 注：样品在这里可以贮存数周，在4℃。此后，沉淀感染ř离心在800×g离心5分钟的BC。用1ml的1×PBS洗两次。 </li><li>重新悬浮颗粒在1毫升0.015％，皂甙。孵育在4℃下15分钟。粒料感染RBC离心800×g离心5分钟。用1ml的1×PBS洗两次。 Repeat.4.3。重悬细胞1ml含1微升（1,000X）的DNA染色的1×PBS。 </li><li>确定寄生虫血症的程度上的流式细胞仪，在488nm激发和〜530纳米（FL-1）发射。 </li><li>第一，栅极血细胞计数基于FSC和SSC流集中在红细胞。然后，通过荧光在FL-1通道测量门控RBC度原虫的（感染RBC）。 注：后期寄生虫是10倍比早期阶段寄生虫多种荧光，和〜100倍以上的未感染RBC。另外，收集在低速趋于降低噪音。最后，该方法相比已经3 H-次黄嘌呤掺入和Giemsa染色，既如先前报道13，和所有的技术GAVË类似的结果。 </li></ol> 5：的miRNA基因融合产品的生物素标记和洗脱<ol><li>直接订购定制合成的RNA寡核苷酸与脱硫生物素（DB-）共价连接于5&#39;端。 </li><li>染未感染RBC具有脱硫生物素 - 缀合（DB-）miRNA与未修饰的miRNA（阴性对照），如在步骤3中指示。 </li><li>感染红细胞转用P.疟原虫寄生虫至0.5％（步骤1.1）的起始寄生虫血症。 </li><li>提取寄生虫核糖核酸4天（96小时）后，如步骤1所述。 </li><li>孵育10微克的寄生虫的RNA用50μl链霉填充珠，通过微量添加，并在4℃下旋转，在微量离心管中旋转1小时。 </li><li>沉淀小珠，在800×g离心30秒，并用20mM的KCl和1 / 1,000 RNA酶抑制剂。 </li><li>洗脱的RNA从珠通过竞争了2mM生物素和用200μl的RNA捕获洗脱缓冲液（20mM氯化钾，1/1，000酶抑制剂和2mM生物素），在4 ℃过夜轮换。 </li><li>决定指出P的富集程度使用SYBR绿定量RT-PCR和微RNA富集（阳性对照）通过定量PCR荧光定量如步骤2所示疟原虫成绩单。 </li></ol> 6：核糖体分离，以确定P的核糖体占用疟原虫 <ol><li>放置5毫升15％的蔗糖溶液（400毫乙酸钾，25mM的钾的HEPES，pH值7.2，15mM的醋酸镁，200μM的放线菌酮，1mM的DTT，1mM的PMSF，40 U / ml的RNA酶抑制剂，和15％的蔗糖）成SW41超速离心管（14×89毫米）。 </li><li>使用5毫升注射器，加入5毫升50％的蔗糖溶液（400毫乙酸钾，25mM的钾的HEPES，pH值7.2，15mM的醋酸镁，200μM的放线菌酮，1mM的DTT，1mM的PMSF，40 U / ml的核糖核酸酶抑制剂，和50％蔗糖）使用长钢针15％的蔗糖的下面，然后取出针。 </li><lI&gt;用封口膜密封的梯度管的顶部，慢慢倾斜管倒向一侧，在稳定的位置，以防止滚动，因此它在桌面上水平地躺着。保持管在其一侧为至少2小时。在等待时，开始步6.5。 </li><li> 2小时后，缓慢地倾斜管背部直立和管转移到冰，允许它冷却至少15分钟前，样品加载（步骤14）。 </li><li>收集足够的疟原虫 -感染的血液阶段的文化产生100-500微克的总RNA，或大约100毫升的3-5％原虫异步文化。添加1/10含有10×（2毫摩尔）环己酰亚胺（CHX）培养基的该卷和在37℃下进行10分钟。 <ol><li>团培养离心500×g离心7分钟的离心分离机（制动器1），然后用室温1×含200μM的CHX PBS洗涤两次。重悬含200微米CHX寄生虫文化在1X PBS，并储存在冰。 </li></ol></l我&gt; <li>估计红细胞粒料的体积。添加裂解缓冲液（400毫乙酸钾，25mM的钾的HEPES，pH值7.2，15mM的醋酸镁，200μM的CHX，1mM的DTT，1mM的PMSF，40 U / ml的RNA酶抑制剂，1％（体积/体积）IGEPAL CA -630和0.5％（重量/体积）DOC）的红细胞沉淀到4.25 ml的终体积。孵育裂解液4 °下进行10分钟，同时旋转。 </li><li>溶胞产物转移到微量离心管，然后在16000×g离心和4离心样品中微量 °下进行10分钟。 </li><li>吹打1.25的冷的0.5M蔗糖缓冲液（400毫乙酸钾，25mM的钾的HEPES，pH值7.2，15mM的醋酸镁，200μM的放线菌酮，1mM的DTT，1mM的PMSF，40 U / ml的RNA酶毫升制备蔗糖垫抑制剂，和0.5M蔗糖）到SW55超速离心管（13×51毫米）。 </li><li>小心地用注射器和小（〜27）号针头的蔗糖垫层的顶层3.75 ml的裂解物上清液（来自步骤7）的。 STORE中的剩余裂解液（〜500微升）在-80°C至提取总RNA水平，如第1所示。 </li><li>加载样本到超速离心机转子，预冷却至4 °C，可容纳13.2毫升管。离心样品在366000×g离心和4℃146分钟。 </li><li>使用移液管，转移上清液成15mL的锥形管中。存储上清液在-80℃的游离（未结合的核糖体）的RNA的RNA分离。 </li><li>重悬核糖体沉淀在500μl核糖体再悬浮缓冲液（400毫乙酸钾，25mM的钾的HEPES，pH值7.2，15mM的醋酸镁，200μM的放线菌酮，1mM的DTT，1mM的PMSF，和40 U / ml的RNA酶抑制剂）的吹打5分钟。 </li><li>离心机在微量样品在16000×g离心和4 &#160; °下进行10分钟。 </li><li>在保持梯度步骤＃4冰上，除去封口膜，然后小心地层渐变USI顶部的核糖体悬浮液NG注射器和小（〜27）号针头。 </li><li>加载围成一个预冷超速离心机转子和离心加载梯度以200,000×g离心和4℃180分钟。当旋转完成后，储存于4离心梯度 °C，直到准备加载到分馏塔。 </li><li>加载一个空的超速离心管放入梯度精馏塔（这可以是在一个先前的运行中使用的管），然后用无RNA酶的水洗涤5分钟，在100×10的速度。 </li><li>期间在步骤6.16洗涤，置紫外吸收检测器的灵敏度。一个很好的出发灵敏度是0.2，但是这可能在稍后奔跑取决于阿254信号（其变化的基础上的寄生虫数等）来进行调节。 </li><li>一旦检测器灵敏度被设置，设置基线信号至零，同时水流过检测器。 </li><li>洗涤该馏分收集后，反向流体流动，直至线是空的，然后对物奥雅纳空超速离心管。 </li><li>运行通过精馏塔60％的蔗糖溶液，直到它离开针头装置。 </li><li>插入包含​​所述第一梯度的管中，在装载室的顶部，并拧紧封，注意不要过度拧紧。然后，刺穿与针管的底部。 </li><li>重置流体流动速度，以12.5×10（由泵前部的流动速度控制旋钮控制），然后设定自动级分收集器以收集18秒的级分（〜330微升/分）在微量离心管中。 </li><li>启动向前流的60％的蔗糖溶液中。 </li><li>当梯度液的首次下降落入离心管中，立即开始双方馏分收集和A 254信号的现场录音。 </li><li>一旦大幅下跌是在A 254的信号观察，对应50％和60％的蔗糖溶液之间的介面，停止流体流动和A 254两种</sUB&gt;录制。 </li><li>存储梯度分数在-80 &#160; °下以后使用。 </li><li>反向液流在100×10的速度，直到60％的蔗糖溶液清空出超速离心管。 </li><li>如果有附加梯度分馏，从装载室取出空超速离心管，并从步骤6.21重启。如果所有的样品都完成后，清洗设备，在步骤6.16和6.19进行。 </li></ol>

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The authors have no competing financial interests to declare.

哈佛商学院在疟疾流行地区最常见的异常血红蛋白之一，主要是因为它提供保护，防止造成P.重症疟疾疟原虫 。该技术需要在P的基因调控的人类微小RNA的作用表征恶性详述整个手稿。通过提取总RNA以这样的方式，以包括所有的小RNA，并通过执行一个相对简单的寄生虫溶胞步骤，这些融合的RNA能够通过各种独立技术来鉴定。 这些步骤都比较简单，但?...

疟疾，造成疟原虫属顶复门寄生虫，是人类最常见的寄生虫病，感染全球大约200万人，每年造成周围60万人死亡1。感染人类的五个疟原虫物种的，最相关的人类疾病是P.疟原虫和P.间日疟原虫 ，由于严重疟疾并发症其广泛分布和潜力。疟原虫的生命周期既需要蚊子和人类的感染。当受感染的蚊子叮咬人类，寄生虫通过血液循环到达肝脏，在首轮的复制发生移动。后从主机肝裂殖子破裂，它们感染附近的红血细胞，发起或者无性或有性复制。复制的无性阶段，持续48小时在体育疟原虫，是本研究的焦点，因为它是最发作的同时在源咏叹调 ​​症状和容易概括体外 。 虽然一些公共卫生措施，包括改善抗疟疾疗法，已经有所减少全球疟疾负担，抗药性疟原虫的持续出现，呈现为疟疾防治工作中的问题。这可能意味着新的治疗方法的一个领域是研究遗传变异如何不同赋予抵抗疟疾。在疟疾流行的地区，各种红细胞多态性的现象相当普遍2,3。这些突变，镰状细胞是也许是最突出，通常与对有症状疟疾感染4发病实质性抗性相关。由它们引起红细胞抵抗疟疾感染的基本机制是不完全的了解。寄生红细胞与血红蛋白突变，受增强吞噬功能，通过增强细胞的刚性和脱水，这与减少入侵体育相关疟原虫 5。 HBC的等位基因也影响蛋白质表达在红细胞表面和与细胞骨架的重塑，进一步抑制寄生虫发育6,7。最后，P.疟原虫生长不佳内纯合镰刀（HBSS）红细胞8,9 体外 ，提示疟疾电阻的固有红细胞因素。然而，虽然所有这些机制似乎发挥作用，但它们不能完全解释后面疟疾镰状细胞抗性的机制。 一个潜在的一套红细胞因素仍然知之甚少是成熟红细胞中的miRNA存在的大池。微RNA是小的非编码RNA，19-25个核苷酸的大小，其中由3&#39;UTR内的碱基配对介导的翻译和/或靶mRNA的稳定性。他们有牵连的哺乳动物的免疫反应的控制，包括抑制Ò˚F病毒复制10，并分别显示出赋予对病毒在植物中它们也已显示出调节一些红细胞过程，包括红细胞生成11,12和铁代谢13。以前的研究确定了红细胞的miRNA，其表达显着改变的HBSS红细胞14,15的丰富和多样化的人口。由于成熟的红细胞缺乏积极的转录和翻译，这些红细胞miRNA的功能作用尚不清楚。由于显著物质交换的宿主细胞和P之间发生的红细胞内发育周期（IDC）16中疟原虫，有人推测的HbS红细胞内改变的miRNA谱可以直接有助于细胞内在疟疾电阻。 这些研究最终导致管道的发展分离，鉴定和功能上的米内研究人类miRNA的作用alaria寄生虫P.疟原虫，这表明这些宿主/人miRNA的最终共价保险丝然后平移地抑制寄生虫mRNA转录物17。这提供了通过转拼形成的第一跨物种的嵌合转录物的示例，并且暗示，这种miRNA的体mRNA融合物可以在其它物种中，包括其他寄生虫可以发生。所有锥虫的mRNA为反式剪接与拼接领袖（SL）来调节多顺反子转录物18的分离。由于P.疟原虫缺乏直系同源对的Dicer /阿戈19,20，它是可能的红细胞的miRNA劫持在巴斯德类似SL机械恶性融入靶基因。最近在体育研究疟原虫有实际上表示的5&#39;剪接前导序列21的存在。这项研究详细介绍了导致人类寄生虫的miRNA基因融合基因的发现，这些方法包括转录和translational调节技术。这些方法的总体目标是研究的小RNA在P的基因调控，表型和翻译潜在的影响疟原虫成绩单。 人类寄生虫嵌合成绩单初步查明依赖的RNA分析技术，如实时PCR，转录组测序和EST库的采集，其中包括总额和小RNA，而不是用技术只孤立的小RNA使用。分离所有的RNA一起在一个大的池，而不是分开，允许两个易位人类小RNA在寄生虫的标识以及这些小RNA序列的存在作为较大序列的一部分。然后，这需要这些融合mRNA的翻译状态的分析，以确定这些融合的功能性后果。 而在寄生虫的基因组的表征广泛的努力ð转录纷纷加入到寄生虫的生物学22-25的了解，远不如所了解的mRNA的转录组跨越P的生命周期中的翻译调控疟原虫 26。寄生虫的蛋白质组的这种有限的理解已经阻碍了双方的了解寄生虫的生物学，并确定为下一代抗疟疾治疗的新目标的能力。寄生虫的细胞生物学的理解，这仍存在差距主要是由于缺乏足够的技术来研究翻译调控的P.疟原虫 。最近的一篇论文描述了使用P的核糖体足迹的恶性确定全球翻译状态21。一个公认的转录的平移电位测定是由多核糖仿形测定相关的核糖体的数目。然而，当这种技术应用到P.疟原虫&lt;/ em&gt;的，它是无法收回大部分多核糖体和捕获主要monosomes。近日，几组27,28优化了P.疟原虫多核糖体的技术，通过同时裂解红细胞和寄生虫保存多核糖体和它们在宿主红细胞28无性发育过程中表征这些疟原虫的核糖体占用和翻译潜能。 总的来说，这些方法证明人类miRNA与寄生虫mRNAs的所观察到的融合调制那些融合的mRNA，其使用先前报道的方法27表明的寄生虫蛋白质翻译，并且是在疟疾的HBA阻力的主要决定因素和HBSS红细胞17。这些方法将在任何系统，查找以确定和功能上探索RNA剪接事件是有用的，这些融合的RNA是否在第疟原虫或其他真核系统。

<table border="0" cellpadding="0" cellspacing="0" width="651">
	<tbody>
		<tr height="28">
			<td height="28" style="height:28px;width:651px;">REAGENTS-All reagents must be RNAse-free.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Diethyl pyrocarbonate (DEPC; Sigma, cat. no. D5758)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DEPC-treated water (see REAGENT SETUP)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Yoyo-1 DNA fluorescent dye (Thermo Fisher)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gene Pulser II (or comparible) electroporator (Bio-Rad)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.2 cm Electroporation cuvettes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4 M potassium acetate (Mallinckrodt, cat. no. 6700)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2 M potassium HEPES (pH 7.2; Sigma, cat. no. H0527)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 M magnesium acetate (Sigma, cat. no. M-2545)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 M dithiothreitol (DTT; Research Products International, cat. no. D11000)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100 mM phenylmethylsulfonate fluoride (PMSF; dissolved in isopropanol; Sigma, cat. no. P7626)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10% (v/v) Igepal CA-630 (Sigma-Aldrich, cat. no. I3021)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10% (w/v) sodium deoxycholate (DOC; Sigma, cat. no. D-6750)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cycloheximide (CHX; Sigma, cat. no. C-7698)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose (Mallinckrodt, cat. no. 8360-04)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNAseOUT (Invitrogen, cat. no. 100000840)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI-1640 w/ L-glutamine (Cellgro, cat. no. 10-040-CV)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10% AlbuMAX I (w/v in sterile water) (Invitrogen, cat. no. 11020-039)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gentamicin (Gibco, cat. no. 15750-060))</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HT supplement (100x) (Invitrogen, cat. no. 11067030)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">45% (w/v) sucrose (Sigma, cat no. G8769)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 M HEPES buffer (Gibco, cat. no. 15630-080)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1x phosphate buffered saline (PBS; Cellgro, cat. no. 2109310CV)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Corning 500 ml vacuum filter flask (VWR, cat. no. 430769)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass slides (VWR, cat. no. 48311-702)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Giemsa stain (50X) (VWR, cat no. m708-01)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mirVana miRNA isolation kit (Ambion, ThermoFisher Scientific).</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5&#39; Biotin conjugated mature miRNA (sequence varies by miRNA of interest) (Dharmacon)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biotin powder (Sigma-Aldrich)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1M Potassium Chloride</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptavidin Sepharose High Performance Beads (GE Healthcare)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ribosome resuspension buffer (see REAGENT SETUP)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lysis buffer (see REAGENT SETUP)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15% (w/v) sucrose gradient solution (see REAGENT SETUP)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50% (w/v) sucrose gradient solution (see REAGENT SETUP)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5 M sucrose cushion (see REAGENT SETUP)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">60% (w/v) sucrose chase solution (see REAGENT SETUP)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plasmodium cell culture media (see REAGENT SETUP)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA capture Wash Buffer (see REAGENT SETUP)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA Elution Buffer (see REAGENT SETUP)</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;">REAGENT SETUP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Solutions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plasmodium cell culture media</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">500 ml RPMI-1640 w/ L-glutamine</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5 ml gentamicin</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5 ml HT supplement</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2.5 ml 45% glucose</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">25 ml 10% AlbuMAX I</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">12.5 ml 1 M HEPES</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile filter with 0.2 mm vacuum filter flask.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plasmodium cell culture media containing 10x CHX</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Same recipe as Plasmodium cell culture media above, with the addition of:</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2 mM cycloheximide</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Media is prepared as normal, then add CHX to a final concentration of 2 mM and filter.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1x PBS containing cycloheximide</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Cycloheximide is added fresh to 1x PBS to a final concentration of 200 mM, and kept at 4 OC until use.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ribosome resuspension buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">400 mM potassium acetate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">25 mM potassium HEPES, pH 7.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 mM magnesium acetate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">200 mM cycloheximide (add fresh)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 mM DTT (add fresh)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 mM PMSF (add fresh)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">40 U/ml RNaseOUT (add fresh)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lysis buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Same recipe as ribosome resuspension buffer above, with the addition of:</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1% (v/v) Igepal CA-630</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5% (w/v) DOC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose solutions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Same recipe as ribosome resuspension buffer above, with the addition of varying amounts of sucrose:</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15% (w/v) sucrose to make 15% sucrose gradient solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50% (w/v) sucrose to make 50% sucrose gradient solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5 M sucrose to make 0.5 M sucrose cushion</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">As above, cycloheximide, DTT, PMSF, and RNaseOUT must be added fresh.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">The 60% sucrose chase solution requires only sucrose and water, no other components</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">60% (w/v) sucrose in DEPC-treated water to make 60% sucrose chase solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA Capture Wash Buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">20mM&#160; KCl</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5unit/ml Rnase Out (Invitrogen)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA Capture Elution Buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Same recipe as RNA Capture Wash Buffer, with the addition of:</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2mM Biotin</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;">EQUIPMENT</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SW55 Ti ultracentrifuge rotor (Beckman, cat. no. 342194)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SW41 Ti ultracentrifuge rotor (Beckman, cat. no. 331362)</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Ultra-ClearTM &#189; x 2 in (13 x 51 mm) ultracentrifuge tubes for the SW55 (Beckman, cat. no. 344057)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyallomer 9/16 x 3 &#189; in (14 x 89 mm) ultracentrifuge tubes for the SW41 (Beckman, cat. no. 331372)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L8-80M ultracentrifuge (Beckman)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Steel blunt syringe needle, 4 inch, 16G (Aldrich, cat. no. Z261378)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 ml syringe with 27G&#189; needle (Becton Dickinson, cat no. 309623)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5 ml syringe (Becton Dickinson, cat. no. 309646)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Parafilm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tube rotator, end-over-end</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microcentrifuge, refrigerated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Density gradient fractionation system (Teledyne Isco, cat. no. 69-3873-179)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TracerDAQ (Measurement Computing)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eppendorf 5810R centrifuge</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eppendorf A-4-81 rotor (Eppendorf, cat. no. 022638807)</td>
		</tr>
	</tbody>
</table>

methods to investigate the regulatory role of small rnas and ribosomal occupancy of plasmodium falciparum

遗传变异负责镰状细胞等位基因（HBS）使红细胞的疟原虫，P.抗感染疟原虫 。这个阻力，这是众所周知的是多因素的分子基础，仍然不完全理解。最近的研究发现，红细胞微RNA，一旦转移进入疟原虫的差异表达，同时影响基因调控和寄生虫生长。这些miRNA后来所示，通过5&#39;RNA融合寄生虫mRNA的谨慎的子集形成了嵌合体RNA转录抑制mRNA的翻译。在这里，中使用的技术来研究基因调控和P的平移潜在的功能作用和假定机制底层红细胞的microRNA 疟原虫，包括改性合成微小RNA转染到宿主红细胞，进行详细说明。最后，一​​个多核糖梯度法是用来确定TRA的程度nslation这些成绩单。一起，这些技术使我们能够证明红细胞微RNA的失调水平有助于镰状红细胞的细胞内在疟疾电阻。

人类的microRNA在P的全球分析疟原虫 这里介绍的技术被用来提取寄生虫微小RNA在多种条件。有一点要注意的是，在32，这往往充当了两个本文以及原研29提出的微小RNA数据的参考点指示进行RNA提取的未感染的红细胞。这些以往的研究也表明，HBSS红细胞具有一定的miRNA中，miR-451特别是更大的水平。上面所指出的技术足以证明在哈佛商学院（HBA卡或HBSS?...

Watch this Scientific Journal Video about Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum at JoVE.com

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts.

方法调查小RNA和核糖体占用的调控作用<em&gt;恶性疟原虫</em

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...

methods-to-investigate-regulatory-role-small-rnas-ribosomal-occupancy

Research

JoVE Journal

Immunology and Infection

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

8.8K 次观看.  Duke University School of Medicine. Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts. 

视频: Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

选择恶性疟原虫</emCytoadhesion>寄生虫对人脑血管内皮细胞

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

科研

免疫与感染

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

寄生蜂简介<em&gt;果蝇</em&gt;和Antiparasite的免疫反应的

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

一个<em&gt;在体外</em&gt;共感染模型研究<em&gt;恶性疟原虫</em人原发性单核细胞源性的免疫细胞-HIV-1相互作用

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most 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 ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

脑幽静的白细胞的分离与分析<em&gt;伯氏疟原虫</emANKA感染的小鼠

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the &asymp;40% limit, the investigator usually keeps the percentage at around 10%. In many cases it is necessary to isolate the parasite-containing red blood cells (RBCs) from the uninfected ones, to enrich the culture and proceed with a given experiment. 
When P. falciparum infects the erythrocyte, the parasite degrades and feeds from haemoglobin 2, 3. However, the parasite must deal with a very toxic iron-containing haem moiety 4, 5. The parasite eludes its toxicity by transforming the haem into an inert crystal polymer called haemozoin 6, 7. This iron-containing molecule is stored in its food vacuole and the metal in it has an oxidative state which differs from the one in haem 8. The ferric state of iron in the haemozoin confers on it a paramagnetic property absent in uninfected erythrocytes. As the invading parasite reaches maturity, the content of haemozoin also increases 9, which bestows even more paramagnetism on the latest stages of P. falciparum inside the erythrocyte.
Based on this paramagnetic property, the latest stages of P. falciparum infected-red blood cells can be separated by passing the culture through a column containing magnetic beads. These beads become magnetic when the columns containing them are placed on a magnet holder. Infected RBCs, due to their paramagnetism, will then be trapped inside the column, while the flow-through will contain, for the most part, uninfected erythrocytes and those containing early stages of the parasite.
Here, we describe the methodology to enrich the population of late stage parasites with magnetic columns, which maintains good parasite viability 10. After performing this procedure, the unattached culture can be returned to an incubator to allow the remaining parasites to continue growing.

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

The paramagnetic properties of hemozoin are used to isolate late stages of Plasmodium falciparum-infected red blood cells growing in culture. The method is simple and fast and does not affect the subsequent invasive capabilities of the parasites.

分离<em&gt;恶性疟原虫</em&gt;晚期感染的红细胞由磁性装置

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

高产净化<em&gt;恶性疟原虫</em&gt;裂殖子适用于Opsonizing抗体检测

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

基因分型<em&gt;金黄色葡萄球菌</em&gt;由核糖体垫片的PCR（RS-PCR）

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct a tree to show the evolutionary relationship among different taxa (classification units) at the molecular level. Phylogenetic analysis can also be used to investigate domain relationships within an individual taxon, particularly for organisms that have undergone substantial change in morphology and physiology, but for which researchers lack fossil evidence due to the organisms&#39; long evolutionary history or scarcity of fossilization.
In this text, a detailed protocol is described for using the phylogenetic method, including amino acid sequence alignment using Clustal Omega, and subsequent phylogenetic tree construction using both Maximum Likelihood (ML) of Molecular Evolutionary Genetics Analysis (MEGA) and Bayesian Inference via MrBayes. To investigate the origin of eukaryotic Sugars Will Eventually be Exported Transporters (SWEET) genes, 228 SWEETs including 35 SWEET proteins from unicellular eukaryotes and 57 SemiSWEET proteins from prokaryotes were analyzed. Interestingly, SemiSWEETs were found in prokaryotes, but SWEETs were found in eukaryotes. Two phylogenetic trees constructed using theoretically distinct methods have consistently&#160;suggested that the first eukaryotic SWEET gene might stem from the fusion of a bacterial SemiSWEET gene and an archaeal SemiSWEET gene.&#160;It is worth noting that one should be cautious to draw a conclusion based only on phylogenetic analysis, although it is useful to explain the underlying relationship between different taxa, which is difficult or even impossible to discern through experimental means.

A method of constructing a phylogenetic tree based on sequence homology of SWEETs from eukaryotes and SemiSWEETs from prokaryotes is described. Phylogenetic analysis is a useful tool for explaining the evolutionary relatedness between homologous proteins or genes from different organism groups.

应用系统进化分析研究真核基因的起源

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct ...

Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to eradicate tumors. However, its application is limited by the development of graft-versus-host disease (GVHD), a major complication of BMT. GVHD is evoked when T-cells in the donor grafts recognizealloantigen expressed by recipient cells and mount unwanted immunological attacks against recipient healthy tissues. Thus, traditional therapies are designed to suppress donor T-cell alloreactivity. However, these approaches substantially impair the GVL effect so that the recipient&#39;s survival is not improved. Understanding the effects of therapeutic approaches on BMT, GVL, and GVHD, is thus essential. Due to the antigen-presenting and cytokine-secreting capacities to stimulate donor T-cells, recipient dendritic cells (DCs) play a significant role in the induction of GVHD. Therefore, targeting recipient DCs becomes a potential approach for controlling GVHD. This work provides a description of a novel BMT platform to investigate how host DCs regulate GVH and GVL responses after transplantation. Also presented is an effective BMT model to study the biology of GVHD and GVL after transplantation.

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation. Dendritic cells play a critical role in the pathogenesis of graft-versus-host disease. The current article describes a novel bone marrow transplantation platform to investigate the role of dendritic cells in the development of graft-versus-host disease and the graft-versus-leukemia effect.

骨髓移植平台研究树突状细胞在移植与宿主疾病中的作用

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to ...