我们感谢艾利森理查德博士，前任董事荧光配体的合成，对生物技术研究Interdisciplinaray中心，用友为配体催化肽合成，和Socolovsky提供荧光显微镜显微镜设施LSU肽设施。经费是由美国国防部SERDP探索发展计划（SEED），能源和环境保护局，生物AgCenter跨学科的团队计划和路易斯安那州。

实验1：提取白蚁肠道原虫在厌氧条件下<ol><li>在手套箱使用风扇框（腼腆实验室）不断通过干燥剂和D型催化剂Stak教材套分发的空气来控制湿度和氧气含量和消除不均匀的温度。手套箱填写的氮为20至30分钟的连续流。氧含量监测氧传感器1小时（C -平方，公司）用氮气以达到和保持无氧条件下，在需要的时候。 </li><li>准备Trager ü媒体（Trager 1934年），并调整pH至7.0。喷雾用2.5％的氢，5％的二氧化碳和92.5％的氮气1 h，以除去氧气残留的混合物在手套箱的过滤消毒媒体。 </li><li> Silanize所有材料，包括显微镜载玻片，离心管，吸液管，玻璃器皿等使用Sigmacote的实验中使用，以防止吸附原生动物或表面的肽（Sigmacote，适马，＃SL - 2，http://www.sigmaaldrich.com/等/了mediaLib / DOCS / Sigma/Product_Information_Sheet/1/sl2pis.Par.0001.File.tmp/sl2pis.pdf）。 </li><li>由于白蚁肠道原虫是严格厌氧的生物，他们绝不能暴露于氧气。因此，以下步骤是在手套箱（见1.1）的厌氧条件下进行。随着钳淹没在70％的乙醇，轻轻地约10秒的漩涡，以除去表面的污染物白蚁工人的整个身体。 </li><li>工人从乙醇中取出，并允许干就清理了约20秒Kimwipe使用无菌的细尖镊子举行工人的腹部和另一双钳抓取腹部的一角，轻轻一拉成45度角向上或向下的肠道。如果在一条直线的角度和用力过猛拉肠是很容易掰开。将在100μLTrager ü载玻片媒体下降10胆量。 </li><li>皮尔斯与无菌精细解剖探头对释放的胆量原生动物和轻轻地用200μL移液器转移成1毫升的离心管中含有900μLTrager ü媒体肠道内容。允许肠壁片段（5秒），转移900μL上清到一个新的管沉淀后。 </li><li>转移10μL原生动物文化sigmacoted显微镜幻灯片和检查的条件显微镜下放大200 ×原生动物。 </li><li>准备有氧原生动物四膜虫 ， 变形虫 SP， 眼虫藻， 草履虫 SP的控制文化。 （北卡罗来纳州生物供应公司，伯灵顿，数控），以及一个由供应商推荐的培养基中的大肠杆菌过夜培养。 </li></ol>实验2：添加配体与荧光染料，原生动物和细菌培养测试表面的细胞膜和细胞壁结合我们以前使用噬菌体展示库（New England Biolabs公司公司，伊普斯维奇，马），以确定绑定到原虫（协议http://www.neb.com/nebecomm/ManualFiles/manualE8110.pdf提供）19七肽序列。一个与肽序列（ALNLTLH）显示，从锥虫布氏膜公认的糖蛋白相似的配体合成并加上C -末端的荧光探针（EDANS，5 - （（2 -氨基乙基）氨基）萘1 -磺酸酸的λmax= 341 nm处，λem= 471 nm处）通过固态肽合成中使用EDANS NovaTag的树脂（EMD Biosciences公司）（SSPS）。在这里，我们展示的配体结合的原生动物从白蚁肠道和其他自由生活的原生动物，而不是细菌分离。 <ol><li>文化的原生动物中所述地契。 1。原虫12小时修复10％的甲醛在4 ° C </li><li>离心原生动物的解决方案（30 XG，10分钟），弃去上清液，并包含固定原虫1毫升Trager ü媒体两次洗颗粒。重新悬浮颗粒在1毫升Trager ü媒体。此外，修复其他原生动物和 E。控制大肠杆菌的细菌。 </li><li>孵育1-2 h合成的配体的解决方案与荧光染料EDANS在终浓度为50微米（水配制）耦合固定微生物。的配位溶解于水比Trager ü媒体。 </li><li> 400 X放大倍率在341 nm处的吸光度最大，在471纳米的蓝色区域排放在荧光显微镜下观察微生物。 </li></ol>实验3：测试protozoacidal配体的活动， 再加上在体外裂解肽（原生动物文化） 一个共轭配体和裂解肽赫卡特（Mutwiri 等，2000）曾在路易斯安那州立大学蛋白质设施的合成。 <ol><li> Silanize材料，并准备在无氧的手套箱一个白蚁肠道原虫文化小号在进出口描述。 1。 </li><li>准备需氧控制微生物（如大肠杆菌和自由生活的原生动物物种T.膜虫 ）文化。 </li><li>在手套箱，吸管6 198原生动物文化微升到0.5毫升的Eppendorf管分装的厌氧环境。加入2μL的配体催化肽100μmol解决方案白蚁肠道原虫文化等分的一半（最终浓度为1微米）。加入2μL水的等分的另一半（对照组）。 </li><li>在台上，准备E.类似等分大肠杆菌和T.膜虫与1微米裂解肽，配体催化肽或水。 </li><li> 1小时后，转移10μL每个原生动物文化的幻灯片。比较显微镜下放大200 ×控制原虫治疗生存。 </li><li> 1小时后，板约100μL。 10-4稀释的E. BHI培养大肠杆菌培养于37℃过夜。比较板块上的菌落形成单位的数量。 </li></ol>实验4：配体注射到白蚁后肠中加上一个荧光染料<ol><li>拉针（GD - 1型，1 × 900毫米），采用了双级耐热等级（65和48），获得一个针尖大小为20-30微米的一个Narishige PC - 10玻璃微管拉拔。确认使用千分尺的显微镜下，通过测量笔尖的大小。 </li><li>与CA填充一针。 30μL荧光标记的配体50微米的悬浮于水中，使用附加的注射器。另一针装满水的控制。将一个微量（0.5μL能力和32毫米的长度，德拉蒙德科学公司）在显微持有人。将针在第二显微注射系统持有人。设置初始注入参数约1秒脉冲长度和10-12 PSI注射。进入微针慢慢推进，注入的解决方案，使用踏板驱动的高速电子喷射系统连接到一个控制氮气流量，从而保证一个恒定的量是可重复注射用脉冲长度控制。注射后，取出微量，并记录注射使用游标卡尺的解决方案的长度。计算从已知参数的微注射量。调整压力的氮气和驱逐0.3μL的解决方案，在单次注射的脉冲长度。 </li><li>挤压与软钳白蚁工人腹部，直肠，以消除任何排泄物目前的结束。固定白蚁工人心寒冰浴5分钟。 </li><li> 100μL枪头，用手术刀刀片，切断了白蚁工人的接收器。切尖长度为10 - 12毫米，根据白蚁的大小和使用。 </li><li>将接收到的显微。放在培养皿上背侧的工人和工人头到接收器的使用氮吸泵吸，所以，从接收的工人伸出总站。 </li><li>手持接收机中的白蚁，仔细提前填写使用的显微针插入工人肛门。注入0.3μL的解决方案（荧光标记的配体或控制的水）。 </li><li>放在单独的培养皿中，配体或水注入湿滤纸的工人和他们保持在26 ± 2 ° C，与78％RH </li><li>从注入白蚁消灭胆量和收集原虫24 h后如上图所示在进出口。 1。修复并观察原虫在进出口。 2。 </li></ol>实验5：测试protozoacidal配体的活动， 再加上在体内的裂解肽（注射到白蚁后肠） <ol><li> Silanize作为进出口描述的材料。 1。 </li><li>准备一个500微米解决方案在水中的配体催化肽。 </li><li>按照步骤3.1），通过3.4）准备玻璃针，接收器和白蚁工人。 </li><li>继在3.5中描述的方法）注入0.3μL的配体催化肽溶液（治疗）或水（对照组）20白蚁工人肠。 </li><li>几个工人举行24小时的白蚁，消灭胆量，并在显微镜下观察肠道内容，如3.6中所述）。和3.7）。 </li><li>只要在白蚁肠道原虫的死亡被证实，其余处理的白蚁和控制，在培养皿中保持湿润滤纸，每天观察死亡率。 </li></ol>代表性的成果： 实验1：通常情况下，前肠，中肠和后肠是在一块时遵循正确的程序（图1a）。原虫肠厌氧部分驻留在高密度，可释放的肠穿孔钳（图1b 1＆2）。最大的原佐阿种在台湾的地下白蚁肠道是纺锤状的体育grassii，这是长200-300微米，宽150微米，可以用肉眼看到。第二个最大的品种是梨状H。 hartmanni（50-140微米长，宽30-80微米）。最小的品种是圆锥状S. leidyi（15-50微米长，宽8-30微米;丽等人 1983年） 。如图2所示的原生动物物种。 最佳培养条件下，从台湾地下白蚁肠道原虫三个孤立的物种将保持健康地活着，至少72 h在厌氧Trager ü媒体（图3a）。然而，如果不是最佳的培养条件原生动物会死快。如果有氧气残留在媒体中，原生动物的运动将立即停止。如果渗透压过高或细胞膜的完整性受到损害原生动物将凸出的细胞破裂（图3b）膜表面。如果渗透压过低或膜受到损害，原生动物将萎缩，萎缩（图3c）。 实验2：我们证实，再加上配体绑定到所有三个品种从台湾地下白蚁后肠中检出密度原生动物的荧光探针。配体结合发生在整个细胞表面（图4）。结合位点都集中在axostyle（表微管）在P.核原生动物前地区grassii。 我们观察到一些修修补补的自体荧光的原生动物摄入的木颗粒。然而，自体荧光通常是便于识别的配体的特异性结合，因为没有从表面上看，axostyle和细胞核（图4）自体荧光。 我们也发现在所有测试的自由生活的有氧原生动物物种（图5），这表明，配体结合结构通用，以原生动物荧光。然而，没有观察到配体结合为E.大肠杆菌 。 实验3：一个配体催化肽微米杀害所有三个从台湾地下白蚁工作者的肠道和自由生活的T.的原生动物物种膜虫在体外 ，在不到10分钟，而对照停留活着。图6显示了膜的完整性与配体催化肽治疗的白蚁肠道原虫逐渐丧失。膜隆起和断裂，原生动物走向枯萎和死亡。在E.数量无明显差异治疗的配体催化肽和水之间的大肠杆菌菌落。然而，没有配体的裂解肽减少大肠杆菌的数量大肠杆菌菌落（图7）。这表明，配体的附着在一定程度上保护非目标微生物，从裂解。 实验4：当0.3μL50μM荧光标记的配体注入白蚁工人肠，结合到 P grassii，S. leidyi和H 。 hartmanni被确认通过荧光显微镜类似进出口。 2（图4）。白蚁肠道组织没有显示出荧光。 实验5：0.3μL500微米配体催化肽注射液的所有三个物种在台湾的地下白蚁肠道原虫在24小时内死亡。白蚁死亡后10天之内失去他们的共生原生动物。此前，Husseneder和Collier（2009）注入白蚁胆量裂解肽的浓度相同。没有连接的配体，它把更多的时间，直到原虫在肠道内（72小时）和白蚁死亡（6周）。这表明，配体提高效率，最有可能通过有约束力的裂解肽原虫的裂解肽protozoacidal。 <img src="/files/ftp_upload/2190/2190fig1.jpg" alt="图1" /> 图1。答：台湾在一个幻灯片显示肠道的主要部分（前，中，后肠）的地下白蚁肠道; B 1＆2：肠刺穿钳释放含有原虫肠道内容。 <img src="/files/ftp_upload/2190/2190fig2.jpg" alt="图2" /> 图2。种的鞭毛原虫在台湾地下白蚁后肠发现：一）Pseudotrichonympha grassii，B）Holomastigotoides hartmanni，和c）Spirotrichonympha leidyi。 <img src="/files/ftp_upload/2190/2190fig3.jpg" alt="图3" /> 图3。一）健康文化的原生动物，原生动物，B）与膜膨出原生动物，C）干瘪的原生动物。 <img src="/files/ftp_upload/2190/2190fig4.jpg" alt="图4" /> 图4。确认配体结合，再加上白蚁肠道荧光探针原虫（从上到下：P. grassii，H. hartmanni，S. leydi），与荧光标记的配体和未经处理的对照（自体荧光显示）的处理。 <img src="/files/ftp_upload/2190/2190fig5.jpg" alt="图5" /> 图5。配体结合自由生活的有氧原虫，一） 变形虫 ， 四膜虫 ，B）C） 眼虫 ，和D） 草履虫 。 <img src="/files/ftp_upload/2190/2190fig6.jpg" alt="图6" /> 图6。治疗a）水（对照组）和B）1微米的配体催化肽原生动物。 <img src="/files/ftp_upload/2190/2190fig7.jpg" alt="图7" /> 图7。 大肠杆菌大肠杆菌菌落板（10 -4稀释）：）处理与水（对照组），B）治疗1微米配体催化肽，C）1微米裂解肽治疗。

<ol>
	<li>Hansel, W., Leuschner, C., Enright, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjugates+of+lytic+peptides+and+LHRH+or+&#946;CG+target+and+cause+necrosis+of+prostate+cancers+and+metastases.">Conjugates of lytic peptides and LHRH or &#946;CG target and cause necrosis of prostate cancers and metastases.</a> Mol. Cell. Endocrinol. 269, 26-33 (2007).</li><li>Husseneder, C., Collier, R. E., Bourtzis, K., Miller, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paratransgenesis+for+termite+control.">Paratransgenesis for termite control.</a> Insect Symbiosis. 3, 361-376 (2009).</li><li>Husseneder, C., Grace, J. K., Oishi, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+genetically+engineered+bacteria+(Escherichia+coli)+to+monitor+ingestion,+loss+and+transfer+of+bacteria+in+termites.">Use of genetically engineered bacteria (Escherichia coli) to monitor ingestion, loss and transfer of bacteria in termites.</a> Curr. Microbiol. 50, 119-123 (2005).</li><li>Husseneder, C., Grace, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+engineered+termite+gut+bacteria+deliver+and+spread+foreign+genes+in+termite+colonies.">Genetically engineered termite gut bacteria deliver and spread foreign genes in termite colonies.</a> Appl. Microbiol. Biotechnol. 68, 360-367 (2005).</li><li>Javadpour, M. M., Juban, M. M., Lo, W. C., Bishop, S. M., Alberty, J. B., Cowell, S. M., Becker, C. L., Mc Laughlin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+antimicrobial+peptides+with+low+mammalian+cell+toxicity.">De novo antimicrobial peptides with low mammalian cell toxicity.</a> J. Med. Chem. 39, 3107-3113 (1996).</li><li>Lai, P. Y., Tamashiro, M., Fuji, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abundance+and+distribution+of+the+three+species+of+symbiotic+protozoa+in+the+hindgut+of+Coptotermes+formosanus+(Isoptera).">Abundance and distribution of the three species of symbiotic protozoa in the hindgut of Coptotermes formosanus (Isoptera).</a> Proc. Haw. Entomol. Soc. 24, 271-276 (1983).</li><li>Leuschner, C., Hansel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+disrupting+lytic+peptides+for+cancer+treatments.">Membrane disrupting lytic peptides for cancer treatments.</a> Curr. Pharm. Des. 10, 2299-2310 (2004).</li><li>Mutwiri, G. K., Henk, W. G., Enright, F. M., Corbeil, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+the+Antimicrobial+Peptide,+d-Hecate,+on+Trichomonads.">Effect of the Antimicrobial Peptide, d-Hecate, on Trichomonads.</a> J. Parasitol. 86, 1355-1359 (2000).</li><li>Trager, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cultivation+of+a+cellulose-digesting+flagellate,+Trichomonas+termopsidis,+and+of+certain+other+termite+protozoa.">The cultivation of a cellulose-digesting flagellate, Trichomonas termopsidis, and of certain other termite protozoa.</a> Biol. Bull. 66, 182-190 (1934).</li></ol>

配体裂解肽已成功地用于有效地定位和摧毁癌细胞（汉塞尔和Leuschner 2004年， 汉塞尔等2007年） 。基于这种理念，我们开发了一个七肽配体结合的表面，在台湾地下白蚁肠道原虫，再加上它与目标裂解肽摧毁这些预留的纤维素消化白蚁肠道共生，实现白蚁控制（2009年Husseneder和科利尔）。 我们成功地证实的3种在台湾地下白蚁在体外和体内肠道原虫使用荧光...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Sigmacote</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>SL-2</td>
<td>&nbsp;</td>
<tr>
<td>EDANS</td>
<td>&nbsp;</td>
<td>Novabiochem</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Anaerobic glove box</td>
<td>&nbsp;</td>
<td>Coy Laboratories, Inc.</td>
<td>Custom made</td>
<td>&nbsp;</td>
<tr>
<td>Intellus environmental controller</td>
<td>&nbsp;</td>
<td>Percival</td>
<td>I36NL</td>
<td>&nbsp;</td>
<tr>
<td>PC-10 Glass micropipette puller</td>
<td>&nbsp;</td>
<td>Narishige Scientific Instrument Lab</td>
<td>PC-10</td>
<td>&nbsp;</td>
<tr>
<td>Glass needles (Model GD-1, 1 X 900 mm)</td>
<td>&nbsp;</td>
<td>Narishige Scientific Instrument Lab</td>
<td>GD-1</td>
<td>&nbsp;</td>
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<td>Leitz micromanipulators</td>
<td>&nbsp;</td>
<td>Vermont Optechs, Inc.</td>
<td>ACS01</td>
<td>&nbsp;</td>
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<td>Microinjector</td>
<td>&nbsp;</td>
<td>Tritech Research, Inc.</td>
<td>MINJ-1</td>
<td>&nbsp;</td>
<tr>
<td>Microcaps</td>
<td>&nbsp;</td>
<td>Drummond Scientific Company</td>
<td>1-000-0005</td>
<td>&nbsp;</td>
<tr>
<td>LEICA fluorescence imaging system</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>DMRxA2</td>
<td>&nbsp;</td>
<tr>
<td>LEICA dissecting scope</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>MZ16</td>
<td>&nbsp;</td>
<tr>
<td>LEICA microscope</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>DMLB</td>
<td>&nbsp;</td>
<tr>
<td>Olympus dissecting scope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>SZ61</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

testing protozoacidal activity of ligand-lytic peptides against termite gut protozoa in vitro (protozoa culture) and in vivo (microinjection into termite hindgut)

我们正在开发一种新的方法，地下白蚁控制，这将导致减少对化学农药的使用的依赖。地下白蚁是依赖于工人的hindguts原生动物有效地消化木材。裂解肽已被证明杀死多种原虫（Mutwiri 等，2000），并在台湾地下白蚁， 家白蚁 （2009年Husseneder和科利尔）的肠道原虫。裂解肽真核生物的非特异性免疫系统的一部分，并破坏微生物膜（2004年Leuschner和汉塞尔）。大多数裂解肽不太可能损害高等真核生物，因为他们不影响电中性的含胆固醇高等真核生物（Javadpour 等，1996年）的细胞膜。裂解肽行动，才能有针对性地增加一个配体的特定的细胞类型。例如，汉塞尔等人 （2007）报道与癌症细胞膜受体的配体结合的裂解肽可以用来摧毁乳腺癌细胞，而单 ​​独或与非特异性肽结合的裂解肽效果不理想。也有裂解肽结合人体激素对前列腺癌和睾丸癌的细胞（2004年Leuschner和汉塞尔）有针对性的破坏肿瘤细胞的受体结合。 在这篇文章中，我们目前的技术用来证明一个裂解肽，再加一个七肽配体的结合从台湾地下白蚁的肠道原虫膜表面（赫卡特）protozoacidal活动。这些技术包括从白蚁工人的肠道摘除，厌氧培养肠道原虫（Pseudotrichonympha grassii，Holomastigotoides hartmanni Spirotrichonympha leidyi），用荧光染料标记的配体结合白蚁肠道原虫和其他自由生活的原生动物而不是细菌或肠道组织的微观确认。我们也表现出相同的配体，再加上一个裂解肽，有效地杀死白蚁肠道原虫在体外（原生动物文化）和体内（微量注射到工人肠），但低于单独的裂解肽bacteriacidal。在不到两个星期的白蚁死亡原虫导致的损失。 在未来，我们通过一个“特洛伊木马”，以表达配体催化肽，杀白蚁肠道内的原虫，并随后在殖民地杀死白蚁白蚁殖民地生存在白蚁后肠和传播的微生物，可以将基因工程。配体裂解肽，也可用于对原虫的药物发展。

Watch this Scientific Journal Video about Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro Protozoa Culture and in vivo Microinjection into Termite Hindgut

Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro Protozoa Culture and in vivo Microinjection into Termite Hindgut

我们目前用于演示程序，配体结合到膜表面的纤维素消化在使用荧光显微镜和配体与裂解肽台湾地下白蚁肠道原虫杀原虫这些<em&gt;在体外</em&gt;（厌氧原虫文化）<em&gt;在体内</em&gt;（注入白蚁后肠）。

廉政白蚁肠道原生动物催化配体肽的Protozoacidal活性测试在体外（原生动物文化）在体内（显微注射到白蚁后肠）

We are developing a novel approach to subterranean termite control that would lead to reduced reliance on the use of chemical pesticides. Subterranean ...

testing-protozoacidal-activity-ligand-lytic-peptides-against-termite

Research

JoVE Journal

Biology

Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro (Protozoa Culture) and in vivo (Microinjection into Termite Hindgut)

14.8K 次观看.  Louisiana State University Agricultural Center. 我们目前用于演示程序，配体结合到膜表面的纤维素消化在使用荧光显微镜和配体与裂解肽台湾地下白蚁肠道原虫杀原虫这些在体外（厌氧原虫文化）在体内（注入白蚁后肠）。

视频: Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro Protozoa Culture and in vivo Microinjection into Termite Hindgut

Extracting DNA from the Gut Microbes of the Termite (Zootermopsis Angusticollis) and Visualizing Gut Microbes

Termites are among the few animals known to have the capacity to subsist solely by consuming wood.  The termite gut tract contains a dense and species-rich microbial population that assists in the degradation of lignocellulose predominantly into acetate, the key nutrient fueling termite metabolism (Odelson &amp; Breznak, 1983).  Within these microbial populations are bacteria, methanogenic archaea and, in some ("lower") termites, eukaryotic protozoa. Thus, termites are excellent research subjects for studying the interactions among microbial species and the numerous biochemical functions they perform to the benefit of their host.  The species composition of microbial populations in termite guts as well as key genes involved in various biochemical processes has been explored using molecular techniques (Kudo et al., 1998; Schmit-Wagner et al., 2003; Salmassi &amp; Leadbetter, 2003). These techniques depend on the extraction and purification of high-quality nucleic acids from the termite gut environment.  The extraction technique described in this video is a modified compilation of protocols developed for extraction and purification of nucleic acids from environmental samples (Mor  et al., 1994; Berthelet et al., 1996; Purdy et al., 1996; Salmassi &amp; Leadbetter, 2003; Ottesen et al. 2006) and it produces DNA from termite hindgut material suitable for use as template for polymerase chain reaction (PCR).

Extracting DNA from the Gut Microbes of the Termite Zootermopsis Angusticollis and Visualizing Gut Microbes

This video illustrates the technique for extracting DNA from the species of microbes resident in the termite hindgut. The preparation of a wet mount slide, which is useful for visualizing the gut microbial community is also illustrated, and a tour through the species-rich gut environment is given.

从白蚁肠道微生物（Zootermopsis Angusticollis）中提取的DNA和可视化肠道微生物

Termites are among the few animals known to have the capacity to subsist solely by consuming wood.  The termite gut tract contains a dense and ...

科研

生物学

Investigating the Microbial Community in the Termite Hindgut - Interview

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes.     The symbiotic relationship existing between the host insect and lignocellulose-degrading gut microbes is explained, as well as the industrial uses of these microbes for degrading plant biomass and generating biofuels.

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes. The symbiotic relationship existing between the host insect and lignocellulose-degrading gut microbes is explained, as well as the industrial uses of these microbes for degrading plant biomass and generating biofuels.

调查白蚁后肠中的微生物的社区 - 面试

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes.     The ...

Layers of Symbiosis - Visualizing the Termite Hindgut Microbial Community

Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different species found nowhere else on Earth.  Jared reveals that the symbiosis exhibited by this system is multi-layered and involves not only a relationship between the termite and its gut inhabitants, but also involves a complex web of symbiosis among the gut microbes themselves.

Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different species found nowhere else on Earth. Jared reveals that the symbiosis exhibited by this system is multi-layered and involves not only a relationship between the termite and its gut inhabitants, but also involves a complex web of symbiosis among the gut microbes themselves.

共生层 - 可视化的白蚁后肠微生物群落

Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different ...

Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When an uninfected aphid feeds on an infected plant it contracts the virus through the plant phloem. Once ingested, the virus must pass from the insect gut to the hemolymph (the insect  blood ) and then must pass through the salivary gland, in order to be transmitted back to a new plant. An aphid may take up different viruses when munching on a plant, however only a small fraction will pass through the gut and salivary gland, the two main barriers for transmission to infect more plants. In the lab, we use physalis plants to study PLRV transmission. In this host, symptoms are characterized by stunting and interveinal chlorosis (yellowing of the leaves between the veins with the veins remaining green). The video that we present demonstrates a method for performing aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut is preventing viral transmission.
The video that we present demonstrates a method for performing Aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut or salivary gland is preventing viral transmission.

Aphids are effective transmitters of plant viruses. Aphid microinjection of virus, the procedure we will show you today, is a technique allowing researchers to inject virus directly into the hemocoel of the aphid, bypassing the gut, one of the 2 major barriers for virus transmission in a circulative manner. The same technique is also used to inject dsRNA for RNAi.

测试使用微量注射蚜虫病毒传播的生理障碍

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When ...

In vivo and in vitro Studies of Adaptor-clathrin Interaction

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs are distinguished by a polyhedral lattice of clathrin that coats the vesicle membrane and serves as a mechanical scaffold. Clathrin coats are assembled during vesicle formation from individual clathrin triskelia , the soluble form of clathrin composed of three heavy and three light chain subunits7,8. Because the triskelion does not have the ability to bind to the membrane directly, clathrin-binding adaptors are critical to link the forming clathrin lattice to the membrane through association with lipids and/or membrane proteins9. Adaptors also package transmembrane protein cargo, such as receptors, and can interact with each other and with other components of the CCV formation machinery9.
Over twenty clathrin adaptors have been described, several are involved in clathrin mediated endocytosis and others localize to the trans Golgi network or endosomes9. With the exception of HIP1R (yeast Sla2p), all known clathrin adaptors bind to the N-terminal -propeller domain of the clathrin heavy chain9. Clathrin adaptors are modular proteins consisting of folded domains connected by unstructured flexible linkers. Within these linker regions, short binding motifs mediate interactions with the clathrin N-terminal domain or other components of the vesicle formation machinery9. Two distinct clathrin-binding motifs have been defined: the clathrin-box and the W-box9. The consensus clathrin-box sequence was originally defined as L[L/I][D/E/N][L/F][D/E]10 but variants have been subsequently discovered11. The W-box conforms to the sequence PWxxW (where x is any residue).
Sla1p (Synthetic Lethal with Actin binding protein-1) was originally identified as an actin associated protein and is necessary for normal actin cytoskeleton structure and dynamics at endocytic sites in yeast cells12. Sla1p also binds the NPFxD endocytic sorting signal and is critical for endocytosis of cargo bearing the NPFxD signal13,14. More recently, Sla1p was demonstrated to bind clathrin through a motif similar to the clathrin box, LLDLQ, termed a variant clathrin-box (vCB), and to function as an endocytic clathrin adaptor15. In addition, Sla1p has become a widely used marker for the endocytic coat in live cell fluorescence microscopy studies16. Here we use Sla1p as a model to describe approaches for adaptor-clathrin interaction studies. We focus on live cell fluorescence microscopy, GST-pull down, and co-immunoprecipitation methods.

In vivo and in vitro Studies of Adaptor-clathrin Interaction

Clathrin-mediated endocytosis depends on adaptor proteins that coordinate cargo selection and clathrin coat assembly. Here we describe procedures to study adaptor-clathrin physical interaction and live cell imaging approaches using as a model the yeast endocytic adaptor protein Sla1p.

适配器，网格蛋白相互作用在体内和体外研究

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

激活细胞凋亡的细胞色素的细胞质显微注射 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

在细胞培养中缺氧诱导和测试

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of neurulation, migrates extensively towards various organs, and differentiates into many types of derivatives (neurons, glia, cartilage and bone, pigmented and endocrine cells). In this protocol, we describe how to dissect the premigratory cranial NC from Xenopus laevis embryos, in order to study NC development in vivo and in vitro. The frog model offers many advantages to study early development; abundant batches are available, embryos develop rapidly, in vivo gain and loss of function strategies allow manipulation of gene expression prior to NC dissection in donor and/or host embryos. The NC explants can be plated on fibronectin and used for in vitro studies. They can be cultured for several days in a serum-free defined medium. We also describe how to graft NC explants back into host embryos for studying NC migration and differentiation in vivo.

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

This protocol describes how to dissect premigratory cranial neural crest (NC) from Xenopus laevis neurulas. These explants can be plated on fibronectin and cultured in vitro, or grafted back into host embryos. This technique allows studying the mechanisms of NC epithelium-to-mesenchyme transition, migration, and differentiation.

剖析<em&gt;非洲爪蟾</em&gt;神经嵴的<em&gt;在体外</em&gt;外植体培养或<em&gt;在体内</em&gt;移植

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of ...

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA (dsDNA) which has the requisite characteristics to stimulate the cytoplasmic DNA sensing pathways can be generated cheaply and with ease. By the concatemerization of short, synthetic oligonucleotides (which lack CpG motifs), dsDNA can be generated to be of sufficient length to activate the cytosolic DNA sensing pathway. This protocol involves blunt end ligation of the oligonucleotides in the presence of polyethylene glycol (PEG), which provides an environment for efficient ligation to occur. The dsDNA concatemers can be used, following purification by phenol/chloroform extraction, to simulate the innate immune response in vitro by standard transfection protocols. This DNA can also be used to stimulate innate immunity in vivo by intradermal injection into the ear pinna of a mouse, for example. By standardizing the concatemerization process and the subsequent stimulation protocols, a reliable and reproducible activation of the innate immune system can be produced.

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

The aim of the protocol is to use optimal methods for stimulating the cytoplasmic DNA sensing pathways in cells and in vivo. This is achieved by improving the generation of long, double-stranded DNA during blunt-end ligation. Cells or mice are then transfected using a lipid transfection reagent.

细胞质DNA的检测途径的刺激<em&gt;在体外</em&gt;和<em&gt;体内</em

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA ...

In Vivo Microinjection and Electroporation of Mouse Testis

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis.
The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV).
For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular&#160;cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red&#160;fluorescence, illustrating transfection success.
Generally, this protocol can be employed for delivering DNA- or RNA- constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques.

In Vivo Microinjection and Electroporation of Mouse Testis

This article describes microinjection and electroporation of mouse testis in&#160;vivo as a&#160;transfection technique for testicular mouse cells to study unique processes of spermatogenesis. The presented protocol involves steps of glass capillary preparation, microinjection via the efferent duct, and transfection by electroporation.

在小鼠睾丸活体显微注射和电穿孔

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...