感谢海南省自然科学基金（批准号：323QN262）、国家自然科学基金（批准号31701843和32172483）、江苏省农业科技创新基金（批准号：323QN262）、国家自然科学基金（批准号：、、国家自然科学基金（批准号、、国家自然科学基金CX（22）3012 和 CX（21）3008）、江苏省“双创医生”基金会（批准号202030472）和南京农业大学创业基金（批准号804018）。

1. 昆虫饲养
<ol><li>在26±1°C的温度和40%±10%的相对湿度下，在玉米粉上饲喂 Corcyra cephalonica 。</li>
	<li>以头孢芽卵为寄主，在室内繁殖吉林昆虫的T. dendrolimi菌株。在果蝇管中饲料黄蜂成虫10%蔗糖水，温度为26±1°C，相对湿度为70%±10%，光照（L）：深色（D）周期为14小时：10小时。</li>
</ol>2、聚乙烯塑料薄膜蛋卡的制备
<ol><li>取长 16 厘米、宽 12 厘米、厚 20 μm 的聚乙烯塑料薄膜。根据96孔的标准PCR板布局，使用玻璃研磨棒压出30个直径为2-3mm，高度约为3mm的半圆形突起。 注意： 使用玻璃研磨棒压出 30 个半圆形突起的过程需要注意压力，因为压力太大会刺穿塑料并污染提取的无毒研棒。</li>
	<li>通过将两面暴露在紫外线（UV）下1小时来对压制的聚乙烯塑料薄膜进行消毒。</li>
	<li>在半圆形表面上加入少量10%聚乙烯醇。</li>
</ol>3. 木滴虫 寄生
<ol><li>CO2麻醉后，将T. dendrolimi雌性黄蜂放入收集箱中，黄蜂数量为~3000。</li>
	<li>将薄膜蛋卡的凸面朝向收集盒，并用橡皮筋固定边缘。</li>
	<li>向每个半圆形突起加入 4 μL 氨基酸溶液（6 g/L 亮氨酸、4 g/L 苯丙氨酸、4.25 g/L 组氨酸）。用长 16 厘米、宽 12 厘米的扁平聚乙烯塑料薄膜覆盖。用橡皮筋用两张塑料片紧紧地盖住收集箱。</li>
	<li>让 T. dendrolimi 黄蜂自由寄生4-8小时，并通过湿棉提供10%的蔗糖水。</li>
</ol>4. 采集 T. dendrolimi 毒液
<ol><li>从人工卵卡的内突处获得寄生的氨基酸溶液，并将其转移到1.5mL试管的盖子上。</li>
	<li>用直径为 10 毫米的 25 μm 尼龙网盖住管盖，紧固尼龙网和离心管。使用小型离心机（1360× g）将离心管直立放置以进行短离心10秒，并收集过滤的溶液（~100μLT . dendrolimi 毒液）。</li>
	<li>使用二辛可宁酸（BCA）测定试剂盒（材料表）测量收集的T. dendrolimi毒液的浓度。</li>
	<li>将毒液储存在-80°C以进行进一步分析。</li>
</ol>5. SDS-PAGE分析
<ol><li>将30μLT . dendrolimi 毒液加入10μL4x十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS-PAGE）样品上样缓冲液（材料表）中，并在95°C下加热10分钟。</li>
	<li>在130 V下进行SDS-PAGE凝胶电泳120分钟。</li>
	<li>使用蛋白质染色装置（材料表）对SDS-PAGE凝胶进行染色和脱色。</li>
</ol>

Trichogramma dendrolimi 和毒液提取的人工宿主寄生

<ol>
	<li>Pennacchio, F., Strand, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+developmental+strategies+in+parasitic+hymenoptera.">Evolution of developmental strategies in parasitic hymenoptera.</a> Annual Review of Entomology. 51, 233-258 (2006).</li><li>Yan, Z. C., Ye, X. H., Wang, B. B., Fang, Q., Ye, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+advances+on+composition,+function+and+evolution+of+venom+proteins+in+parasitoid+wasps.">Research advances on composition, function and evolution of venom proteins in parasitoid wasps.</a> Chinese Journal of Biological Control. 33 (1), 1-10 (2017).</li><li>Asgari, S., Rivers, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Venom+proteins+from+endoparasitoid+wasps+and+their+role+in+host-parasite+interactions.">Venom proteins from endoparasitoid wasps and their role in host-parasite interactions.</a> Annual Review of Entomology. 56, 313-335 (2011).</li><li>Moreau, S. J. M., Guillot, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+and+prospects+on+biosynthesis,+structures,+and+functions+of+venom+proteins+from+parasitic+wasps.">Advances and prospects on biosynthesis, structures, and functions of venom proteins from parasitic wasps.</a> Insect Biochemistry and Molecular Biology. 35 (11), 1209-1223 (2005).</li><li>Yan, Z. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+venom+serpin+splicing+isoform+of+the+endoparasitoid+wasp+Pteromalus+puparum+suppresses+host+prophenoloxidase+cascade+by+forming+complexes+with+host+hemolymph+proteinases.">A venom serpin splicing isoform of the endoparasitoid wasp Pteromalus puparum suppresses host prophenoloxidase cascade by forming complexes with host hemolymph proteinases.</a> Journal Biological Chemistry. 292 (3), 1038-1051 (2017).</li><li>Woelke, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Description+and+biology+of+two+new+egg+parasitoid+species+(Hymenoptera:+Trichogrammatidae)+reared+from+eggs+of+Heliconiini+butterflies+(Lepidoptera:+Nymphalidae:+Heliconiinae)+in+Panama.">Description and biology of two new egg parasitoid species (Hymenoptera: Trichogrammatidae) reared from eggs of Heliconiini butterflies (Lepidoptera: Nymphalidae: Heliconiinae) in Panama.</a> Journal of Natural History. 53 (11-12), 639-657 (2019).</li><li>Zang, L. S., Wang, S., Zhang, F., Desneux, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+control+with+Trichogramma+in+China:+History,+present+status,+and+perspectives.">Biological control with Trichogramma in China: History, present status, and perspectives.</a> Annual Review of Entomology. 66, 463-484 (2021).</li><li>Nettles, W. C. J., Morrison, R. K., Xie, Z. N., Ball, D., Shenkir, C. A., Vinson, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+action+of+potassium+chloride+and+magnesium+sulfate+on+parasitoid+wasp+oviposition.">Synergistic action of potassium chloride and magnesium sulfate on parasitoid wasp oviposition.</a> Science. 218, 4568 (1982).</li><li>Tilden, R. L., Ferkovich, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kairomonal+stimulation+of+oviposition+into+an+artificial+substrate+by+the+endoparasitoid+Microplitis+croceipes+(Hymenoptera)Braconidae).">Kairomonal stimulation of oviposition into an artificial substrate by the endoparasitoid Microplitis croceipes (Hymenoptera)Braconidae).</a> Annals of the Entomological Society of America. 81 (1), 152-156 (1988).</li><li>Xie, Z. N., Li, L., Xie, Y. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+culture+of+Habrobracon+hebetor.">In vitro culture of Habrobracon hebetor.</a> Chinese Journal of Biological Control. 5 (2), 49-51 (1989).</li><li>Han, S. T., Liu, W. H., Li, L. Y., Chen, Q. X., Zeng, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breeding+Trichogramma+ostriniae+with+artificial+eggs.">Breeding Trichogramma ostriniae with artificial eggs.</a> Journal of Environmental Entomology. 21 (1), 9-12 (1999).</li><li>Li, L. Y., Chen, Q. X., Liu, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oviposition+behavior+of+twelve+species+of+Trichogramma+and+its+influence+on+the+efficiency+of+rearing+them+in+vitro.">Oviposition behavior of twelve species of Trichogramma and its influence on the efficiency of rearing them in vitro.</a> Journal of Environmental Entomology. 11 (1), 31-35 (1989).</li><li>Xing, J. Q., Li, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rearing+of+an+egg+parasite+Anastatus+japonicus+Ashmead+in+vitro.">Rearing of an egg parasite Anastatus japonicus Ashmead in vitro.</a> Acta Entomologica Sinica. 33 (2), 166-173 (1990).</li><li>Moreau, S. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#34;It+stings+a+bit+but+it+cleans+well&#34;:+Venoms+of+Hymenoptera+and+their+antimicrobial+potential.">&#34;It stings a bit but it cleans well&#34;: Venoms of Hymenoptera and their antimicrobial potential.</a> Journal of Insect Physiology. 59 (2), 186-204 (2013).</li><li>Moreau, S. J. M., Asgari, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Venom+proteins+from+parasitoid+wasps+and+their+biological+function.">Venom proteins from parasitoid wasps and their biological function.</a> Toxins. 7 (7), 2385-2412 (2015).</li></ol>

提交人没有什么可披露的，也没有相互竞争的经济利益。

在这里，我们提出了一种使用人工宿主从T. dendrolimi中提取毒液的方法。毒液采集实验的要点如下。（1）在制备过程中，必须用适当浓度的CO2迅速麻醉树状枸杞。如果 CO2 浓度过低，则不足以快速麻醉 Trichogramma。相反，如果浓度过高，Trichogramma可能会死亡，从而降低它们寄生在人工宿主上的能力。（2）必须保证氨基酸溶液的无菌性，因为氨基酸溶液的污...

寄生蜂是寄生膜翅目昆虫，是生物防治的重要资源1。寄生蜂种类繁多，估计有超过 600,000 种2.寄生蜂的多样性远远超过其他有毒节肢动物，如蛇、锥形蜗牛、蜘蛛、蝎子和蜜蜂。毒液是寄生蜂的重要寄生因子。为了成功寄生，将毒液注射到宿主体内，调节宿主的行为、免疫、发育和新陈代谢3.此外，寄生蜂的毒液在其分子结构、靶点和功能上表现出显著的多样性，反映了与宿主的复杂协同进化。因此，寄生虫毒液是一种有价值且被低估的活性分子资源，用于杀虫或医疗目的4.与蛇、锥形蜗牛、蜘蛛、蝎子和蜜蜂的毒液不同，寄生蜂毒液不能通过直接刺激或电刺激收集5.目前提取寄生蜂毒液的方法是解剖毒液库。然而，寄生蜂通常很小，解剖寄生蜂需要很高的技术技能。因此，如果我们能找到一种高效方便地收集寄生蜂毒液的方法，对研究寄生蜂的毒液将有很大的帮助。
毛螨（膜翅目：Trichogrammatidae）是微小（~0.5毫米长）寄生蜂属 6.这些黄蜂是使用最广泛的生物防治剂之一，特别是针对农业和森林中各种鳞翅目害虫的卵。例如，中国应用最广泛的木霉菌种之一——枸杞已被广泛用于防治多种农林害虫，如石斛、毛竹、桫椤等。先前的研究表明，Trichogramma黄蜂可以将其卵注射到人工宿主中7。可以使用蜡8、琼脂9、封口膜10 和塑料薄膜11 等材料创建人工宿主。在人工宿主中诱导足够的Trichogramma产卵的溶液可以很简单，例如氨基酸或无机盐12。基于树状毛蛾能寄生人工寄主的特点，本研究提供了一种利用人工寄主从寄生蜂中提取毒液的新方法。这种方法旨在解决当前提取技术中产量低、纯度低和易受污染的缺点。利用该方法，可提取大量高纯度的石栖毒液，满足了杀虫或医用生物活性分子的科学研究和筛选需求。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 &#956;m Nylon Net</td>
			<td>Millipore</td>
			<td>NY1002500</td>
			<td>For filtering the eggs</td>
		</tr>
		<tr>
			<td>10% Polyvinyl alcohol</td>
			<td>Aladdin</td>
			<td>P139533</td>
			<td>For attractting&#160; T. dendrolimi&#160; to lay eggs</td>
		</tr>
		<tr>
			<td>10% Sucrose water</td>
			<td>Sinopharm Chemical Reagent&#160;</td>
			<td>10021463</td>
			<td>Feed Trichogramma dendrolimi</td>
		</tr>
		<tr>
			<td>4x LDS loading buffer</td>
			<td>Ace Hardware</td>
			<td>B23010301</td>
			<td>SDS-PAGE</td>
		</tr>
		<tr>
			<td>Collection box</td>
			<td>Deli</td>
			<td>8555</td>
			<td>Container for T. dendrolimi parasitism</td>
		</tr>
		<tr>
			<td>Future PAGE&#160; 4&#8211;12% (12 wells)</td>
			<td>Ace Hardware</td>
			<td>J70236502X</td>
			<td>SDS-PAGE</td>
		</tr>
		<tr>
			<td>GenScript eStain L1 protein staining apparatus</td>
			<td>GenScript</td>
			<td>L00753</td>
			<td>SDS-PAGE</td>
		</tr>
		<tr>
			<td>Glass grinding rod&#160;&#160;</td>
			<td>Applygen</td>
			<td>tb6268</td>
			<td>Semicircular protrudations&#160;</td>
		</tr>
		<tr>
			<td>L- Leucine</td>
			<td>Solarbio</td>
			<td>L0011</td>
			<td>Artificial host components</td>
		</tr>
		<tr>
			<td>L-Histidine</td>
			<td>Aladdin</td>
			<td>A2219458</td>
			<td>Artificial host components</td>
		</tr>
		<tr>
			<td>L-Phenylalanine</td>
			<td>Solarbio</td>
			<td>P0010</td>
			<td>Artificial host components</td>
		</tr>
		<tr>
			<td>Mini-Centrifuges</td>
			<td>Scilogex</td>
			<td>D1008</td>
			<td>Centrifuge</td>
		</tr>
		<tr>
			<td>MOPS-SDS running buffer</td>
			<td>Ace Hardware</td>
			<td>B23021</td>
			<td>SDS-PAGE</td>
		</tr>
		<tr>
			<td>Omni-Easy Instant BCA protein assay kit</td>
			<td>Shanghai Yamay Biomedical Technology&#160;</td>
			<td>ZJ102</td>
			<td>For esimation of venom protein concentration</td>
		</tr>
		<tr>
			<td>PCR plate layout of 96 holes</td>
			<td>Thermo Fisher</td>
			<td>AB1400L</td>
			<td>Semicircular protrudations&#160;</td>
		</tr>
		<tr>
			<td>Polyethylene plastic film</td>
			<td>Suzhou Aopang Trading &#160;</td>
			<td>001c5427</td>
			<td>Artificial egg card</td>
		</tr>
		<tr>
			<td>Prestained color protein marker(10&#8211;180 kDa)</td>
			<td>YiFeiXue Biotech</td>
			<td>YWB007</td>
			<td>SDS-PAGE</td>
		</tr>
		<tr>
			<td>Rubber band</td>
			<td>Guangzhou qianrui biology science and technology</td>
			<td>009</td>
			<td>Tighten the plastic film and the collection box</td>
		</tr>
		<tr>
			<td>Silicone rubber septa mat, 96-well, round hole</td>
			<td>Sangon Biotech</td>
			<td>F504416-0001</td>
			<td>Semicircular protrudations&#160;</td>
		</tr>
	</tbody>
</table>

extracting venom from the parasitoid wasp trichogramma dendrolimi using an artificial host

寄生蜂是一类多样化的膜翅目昆虫，是害虫生物防治的宝贵资源。为了确保成功寄生，寄生蜂将毒液注射到宿主体内，以抑制宿主的免疫力，调节宿主的发育、新陈代谢甚至行为。据估计，寄生蜂的种类超过 600,000 种，其多样性超过了其他有毒动物，如蛇、锥形蜗牛和蜘蛛。寄生蜂毒液是一种未被充分开发的生物活性分子来源，在害虫防治和医学方面具有潜在应用。然而，由于无法使用直接或电刺激，并且由于其体积小而难以解剖，因此收集寄生虫毒液具有挑战性。 Trichogramma 是一种微小的 （~0.5 毫米） 卵寄生蜂属，广泛用于农业和森林中鳞翅目害虫的生物防治。在这里，我们报道了一种使用人工宿主从 T. dendrolimi 中提取毒液的方法。这些人工宿主是用聚乙烯薄膜和氨基酸溶液产生的，然后接种有 毛滴虫 蜂进行寄生。随后收集并浓缩了毒液。这种方法能够提取大量的 毛滴虫 毒液，同时避免由解剖引起的其他组织的污染，这是毒液库解剖方案中的常见问题。这种创新方法促进了 Trichogramma 毒液的研究，为新的研究和潜在应用铺平了道路。

使用蛋白质检测试剂盒测量代表性毒液样品的蛋白质浓度，结果如 表1所示。结果表明，该方法采集的毒液蛋白浓度范围为0.35 μg/μL至0.46 μg/μL，而氨基酸溶液阴性对照的蛋白浓度仅为0.03 μg/μL至0.05 μg/μL。该方法采集的毒液蛋白浓度远高于阴性对照，说明该方法能较好地采集寄生蜂的毒液。此外，寄生时间和浓度之间没有特定的相关性，因为不同批次的寄生蜂可能具有不同的活力。...

Watch this Scientific Journal Video about Efficient Venom Extraction Method from Trichogramma Parasitoid Wasps at JoVE.com

作者聚焦：Trichogramma 寄生黄蜂的高效毒液提取方法 

在这里，我们提出了一种使用由聚乙烯薄膜和氨基酸溶液制成的人工宿主从 木 霉中提取毒液的方案。

使用人工宿主从寄生蜂 Trichogramma dendrolimi 中提取毒液

Parasitoid wasps are a diverse group of hymenopteran insects that serve as invaluable resources for pest biocontrol. To ensure successful parasitism, ...

缺乏有效的毒液提取方法往往限制了对寄生蜂毒液的研究，特别是在微小的寄生蜂中。本研究提供了一种有效的提取毛滴虫毒液的方法，并对毛滴虫毒液进行后续研究，如蛋白质补偿和毒液功能分析。以前，没有交换寄生蜂毒液的方法需要区分毒液储备。然而，寄生蜂很小。不仅区分毒液的技术要求很高，而且在解剖过程中其他组织的延续也很常见。我们的常规方法使用人工宿主，从而避免了此类问题。未来，我们将专注于Trichogramma毒液的农业和医学应用。我们的目标是利用特定的杀虫毒液基因来控制害虫。同时，我们计划研究Trichogramma毒液在方法学方面的未开发潜力，特别是其在增强免疫治疗技术和发现新型抗生素分子方面的预期效用。

extracting-venom-from-parasitoid-wasp-trichogramma-dendrolimi-using

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

Extracting Venom from the Parasitoid Wasp Trichogramma dendrolimi Using an Artificial Host

1.4K 次观看.  Nanjing Agricultural University. 缺乏有效的毒液提取方法往往限制了对寄生蜂毒液的研究，特别是在微小的寄生蜂中。本研究提供了一种有效的提取毛滴虫毒液的方法，并对毛滴虫毒液进行后续研究，如蛋白质补偿和毒液功能分析。以前，没有交换寄生蜂毒液的方法需要区分毒液储备。然而，寄生蜂很小。不仅区分毒液的技术要求很高，而且在解剖过程中其他组织的延续也很常见。我们的常规方法使?...

视频: 作者聚焦：Trichogramma 寄生黄蜂的高效毒液提取方法 

Mimicking the Function of Signaling Proteins: Toward Artificial Signal Transduction Therapy

Signal transduction pathways, which control the response of cells to various environmental signals, are mediated by the function of signaling proteins that interact with each other and activate one other with high specificity. Synthetic agents that mimic the function of these proteins might therefore be used to generate unnatural signal transduction steps and consequently, alter the cell's function. We present guidelines for designing 'chemical transducers' that can induce artificial communication between native proteins. In addition, we present detailed protocols for synthesizing and testing a specific 'transducer', which can induce communication between two unrelated proteins: platelet-derived growth-factor (PDGF) and glutathione-S-transferase (GST). The way by which this unnatural PDGF-GST communication could be used to control the cleavage of an anticancer prodrug is also presented, indicating the potential for using such systems in 'artificial signal transduction therapy'. This work is intended to facilitate developing additional 'transducers' of this class, which may be used to mediate intracellular protein-protein communication and consequently, to induce artificial cell signaling pathways.

We present guidelines for developing synthetic 'chemical transducers' that can induce communication between naturally unrelated proteins. In addition, detailed protocols are presented for synthesizing and testing a specific 'transducer' that enables a growth factor to activate a detoxifying enzyme and consequently, to regulate the cleavage of an anticancer prodrug.

模拟信号蛋白的功能:走向人工信号转导疗法

Signal transduction pathways, which control the response of cells to various environmental signals, are mediated by the function of signaling proteins ...

科研

生物化学

Structural Information from Single-molecule FRET Experiments Using the Fast Nano-positioning System

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. Thereby, multiple smFRET measurements are used to localize an unknown dye position inside a protein complex by means of trilateration. In order to obtain quantitative information, the Nano-Positioning System (NPS) uses probabilistic data analysis to combine structural information from X-ray crystallography with single-molecule fluorescence data to calculate not only the most probable position but the complete three-dimensional probability distribution, termed posterior, which indicates the experimental uncertainty. The concept was generalized for the analysis of smFRET networks containing numerous dye molecules. The latest version of NPS, Fast-NPS, features a new algorithm using Bayesian parameter estimation based on Markov Chain Monte Carlo sampling and parallel tempering that allows for the analysis of large smFRET networks in a comparably short time. Moreover, Fast-NPS allows the calculation of the posterior by choosing one of five different models for each dye, that account for the different spatial and orientational behavior exhibited by the dye molecules due to their local environment.
Here we present a detailed protocol for obtaining smFRET data and applying the Fast-NPS. We provide detailed instructions for the acquisition of the three input parameters of Fast-NPS: the smFRET values, as well as the quantum yield and anisotropy of the dye molecules. Recently, the NPS has been used to elucidate the architecture of an archaeal open promotor complex. This data is used to demonstrate the influence of the five different dye models on the posterior distribution.

We present the setup and experimental procedure to obtain smFRET data from large donor-acceptor networks with a TIRF microscope. The step-by-step analysis of these measurements with the Bayesian inference software Fast-NPS yields high-resolved structural information via the application of adapted dye models.

从单分子结构信息FRET实验使用快速纳米定位系统

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. ...

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.

We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.

对于高度纯化RNA的种子定量转录组分析的隔离中使用的有效方法

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an essential step in basic and applied science. MicroScale Thermophoresis (MST) is a sensitive biophysical method to obtain this important information. Relying on a physical effect called thermophoresis, which describes the movement of molecules through temperature gradients, this technology allows for the fast and precise determination of binding parameters in solution and allows the free choice of buffer conditions (from buffer to lysates/sera). MST uses the fact that an unbound molecule displays a different thermophoretic movement than a molecule that is in complex with a binding partner. The thermophoretic movement is altered in the moment of molecular interaction due to changes in size, charge, and hydration shell. By comparing the movement profiles of different molecular ratios of the two binding partners, quantitative information such as binding affinity (pM to mM) can be determined. Even challenging interactions between molecules of small sizes, such as aptamers and small compounds, can be studied by MST. Using the well-studied model interaction between the DH25.42 DNA aptamer and ATP, this manuscript provides a protocol to characterize aptamer-small molecule interactions. This study demonstrates that MST is highly sensitive and permits the mapping of the binding site of the 7.9 kDa DNA aptamer to the adenine of ATP.

MicroScale Thermophoresis (MST) is a sensitive technology to characterize aptamer-target interactions. This manuscript describes an MST protocol to characterize aptamer-small molecule interactions.

映射的适体对ATP的结合部位使用MicroScale程序热泳

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an ...

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

从真核细胞蛋白质复合体串联亲和纯化

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 &#197; structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

This paper presents a protocol for processing cryo-EM images using the software suite SPHIRE. The present protocol can be applied for nearly all single particle EM projects that target near-atomic resolution.

使用SPHIRE的电子冷冻显微镜图像的高分辨率单粒子分析

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations&#39; survivability.

体外肠道宿主-微生物界面合成细菌联合的可行性评价

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

Manipulating Living Cells to Construct Stable 3D Cellular Assembly Without Artificial Scaffold

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the importance of 3-dimensional (3D) cellular assemblies in these fields. Artificial scaffolds have often been used to construct 3D cellular assemblies. However, the scaffolds used to construct cellular assemblies are sometimes toxic and may change the properties of the cells. Thus, it would be beneficial to establish a non-toxic method for facilitating cell-cell contact. In this paper, we introduce a novel method for constructing stable cellular assemblies by using optical tweezers with dextran. One of the advantages of this method is that it establishes stable cell-to-cell contact within a few minutes. This new method allows the construction of 3D cellular assemblies in a natural hydrophilic polymer and is expected to be useful for constructing next-generation 3D single-cell assemblies in the fields of regenerative medicine and tissue engineering.

We demonstrate a novel method for constructing a single-cell-based 3-dimensional (3D) assembly without an artificial scaffold.

操纵活细胞构建稳定的3D 蜂窝组件, 无需人工支架

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

翻译核糖体亲性纯化(TRAP),用于从体内内皮细胞中分离RNA

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.