We acknowledge financial support from the Natural Science Foundation of Hainan Province (Grant no. 323QN262), the National Natural Science Foundation of China (Grant no. 31701843 and 32172483), the Jiangsu Agriculture Science and Technology Innovation Fund (Grant No. CX(22)3012 and CX(21)3008), the &#34;Shuangchuang Doctor&#34; Foundation of Jiangsu Province (Grant No. 202030472), and the Nanjing Agricultural University startup fund (Grant No. 804018).

1. Insect rearing
<ol>
	<li>Feed Corcyra cephalonica on corn flour at a temperature of 26 &#177; 1&#176;C and a relative humidity of 40% &#177; 10%.</li>
	<li>Breed T. dendrolimi strain from the Jilin insectary indoors using the eggs of Corcyra cephalonica as hosts. Feed wasp adults 10% sucrose water in Drosophila tubes at a temperature of 26 &#177; 1 &#176;C, relative humidity of 70% &#177; 10%, light (L): dark (D) period of 14 h: 10 h.</li>
</ol>
2. Preparation of polyethylene plastic film egg cards
<ol>
	<li>Take a polyethylene plastic film with a length of 16 cm, a width of 12 cm, and a thickness of 20 &#181;m. Press out 30 semicircular protrusions with a diameter of 2-3 mm and a height of approximately 3 mm using a glass grinding rod according to the standard PCR plate layout of 96 holes. 
	NOTE: The process of pressing out 30 semicircular protrusions using a glass grinding rod needs to be done paying attention to the pressure because too hard a press will puncture the plastic and contaminate the extracted venomless grinding rod.</li>
	<li>Disinfect the pressed polyethylene plastic film by exposing both sides to ultraviolet (UV) light for 1 h.</li>
	<li>Add a small amount of 10% polyvinyl alcohol to the semicircular surface.</li>
</ol>
3. Trichogramma dendrolimi parasitism
<ol>
	<li>After CO2 anesthesia, place T. dendrolimi female wasps into a collection box, and the number of wasps was ~3000.</li>
	<li>Place the convex side of the film egg card toward the collection box and secure the edges with a rubber band.</li>
	<li>Add 4 &#181;L of amino acid solution (6 g/L leucine, 4 g/L phenylalanine, 4.25 g/L histidine) to each semicircular protrusion. Cover it with a flat polyethylene plastic film 16 cm long and 12 cm wide. Use a rubber band to tightly cover the collection box with two sheets of plastic.</li>
	<li>Let T. dendrolimi wasps parasitize freely for 4-8 h and provide 10% sucrose water through wetted cotton.</li>
</ol>
4. Collecting T. dendrolimi venom
<ol>
	<li>Obtain the parasitized amino acid solution from the inner protrusion of the artificial egg card and transfer it to the cap of 1.5 mL tubes.</li>
	<li>Cover the tube cap with a 10 &#181;m nylon net with a 25 mm diameter, fasten the nylon net and centrifuge tube tightly. Place the centrifuge tube upright for short centrifugation using a mini-centrifuge (1360 x g) for 10 s and collect the filtered solution (~100 &#181;L of T. dendrolimi venom).</li>
	<li>Measure the concentration of collected T. dendrolimi venom using a Bicinchoninic acid (BCA) assay kit (Table of Materials).</li>
	<li>Store the venom at -80 &#176;C for further analyses.</li>
</ol>
5. SDS-PAGE analyses
<ol>
	<li>Add 30 &#181;L of T. dendrolimi venom to 10 &#181;L of 4x sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer (Table of Materials) and heat at 95 &#176;C for 10 min.</li>
	<li>Perform SDS-PAGE gel run at 130 V for 120 min.</li>
	<li>Stain and decolorize the SDS-PAGE gel using the protein staining apparatus (Table of Materials).</li>
</ol>

Artificial Host Parasitization by Trichogramma dendrolimi and Venom Extraction

<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>

The author has nothing to disclose and no competing financial interests.

Here, we present a method for extracting venom from T. dendrolimi using artificial hosts. The key points in the venom collection experiment are as follows. (1) During preparation, T. dendrolimi must be anesthetized rapidly with an appropriate concentration of CO2. If the CO2 concentration is too low, it will be insufficient to anesthetize the Trichogramma quickly. Conversely, if the concentration is too high, Trichogramma may die, reducing their ability to parasit...

Parasitoid wasps are parasitic hymenopteran insects that are important resources for biological control1. There is a wide variety of parasitoid wasps, with over 600,000 estimated species2. The diversity of parasitoid wasps far exceeds that of other venomous arthropods, such as snakes, cone snails, spiders, scorpions, and bees. Venom is an important parasitic factor in parasitoid wasps. For successful parasitism, venom is injected into the host, modulating the host&#39;s behavior, immunity, development, and metabolism3. Moreover, the venom of parasitoid wasps displays remarkable diversity in its molecular structures, targets, and functions, reflecting complex coevolution with their hosts. Thus, parasitoid venom is a valuable and underappreciated resource of active molecules for insecticidal or medical purposes4. Unlike the venom of snakes, cone snails, spiders, scorpions, and bees, parasitoid wasp venom cannot be collected by direct stimulation or electrical stimulation5. The current method of extraction of parasitoid wasp venom is to dissect the venom reservoir. However, parasitoid wasps are often small, and dissection of parasitoid wasps requires high technical skills. Therefore, if we can find a way to collect the venom of parasitoid wasps efficiently and conveniently, it will be of great help to research the venom of parasitoid wasps.
Trichogramma (Hymenoptera: Trichogrammatidae) is a genus of tiny (~0.5 mm long) parasitoid wasps6. These wasps are among the most widely used biocontrol agents, particularly targeting eggs of various lepidopteran pests in both agriculture and forests. For example, T. dendrolimi, one of the most widely used Trichogramma species in China, has been extensively applied to control a variety of agricultural and forestry pests, such as Dendrolimus superans, Ostrinia furnacalis, and Chilo suppressalis. Previous studies showed that Trichogramma wasps could inject their eggs into artificial hosts7. Artificial hosts can be created using materials such as wax8, agar9, Parafilm10, and plastic film11. The solution in artificial hosts that induces sufficient oviposition for Trichogramma can be simple, such as amino acids or inorganic salts12. Based on the characteristic that T. dendrolimi can parasitize artificial hosts, this study provides a new method for extracting venom from parasitoid wasps using artificial hosts. This approach aims to address the shortcomings of low yield, low purity, and susceptibility to contamination in current extraction techniques. By using this method, a large amount of high-purity venom from T. dendrolimi can be extracted, which meets the needs of scientific research and screening of bioactive molecules for insecticidal or medical purposes.

<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

Parasitoid wasps are a diverse group of hymenopteran insects that serve as invaluable resources for pest biocontrol. To ensure successful parasitism, parasitoid wasps inject venom into their hosts to suppress their hosts&#39; immunity, modulate hosts&#39; development, metabolism, and even behavior. With over 600,000 estimated species, the diversity of parasitoid wasps surpasses that of other venomous animals, such as snakes, cone snails, and spiders. Parasitoid wasp venom is an underexplored source of bioactive molecules with potential applications in pest control and medicine. However, collecting parasitoid venom is challenging due to the inability to use direct or electrical stimulation and the difficulty in dissection because of their small size. Trichogramma is a genus of tiny (~0.5 mm) egg parasitoid wasps that are widely used for the biological control of lepidopteran pests in both agriculture and forests. Here, we report a method for extracting venom from T. dendrolimi using artificial hosts. These artificial hosts are created with polyethylene film and amino acid solutions and then inoculated with Trichogramma wasps for parasitism. The venom was subsequently collected and concentrated. This method enables the extraction of large amounts of Trichogramma venom while avoiding contamination from other tissues caused by dissection, a common issue in venom reservoir dissection protocols. This innovative approach facilitates the study of Trichogramma venom, paving the way for new research and potential applications.

The protein concentration of representative venom samples was measured using the protein assay kit, with the results presented in Table 1. The results showed that the concentration of venom protein collected by this method ranged from 0.35 &#181;g/&#181;L to 0.46 &#181;g/&#181;L, while the negative control of amino acid solution only had a protein concentration of 0.03 &#181;g/&#181;L to 0.05 &#181;g/&#181;L. The concentration of venom protein collected by this method is much higher than that of negative...

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

Author Spotlight: Efficient Venom Extraction Method from Trichogramma Parasitoid Wasps 

Here, we present a protocol for extracting venom from Trichogramma dendrolimi using an artificial host created with polyethylene film and amino acid solution.

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

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

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

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

1.4K Views.  Nanjing Agricultural University. Here, we present a protocol for extracting venom from Trichogramma dendrolimi using an artificial host created with polyethylene film and amino acid solution.

Video: Author Spotlight: Efficient Venom Extraction Method from Trichogramma Parasitoid Wasps 

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.