We gratefully acknowledge Agriculture and Agri-Food Canada for the funding under which this research was made possible (projects J-001843 and J-002021). We thank Brett van Heyningen for filming the video content for this protocol. We would also like to thank former graduate students (Jennifer Vacon and Mark Nabuurs) for their insights into the methods described in this manuscript.

The details of the reagents and the equipment used in this study are listed in the Table of Materials.
1. Selecting the microbial biocontrol candidates
<ol>
	<li>Using competition plate assays, select microbial isolates that display antagonism against the pathogen of interest. 
	NOTE: Nine Bacillus isolates that exhibited antagonism against S. sclerotiorum were selected, including Bacillus atrophaeus, mojavensis and subtilis species, to demonstrate this protocol.</li>
</ol>
2. Media preparation
<ol>
	<li>Prepare potato dextrose agar (PDA) by dissolving 39 g of medium per liter of water.</li>
	<li>Prepare Pseudomonas F agar by adding 45 g of media per liter of water.</li>
	<li>Prepare potato dextrose broth (PDB) with 1% agar by adding 24 g of medium, and 0.24 g agar per liter of water.</li>
	<li>Prepare yeast-glucose-manganese (YGM) broth. Create a buffer solution consisting of 2.5 g of KH2PO4 and 2.5 g K2HPO4 in 100 mL of water, and a salt solution consisting of 0.58 g of MnSO4.H20, 0.5 g of NaCl and 0.05 g of FeSO4.7H20 in 100 mL of water.
	<ol>
		<li>Next, add 1 g of yeast extract, 1.25 g of dextrose, 400 mL of water, 5 mL of the buffer solution, 5 mL of the salt solution and 90 mL of water to ensure that the metal salts do not precipitate in solution.</li>
	</ol>
	</li>
	<li>Autoclave-sterilize the media, pour the agar into 100 x 15 mm Petri plates, and let all cool to room temperature before use.</li>
</ol>
3. Pathogen preparation
<ol>
	<li>Culture five plates of Sclerotinia sclerotiorum on PDA (prepared in step 2.1) and incubate at 25 &#176;C for 3 days.</li>
</ol>
4. Natural product extract preparation
<ol>
	<li>Culture each bacterial isolate on Pseudomonas F Agar (prepared in step 2.2) and grow until individual colonies are observed.</li>
	<li>Subculture single bacterial colonies into 25 mL of YGM media in centrifuge or culture tubes and incubate with shaking for 3 days.</li>
	<li>Transfer 5 mL of aliquots of each extract into 1 L of YGM and incubate in the same conditions for another 3 days.</li>
	<li>Centrifuge each culture at 3000 x g for 15 min at 25 &#176;C to pellet the cells.</li>
	<li>Decant and wash the supernatant three times with ethyl acetate to extract metabolites from the culture medium.</li>
	<li>Combine and dry the ethyl acetate layer of each using rotary evaporation.</li>
	<li>Re-dissolve the extract in minimal methanol, transfer it to a small scintillation vial, and dry it again by rotary evaporation. 
	NOTE: If the extract dries to an oily or resinous material, lyophilize the material to obtain a dry powder that can be accurately weighed.</li>
</ol>
5. TLC plate preparation
<ol>
	<li>Prepare the 20 cm x 20 cm TLC plate by drawing a line 2 cm from the bottom and 2 cm from the top of the plate. On the bottom line, place nine dots 2 cm apart. Above the top line, place four dots 4 cm apart.</li>
	<li>Dissolve 5 mg of each extract in 15 &#181;L of methanol and apply 5 &#181;L of each extract on the nine dots marked on the bottom line using a pipette. Let each extract spot dry on the TLC plate and apply another 5 &#181;L aliquot to the same spot. Repeat until all of the material is loaded onto the TLC plate.</li>
	<li>Develop the TLC plate in a developing tank using a 1:2 solution of dichloromethane and methanol until the solvent reaches the line marked 2 cm from the top of the plate (approximately 45 min).</li>
	<li>Once developed, remove the TLC plate and apply a series of positive controls on the four dots marked above the solvent line. Four concentrations of the same control are used. For S. scl., 0.5 &#181;g/mL, 5 &#181;g/mL, 50 &#181;g/mL, and 500 &#181;g/mL of Hygromycin B are positive controls.</li>
	<li>Before all solvent evaporates from the plate, place it in an ethanol-sterilized TLC plate box.</li>
</ol>
6. Direct bioautography assay
<ol>
	<li>Autoclave the materials to be used in the assay (glass components of the chromatography sprayer, metal spatula, four sheets of paper towel, cellulose or filter paper, water, and media) in a large beaker covered with tinfoil. 
	NOTE: Paper materials (paper towel, cellulose paper) should be wrapped in tin foil to avoid becoming wet in the autoclave.</li>
	<li>Connect an air source, pressure gauge, and chromatography sprayer using ethanol-sterilized PTFE tubing. Connect a HEPA filter between the chromatography sprayer and the pressure gauge.</li>
	<li>Collect the mycelial mat of the five pathogen plates using the sterile metal spatula into a 50 mL centrifuge tube using a sterile spatula. 
	NOTE: Use a small volume of liquid broth to help dislodge the mycelia if needed, and transfer with a sterile pipette.</li>
	<li>Add 25 mL of PDB amended with agar and sterile glass beads to the 50 mL centrifuge tube and vortex for 5 min to break up the mycelial mat.</li>
	<li>Transfer the mycelial suspension to a chromatography sprayer using a sterile syringe with a needle tip to ensure large pieces of mycelia will not clog the chromatography sprayer.</li>
	<li>Place the previously developed TLC plates on sterile paper towels in a laminar flow hood to reduce hand contact with the plate while applying the media suspension.</li>
	<li>Increase the air pressure to approximately 4 bars and apply three coats of the suspension to the TLC plate, allowing the plate to dry completely between applications. 
	NOTE: Three layers were found to be the optimal amount. Less than this, and the pathogen does not have enough media to grow on. More than this, the media is too thick, which may not allow for pathogen contact with active metabolites.</li>
	<li>Add 500 mL of sterile water into a sterilized plastic plate box and place sterilized folded cellulose sheets or filter paper on each side to hold moisture.</li>
	<li>Place the completed assay plate onto four empty Petri dishes in the box.</li>
	<li>Incubate the assay for 3 days to 1 week or until an even layer of mycelia grows evenly across the TLC plate except for around the positive controls and zone of inhibition (ZOIs).</li>
</ol>
7. Liquid chromatography mass spectrometry analysis
<ol>
	<li>Image the completed assay using photography.</li>
	<li>Calculate the retention factor for each zone of inhibition by dividing the distance from the bottom line to the ZOI by the distance from the bottom line to the top line.</li>
	<li>Scrape the silica off the inhibition zones and place it into microcentrifuge tubes.</li>
	<li>Add 500 &#181;L of methanol to each tube and vortex to extract the metabolites from the silica.</li>
	<li>Centrifuge at 8,500 x g at 20 &#176;C for 10 min, and transfer the supernatant to a small vial.</li>
	<li>Evaporate to dryness by rotary evaporation or under a stream of nitrogen.</li>
	<li>Re-suspend the extract in 50 &#181;L of methanol in an LC vial.</li>
	<li>Analyze the metabolites in the zone of inhibition by LC-MS and compare them to the metabolites in the crude extract to determine which metabolites in the crude extract are responsible for the inhibition of the pathogen16.</li>
	<li>Using the genus and species of the source microorganism and the masses identified in the ZOI, search the literature and relevant databases such as Antibase and the Dictionary of Natural Products to identify the metabolites.</li>
</ol>

Using TLC-Direct Bioautography Assay to Identify Natural Products for Biocontrol

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resilience+in+agriculture+through+crop+diversification:+Adaptive+management+for+environmental+change.">Resilience in agriculture through crop diversification: Adaptive management for environmental change.</a> BioSci. 61 (3), 183-193 (2011).</li><li>Piquerez, S. J., Harvey, S. E., Beynon, J. L., Ntoukakis, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+crop+disease+resistance:+Lessons+from+research+on+arabidopsis+and+tomato.">Improving crop disease resistance: Lessons from research on arabidopsis and tomato.</a> Front Plant Sci. 5, 671 (2014).</li><li>Damalas, C., Koutroubas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+and+recent+developments+in+biopesticide+use.">Current status and recent developments in biopesticide use.</a> Agriculture. 8 (1), 13 (2018).</li><li>Syed Ab Rahman, S. F., Singh, E., Pieterse, C. M. J., Schenk, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+microbial+biocontrol+strategies+for+plant+pathogens.">Emerging microbial biocontrol strategies for plant pathogens.</a> Plant Sci. 267, 102-111 (2018).</li><li>Tudi, M., 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=Exposure+routes+and+health+risks+associated+with+pesticide+application.">Exposure routes and health risks associated with pesticide application.</a> Toxics. 10 (6), 335 (2022).</li><li>Santos, E. N., 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=Bacillus+thuringiensis:+From+biopesticides+to+anticancer+agents.">Bacillus thuringiensis: From biopesticides to anticancer agents.</a> Biochimie. 192, 83-90 (2022).</li><li>Kumar, J., Ramlal, A., Mallick, D., Mishra, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+some+biopesticides+and+their+importance+in+plant+protection+for+commercial+acceptance.">An overview of some biopesticides and their importance in plant protection for commercial acceptance.</a> Plants. 10 (6), 1185 (2021).</li><li>Marrone, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pesticidal+natural+products+-+status+and+future+potential.">Pesticidal natural products - status and future potential.</a> Pest Manag Sci. 75 (9), 2325-2340 (2019).</li><li>Ayilara, M. S., 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=Biopesticides+as+a+promising+alternative+to+synthetic+pesticides:+A+case+for+microbial+pesticides,+phytopesticides,+and+nanobiopesticides.">Biopesticides as a promising alternative to synthetic pesticides: A case for microbial pesticides, phytopesticides, and nanobiopesticides.</a> Front. Microbiol. 14, 1040901 (2023).</li><li>Abdelaziz, A. M., 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=Biocontrol+of+soil+borne+diseases+by+plant+growth+promoting+rhizobacteria.">Biocontrol of soil borne diseases by plant growth promoting rhizobacteria.</a> Trop. Plant Pathol. 48 (2), 105-127 (2023).</li><li>Zhao, Z., Liu, D., Ruan, L., Wang, T., Liang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+mechanism+of+Bacillus+amyloliquefaciens+SC-B15+and+its+application+in+cereal+mildewproof+and+grape+preservation.">Antifungal mechanism of Bacillus amyloliquefaciens SC-B15 and its application in cereal mildewproof and grape preservation.</a> Food Biosci. 56, 103287 (2023).</li><li>Jamshidi-Aidji, M., Dimkic, I., Ristivojevic, P., Stankovic, S., Morlock, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect-directed+screening+of+bacillus+lipopeptide+extracts+via+hyphenated+high-performance+thin-layer+chromatography.">Effect-directed screening of bacillus lipopeptide extracts via hyphenated high-performance thin-layer chromatography.</a> J Chromatogr A. 1605, 460366 (2019).</li><li>Prichystal, J., Schug, K. A., Lemr, K., Novak, J., Havlicek, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+analysis+of+natural+products.">Structural analysis of natural products.</a> Anal Chem. 88 (21), 10338-10346 (2016).</li><li>De Souza, C. G., 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=Simultaneous+quantification+of+lipopeptide+isoforms+by+UPLC-MS+in+the+fermentation+broth+from+Bacillus+subtilis+CNPMS22.">Simultaneous quantification of lipopeptide isoforms by UPLC-MS in the fermentation broth from Bacillus subtilis CNPMS22.</a> Anal Bioanal Chem. 410 (26), 6827-6836 (2018).</li><li>Dewanjee, S., Gangopadhyay, M., Bhattacharya, N., Khanra, R., Dua, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioautography+and+its+scope+in+the+field+of+natural+product+chemistry.">Bioautography and its scope in the field of natural product chemistry.</a> J Pharm Anal. 5 (2), 75-84 (2015).</li><li>Choma, I., Jesionek, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLC-direct+bioautography+as+a+high+throughput+method+for+detection+of+antimicrobials+in+plants.">TLC-direct bioautography as a high throughput method for detection of antimicrobials in plants.</a> Chromatography. 2 (2), 225-238 (2015).</li><li>Attia, R., 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=Thin-layer+chromatography-bioautographic+method+for+the+detection+of+arginase+inhibitors.">Thin-layer chromatography-bioautographic method for the detection of arginase inhibitors.</a> J Sep Sci. 43 (12), 2477-2486 (2020).</li><li>Legerska, B., Chmelova, D., Ondrejovic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLC-bioautography+as+a+fast+and+cheap+screening+method+for+the+detection+of+alpha-chymotrypsin+inhibitors+in+crude+plant+extracts.">TLC-bioautography as a fast and cheap screening method for the detection of alpha-chymotrypsin inhibitors in crude plant extracts.</a> J Biotechnol. 313, 11-17 (2020).</li><li>Agatonovic-Kustrin, S., Doyle, E., Gegechkori, V., Morton, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-performance+thin-layer+chromatography+linked+with+(bio)assays+and+FTIR-ATR+spectroscopy+as+a+method+for+discovery+and+quantification+of+bioactive+components+in+native+Australian+plants.">High-performance thin-layer chromatography linked with (bio)assays and FTIR-ATR spectroscopy as a method for discovery and quantification of bioactive components in native Australian plants.</a> J Pharm Biomed Anal. 184, 113208 (2020).</li><li>Hilaire, V., 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=New+method+for+screening+anti-leishmania+compounds+in+plants+extracts+by+HPTLC-bioautography.">New method for screening anti-leishmania compounds in plants extracts by HPTLC-bioautography.</a> J Chromatogr B. 1188, 123061 (2022).</li><li>Stankovic, J., 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=HPTLC-direct+bioautography-guided+isolation+of+isogeranic+acid+as+the+main+antibacterial+constituent+of+Artemisia+santonicum+essential+oil.">HPTLC-direct bioautography-guided isolation of isogeranic acid as the main antibacterial constituent of Artemisia santonicum essential oil.</a> J Serb Chem Soc. 84 (12), 1355-1365 (2019).</li><li>Su, Y., Qi, Y., Cai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+sporulation+in+plant+pathogenic+fungi.">Induction of sporulation in plant pathogenic fungi.</a> Mycology. 3, 195-200 (2012).</li><li>Wedge, E., Nagle, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+2D-TLC+bioautography+method+for+the+discovery+of+novel+antifungal+agents+to+control+plant+pathogens.">A new 2D-TLC bioautography method for the discovery of novel antifungal agents to control plant pathogens.</a> J Nat Prod. 63, 1050-1054 (2000).</li></ol>

Dr. Susan Boyetchko passed away prior to the submission of this work (Feb 8, 2023). All other authors have declared to have no conflicts of interest.

TLC-DB is a valuable and well-established NP research tool and a simple and inexpensive alternative to microplate bioassay-guided isolation methods17. It requires minimal time and material resources compared to microplate assays, which require metabolite separation using liquid chromatography techniques. It is a highly versatile assay that can be used to detect antibacterial, antifungal, anti-parasitic, and antioxidant NPs in addition to enzyme inhibitors18,19,20,21,22,23. Though most commonly used for detecting and identifying bioactive phytochemicals, the same method can be applied for bacterial NPs as explored in this protocol18. Additionally, the protocol can be optimized for use with a variety of NP sources and target pathogens to aid in discovering and assessing novel bioactive NPs.
The media used for bacterial growth and solvent used for extraction can greatly impact natural product discovery results. The media used in this protocol is optimized for bacteria that produce cyclic lipopeptides, including Pseudomonas and Bacillus spp. but other media and nutrient sources should be considered if exploring other genera. One may choose to complete TLC-DB using the same microorganism grown in a range of media to evaluate the full breadth of metabolite diversity from one isolate or use identical growth conditions for a wider range of isolates. The choice of extraction solvent also impacts the natural products detected. It is generally understood that most bioactive NPs have low to moderate polarity, making ethyl acetate a suitable choice due to its low boiling point, which makes it easy to remove. However, if one also wishes to examine the polar and non-polar fractions, multiple extractions with other solvents can be carried out. Alternatively, the cell-free extract can be lyophilized and used in the assay to see all metabolites released into the media. However, more material will often need to be loaded onto the TLC plate to account for the dried media components in the lyophilized material. Similarly, if this method is used with a different pathogen, such as inoculum, the media used and incubation conditions must be optimized to obtain the 5 agar plates of mycelium used for the TLC-DB assay.
TLC-DB is advantageous compared to contact and immersion bioautography as it uses the thinnest layer of agar and inoculum, minimizing the reliance on the diffusion of metabolites into the agar layer, which can allow smaller amounts of natural products to induce pathogen inhibition17. Previously published findings using TLC bioautographic methods have used a spore suspension of the pathogen to complete the assay17. Although this does allow for precise control over the suspension concentration, it can be exceedingly difficult and time-consuming to induce the sporulation of certain fungi24. This modification greatly simplifies the assay and allows for the completion of the assay using fungal pathogens that are difficult to sporulate and may have previously been avoided for this method.
The mass of bacterial extract used in the assay can impact results. If too little extract is applied to the TLC plate, it is possible that the minimum inhibitory concentration of an active compound will not be surpassed, and bioactivity will not be detected. As a result, in some cases, and as seen in Figure 1 and Figure 2, it is worthwhile to overload the TLC plate, compromising separation for the ability to easily detect activity. Similarly, the pathogen load sprayed onto the plate must not be too low, as there will not be enough media applied to support pathogen growth. This method can be easily tuned to accommodate a variety of bacterial extracts and pathogens, and the quantities of microbial extract and inoculum outlined in the protocol have ensured bioactivity can be detected for multiple pathogens and microbial extracts. If no zones of inhibition are observed upon the completion of the assay, it may indicate one of the following. First, active metabolites may not be present in the extract applied due to incompatible media being used for bacterial growth or due to the activity of the bacteria not being a result of the NP production. A disc diffusion assay with the extract can be completed to confirm or deny the existence of active NPs in the extract. If the disk diffusion assay shows no pathogen suppression, other media can be tested to determine if other conditions produce bioactive NPs. If the disk diffusion assay does indicate that the extract suppresses the pathogen, then a larger mass of the bacterial extract may need to be applied to the TLC plate, in which case another assay can be attempted.
Comparing metabolites in the ZOI to the crude extract is essential for identifying active NPs. In the assay, metabolites from the crude extract can be metabolized or modified by the pathogen, which can be observed via LC-MS. Thus, only metabolites that occur in both the crude extract and the ZOI can be considered as NPs produced by the bacteria under study. If one cannot correlate the metabolites extracted from the ZOI to metabolites in the crude extract, a TLC plate can be prepared, as described in step 5. Without completing the bioautography assay, extract the metabolites from the TLC plate at the same retention time observed in the completed assay. This should allow for an easier correlation between the metabolites in the ZOIs and crude extract, causing pathogen suppression.
One drawback of TLC-DB is that the resolution of TLC is considerably less than that achieved when using traditional microwell screening techniques, which require liquid chromatography for separation. Thus, it is common for multiple metabolites to exist in the zone of inhibition where some metabolites may not be contributing to the bioactivity. This issue can be further caused by the practice of overloading the TLC plate to observe bioactivity more clearly. Recent work has been published using high-performance TLC (HP-TLC), which greatly improves resolution and can allow for the automation of TLC development that is otherwise impossible when using conventional TLC14,21,22,23. Also, plates can be developed in a second dimension (2D-TLC) to further separate metabolites with similar retention times. That being said, one should evaluate whether the increased time and material cost is a worthwhile compromise for increased resolution obtained from HP- and 2D-TLC25.

Fungal agricultural pathogens cause significant losses in crop quality and yields worldwide, contributing to the economic and supply challenges of a stable global food production system1,2. Pathogen damage can be prevented by breeding cultivars resistant to infection and using integrated crop management systems, including crop rotations and land management practices to suppress pathogen proliferation3,4. Though these methods reduce damage to crops, chemical pesticides are generally used in tandem to actively kill fungal reproductive structures in the field and further prevent damage and yield reductions. Although effective, chemical pesticide usage has many drawbacks, including damage to surrounding ecosystems, a decline in soil fertility, associated human health risks, and the development of pathogen resistance, the latter causing higher doses of pesticides to be required each year5,6,7.
Microbial-based pest and pathogen management products have long been considered potential alternatives or complementary to synthetic pesticides. Since the early 1900s, Bacillus thuringiensis has been widely used to control agricultural pests and pathogens as a seed treatment, as a foliar spray, and in direct soil treatment8. Such products have been named biopesticides and are characterized as naturally occurring microorganisms or biochemicals that can kill, suppress, or reduce the vigor of a target pest or pathogen. Biopesticides can control the growth of a pathogen through various mechanisms but most commonly do so through the secretion of secondary metabolites9. Secondary metabolites, often referred to as natural products, are not involved in primary metabolism but are produced as an evolutionary advantage to outcompete other microorganisms10.
Biopesticides offer many advantages over their synthetic counterparts. They pose a low toxicity risk to the environment, fauna, and humans compared to synthetic pest management products9,10. Since most biopesticides have existed naturally in the environment for millennia, biodegradation pathways for microbial metabolites exist in the environment, limiting the possibility of soil or ecosystem contamination and reducing residence times that contribute to synthetic pesticides being so environmentally destructive11. Additionally, many biopesticides used for mitigating pathogen infection also exhibit plant growth-promoting properties, which can increase nutrient bioavailability and induce plant systemic resistance12.
Most commonly, biopesticides are applied in the form of microbial inoculum, and NPs are secreted by live microorganisms in situ12,13. In such a case, identifying the source of the activity of a biopesticide is of great value. Doing so provides insight into the mechanism of action of the biopesticide, aids with building a case for the protection of a microorganism with a patent, and can have a significant scientific impact if their structures are novel. Most importantly, however, identification of the bioactive source informs formulation possibilities for a downstream biopesticide product. If the NP itself is active, one can use the microorganism as a biomolecule factory for large-scale biopesticide production. Additionally, many NPs that have been explored for biocontrol also have potential applications in human medicine, making them even more economically valuable8.
Thin layer chromatography-direct bioautography (TLC-DB) assays are an inexpensive and straightforward method for the bioactivity-guided isolation and identification of biopesticidal metabolites. Although the technique is commonly used for separating bioactivity testing of phytochemicals from crude plant extracts, it also has great potential for the analysis of microbial extracts14. TLC provides fast and inexpensive separation of NPs in a crude microbial extract, and after coating with a media pathogen suspension, zones that contain active metabolites are easily visualized. Those zones can be scraped from the plate and extracted for chemical analysis by ultra-high-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) to identify known metabolites. Metabolites that do not match previously reported compounds can be isolated in larger quantities via liquid chromatography to undergo structure elucidation studies using techniques such as nuclear magnetic resonance spectroscopy and X-ray crystallography15.
This article describes a model system for discovering and identifying biopesticide NPs using TLC-DB with Bacillus spp. and the agricultural pathogen Sclerotinia sclerotiorum. Figure 1 provides a schematic overview of the TLC-DB procedure.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66967/66967fig01.jpg" /> 
Figure 1: Schematic overview of steps 4-7 of the procedure of TLC-DB. <a href="https://www.jove.com/files/ftp_upload/66967/66967fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5-5 mL single channel Pipette</td>
			<td>VWR</td>
			<td>CA11020-004</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Thin Layer Chromatography Sprayer</td>
			<td>VWR</td>
			<td>KT422530-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>100 x 15 mm Petri plates</td>
			<td>VWR</td>
			<td>89038-970</td>
			<td></td>
		</tr>
		<tr>
			<td>100-1000 &#181;L pipette tips</td>
			<td>VWR</td>
			<td>76322-164</td>
			<td></td>
		</tr>
		<tr>
			<td>100-1000 &#181;L single channel pipette</td>
			<td>VWR</td>
			<td>76169-240</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL sterile centrifuge tubes</td>
			<td>VWR</td>
			<td>CA21008-918</td>
			<td></td>
		</tr>
		<tr>
			<td>1 L glass bottle</td>
			<td>Millipore Sigma</td>
			<td>CLS13951L</td>
			<td>Must be autoclave safe</td>
		</tr>
		<tr>
			<td>1 mL sterile syringe with needle</td>
			<td>Thomas Scientific</td>
			<td>8935L75</td>
			<td>Detachable needle is recommended</td>
		</tr>
		<tr>
			<td>2 mL Microcentrifuge tube</td>
			<td>VWR</td>
			<td>87003-298</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL sterile centrifuge tubes</td>
			<td>VWR</td>
			<td>CA21008-940</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL pipette tips</td>
			<td>VWR</td>
			<td>CA11020-008</td>
			<td></td>
		</tr>
		<tr>
			<td>7 mL scintilation vials</td>
			<td>VWR</td>
			<td>76538-962</td>
			<td></td>
		</tr>
		<tr>
			<td>95% ethanol</td>
			<td>Thermo Fisher Scientific</td>
			<td>A412-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>Cole-Parmer</td>
			<td>UZ-01850-34</td>
			<td>8 L, 115 VAC</td>
		</tr>
		<tr>
			<td>Bacteriological agar</td>
			<td>VWR</td>
			<td>97064-336</td>
			<td></td>
		</tr>
		<tr>
			<td>bin</td>
			<td>Thomas Scientific</td>
			<td>1216H91</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>VWR</td>
			<td>BDH9230-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane &#8805;99.8% ACS</td>
			<td>VWR</td>
			<td>BDH1113-4LG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Acetate &#8805;99.8% ACS</td>
			<td>VWR</td>
			<td>BDH1123-4LG</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>VWR</td>
			<td>CA28297-846</td>
			<td></td>
		</tr>
		<tr>
			<td>Grinding Beads</td>
			<td>VWR</td>
			<td>12621-148</td>
			<td></td>
		</tr>
		<tr>
			<td>Hygromycin B</td>
			<td>VWR</td>
			<td>CA80501-074</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron Sulfate Heptahydrate</td>
			<td>VWR</td>
			<td>97061-542</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td>CleanTech</td>
			<td>1000-6-A</td>
			<td></td>
		</tr>
		<tr>
			<td>LC-MS</td>
			<td>Waters</td>
			<td>LITR10064178</td>
			<td>UPLC/MS/MS TQD system</td>
		</tr>
		<tr>
			<td>Lyophilizer</td>
			<td>Labconco</td>
			<td>700201000</td>
			<td>Temperature collector&#160;-50 &#176;C&#160;</td>
		</tr>
		<tr>
			<td>Manganese Sulfate Hydrate</td>
			<td>VWR</td>
			<td>CAAA10807-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol &#8805;99.8% ACS</td>
			<td>VWR</td>
			<td>BDH2018-5GLP</td>
			<td></td>
		</tr>
		<tr>
			<td>Paper towel</td>
			<td>VWR</td>
			<td>89402-824</td>
			<td></td>
		</tr>
		<tr>
			<td>Potato Dextrose Agar</td>
			<td>VWR</td>
			<td>CA90000-758</td>
			<td></td>
		</tr>
		<tr>
			<td>Potato Dextrose Broth</td>
			<td>VWR</td>
			<td>CA90003-494</td>
			<td></td>
		</tr>
		<tr>
			<td>Potsasium Phosphate Dibasic</td>
			<td>VWR</td>
			<td>470302-246</td>
			<td></td>
		</tr>
		<tr>
			<td>Potsasium Phosphate Monobasic</td>
			<td>VWR</td>
			<td>470302-252</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure Gauge 6mm Union Straight 0-10 bar (0-145 psi)</td>
			<td>Tameson</td>
			<td>F25U6</td>
			<td></td>
		</tr>
		<tr>
			<td>Pseudomonas F Agar</td>
			<td>VWR</td>
			<td>90003-352</td>
			<td>Also known as Flo Agar</td>
		</tr>
		<tr>
			<td>PTFE Tubing</td>
			<td>Sigma Aldrich</td>
			<td>58697-U</td>
			<td>1/16 inch inner diameter&#160;</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>VWR</td>
			<td>BDH9286-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Spatula</td>
			<td>VWR</td>
			<td>82027-490</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Inoculation loops with needle</td>
			<td>VWR</td>
			<td>76534-512</td>
			<td></td>
		</tr>
		<tr>
			<td>Tinfoil</td>
			<td>Thomas Scientific</td>
			<td>1086F24</td>
			<td>Can be purchased from supermarket</td>
		</tr>
		<tr>
			<td>TLC Silica Gel 60 RP-18 F254S 25 Glass Plates 20 X 20 cm</td>
			<td>Thomas Scientific</td>
			<td>1205Q12</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Pump</td>
			<td>Labconco</td>
			<td>1472100</td>
			<td>98 L/min</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>VWR</td>
			<td>76549-928</td>
			<td>Must accomadate 15 mL and 50 mL centrifuge tubes</td>
		</tr>
		<tr>
			<td>Whatman in-line HEPA-VENT</td>
			<td>Millipore Sigma</td>
			<td>WHA67235000</td>
			<td>10 filters, 1/4 to 3/8 inch inlet/outlet</td>
		</tr>
		<tr>
			<td></td>
			<td>VWR</td>
			<td>97063-370</td>
			<td></td>
		</tr>
	</tbody>
</table>

bioassay-guided identification of natural products for biocontrol by thin layer chromatography-direct bioautography

Thin layer chromatography-direct bioautography (TLC-DB) is a well-established bioassay used to separate and identify natural products (NPs) that are antagonistic against a target pathogen. It is a rapid, inexpensive, and simple option for the bioassay-guided isolation and identification of NPs that hinges on separation by TLC coupled with the direct application of a target pathogen to examine bioactivity. It is typically used for the analysis of bioactive plant extracts, detecting inhibitory activity against bacteria, fungi, and enzymes. That being said, it has great potential in bacterial NP discovery, particularly for evaluating bacterial NPs against pertinent agricultural pathogens, which is valuable for discovering and developing novel biopesticides for the agriculture industry. Furthermore, it is a tunable protocol that could be applied to other target pathogens or sources of NPs in research programs concerning the discovery and identification of bioactive compounds. Herein, we describe a model system for discovering and identifying biopesticide NPs using TLC-DB with Bacillus spp. and the agricultural pathogen Sclerotinia sclerotiorum.

Upon the separation of microbial extracts by TLC, metabolites should be dispersed along the TLC plate vertically. Under visible light, it can be difficult to view metabolites that do not absorb in the visible light range. Thus, imaging under UV light can help view the separation of metabolites, as seen in Figure 2A,B. After the incubation period, the pathogen should appear to grow evenly across the entire plate except over the positive controls and the inhibition zones where active metabolites reside, pictured in Figure 2C.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66967/66967fig02v2.jpg" /> 
Figure 2: Separation of microbial extracts by TLC. (A) Developed TLC plate containing nine Bacillus extracts imaged under visible light and (B) under 320 nm UV light prior to the application of fungal inoculum. (C) Completed bioautography assay with pathogen growth observed across the plate except for where growth is inhibited around the positive controls, and ZOI of each extract. <a href="https://www.jove.com/files/ftp_upload/66967/66967fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Metabolites extracted from the zones of inhibition are analyzed by LC-MS and compared to the crude extract to determine the NPs responsible for the antagonism against the pathogen. Matching metabolites should have the same retention time and molecular ion species to be considered a match. Once the active metabolites are identified, the bacterial culture can be grown in bulk to isolate active metabolites of interest for structural chemistry or biological study.

Author Spotlight: Discovering New Biopesticides from Bioactive Soil Microbe-Derived Natural Products

We describe the use of thin-layer chromatography direct bioautography assay and liquid chromatography-mass spectrometry to identify microbial natural products that display antagonism against fungal pathogens using the pathogen Sclerotinia sclerotiorum and biopesticidal Bacillus isolates as a model system.

Bioassay-Guided Identification of Natural Products for Biocontrol by Thin Layer Chromatography-Direct Bioautography

Thin layer chromatography-direct bioautography (TLC-DB) is a well-established bioassay used to separate and identify natural products (NPs) that are ...

bioassay-guided-identification-natural-products-for-biocontrol-thin

Research

JoVE Journal

Biochemistry

1.3K Views.  Agriculture and Agri-Food Canada. We describe the use of thin-layer chromatography direct bioautography assay and liquid chromatography-mass spectrometry to identify microbial natural products that display antagonism against fungal pathogens using the pathogen Sclerotinia sclerotiorum and biopesticidal Bacillus isolates as a model system.

Video: Author Spotlight: Discovering New Biopesticides from Bioactive Soil Microbe-Derived Natural Products

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological materials. Such approaches have received considerable attention in research on the boundary between living and nonliving matter and have been used to construct artificial cells over the past two decades. In particular, Giant Vesicles (GVs) have often been used as artificial cell membranes. In this paper, we describe the preparation of GVs encapsulating highly packed microspheres as a model of cells containing highly condensed biomolecules. The GVs were prepared by means of a simple water-in-oil emulsion centrifugation method. Specifically, a homogenizer was used to emulsify an aqueous solution containing the materials to be encapsulated and an oil containing dissolved phospholipids, and the resulting emulsion was layered carefully on the surface of another aqueous solution. The layered system was then centrifuged to generate the GVs. This powerful method was used to encapsulate materials ranging from small molecules to microspheres.

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning because it is involved in microtubule assembly and stabilization. Besides neurons, tau is expressed in human breast, prostate, gastric, colorectal, and pancreatic cancers where it shows nearly similar structure and exerts similar functions as the neuronal tau. The amount of tau and its phosphorylation can change its function as a stabilizer of microtubules, and lead to the development of paired helical filaments in different neurodegenerative disorders, such as Alzheimer&#39;s disease. Determining the phosphorylation state of tau and its microtubule-binding characteristics is important. In addition, examining the intracellular localization of tau is important in different diseases. This manuscript details standard protocols for measuring tau phosphorylation and tau binding to microtubules in colorectal cancer cells with or without curcumin and LiCl treatment. These treatments can be used to stop cancer cell proliferation and development. Intracellular localization of tau is examined by using immunohistochemistry and confocal microscopy while using low amounts of antibodies. These assays can be used repetitively for screening compounds that affect tau hyperphosphorylation or microtubule binding. Novel therapeutics used for different tauopathies or related anticancer agents can potentially be characterized using these protocols.

This manuscript describes standard protocols for measuring tau hyperphosphorylation, measuring tau binding to microtubules, and localization of intracellular tau following drug treatments. These protocols can be used repetitively for screening drugs or other compounds that target tau hyperphosphorylation or microtubule binding.

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning ...

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and identification of an active pheromone compound. This method has resulted in the successful characterization of the chemical signals that function as pheromones in a wide range of animal species. Sea lampreys rely on olfaction to detect pheromones that mediate behavioral or physiological responses. We use this knowledge of fish biology to posit functions of putative pheromones and to guide the isolation and identification of active pheromone components. Chromatography is used to extract, concentrate, and separate compounds from the conditioned water. Electro-olfactogram (EOG) recordings are conducted to determine which fractions elicit olfactory responses. Two-choice maze behavioral assays are then used to determine if any of the odorous fractions are also behaviorally active and induce a preference. Spectrometric and spectroscopic methods provide the molecular weight and structural information to assist with the structure elucidation. The bioactivity of the pure compounds is confirmed with EOG and behavioral assays. The behavioral responses observed in the maze should ultimately be validated in a field setting to confirm their function in a natural stream setting. These bioassays play a dual role to 1) guide the fractionation process and 2) confirm and further define the bioactivity of isolated components. Here, we report the representative results of a sea lamprey pheromone identification that exemplify the utility of the bioassay-guided fractionation approach. The identification of sea lamprey pheromones is particularly important because a modulation of its pheromone communication system is among the options considered to control the invasive sea lamprey in the Laurentian Great Lakes. This method can be readily adapted to characterize the chemical communication in a broad array of taxa and shed light on waterborne chemical ecology.

Here, we present a protocol to isolate and characterize the structure, olfactory potency, and behavioral response of putative pheromone compounds of sea lampreys.

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and ...

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used strategy for RBP capture exploits the polyadenylation [poly(A)] of target RNAs, which mostly occurs on eukaryotic mature mRNAs, leaving most binding proteins of non-poly(A) RNAs unidentified. Here we describe the detailed procedures of a recently reported method termed click chemistry-assisted RNA-interactome capture (CARIC), which enables the transcriptome-wide capture of both poly(A) and non-poly(A) RBPs by combining the metabolic labeling of RNAs, in vivo UV cross-linking, and bioorthogonal tagging.

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

A detailed protocol for applying the click chemistry-assisted RNA-interactome capture (CARIC) strategy to identify proteins binding to both coding and noncoding RNAs is presented.

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

Identification of Functional Protein Regions Through Chimeric Protein Construction

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences in a structurally similar protein, in order to determine the functional importance of these regions. Such chimeras are generated by means of a nested PCR protocol using overlapping DNA fragments and adequately designed primers, followed by their expression within a mammalian system to ensure native secondary structure and post-translational modifications. 
The functional role of a distinct region is then indicated by a loss of activity of the chimera in an appropriate readout assay. In consequence, regions harboring a set of critical amino acids are identified, which can be further screened by complementary techniques (e.g. site-directed mutagenesis) to increase molecular resolution. Although limited to cases in which a structurally related protein with differing functions can be found, chimeric proteins have been successfully employed to identify critical binding regions in proteins such as cytokines and cytokine receptors. This method is particularly suitable in cases in which the protein's functional regions are not well defined, and constitutes a valuable first step in directed evolution approaches to narrow down the regions of interest and reduce the screening effort involved.

Structurally related proteins frequently exert distinct biological functions. The exchange of equivalent regions of these proteins in order to create chimeric proteins constitutes an innovative approach to identify critical protein regions that are responsible for their functional divergence.

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological functions in developmental processes, interorganismal interactions, and environmental adaptation. Due to these various bioactivities, many diterpenoids are also of economic importance as pharmaceuticals, food additives, biofuels, and other bioproducts. Advanced genomics and biochemical approaches have enabled a rapid increase in the knowledge of diterpenoid-metabolic genes, enzymes, and pathways. However, the structural complexity of diterpenoids and the narrow taxonomic distribution of individual compounds in often only a single species remain constraining factors for their efficient production. Availability of a broader range of metabolic enzymes now provide resources for producing diterpenoids in sufficient titers and purity to facilitate a deeper investigation of this important metabolite group. Drawing on established tools for microbial and plant-based enzyme co-expression, we present an easily operated and customizable protocol for the enzymatic production of diterpenoids in either Escherichia coli or Nicotiana benthamiana, and the purification of desired products via silica chromatography and semi-preparative HPLC. Using the group of maize (Zea mays) dolabralexin diterpenoids as an example, we highlight how modular combinations of diterpene synthase (diTPS) and cytochrome P450 monooxygenase (P450) enzymes can be used to generate different diterpenoid scaffolds. Purified compounds can be used in various downstream applications, such as metabolite structural analyses, enzyme structure-function studies, and in vitro and in planta bioactivity experiments.

Here we present easy to use protocols for producing and purifying diterpenoid metabolites through the combinatorial expression of biosynthetic enzymes in Escherichia coli or Nicotiana benthamiana, followed by chromatographic product purification. The resulting metabolites are suitable for various studies including molecular structure characterization, enzyme functional studies, and bioactivity assays.

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological ...

A Planarian Motility Assay to Gauge the Biomodulating Properties of Natural Products

A straightforward, controllable means of using the non-parasitic planarian, Dugesia tigrina, a free-living aquatic flatworm, to study the stimulant and withdrawal properties of natural products is described. Experimental assays benefitting from unique aspects of planarian physiology have been applied to studies on wound healing, regeneration, and tumorigenesis. In addition, because planarians exhibit sensitivity to a variety of environmental stimuli and are capable of learning and developing conditioned responses, they can be used in behavioral studies examining learning and memory. Planarians possess a basic bilateral symmetry and a central nervous system that uses neurotransmitter systems amenable to studies examining the effects of neuromuscular biomodulators. Consequently, experimental systems monitoring planarian movement and motility have been developed to examine substance addiction and withdrawal. Because planarian motility offers the potential for a sensitive, easily standardized motility assay system to monitor the effect of stimuli, the planarian locomotor velocity (pLmV) test was adapted to monitor both stimulation and withdrawal behaviors by planarians through the determination of the number of grid lines crossed by the animals with time. Here, the technique and its application are demonstrated and explained.

Planarian motility is used to gauge the stimulant and withdrawal properties of natural products when compared to the movement of the animals in spring water alone.

A straightforward, controllable means of using the non-parasitic planarian, Dugesia tigrina, a free-living aquatic flatworm, to study the stimulant and ...