Name: a planarian motility assay to gauge the biomodulating properties of natural products
Uploaded: 2020-05-30T14:00:00
Description: Watch this Scientific Journal Video about A Planarian Motility Assay to Gauge the Biomodulating Properties of Natural Products at JoVE.com

The authors wish to acknowledge the office of Institutional Advancement, and the Morrisville College Foundation for a publication grant to support this work, as well as the SUNY Morrisville Collegiate Science and Technology Entry Program (CSTEP) for their ongoing assistance and support of undergraduate research at SUNY Morrisville. We also wish to thank Sophia Hutchens for helpful comments on the technique described.

1. Planarian husbandry
<ol>
	<li>Use planarians purchased from a biological supply company or wild-caught if needed. The planarians used in this protocol are Dugesia tigrina, as listed in the supply list. This species is also referred to as Girardia tigrina31. Other aquatic planarian species are also acceptable2,3. 
	NOTE: The protocol described is geared to animals purchased from a biological supply company. This ...

<ol>
	<li>Moustakas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guarana+provides+additional+stimulation+over+caffeine+alone+in+the+planarian+model.">Guarana provides additional stimulation over caffeine alone in the planarian model.</a> PloS One. 10 (4), 0123310 (2015).</li><li>Raffa, R. B., Valdez, 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=Cocaine+withdrawal+in+Planaria.">Cocaine withdrawal in Planaria.</a> European Journal of Pharmacology. 430 (1), 143-145 (2001).</li><li>Raffa, R. B., Holland, L. J., Schulingkamp, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+dopamine+D2+antagonist+activity+using+invertebrate+(Planaria)+locomotion+as+a+functional+endpoint.">Quantitative assessment of dopamine D2 antagonist activity using invertebrate (Planaria) locomotion as a functional endpoint.</a> Journal of Pharmacology and Toxicological Methods. 45 (3), 223-226 (2001).</li><li>Thum&#233;, I. S., Frizzo, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sertraline+induces+toxicity+and+behavioral+alternations+in+planarians.">Sertraline induces toxicity and behavioral alternations in planarians.</a> Biomedical Research International. 2017, 5792621 (2017).</li><li>Aggarwal, 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=Identification+of+a+novel+allosteric+modulator+of+the+human+dopamine+transporter.">Identification of a novel allosteric modulator of the human dopamine transporter.</a> ACS Chemical Neuroscience. 10 (8), 3718-3730 (2019).</li><li>Zhang, C., Tallarida, C. S., Raffa, R. B., Rawls, 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=Sucrose+produces+withdrawal+and+dompamine-sensitive+reinforcing+effects+in+planarians.">Sucrose produces withdrawal and dompamine-sensitive reinforcing effects in planarians.</a> Physiology &amp; Behavior. , 8-13 (2013).</li><li>Zewde, 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=PLDT+(planarian+light/dark+test):+an+invertebrate+assay+to+quantify+defensive+responding+and+study+anxiety-like+effects.">PLDT (planarian light/dark test): an invertebrate assay to quantify defensive responding and study anxiety-like effects.</a> Journal of Neuroscience Methods. 293, 284-288 (2018).</li><li>Risse, B., Otto, N., Berh, D., Jiang, X., Kl&#228;mbt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FIM+Imaging+and+FIMtrack:+two+new+tools+allowing+high-throughput+and+cost+effective+locomotion+analysis.">FIM Imaging and FIMtrack: two new tools allowing high-throughput and cost effective locomotion analysis.</a> Journal of Visualized Experiments. (94), e52207 (2014).</li><li>Inoue, T., Hoshino, H., Yamashita, T., Shimoyama, S., Agata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planarian+shows+decision-making+behavior+in+response+to+multiple+stimuli+by+integrative+brain+function.">Planarian shows decision-making behavior in response to multiple stimuli by integrative brain function.</a> Zoological Letters. 1, 7 (2015).</li><li>Hastrom, D., Cochet-Escartin, O., Zhang, S., Khuu, C., Collins, E. M. 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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=Planarians+as+models+of+cadmium-induced+neoplasia+provide+measurable+benchmarks+for+mechanistic+studies.">Planarians as models of cadmium-induced neoplasia provide measurable benchmarks for mechanistic studies.</a> Ecotoxicology and Environmental Safety. 142, 544-554 (2017).</li><li>Van Roten, A., 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+carcinogenic+trigger+to+study+the+function+of+tumor+suppressor+genes+in+Schmedtea+mediterranea.">A carcinogenic trigger to study the function of tumor suppressor genes in Schmedtea mediterranea.</a> Disease Models and Mechanisms. 11 (9), 032573 (2018).</li><li>Mason, P. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electrical+response+of+the+planarian+ocellus.">The electrical response of the planarian ocellus.</a> Journal of General Physiology. 51 (2), 255-260 (1968).</li><li>Inoue, T., Yamashita, T., Agata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermosensory+signaling+by+TRPM+is+processed+by+brain+serotonergic+neurons+to+produce+planarian+thermotaxis.">Thermosensory signaling by TRPM is processed by brain serotonergic neurons to produce planarian thermotaxis.</a> The Journal of Neuroscience. 34 (47), 15701-15714 (2014).</li><li>Byrne, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+ethanol+on+negative+phototaxis+and+motility+in+brown+planarians+(Dugesia+tigrina).">Effects of ethanol on negative phototaxis and motility in brown planarians (Dugesia tigrina).</a> Neuroscience Letters. 685, 102-108 (2018).</li><li>de Sousa, 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=Transcriptomic+analysis+of+planarians+under+simulated+microgravity+or+8g+demonstrates+that+alteration+of+gravity+induces+genomic+and+cellular+alterations+that+could+facilitate+tumoral+transformation.">Transcriptomic analysis of planarians under simulated microgravity or 8g demonstrates that alteration of gravity induces genomic and cellular alterations that could facilitate tumoral transformation.</a> International Journal of Molecular Sciences. 20 (3), 720 (2019).</li><li>Best, J. B., Rubinstein, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maze+learning+and+associated+behavior+in+planaria.">Maze learning and associated behavior in planaria.</a> Journal of Comparative and Physiological Psychology. 55, 560-566 (1962).</li><li>Shomrat, T., Levin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+training+paradigm+reveals+long-term+memory+in+planarians+and+its+persistence+through+head+regeneration.">An automated training paradigm reveals long-term memory in planarians and its persistence through head regeneration.</a> The Journal Experimental Biology. 216, 3799-3810 (2013).</li><li>Robarts-Galbraith, R. H., Newmark, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+organ+trail:+insights+into+organ+regeneration+in+the+planarian.">On the organ trail: insights into organ regeneration in the planarian.</a> Current Opinion in Genetics &amp; Development. 32, 37-46 (2015).</li><li>Ivancovic, 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=Model+systems+for+regeneration:+planarians.">Model systems for regeneration: planarians.</a> Development. 146 (17), 167684 (2019).</li><li>Herath, S., Lobo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-inhibition+of+Turing+patterns+explains+the+self-organized+regulatory+mechanism+of+planarian+fission.">Cross-inhibition of Turing patterns explains the self-organized regulatory mechanism of planarian fission.</a> Journal of Theoretical Biology. 485, 110042 (2019).</li><li>Itoh, M. T., Shinozawa, T., Sumi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythms+of+melatonin-synthesizing+enzyme+activities+and+melatonin+levels+in+planarians.">Circadian rhythms of melatonin-synthesizing enzyme activities and melatonin levels in planarians.</a> Brain Research. 830 (1), 165-173 (1999).</li><li>Itoh, M. T., Igarashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythm+of+serotonin+levels+in+planarians.">Circadian rhythm of serotonin levels in planarians.</a> Neuroreports. 11 (3), 473-476 (2000).</li><li>Hinrichsen, R. D., 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=Photosensitivity+and+motility+in+planarian+Schmedtea+mediterranea+vary+diurnally.">Photosensitivity and motility in planarian Schmedtea mediterranea vary diurnally.</a> Chronobiology International. 36 (12), 1789-1793 (2019).</li><li>Raffa, R. B., Desai, P. <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+quantification+of+cocaine+withdrawal+signs+in+planaria.">Description and quantification of cocaine withdrawal signs in planaria.</a> Brain Research. 1032 (1-2), 200-202 (2005).</li><li>Pag&#225;n, O. 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=A+cembranoid+from+tobacco+prevents+the+expression+of+induced+withdrawal+behavior+in+planarian+worms.">A cembranoid from tobacco prevents the expression of induced withdrawal behavior in planarian worms.</a> European Journal of Pharmacology. 615 (1-3), 118-124 (2009).</li><li>Rawls, S. M., Patil, T., Yuvasheva, E., Raffa, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+evidence+that+drugs+of+abuse+produce+behavioral+sensitization+and+cross-sensitization+in+planarians.">First evidence that drugs of abuse produce behavioral sensitization and cross-sensitization in planarians.</a> Behavioural Pharmacology. 21 (4), 301-313 (2010).</li><li>Venturini, 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=A+pharmacological+study+of+dopaminergic+receptors+in+planaria.">A pharmacological study of dopaminergic receptors in planaria.</a> Neuropharmacology. 28 (12), 1377-1382 (1989).</li><li>Ouyang, K., 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=Behavioral+effects+of+Spenda,+Equal+and+sucrose:+Clues+from+planarians+on+sweeteners.">Behavioral effects of Spenda, Equal and sucrose: Clues from planarians on sweeteners.</a> Neuroscience Letters. 636, 213-217 (2017).</li><li>Pag&#225;n, O. R., Montgomery, E., Deats, S., Bach, D., Baker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+nicotine-induced,+curare-sensitive,+behavior+in+planarians.">Evidence of nicotine-induced, curare-sensitive, behavior in planarians.</a> Neurochemical Research. 40 (10), 2087-2090 (2015).</li><li>Shibata, N., Agata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+in+planarians:+feeding+and+injection+of+synthetic+dsRNA.">RNA interference in planarians: feeding and injection of synthetic dsRNA.</a> Methods in Molecular Biology. 1774, 455-466 (2018).</li><li>Pag&#225;n, O. 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=Reversal+of+cocaine-induced+planarian+behavior+by+parthenolide+and+related+sesquiterpene+lactones.">Reversal of cocaine-induced planarian behavior by parthenolide and related sesquiterpene lactones.</a> Pharmacology Biochemistry and Behavior. 89 (2), 160-170 (2008).</li><li>Vouga, A., 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=Stereochemistry+and+neuropharmacology+of+a+'bath+salt'+cathinone:+S-enantiomer+of+mephedrone+reduces+cocaine-induced+reward+and+withdrawal+in+invertebrates.">Stereochemistry and neuropharmacology of a 'bath salt' cathinone: S-enantiomer of mephedrone reduces cocaine-induced reward and withdrawal in invertebrates.</a> Neuropharmacology. 91, 109-116 (2015).</li><li>Chan, J. D., Marchant, J. 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A straightforward and accessible planarian motility assay is described to determine the stimulant and withdrawal effects of natural products. As a behavioral model it is necessary to have stringent protocols for scoring movement and clear definitions of any behaviors to standardize observations between different experimenters. The ideas presented offer a demonstration of how this can be achieved. Each laboratory using this protocol should adapt the presented information to suit the effects of the particular product being...

The protocol described uses planarian motility to provide a means of assessing the biomodulating effects of natural substances. It was specifically adapted to determine if these substances function as stimulants, and if they then were associated with a measurable withdrawal behavior1. This assay, known as the planarian locomotor velocity (pLmV) test, was first used to test known pharmacological agents2,3. The application of this planarian motility-based assay has since grown in popularity and has been adopted by different laboratories interested in substances other than natural products4,5. For this assay, a planarian is placed in a Petri dish containing spring water or spring water containing a dissolved biomodulator. Because the dish itself is placed on graph paper, the number of grid lines crossed by the animal with time as it moves about the container can be used to determine&#160;the rate of movement in each condition. The light/dark test, otherwise referred to as the conditioned place preference test (CPP), is another variation on the theme of monitoring planarian motility, and assesses how quickly the animals respond and migrate to a darkened environment6,7. Video tracking of planarian movements can also be analyzed using computer programs and center of mass (COM) tracking8,9,10,11.
Using the planarian as an animal model for such studies offers several advantages over other animals in that the experimenter can easily control the assay environment. Specifically, starving the planarians before experimentation can prevent their exposure to other nutritional or pharmacological agents that might otherwise confound the results, and the specific biomodulator under investigation can be introduced to the planarians simply by adding it directly to the culture water, thereby standardizing exposure. Since planarians have a nervous system and neurotransmitters that are reminiscent of &#8216;higher-order&#8217; animals, the physiology and experimental responses of these animals to neuromuscular stimuli are considered biologically relevant to other organisms12,13,14,15,16. Also, because planarians are relatively inexpensive and straightforward to maintain in the laboratory, they offer an accessible biological model for many investigators.
As an experimental animal, planarians are suitable to a wide range of studies. For example, our group, as well as other investigators use planarians to study tumorigenesis17,18,19. Planarians also exhibit a host of response behaviors to chemical, thermal, gravitational, electrical, photo, and magnetic stimuli that have formed the basis of other assay systems. Some of these effects have been used to study learning and memory in these animals20,21,22,23,24,25,26,27. The primary use of the planarian model in the literature at present focuses on the activity of planarian pluripotent stem cells, called neoblasts, and their role in regeneration28,29,30. Thus, adopting the model described here allows for further study using other planarian-based assays to provide a broader understanding of how natural products and other biomodulators affect the organism.

<table>
	<tbody>
		<tr>
			<td>Bottled Water - 1 Gal.</td>
			<td>Poland Spring</td>
			<td>N/A</td>
			<td>Spring water for planarian culture and to prepare solutions</td>
		</tr>
		<tr>
			<td>Brown Planaria (Dugesia tigrina)</td>
			<td>Carolina Biological Supply Company</td>
			<td>132954</td>
			<td>Brown planaria living (other species are acceptable)</td>
		</tr>
		<tr>
			<td>Flat Paintbrush</td>
			<td>Royal Crafter&#39;s Choice</td>
			<td>9159</td>
			<td>Flat watercolor paintbrushes for cleaning planarian culture containers</td>
		</tr>
		<tr>
			<td>Glass Petri Dish - 10 cm</td>
			<td>Kimax</td>
			<td>N/A</td>
			<td>10 cm diameter (glass) Petri dishes for pLmV assay</td>
		</tr>
		<tr>
			<td>Glass Petri Dish - 5 cm</td>
			<td>Kimax</td>
			<td>N/A</td>
			<td>5 cm and Petri dishes for rinsing planarians during withdrawal experiments and for stimulant habituation</td>
		</tr>
		<tr>
			<td>Grid Paper</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Standard 0.5 cm grid paper for pLmV assay</td>
		</tr>
		<tr>
			<td>iPEVO Visualizer (software)</td>
			<td>iPEVO</td>
			<td>https://www.ipevo.com/software/visualizer</td>
			<td>Document camera software for video capture and recording</td>
		</tr>
		<tr>
			<td>Metalware Set with Support Stand and Retort Ring</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Standard chemistry lab ring stand to hold a cell phone camera if used</td>
		</tr>
		<tr>
			<td>Organic Egg</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Organic egg or beef liver for feeding planarains</td>
		</tr>
		<tr>
			<td>Polycarbonate Bottle w/ Screw-on Cap - 10 mL</td>
			<td>Beckman</td>
			<td>N/A</td>
			<td>Plastic vials to hold 5 to 10 mL volumes for stimulant habituation</td>
		</tr>
		<tr>
			<td>Round Storage Container - 10 cm</td>
			<td>Ziploc</td>
			<td>N/A</td>
			<td>10 cm Round food storage containers for approximately 90 planarians or fewer</td>
		</tr>
		<tr>
			<td>Round Water Paint Brush</td>
			<td>LOEW-Cornell</td>
			<td>N/A</td>
			<td>Small round watercolor paint brushes (numbers 3 to 6) - soft</td>
		</tr>
		<tr>
			<td>Transfer Pipette</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Wide bore (5 mL) plastic transfer pipettes to move planarians</td>
		</tr>
		<tr>
			<td>USB Document Camera</td>
			<td>iPEVO</td>
			<td>CDVU-06IP</td>
			<td>Document camera (or other camera or cell phone camera)</td>
		</tr>
	</tbody>
</table>

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.

The laboratory set up and preparation of the workspace for running the pLmV assay should be completed before experimentation begins. This includes the preparation of the habituation container, rinse container if needed (for withdrawal experiments), Petri dish over laminated grid paper, and properly placed camera (Figure 1). Once all the videos are taken, it is advisable to use a common datasheet to standardize data collection and presentation between investigators (Figur...

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

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 ...

The Planarian Locomotor Velocity Test is a means for quickly assessing stimulant and possible withdrawal effects for a variety of biological modifying agents that might affect motility. This essay is easily controlled and provides a straightforward and accessible method for generating reliable behavioral data in even the most basic research settings. Since the planarian nervous system has a centralized rudimentary brain, a bilateral symmetry and analogous neurotransmitter systems, behavioral studies using these organisms offer preclinical insights into possible vertebrate responses.Experiments can be further affected by physiological pathway modifiers, drugs or even genetically modified planarians to provide insight into the associated biological mechanisms. It is important to have the planarian so that the transfers between containers do not stress them and to standardize the grid line curves between lab members. The best way to obtain consistent results from this behavior essay experiment, is by seeing how experienced users performed them.After setting up a planarian culture, feed the worms ad libitum on a regular twice a week schedule of chopped organic boiled eggs or blended organic beef liver for one to two hours. After feeding, remove any soiled water from the container and use a flat watercolor paint brush to transfer any food debris and slime adhering to the container to a paper towel. Use fresh spring water and gentle swirling or agitation to dislodge the planarians and decamp the worms into a clean container.Slide a Wide Bore Pipette Tip under any planarians remaining adhered to the old container to facilitate their transfer to the new dish and to carefully decant the transfer water. Then, cover the planarians in the new container with fresh spring water. To perform a Planarian Locomotor Velocity Test, first ensure that the five to 10 days starved planarians are fully formed with complete and pigmented heads and tails.If the planarians are ready place one clean 10 centimeter diameter Petri dish onto one piece of pre-laminated grid paper with 0.5 centimeter squares per experimental group and add 20 milliliters of unadulterated spring water for the control group or 20 milliliters of spring water containing the appropriate concentration of the natural product being tested to each dish. Position a camera above the prepared locomotor velocity stimulation dish and add five to 10 milliliters of unadulterated spring water or spring water containing the appropriate concentration of the experimental compound to an appropriately sized habituation container. Next, use a small clean watercolor paint brush to gently transfer one planarian from the culture container to the prepared habituation container and allow the planarian to habituate to the solution in the container for two minutes.Research will allow the planarians to move onto the brush and to carefully lift the animals without forcing them. At the end of the habituation period, use the brush to carefully transfer the planarian to the center of the prepared locomotor velocity stimulation dish containing the same solution as the habituation container and start the camera to record 10 to 11 minutes of movement of the planarian. To test the effects of withdrawal at the end of the habituation period, rinse the planarian in a five centimeter dish containing spring water only.Before transferring the worm to the center of the prepared locomotor velocity dish containing fresh spring water and start the camera to record 10 to 11 minutes of movement. The camera setup allows a clear view of the planarian and the grid paper and permits an accurate assessment of the progress of the animal for the duration of the experiment. The data collection should include the total number of grid lines crossed as well as the cumulative total number of lines crossed per minute of the experiment.To standardize the results from each trial, the planarian locomotor velocity runs can be calculated and plotted as the number of boxes crossed relative to the progress of the matching control worm. As a benchmark, planarians in spring water typically cover approximately 25 boxes in three minutes. A range of test reagent concentrations can be examined to determine planarian motility in response to the test compounds of interest.Withdrawal data can be assessed by quantifying the number of grid lines crossed in spring water relative to the spring water control after the animals were habituated in the bio modulating product being examined. Spring water control planarians move over grid lines for the duration of the experiment. At times however, the test planarians cease crossing grid lines during the essay with behaviors that are characteristic for the product being tested.This suggests that a different method of analysis might be required. Such instances can be tracked and the data can be illustrated as the percentage of the total number of animals exposed to a particular reagent concentration. It is essential to handle the planarian so that they are not damaged during the transfer between dishes.After determining the effects of different concentrations of a natural product, biological pathway modifiers can be added to observe any dynamic changes to the initial results. The Planarian Locomotor Velocity Test was used to test for stimulation and withdrawal using known pharmacological agents. We adapted the system to study these same concepts with natural products.

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Biochemistry

A Rapid Laser Probing Method Facilitates the Non-invasive and Contact-free Determination of Leaf Thermal Properties

Plants can produce valuable substances such as secondary metabolites and recombinant proteins. The purification of the latter from plant biomass can be streamlined by heat treatment (blanching). A blanching apparatus can be designed more precisely if the thermal properties of the leaves are known in detail, i.e., the specific heat capacity and thermal conductivity. The measurement of these properties is time consuming and labor intensive, and usually requires invasive methods that contact the sample directly. This can reduce the product yield and may be incompatible with containment requirements, e.g., in the context of good manufacturing practice. To address these issues, a non-invasive, contact-free method was developed that determines the specific heat capacity and thermal conductivity of an intact plant leaf in about one minute. The method involves the application of a short laser pulse of defined length and intensity to a small area of the leaf sample, causing a temperature increase that is measured using a near infrared sensor. The temperature increase is combined with known leaf properties (thickness and density) to determine the specific heat capacity. The thermal conductivity is then calculated based on the profile of the subsequent temperature decline, taking thermal radiation and convective heat transfer into account. The associated calculations and critical aspects of sample handling are discussed.

A method was developed to determine the specific heat capacity and thermal conductivity of leaf tissue by non-invasive, contact-free near infrared laser probing, which requires less than 1 min per sample.

Plants can produce valuable substances such as secondary metabolites and recombinant proteins. The purification of the latter from plant biomass can be ...

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 ...

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 ...

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, consequently, cardiovascular disease. Although PCSK9 proteolysis is required for its full hypercholesterolemic effect, the evaluation of its proteolytic function is challenging: PCSK9 is only known to cleave itself, undergoes only a single turnover, and after proteolysis, retains its substrate in its active site as an auto-inhibitor. The methods presented here describe an assay which overcomes these challenges. The assay focuses on intermolecular proteolysis in a cell-based context and links successful cleavage to the secreted luciferase activity, which can be easily read out in the conditioned medium. Via sequential steps of mutagenesis, transient transfection, and a luciferase readout, the assay can probe PCSK9 proteolysis under conditions of either genetic or molecular perturbation in a high-throughput manner. This system is well suited for both the biochemical evaluation of clinically discovered missense single-nucleotide polymorphisms (SNPs), as well as for the screening of small-molecule inhibitors of PCSK9 proteolysis.

This protocol presents a method to evaluate the proteolytic activity of an intrinsically low-activity, single turnover protease in a cellular context. Specifically, this method is applied to evaluate the proteolytic activity of PCSK9, a key driver of lipid metabolism whose proteolytic activity is required for its ultimate hypercholesterolemic function.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, ...

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 ...

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A mitochondrial genome of baker&#8217;s yeast encodes eight proteins: three subunits of the cytochrome c oxidase (Cox1p, Cox2p, and Cox3p), three subunits of the ATP synthase (Atp6p, Atp8p, and Atp9p), a subunit of the ubiquinol-cytochrome c oxidoreductase enzyme, cytochrome b (Cytb), and mitochondrial ribosomal protein Var1p. The purpose of the method described here is to specifically label these proteins with 35S methionine, separate them by electrophoresis and visualize the signals as discrete bands on the screen. The procedure involves several steps. First, yeast cells are cultured in a galactose-containing medium until they reach the late logarithmic growth stage. Next, cycloheximide treatment blocks cytoplasmic translation and allows 35S methionine incorporation only in mitochondrial translation products. Then, all proteins are extracted from yeast cells and separated by polyacrylamide gel electrophoresis. Finally, the gel is dried and incubated with the storage phosphor screen. The screen is scanned on a phosphorimager revealing the bands. The method can be applied to compare the biosynthesis rate of a single polypeptide in the mitochondria of a mutant yeast strain versus the wild type, which is useful for studying mitochondrial gene expression defects. This protocol gives valuable information about the translation rate of all yeast mitochondrial mRNAs. However, it requires several controls and additional experiments to make proper conclusions.

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker&#039;s Yeast Saccharomyces cerevisiae

Baker&#8217;s yeast mitochondrial genome encodes eight polypeptides. The goal of the current protocol is to label all of them and subsequently visualize them as separate bands.

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A ...

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of catalytic motor domains, located at opposite ends of the active complex. This unique architecture enables kinesin-5 motors to crosslink and slide apart antiparallel spindle microtubules (MTs), thus providing the outwardly-directed force that separates the spindle poles apart. Previously, kinesin-5 motors were believed to be exclusively plus-end directed. However, recent studies revealed that several fungal kinesin-5 motors are minus-end directed at the single-molecule level and can switch directionality under various experimental conditions. The Saccharomyces cerevisiae kinesin-5 Cin8 is an example of such bi-directional motor protein: in high ionic strength conditions single molecules of Cin8 move in the minus-end direction of the MTs. It was also shown that Cin8 forms motile clusters, predominantly at the minus-end of the MTs, and such clustering allows Cin8 to switch directionality and undergo slow, plus-end directed motility. This article provides a detailed protocol for all steps of working with GFP-tagged kinesin-5 Cin8, from protein overexpression in S. cerevisiae cells and its purification to in vitro single-molecule motility assay. A newly developed method described here helps to differentiate between single molecules and clusters of Cin8, based on their fluorescence intensity. This method enables separate analysis of motility of single molecules and clusters of Cin8, thus providing the characterization of the dependence of Cin8 motility on its cluster size.

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The bi-directional mitotic kinesin-5 Cin8 accumulates in clusters that split and merge during their motility. Accumulation in clusters also changes the velocity and directionality of Cin8. Here, a protocol for motility assays with purified Cin8-GFP and analysis of motile properties of single molecules and clusters of Cin8 is described.

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of ...

Shotgun Lipidomics of Rodent Tissues

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have made lipidomics a powerful analytical tool for monitoring tissue lipidome compositions in homeostatic as well as disease states. This paper presents a step-by-step protocol for a shotgun lipid analysis method that supports the simultaneous detection and quantification of a few hundred molecular lipid species in different tissue and biofluid samples at high throughput. This method leverages automated nano-flow direct injection of a total lipid extract spiked with labeled internal standards without chromatographic separation into a high-resolution mass spectrometry instrument. Starting from sub-microgram amounts of rodent tissue, the MS analysis takes 10 min per sample and covers up to 400 lipids from 14 lipid classes in mouse lung tissue. The method presented here is well suited for studying disease mechanisms and identifying and quantifying biomarkers that indicate early toxicity or beneficial effects within rodent tissues.

Shotgun mass spectrometry-based lipidomics delivers a sensitive quantitative snapshot of a broad spectrum of lipid classes simultaneously in a single measurement from various rodent tissues.

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have ...

Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors

Heat shock protein 90 (Hsp90) is a promising anticancer target because of its chaperoning effect on multiple oncogenic proteins. The activity of Hsp90 is dependent on its ability to hydrolyze adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and free phosphate. The ATPase activity of Hsp90 is linked to its chaperoning function; ATP binds to the N-terminal domain of the Hsp90, and disrupting its binding was found to be the most successful strategy in suppressing Hsp90 function. The ATPase activity can be measured by a colorimetric malachite green assay, which determines the amount of free phosphate formed by ATP hydrolysis. Here, a procedure for determining the ATPase activity of yeast Hsp90 by using the malachite green phosphate assay kit is described. Further, detailed instructions for the discovery of Hsp90 inhibitors by taking geldanamycin as an authentic inhibitor is provided. Finally, the application of this assay protocol through the high-throughput screening (HTS) of inhibitor molecules against yeast Hsp90 is discussed.

The malachite green assay protocol is a simple and cost-effective method to discover heat shock protein 90 (Hsp90) suppressors, as well as other inhibitor compounds against ATP-dependent enzymes.

Heat shock protein 90 (Hsp90) is a promising anticancer target because of its chaperoning effect on multiple oncogenic proteins. The activity of Hsp90 is ...

A Fibrinolytic Enzyme-Based Method to Study Cerebral Thrombus Cell Components

A Comprehensive Approach to Analyze the Cell Components of Cerebral Blood Clots

Cerebral thrombosis, a blood clot in a cerebral artery or vein, is the most common type of cerebral infarction. The study of the cell components of cerebral blood clots is important for diagnosis, treatment, and prognosis. However, the current approaches to studying the cell components of the clots are mainly based on in situ staining, which is unsuitable for the comprehensive study of the cell components because cells are tightly wrapped in the clots. Previous studies have successfully isolated a fibrinolytic enzyme (sFE) from Sipunculus nudus, which can degrade the cross-linked fibrin directly, releasing the cell components. This study established a comprehensive method based on the sFE to study the cell components of cerebral thrombus. This protocol includes clot dissolving, cell releasing, cell staining, and routine blood examination. According to this method, the cell components could be studied quantitatively and qualitatively. The representative results of experiments using this method are shown.

Author Spotlight: A Novel Method for Comprehensive Cell Component Analysis of Cerebral Blood Clots 

This study describes a fast and effective method for the cell component analysis of cerebral blood clots through clot dissolving, cell staining, and routine blood examination.

Cerebral thrombosis, a blood clot in a cerebral artery or vein, is the most common type of cerebral infarction. The study of the cell components of ...