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 assay system has not been tested with wild-caught planarians. However, if wild-caught planarians are used, it is recommended that they be habituated to the water used in the experiments, as well as the laboratory environment for at least 1 week before use.</li>
	<li>Upon arrival, transfer planarians to plastic food storage containers containing clean spring water and keep the lids ajar.</li>
	<li>Maintain planarians in a darkened environment.</li>
	<li>Feed newly delivered planarians after 24&#8211;36 h in their new environment.</li>
	<li>Allow planarians to acclimate to the laboratory at least 1 week prior to experimentation.</li>
	<li>Feed planarians on a regular twice-a-week schedule.
	<ol>
		<li>Allow planarians to feed ad libitum on chopped organic boiled eggs or blended organic beef liver for 1&#8211;2 h.</li>
		<li>Place fed planarians into a clean container after feeding.
		<ol>
			<li>Remove the soiled water off the planarians.</li>
			<li>Use a small flat watercolor paintbrush (number 3&#8211;6) to transfer food debris and slime adhering to the container from around planarians to a paper towel.</li>
			<li>With fresh spring water and gentle swirling or agitation, dislodge planarians and pour them into a clean container.</li>
			<li>Any planarians remaining adhered to the container can be transferred using a round watercolor paintbrush (number 3&#8211;6) or transfer pipette with a wide bore.</li>
			<li>Decant the transfer water.</li>
			<li>Cover the planarians with clean spring water.</li>
			<li>After 24 h, remove the planarians from any expelled food waste by transferring them to a clean container as described above (steps 1.6.2.1&#8211;1.6.2.6).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>To clean containers and utensils used for planarian husbandry, do not use soap or detergent. Clean these items by rinsing them well with clean water (tap water is acceptable) and drying them with a clean cloth or paper towel.</li>
</ol>
2. Preparation of planarians for experiments
<ol>
	<li>Allow newly delivered planarians to acclimate to their environment at least 1 week prior to experimentation.</li>
	<li>Starve planarians for 5&#8211;10 days prior to experimentation.</li>
	<li>Change the culture water at least 1x during the starvation period.</li>
</ol>
3. Planarian locomotor velocity (pLmV) test: Stimulant behaviors
<ol>
	<li>Prior to experimentation, ensure starved planarians are fully formed, with a complete and pigmented head and tail.</li>
	<li>Prepare a glass or plastic 10 cm Petri dish and a habituation container for the pLmV test prior to starting the experiment.
	<ol>
		<li>Place a clean 10 cm diameter Petri dish to be used for the pLmV test on prelaminated grid paper (with 0.5 cm squares).</li>
		<li>Add 20 mL of unadulterated spring water for controls, or spring water containing the appropriate concentration of the natural product being tested, to the 10 cm diameter Petri dish to be used for the pLmV test.</li>
		<li>Position a camera (e.g., cell phone or high-resolution camera) above the prepared 10 cm diameter Petri dish to record planarian motility over the grid paper during the experiment. A ring stand is a convenient way to position the camera at a distance that can record the entire view of the 10 cm Petri dish and grid lines.</li>
		<li>Prepare a habituation container with 5&#8211;10 mL of unadulterated spring water (controls), or spring water containing the appropriate concentration of the natural product being tested. A container similar to a scintillation vial or small 5 cm Petri dish is suitable.</li>
	</ol>
	</li>
	<li>Use a small, clean, flat, or round watercolor paintbrush to gently transfer a planarian from the stock container with spring water to the habituation container having 5&#8211;10 mL of unadulterated spring water or spring water containing the natural product being tested. 
	NOTE: When manipulating planarians for the pLmV assay, use a small, clean, flat, or round watercolor paintbrush (number 3&#8211;6). When moving planarians, the brush should be placed under the animal to lift it gently. To ensure planarians are not damaged when using the brush, the bristles of the brush should not be splayed out under the animal. Spreading out of the bristles could harm the planarian if it is caught between the fibers of the brush. 
	NOTE: A wide bore transfer pipette can also be used for transferring the planarian into a clean and dry habituation container.
	<ol>
		<li>If using the pipette, remove excess water moved with the planarian from the habituation container using the transfer pipette.</li>
		<li>Carefully add the habituation solution (i.e., spring water for controls or spring water containing the concentration of natural product being tested) to the habituation container containing the planarian.</li>
	</ol>
	</li>
	<li>Habituation periods will depend on the stimulation dynamics assessed for the natural product being tested. A habituation time of 2 min proved acceptable to detect stimulation in this work1.</li>
	<li>Following the 2 min habituation period use a watercolor paintbrush to gently transfer the planarian to the center of the prepared 10 cm Petri dish for the pLmV stimulation experiment.</li>
	<li>Start the camera to record the movement of the planarian. Record 10&#8211;11 min of video.</li>
	<li>Prepare the habituation container and the 10 cm Petri dish for the pLmV experiment with fresh solutions for each planarian.</li>
	<li>Use dedicated pipettes, dishes, containers, and paintbrushes for each experimental concentration of the natural product being tested to avoid inadvertently exposing planarians to the wrong solution during experimentation.</li>
	<li>Because planarians exhibit learned behaviors, each planarian (control or test) should only be used once21,22.</li>
</ol>
4. Planarian locomotor velocity (pLmV) test: Withdrawal behaviors
<ol>
	<li>Prior to experimentation, ensure starved planarians are fully formed, with a complete and pigmented head and tail.</li>
	<li>Prepare a 10 cm Petri dish (glass or plastic) for the pLmV experiment, a 5 cm Petri dish (glass or plastic) for rinsing the planarian following habituation, and a habituation container prior to starting the experiment.
	<ol>
		<li>Place a clean 10 cm diameter Petri dish to be used for the pLmV experiment on prelaminated grid paper with 0.5 cm squares).</li>
		<li>Add 20 mL of unadulterated spring water to the 10 cm diameter Petri dish to be used for the pLmV experiment.</li>
		<li>Position a camera above the prepared 10 cm diameter Petri dish as in step 3.2.3 to record planarian motility over the grid paper during the experiment.</li>
		<li>Prepare the planarian rinse container by adding 5 mL of spring water alone to the 5 cm Petri dish.</li>
		<li>Prepare a habituation container with 5&#8211;10 mL of unadulterated spring water (controls) or spring water containing the natural product being tested. A container similar to a scintillation vial or small 5 cm Petri dish (glass or plastic) is suitable.</li>
	</ol>
	</li>
	<li>Use a small, clean, flat, or round watercolor paintbrush to transfer a planarian from spring water to the prepared habituation container having 5&#8211;10 mL of unadulterated spring water (controls) or spring water containing the natural product being tested. Gently move the animal from the stock container to the habituation container. Ensure the planarian is not damaged by the brush. 
	NOTE: When manipulating planarians for the pLmV assay, use a small, clean, flat, or round watercolor paintbrush (number 3&#8211;6). When moving planarians, the brush should be placed under the animal to lift it gently. To ensure planarians are not damaged when using the brush, the bristles of the brush should not be splayed out under the animal. Spreading out of the bristles could harm the planarian if it is caught between the fibers of the brush. 
	NOTE: A wide bore transfer pipette can also be used to transfer the planarian into a clean and dry habituation container.
	<ol>
		<li>If using the pipette, excess water moved with the planarian should be removed from the habituation container using the transfer pipette.</li>
		<li>Carefully add the habituation solution (i.e., unadulterated spring water for controls or spring water containing the natural product being tested) to the habituation container containing the planarian.</li>
	</ol>
	</li>
	<li>Habituation periods for withdrawal will depend on the stimulation dynamics assessed for the natural product being tested; 2&#8211;5 min have proven sufficient.</li>
	<li>Following the habituation period use a watercolor paintbrush to gently transfer the planarian to the prepared 5 cm Petri dish containing spring water to rinse off any natural product from the habituation container. Ensure the planarian is not damaged by the brush.</li>
	<li>Immediately transfer the planarian to the center of the prepared 10 cm Petri dish containing spring water for the pLmV withdrawal experiment. Ensure the planarian is not damaged by the brush.</li>
	<li>Start the camera to record the movement of the planarian. Record 10&#8211;11 min of video.</li>
	<li>Prepare the habituation container, the rinse container, and the 10 cm Petri dish for the pLmV experiment with fresh solutions for each planarian.</li>
	<li>Use dedicated pipettes, dishes, containers, and paintbrushes for each experimental concentration of the natural product being tested to avoid inadvertently exposing planarians to the wrong solution during experimentation.</li>
	<li>Because planarians exhibit learned behaviors, use each planarian (control or test) only once26,27.</li>
</ol>
5. Data analysis
<ol>
	<li>Prepare a data collection table to document the behavior and the motility of planarians as the number of grid lines crossed for each minute during the pLmV run. The table should allow for the accumulated number of lines per minute crossed by the planarian to be documented as well. Include lines for notes and a table of definitions to tally the observation of behaviors during the experimental period, such as &#8216;wander&#8217; and &#8216;stop&#8217; (see Discussion).</li>
	<li>Using the video, count the number of full grid lines crossed by the planarian per minute for 10 min, and record that number on the data table. Typical planarian behavior consists of continuous velocity, forward-directed, horizontal movement, with periodic turns, and without stops.
	<ol>
		<li>Begin to time the experiment at the point that the planarian has moved off the paintbrush used to transfer it to the 10 cm Petri dish. Record this start time.</li>
		<li>To determine when the animal crosses a full grid square, focus on the head and score one line when the head fully crosses a square.</li>
		<li>To score a full grid when the worm moves around the edge of the dish, visualize a distance of 0.5 cm by referring to the lines as they extend out from the margins of the dish. If the planarian crosses the corner of a box, refer to the second line crossed to score one grid line. Again, focus on the head to make these determinations.</li>
		<li>Stop the video after each minute to record the data.</li>
		<li>When restarting the video to count the next minute, if the head of the worm was between grid lines when the video was stopped, record the first line crossed as a full box.</li>
		<li>Score the number of grid lines crossed for 10 min.</li>
	</ol>
	</li>
	<li>If the animal stops moving during a pLmV test and no longer crosses grid lines during the 10 min recording time, document the behavior of the planarian in the behavioral chart (e.g., &#8216;wander&#8217; or &#8216;stop&#8217;). Animals that cease their forward track during the pLmV assay should instead be taken note of and the data presented as a frequency of the total number of animals exposed to that reagent concentration. Coiling or convulsive behavior (known as a C behavior) preventing any forward movement during the habituation period indicates that the concentration of the natural product is not appropriate for use in a pLmV assay because the pLmV assay is motility-based. C-type behaviors can be analyzed using a different type of analysis (see Discussion).</li>
	<li>If possible, test multiple experimental concentrations of the natural product using at least 9&#8211;12 worms on different days and different times of the day if determining the overall effect of the reagent on planarian physiology. However, if researchers endeavor to reduce circadian rhythm-induced variability, experiments can be conducted with constant lighting at a set time of day using worms that are cultured with timed light/dark cycles and set feeding times. Have at least two experimenters involved in the project to allow for the option of having one individual record data, while the second individual counts the grid lines &#8216;blind&#8217; to the conditions used for data collection. Having different individuals involved in data collection, as well as statistical calculations and analysis, also reduces possible bias.</li>
	<li>Calculate grid line counts for each natural product concentration on each day as relative to control counts for each minute so that data from different days, times, and experimenters can be combined. These data can be averaged and then analyzed using Student&#8217;s T-tests. P-values for each test can be assessed per minute compared to the control and between reagent concentrations. ANOVA assessments using data sets derived from different experimental concentrations provide a further method of analysis.</li>
</ol>

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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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+and+functional+genetic+assays+to+manipulate+regeneration+of+the+planarian+Dugesia+japonica.">Pharmacological and functional genetic assays to manipulate regeneration of the planarian Dugesia japonica.</a> Journal of Visualized Experiments. (54), e3058 (2011).</li></ol>

The authors have nothing to disclose.

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...

Watch this Scientific Journal Video about A Planarian Motility Assay to Gauge the Biomodulating Properties of Natural Products at JoVE.com

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 ...

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Biochemistry

5.6K Views.  State University of New York (SUNY) at Morrisville. 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.

Video: A Planarian Motility Assay to Gauge the Biomodulating Properties of 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 ...

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 ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

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'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 ...

Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals and starch in plants. The crystal structures of glucan phosphatase with model glucan substrates reveal distinct glucan-binding interfaces made of DSP and carbohydrate-binding domains. However, quantitative measurements of glucan-glucan phosphatase interactions with physiologically relevant substrates are fundamental to the biological understanding of the glucan phosphatase family of enzymes and the regulation of energy metabolism. This manuscript reports a Concanavalin A (ConA)-based in vitro sedimentation assay designed to detect the substrate binding affinity of glucan phosphatases against different glucan substrates.&#160;As a proof of concept, the dissociation constant (KD) of glucan phosphatase Arabidopsis thaliana Starch Excess4 (SEX4) and amylopectin was determined. The characterization of SEX4 mutants and other members of the glucan phosphatase family of enzymes further demonstrates the utility of this assay to assess the differential binding of protein- carbohydrate interactions. These data demonstrate the suitability of this assay to characterize a wide range of starch and glycogen interacting proteins.

This method describes a lectin-based in vitro sedimentation assay to quantify the binding affinity of glucan phosphatase and amylopectin. This co-sedimentation assay is reliable for measuring glucan phosphatase substrate binding and can be applied to various solubilized glucan substrates.

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals ...

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 ...