This work was supported by Programa de Apoyo a Proyectos de Investigaci&#243;n e Innovaci&#243;n Tecnol&#243;gica (PAPIIT), with a grant number IT200819. The authors acknowledge to Tom Musselman, Rock Paper Editing, LLC, for checking the English grammar of this manuscript; and the technical assistance of Samanta Jim&#233;nez (CICESE, Ensenada), and Juan Manuel Barbosa Castillo (Instituto de Fisiolog&#237;a Celular, UNAM).&#160;We also thank to Dr Augusto C&#233;sar Lizarazo Chaparro (CEPIPSA) for obtaining sheep blood.&#160;We especially thank Dr Jos&#233; Saniger Blesa, ICAT-UNAM,&#160;&#160;for the facilities in his laboratory for the video recording.&#160;

The sea anemones were collected according to guidelines of the National Commission for Aquaculture, Fisheries, and Food of the Federal Government of Mexico (permit number PPF / DGOPTA 07332.250810.4060). Bioethics Committee of the Institute of Biotechnology, National Autonomous University of Mexico approved all the experiments with sea anemones. The sheep blood sample was purchased at the Center for Practical Teaching and Research in Animal Production and Health (CEPIPSA, National Autonomous University of Mexico).
1. Organism collection
<ol>
	<li>Collect sea anemone A. dowii. 
	NOTE: Here, the organisms were collected during low tide periods in the intertidal zone off the coast of Ensenada Baja, California, Mexico (Figure 1A,B).</li>
	<li>Transport the organisms to the laboratory in containers with seawater (Figure 1C).</li>
	<li>In the laboratory, clean the organisms with distilled water by hand to remove the big substrate particles adhering to the body.</li>
	<li>Immediately freeze the organisms at -80 &#176;C in an ultra-freezer for 72 h.</li>
	<li>Place the organisms in special glasses for freeze-drying, with lyophilization conditions of -20 &#176;C, 0.015 psi, for 48 h.</li>
	<li>Store the lyophilized organisms at -20 &#176;C until use.</li>
</ol>
2. Tissue hydration
<ol>
	<li>Reconstitute 20 g dry weight of lyophilized organisms with 60 mL (1/3 w/v) of 50 mM sodium phosphate buffer, pH 7.5, and 1 inhibitor cocktail tablet (Table of Materials).</li>
	<li>In a magnetic stirrer, continuously stir the sample at ~800 rpm, 4 &#176;C, for 12 h.</li>
</ol>
3. Toxin release
<ol>
	<li>To induce cell lysis and nematocyst discharge, freeze the sample at -20 &#176;C for 12 h, and then place the container in water at room temperature (RT) until it thaws up to 90%. Repeat this procedure three times. 
	NOTE: The sample does not thaw 100%; it is crucial to maintain the sample at ~4 &#176;C to prevent protein degradation.</li>
	<li>Observe the nematocyst discharged under a confocal microscope. Take 10 &#181;L of the sample on a slide and place a coverslip over it. Observe under a confocal microscope at 100x and 60x magnification. Here a dye was not necessary.</li>
	<li>Process the images captured in the confocal microscope in ImageJ software (version 1.53c, Wayne Rasband, National Institutes of Health, USA,&#160;https://imagej.nih.gov/ij).</li>
	<li>If the tubule inside the nematocyst uncoils and is outside, the nematocysts are discharged; then, continue with the next step. Otherwise, only conduct one more freeze-thaw cycle.</li>
	<li>To clarify the extract, centrifuge the sample at 25,400 x g for 40 min, 4 &#176;C, recover the supernatant, and centrifuge it again. Repeat this step 3-4 times.</li>
	<li>Recover the supernatant and discard the pellet. The supernatant or crude extract contains the venom components (toxins) and other cell molecules, such as lipids, carbohydrates, and nucleic acids.</li>
</ol>
4. Quantitation of total protein
<ol>
	<li>Determine the protein concentration of the crude extract using a commercial Bradford colorimetric assay15 kit (Table of Materials).</li>
	<li>Dilute the dye reagent (one part of dye with four parts of distilled water).</li>
	<li>Prepare bovine serum albumin (BSA) Fraction V (Table of Materials) solution at 1 mg/mL concentration in phosphate buffer (50 mM sodium phosphate, pH 7.5).</li>
	<li>Prepare the solutions listed in Table 1 in triplicate.</li>
	<li>Vigorously mix each solution in a shaker for 4 s.</li>
	<li>Incubate the samples for 5 min at RT, without shaking.</li>
	<li>Measure absorbance (AU) at 595 nm.</li>
	<li>Plot the averages of the sample BSA absorbances (AU vs. protein concentration), and determine the equation of the graph (Equation 1). 
	<img alt="Equation 1" src="/files/ftp_upload/63630/63630eq01.jpg" style="margin: auto;" />&#160; &#160; &#160;Equation 1 
	where y is the absorbance, m is the slope of the line, x is the protein concentration, and b is the y-intercept. To calculate crude extract concentration, solve for the x as follows: 
	<img alt="Equation 2" src="/files/ftp_upload/63630/63630eq02.jpg" style="margin: auto;" />&#160; &#160; &#160;Equation 2</li>
</ol>
5. Determine polypeptide venom complexity
<ol>
	<li>Assemble the glass container for vertical gel electrophoresis according to the manual.</li>
	<li>Prepare acrylamide mix (30%): 29.2 g of acrylamide, 0.8 g of bis-acrylamide, the final volume of 100 mL. Filter the solution using a 0.45 &#181;m membrane. Store the solution in a dark bottle at 4 &#176;C. Then, prepare the mixture for the resolving gel (Table 2).</li>
	<li>The SDS-PAGE gel consists of a resolving gel and a stacking gel. Prepare the gels as per the solution volumes defined in Table 2. During preparation, ensure to maintain each mix at 4 &#176;C to reduce polymerization time. 
	NOTE: The volume of each mixture varies depending on the brand of equipment used; for this protocol, the volumes correspond to the preparation of 15% acrylamide gel, 0.75 mm thick, for a vertical electrophoresis chamber (Table of Materials).</li>
	<li>After the acrylamide has polymerized, ~10 min, add the mixture of stacking gel.</li>
	<li>Prepare the mixture for the stacking gel, immediately add it to the glass plates, and place a comb to form the wells for loading the samples.</li>
	<li>Once the stacking gel has polymerized (~15 min), remove the comb and place the glass container in a chamber for vertical electrophoresis.</li>
	<li>Place 200 mL of electrode buffer (0.25 M Tris, 0.192 M glycine, 0.1% SDS) in the electrophoresis chamber.</li>
	<li>Analyze the crude extract: Mix 30 &#181;g of crude extract with 5 &#181;L of protein loading buffer (25% glycerol, 15% sodium dodecyl sulfate, 25% 2-mercaptoethanol, 0.125 mM Tris pH 7, bromophenol blue 0.1 mg/mL) to denature proteins. The final volume must be &#60;50 &#181;L.</li>
	<li>Heat at 90 &#176;C for 5 min, cool, and centrifuge for 3 s at 1,400 x g.</li>
	<li>Load the samples in the wells of the stacking gel. Load a standard molecular weight ladder.</li>
	<li>Run electrophoresis at 25 mA constant for 1 h 30 min.</li>
	<li>Remove the glass containers from the electrophoresis chamber to proceed with the staining of the gel proteins.</li>
</ol>
6. Protein staining
<ol>
	<li>Rinse the gel in 50 mL of distilled water to remove debris from the buffer electrode.</li>
	<li>Discard the water.</li>
	<li>To avoid the diffusion of the gel proteins, add the fixation solution (60 mL of ethanol, 20 mL of acetic acid, and 20 mL of distilled water, final volume 100 mL). Incubate at RT for 40 min with shaking at 80 rpm. Decant the solution.</li>
	<li>Add the protein crosslinking solution (15 mL of ethanol, 0.25 mL of glutaraldehyde, 50 mL of distilled water, final volume 65.25 mL), and incubate for 30 min at RT with shaking at 80 rpm. Remove the solution.</li>
	<li>Wash the gel with 100 mL of distilled water for 5 min, and remove the water. Repeat this procedure four times.</li>
	<li>Carry out protein staining with 50 mL of Coomassie brilliant blue R-250 filtered solution (40% methanol, 10% acetic acid, 50% distilled water, 0.1% dye, final volume 100 mL), stirring at 60 rpm in a laboratory rotary oscillator at RT for 15 min.</li>
	<li>Recover the solution, which can be used to stain other gels.</li>
	<li>To eliminate the excess dye, add a destaining solution (40 mL of methanol, 10 mL of acetic acid, 50 mL of distilled water). Keep stirring (80 rpm) at RT until the bands (stained proteins) visualize.</li>
	<li>Keep the gel in 50 mL of distilled water and scan the image in a Gel documentation system.</li>
</ol>
7. Prepare red blood cell solution
<ol>
	<li>Extract 1 mL of blood from the jugular vein of a sheep.</li>
	<li>Remove the needle from the syringe and immediately drain blood into a tube with 50 mL of Alsever solution (0.002 M citric acid, 0.07 M NaCl, 0.1 M dextrose, and 0.027 M sodium citrate as an anticoagulant, pH 7.4).</li>
	<li>Slowly invert the solution three times.</li>
	<li>Keep at 4 &#176;C for transfer to the laboratory.</li>
	<li>Centrifuge at 804 x g for 5 min at 4 &#176;C and discard the supernatant.</li>
	<li>Resuspend the pellet in 30 mL of Alsever solution. Add the solution by sliding it along the tube&#39;s inner walls and, using a Pasteur plastic pipette, slowly resuspend the cells. Centrifuge again at 804 x g for 5 min, 4 &#176;C. Repeat this procedure until the supernatant is clarified.</li>
	<li>Add 15 mL of Alsever solution; resuspend the pellet slowly with a Pasteur plastic pipette.</li>
	<li>Maintain the final volume of Alsever solution to achieve a concentration of 2 x 106 cells/mL, approximately. Then, use a Neubauer chamber or hemocytometer to count the cells. 
	NOTE: Count the erythrocytes in the hemacytometer slide (Improved Neubauer). The slide is a 30 mm x 70 mm x 4 mm slide. The center has two silver footplates (Figure 2A) (one top and one bottom grid), each 3 mm x 3 mm, and two channels on the sides of the grid. Counting cells is in the corner and center squares (red squares in Figure 2B). The optimal concentration of cells for counting should be in the order of 106 cells. The central box has a surface area of 0.1 cm2, which is equivalent to 0.0001 mL.</li>
	<li>Place a coverslip over the slide.</li>
	<li>Place 10 &#181;L of the sample between the slide and the coverslip and wait for the sample to disburse.</li>
	<li>In an optical microscope (magnification 60x), perform the count in the first square box. Count only the cells in the center grid and those that touch the upper or the left margin of the box.</li>
	<li>Determine cell concentration using the following equation: 
	<img alt="Equation 3" src="/files/ftp_upload/63630/63630eq03v4.jpg" style="margin: auto;" /> Equation 3 
	If the sample is very concentrated and dilution is required, then use the following equation: 
	<img alt="Equation 4" src="/files/ftp_upload/63630/63630eq04v3.jpg" style="margin: auto;" /> Equation 4 
	NOTE: When taking the erythrocyte solution for the hemolysis test, slowly homogenize the solution with a plastic Pasteur pipette.</li>
	<li>Perform the steps in triplicate to reduce error.</li>
</ol>
8. Hemolysis assay
<ol>
	<li>Prepare the solution mixtures (in triplicates) indicated in Table 3 in conical 96-well plates. 
	NOTE: Mix the erythrocytes slowly to maintain a homogeneous suspension.</li>
	<li>Incubate for 1 h at 37 &#176;C, without shaking.</li>
	<li>Centrifuge the plate at 804 x g for 5 min at 4 &#176;C.</li>
	<li>Recover the supernatant in another 96-well plate with a flat bottom.</li>
	<li>If the crude extract contains cytolysins, erythrocytes will lyse and release hemoglobin. Read the absorbance at 415 nm.</li>
	<li>Calculate hemolysis percentage, using the following equation: 
	%&#160;Hemolysis = ((Acrude extract-AAlsever buffer) / (AH2O-AAlsever)) x 100 
	NOTE: Acrude extract, AAlsever buffer, and AH2O correspond to the absorbance of erythrocytes with the crude extract, absorbance of Alsever solution, and absorbance of distilled water, respectively.</li>
	<li>Perform a hemolysis test before and after each freezing and thawing cycle with 50 &#181;g of total protein from the crude extract.</li>
	<li>Determine the number of freezing and thawing cycles required to reach 100% hemolytic activity.</li>
	<li>Calculate the amount of extract that produces hemolysis in 50% of the erythrocytes (HU50); follow the same protocol described in Table 3, increase the amount of sea anemone crude extract, and design the plot with sigmoidal adjustments using appropriate software (e.g., Origin, Table of Materials).</li>
</ol>
9. Phospholipase assay
<ol>
	<li>Wash one chicken egg with 1% SDS in distilled water.</li>
	<li>Separate the egg yolk from the egg white under sterile conditions.</li>
	<li>Prepare 50 mL of 0.86% NaCl solution, and filter through a 0.22 &#181;m filter. Then, prepare Solution A: Add 12 mL of egg yolk and 36 mL of 0.86% NaCl solution.</li>
	<li>Prepare Solution B: Mix 300 mg of agarose in 50 mL of buffer (50 mM Tris, pH 7.5), filter through a 0.22 &#181;m filter. Heat the solution in a microwave until boiling. Cool down in warm water to reach 43-45&#176;C.</li>
	<li>Prepare Solution C: Prepare 10 mM CaCl2 and filter it with a 0.22 &#181;m filter.</li>
	<li>Prepare Solution D: 10 mg of rhodamine 6G in 1 mL of distilled water.</li>
	<li>Under sterile conditions (laminar flow hood), add 500 &#181;L of solutions A and C to solution B and 100 &#181;L of solution D, mix, and pour 25 mL into each Petri dish (90 x 15 mm).</li>
	<li>Wait for the solution to solidify under sterile conditions (30 min).</li>
	<li>Make wells (~2-3 mm diameter) with a thin tube.</li>
	<li>Add a total of 20 &#181;L of phosphate buffer (negative control) in one well and 20 &#181;L of a determined phospholipase (positive control) in another well.</li>
	<li>Place different amounts of the crude extract protein, 5, 15, 25, 35, 45 &#181;g, in the remaining wells, each in a final volume of 20 &#181;L.</li>
	<li>Wait for the agar to adsorb all the samples (30 min).</li>
	<li>Incubate at 37 &#176;C for 20 h.</li>
	<li>If a halo forms around the well, this indicates phospholipase activity. Measure the halo formed with a vernier caliper. 
	NOTE: Perform the experiment in triplicate (steps from 9.1-9.14).</li>
</ol>

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The authors declare that they have no competing interest in relation to the publication of this paper.

The high demand for new compounds with applications in different fields of science and industry has led to the study of venom. Venom represents a rich source of molecules that serves as a template for generating new molecular tools. However, the complexity of these venoms requires the implementation and combination of various methods to obtain and study them.
Here, we show a method for obtaining and analyzing the venom of the sea anemone Anthopleura dowii, Verrill 1869, which can be u...

Marine animals are a rich source of biologically active compounds. In recent decades, the composition of sea anemone venom has attracted scientific attention since it comprises a diversity of polypeptides with hemolytic, cytotoxic, enzymatic (phospholipase, protease, chitinase), and neurotoxic activity and inhibitory effects on proteolytic activity1. In addition, these polypeptides are potential sources for the development of molecular tools in biotechnological and therapeutical use2,3.
There are few reports about sea anemone venom and its molecular components due to the complexity of obtaining the venom, even isolation, and characterization of toxins. The extraction methods used in the reports involved lysis and emptying the contents of cells that are related and unrelated to the venom production1.
A particular characteristic in all cnidarians is the absence of a system for production and release of the venom centralized in a single anatomical region. Instead, the nematocysts are structures that keep the venom4,5. Other types of cells, called epidermal gland cells, also secrete toxins and are also distributed throughout the body of sea anemones6.
The first and most crucial challenge in obtaining the venom is the generation of an extract with sufficient manipulation in subsequent processes, without the inactivation or degradation of labile proteins. Next, the cells must be lysed, and the components-in this case, polypeptides must be efficiently and quickly extracted, avoiding proteolysis and hydrolysis while eliminating other cellular components7.
Different methods are used to obtain the crude extract of a sea anemone; some involve sacrificing the organism while others allow it to be kept alive. Methods that imply the use of the organism&#180;s whole body allow for the release of most toxins from the venom8, compared to methods that keep organisms alive, which extract only some components of the venom9. The preparation of an extract requires evaluating the presence and potency of a substance of interest through a specific bioassay, which includes strategies to observe the pharmacological effects by in vivo or in vitro methods10.
Sea anemone venom contains cytolytic polypeptides, pore-forming toxins (PFTs)11, and phospholipases12; these molecules are models in the study of protein-lipid interaction, molecular tools in cancer therapy, and biosensors based on nanopore3. The classification of sea anemone PFTs is carried out according to their size or molecular weight, from 5 kDa to 80 kDa. The 20 kDa PFT, the most studied and known as actinoporins11, is of particular interest for its biomedical potential in the development of molecular tools for possible applications as anticancer, antimicrobial, and nanopore-based biosensors. Another cytolysin, including phospholipases, specifically phospholipase A2 (PLA2)13, releases a fatty acid due and hydrolyzes phospholipids, destabilizing the cell membrane. Due to this mechanism of action, PLA2 promises to be an essential model for the study and applications in inflammatory diseases. It could serve as a model for studies of lipid behavior in the cell membrane14.
Here, we describe an efficient protocol for obtaining the crude extract from sea anemone Anthopleura dowii Verrill, 1869, and detecting hemolysins and phospholipases. Both are relevant toxins that could be used as a template to design new molecular tools.

<table><tbody><tr><td>15 mL conical centrifuge tube</td><td>Corning</td><td>430766</td><td></td></tr><tr><td>2-Bromophenol blue</td><td>Sigma</td><td>B75808</td><td></td></tr><tr><td>2-mercaptoetanol</td><td>Sigma-Aldrich</td><td>M6250-100ML</td><td></td></tr><tr><td>50 mL conical centrifuge tubes</td><td>Corning</td><td>430828</td><td></td></tr><tr><td>Acetic Acid Glacial</td><td>J.T. Baker</td><td>9515-03</td><td></td></tr><tr><td>Acrylamide</td><td>Promega</td><td>V3115</td><td></td></tr><tr><td>Agarose</td><td>Promega</td><td>V3125</td><td></td></tr><tr><td>Bisacrylamide</td><td>Promega</td><td>V3143</td><td></td></tr><tr><td>Bovine Serum Albumin Fraction V</td><td>Sigma</td><td>A3059-100G</td><td></td></tr><tr><td>Bradford Protein Assays</td><td>Bio-Rad</td><td>5000006</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich</td><td>C3306</td><td></td></tr><tr><td>Cell culture plates 96 well, V-bottom</td><td>Corning</td><td>3894</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804R</td><td></td></tr><tr><td>Centrifuge tubes</td><td>Corning</td><td>CLS430829</td><td></td></tr><tr><td>ChemiDoc MP system</td><td>Bio-Rad</td><td>1708280</td><td></td></tr><tr><td>Citric acid</td><td>Sigma-Aldrich</td><td>251275</td><td></td></tr><tr><td>Clear flat.bottom 96-Well Plates</td><td>Thermo Scientific</td><td>3855</td><td></td></tr><tr><td>Coomassie Brilliant Blue G-250</td><td>Bio-Rad</td><td>#1610406</td><td></td></tr><tr><td>Coomassie brilliant blue R-250</td><td>Bio-Rad</td><td>1610400</td><td></td></tr><tr><td>Dextrose</td><td>J.T. Baker</td><td>1916-01</td><td></td></tr><tr><td>Ductless Enclosure</td><td>Labconco</td><td>Vertical</td><td>https://imagej.nih.gov/ij ImageJ 1.53c</td></tr><tr><td>Gel Doc EZ</td><td>Bio Rad.</td><td></td><td>Gel Documentation System</td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516-4L</td><td></td></tr><tr><td>Hemocytometer</td><td>Marienfeld</td><td>650030</td><td></td></tr><tr><td>ImageJ (Software)</td><td>NIH, USA</td><td>Version 1.53c</td><td></td></tr><tr><td>Incubator 211</td><td>Labnet</td><td>I5211 DS</td><td></td></tr><tr><td>Methanol</td><td>J.T. Baker</td><td>9049-03</td><td></td></tr><tr><td>Mini-PROTEAN tetra cell</td><td>Bio-Rad</td><td>1658000EDU</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>J.T. Baker</td><td>3824-01</td><td></td></tr><tr><td>NaCl</td><td>J.T. Baker</td><td>3624-01</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;.H&lt;sub&gt;2&lt;/sub&gt;O</td><td>J.T. Baker</td><td>3818-05</td><td></td></tr><tr><td>Origin software</td><td></td><td>version 9</td><td>To design the plot with sigmoidal adjustments</td></tr><tr><td>Petridish</td><td>Falcon</td><td>351007</td><td></td></tr><tr><td>Pipetman kit</td><td>Gilson</td><td>F167380</td><td></td></tr><tr><td>Precast mini gel</td><td>BioRad</td><td>1658004</td><td></td></tr><tr><td>Prestained Protein Ladder</td><td>Thermo Scientific</td><td>26620</td><td></td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Roche</td><td>11836153001</td><td></td></tr><tr><td>Protein Assay Dye Reagent Concentrate</td><td>Bio-Rad</td><td>5000006</td><td></td></tr><tr><td>Rhodamine 6G</td><td>Sigma-Aldrich</td><td>252433</td><td></td></tr><tr><td>SDS</td><td>Sigma-Aldrich</td><td>L4509</td><td></td></tr><tr><td>Sodium citrate dihydrate</td><td>JT Baker</td><td>3646-01</td><td></td></tr><tr><td>Spectrophotometer</td><td>THERMO SCIENTIFIC</td><td>G10S UV-VIS</td><td></td></tr><tr><td>Tris Base</td><td>Sigma-Aldrich</td><td>77-86-1</td><td></td></tr><tr><td>Volt Power Supply</td><td>Hoefer</td><td>PS300B</td><td></td></tr></tbody></table>

identification of hemolytic and phospholipase activity in crude extracts from sea anemones by straightforward bioassays

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential for designing new molecular tools. Sea anemone venom locates in glandular cells from ectoderm and sub-cellular structures called nematocysts, both of which are distributed throughout the sea anemone body. This characteristic implies challenges because the cells and nematocyst must be lysed to release the venom components with other non-toxic molecules. Therefore, first, the venom is derived from a crude extract (mixture of different and diverse molecules and tissue debris). The next step is to detect polypeptides with specific bioactivities. Here, we describe an efficient strategy to obtain the sea anemone crude extract and bioassay to identify the presence of cytolysins. The first step involves inexpensive and straightforward techniques (stirred and freeze-thaw cycle) to release cytolysins. We obtained the highest cytolytic activity and protein (~500 mg of protein from 20 g of dry weight). Next, the polypeptide complexity of the extract was analyzed by SDS-PAGE gel detecting proteins with molecular weights between 10 kDa and 250 kDa. In the hemolytic assay, we used sheep red blood cells and determined HU50 (11.1 &plusmn; 0.3 &#181;g/mL). In contrast, the presence of phospholipases in the crude extract was determined using egg yolk as a substrate in a solid medium with agarose. Overall, this study uses an efficient and inexpensive protocol to prepare the crude extract and applies replicable bioassays to identify cytolysins, molecules with biotechnological and biomedical interests.

The representative results of the protocol used to obtain the crude extract of sea anemone showed that combining two techniques (agitation and cycles of freezing and thawing) produced an efficient discharge of nematocysts, and the total amount of protein was 500 mg (8 mg/mL) (Figure 3).
The crude extract&#39;s protein complexity could be observed from 10 kDa and greater than 250 kDa through SDS-PAGE electrophoresis. In addition, cytolysins were detected in the mol...

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Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays

Here, we describe a protocol to obtain crude venom extract from sea anemone and detect its hemolytic and phospholipase activity.

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential ...

identification-hemolytic-phospholipase-activity-crude-extracts-from

Research

JoVE Journal

Biochemistry

2.7K Views.  Universidad Nacional Autónoma de México. Zeeanemoon toxine interactie moet het minste aantal organismen en efficiënte protocollen gebruiken om een grote diversiteit en overvloed van de toxines en snelle identificatie van een specifiek toxine te verkrijgen. We combineerden twee efficiënte technieken om het ruwe extract te bereiden met een grote hoeveelheid eiwit en beschrijven reproduceerbare, specifieke, snelle en goedkope testen om hemolyse en fosfolic basen in het zeeanemoongif te identificeren. De methode kan worden gebruikt in...

Video: Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays

Defining Substrate Specificities for Lipase and Phospholipase Candidates

Microorganisms produce a wide spectrum of (phospho)lipases that are secreted in order to make external substrates available for the organism. Alternatively, other (phospho)lipases may be physically associated with the producing organism causing a turnover of intrinsic lipids and frequently giving rise to a remodeling of the cellular membranes. Although potential (phospho)lipases can be predicted with a number of algorithms when the gene/protein sequence is available, experimental proof of the enzyme activities, substrate specificities, and potential physiological functions has frequently not been obtained. This manuscript describes the optimization of assay conditions for prospective (phospho)lipases with unknown substrate specificities and how to employ these optimized conditions in the search for the natural substrate of a respective (phospho)lipase. Using artificial chromogenic substrates, such as p-nitrophenyl derivatives, may help to detect a minor enzymatic activity for a predicted (phospho)lipase under standard conditions. Having encountered such a minor enzymatic activity, the distinct parameters of an enzyme assay can be varied in order to obtain a more efficient hydrolysis of the artificial substrate. After having determined the conditions under which an enzyme works well, a variety of potential natural substrates should be assayed for their degradation, a process that can be followed employing distinct chromatographic methods. The definition of substrate specificities for new enzymes, often provides hypotheses for a potential physiological role of these enzymes, which then can be tested experimentally. Following these guidelines, we were able to identify a phospholipase C (SMc00171) that degrades phosphatidylcholine to phosphocholine and diacylglycerol, in a crucial step for the remodeling of membranes in the bacterium Sinorhizobium meliloti upon phosphorus-limiting conditions of growth. For two predicted patatin-like phospholipases (SMc00930 and SMc01003) of the same organism, we could redefine their substrate specificities and clarify that SMc01003 is a diacylglycerol lipase.

Many predicted (phospho)lipases are poorly characterized with regard to their substrate specificities and physiological functions. Here we provide a protocol to optimize enzyme activities, search for natural substrates, and propose physiological functions for these enzymes.

Microorganisms produce a wide spectrum of (phospho)lipases that are secreted in order to make external substrates available for the organism. ...

A Fluorescence-based Assay of Phospholipid Scramblase Activity

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and biochemically verified was opsin, the apoprotein of the photoreceptor rhodopsin. Rhodopsin is a G protein-coupled receptor localized in rod photoreceptor disc membranes of the retina where it is responsible for the perception of light. Rhodopsin&#39;s scramblase activity does not depend on its ligand 11-cis-retinal, i.e., the apoprotein opsin is also active as a scramblase. Although constitutive and regulated phospholipid scrambling play an important role in cell physiology, only a few phospholipid scramblases have been identified so far besides opsin. Here we describe a fluorescence-based assay of opsin&#39;s scramblase activity. Opsin is reconstituted into large unilamellar liposomes composed of phosphatidylcholine, phosphatidylglycerol and a trace quantity of fluorescent NBD-labeled PC (1-palmitoyl-2-{6-[7-nitro-2-1,3-benzoxadiazole-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine). Scramblase activity is determined by measuring the extent to which NBD-PC molecules located in the inner leaflet of the vesicle are able to access the outer leaflet where their fluorescence is chemically eliminated by a reducing agent that cannot cross the membrane. The methods we describe have general applicability and can be used to identify and characterize scramblase activities of other membrane proteins.

We describe a fluorescence-based assay to measure phospholipid scrambling in large unilamellar liposomes reconstituted with opsin.

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and ...

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

Environment

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

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

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

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

Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom body (MB) is a higher brain center proposed to be the neural substrate of complex honeybee behaviors. Although previous studies identified genes and proteins that are differentially expressed in the MBs and other brain regions, the activities of the proteins in each region are not yet fully understood. To reveal the functions of these proteins in the brain, pharmacologic analysis is a feasible approach, but it is first necessary to confirm that pharmacologic manipulations indeed alter the protein activity in these brain regions.
We previously identified a higher expression of genes encoding phospholipase C (PLC) in the MBs than in other brain regions, and pharmacologically assessed the involvement of PLC in honeybee behavior. In that study, we biochemically tested two pharmacologic agents and confirmed that they decreased PLC activity in the MBs and other brain regions. Here, we present a detailed description of how to detect PLC activity in honeybee brain homogenate. In this assay system, homogenates derived from different brain regions are reacted with a synthetic fluorogenic substrate, and fluorescence resulting from PLC activity is quantified and compared between brain regions. We also describe our evaluation of the inhibitory effects of certain drugs on PLC activity using the same system. Although this system is likely affected by other endogenous fluorescence compounds and/or the absorbance of the assay components and tissues, the measurement of PLC activity using this system is safer and easier than that using the traditional assay, which requires radiolabeled substrates. The simple procedure and manipulations allow us to examine PLC activity in the brains and other tissues of honeybees involved in different social tasks.

To test the inhibitory effects of pharmacologic agents on phospholipase C (PLC) in different regions of the honeybee brain, we present a biochemical assay to measure PLC activity in those regions. This assay could be useful for comparing PLC activity among tissues, as well as among bees exhibiting different behaviors.

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom ...

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

Biology

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found to represent a novel lipid transfer protein (LTP) group in yeast and human cells. They transfer phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) via PS/phosphatidylinositol 4-phosphate (PI(4)P) exchange cycles. This finding allows a better understanding of how PS, which is critical for signaling processes, is distributed throughout the cell and the investigation of the link between this process and phosphoinositide (PIP) metabolism. The development of new fluorescence-based protocols has been instrumental in the discovery and characterization of this new cellular mechanism in vitro at the molecular level. This paper describes the production and the use of two fluorescently labelled lipid sensors, NBD-C2Lact and NBD-PHFAPP, to measure the ability of a protein to extract PS or PI(4)P and to transfer these lipids between artificial membranes. First, the protocol describes how to produce, label, and obtain high-purity samples of these two constructs. Secondly, this paper explains how to use these sensors with a fluorescence microplate reader to determine whether a protein can extract PS or PI(4)P from liposomes, using Osh6p as a case study. Finally, this protocol shows how to accurately measure the kinetics of PS/PI(4)P exchange between liposomes of defined lipid composition and to determine lipid transfer rates by fluorescence resonance energy transfer (FRET) using a standard fluorometer.

Here, we describe protocols using fluorescent lipid sensors and liposomes to determine whether a protein extracts and transports phosphatidylserine or phosphatidylinositol 4-phosphate in vitro.

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found ...

Lethality Bioassay Using Artemia salina L.

Natural products have been used since ancient times to produce medicines. Nowadays, there are plenty of chemotherapeutic drugs obtained from natural sources and used against a plethora of diseases. Unfortunately, most of these compounds often display systemic toxicity and adverse effects. In order to better evaluate the tolerability of selected potentially bioactive samples, brine shrimp (Artemia salina) is generally used as a model in lethality studies. The A. salina test is based on the ability of the studied bioactive compounds to kill the microcrustaceans in their larval stage (nauplii). This method represents a convenient starting point for cytotoxicity studies, as well as for the general toxicity screening of synthetic, semisynthetic, and natural products. It can be considered a simple, quick, and low-cost assay, compared to many other assays (in vitro cells or yeast strains, zebrafish, rodents) generally suitable for the aforementioned purposes; moreover, it can be easily performed even without any specific training. Overall, A. salina assay represents a useful tool for the preliminary toxicity evaluation of selected compounds and the bio-guided fractionation of natural product extracts.

Lethality Bioassay Using Artemia salina L.

This work aims to evaluate and review the Artemia salina lethality bioassay procedure, also identified as brine shrimp lethality assay. This simple and cheap method gives information about the general toxicity (considered as a preliminary toxicity evaluation) of samples, namely, natural products.

Natural products have been used since ancient times to produce medicines. Nowadays, there are plenty of chemotherapeutic drugs obtained from natural ...

Chemistry

Endotoxin Activity Assay for the Detection of Whole Blood Endotoxemia in Critically Ill Patients

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis and septic shock. The early identification of an infectious process that is rapidly evolving to a critical illness might prompt a quicker and more intensive treatment, thereby potentially leading to better patient outcomes. The Endotoxin Activity (EA) assay can be used at the bedside as a reliable biomarker of systemic endotoxemia. The detection of elevated endotoxin activity levels has been repeatedly shown to be associated with an increased disease severity in patients with sepsis and septic shock. The assay is quick and easy to perform. Briefly, after sampling, an aliquot of whole blood is mixed with an anti-endotoxin antibody and with added LPS. Endotoxin activity is measured as the relative oxidative burst of primed neutrophils as detected by chemioluminescence. The assay&#39;s output is expressed on a scale from 0 (absent) to 1 (maximal) and categorized as &#8220;low&#8221; (&#60;0.4 units), &#8220;intermediate&#8221; (0.4&#8211;0.59 units), or &#8220;high&#8221; (&#8805;0.6 units). The detailed methodology and rationale for the implementation of the EA assay are reported in this manuscript.

We hereby present a protocol to measure at the bedside the endotoxin activity of human whole blood samples. The Endotoxin Activity assay is a simple test to perform and may be a useful biomarker in critically ill patients with sepsis.

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis ...

Medicine

Assessing the Putative Anticryptococcal Properties of Crude and Clarified Extracts from Mollusks

Cryptococcus neoformans is an encapsulated human fungal pathogen with a global distribution that primarily infects immunocompromised individuals. The widespread use of antifungals in clinical settings, their use in agriculture, and strain hybridization have led to increased evolution of resistance. This rising rate of resistance against antifungals is a growing concern among clinicians and scientists worldwide, and there is heightened urgency to develop novel antifungal therapies. For instance, C. neoformans produces several virulence factors, including intra- and extra-cellular enzymes (e.g., peptidases) with roles in tissue degradation, cellular regulation, and nutrient acquisition. The disruption of such peptidase activity by inhibitors perturbs fungal growth and proliferation, suggesting this may be an important strategy for combating the pathogen. Importantly, invertebrates such as mollusks produce peptidase inhibitors with biomedical applications and anti-microbial activity, but they are underexplored in terms of their usage against fungal pathogens. In this protocol, a global extraction from mollusks was performed to isolate potential peptidase inhibitors in crude and clarified extracts, and their effects against classical cryptococcal virulence factors were assessed. This method supports the prioritization of mollusks with antifungal properties and provides opportunities for the discovery of anti-virulence agents by harnessing the natural inhibitors found in mollusks.

The human fungal pathogen Cryptococcus neoformans produces a variety of virulence factors (e.g., peptidases) to promote its survival within the host. Environmental niches represent a promising source of novel natural peptidase inhibitors. This protocol outlines the preparation of extracts from mollusks and the assessment of their effect on fungal virulence factor production.

Cryptococcus neoformans is an encapsulated human fungal pathogen with a global distribution that primarily infects immunocompromised individuals. The ...

Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays

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