Name: in vitro digestion of emulsions in a single droplet via multi subphase exchange of simulated gastrointestinal fluids
Uploaded: 2022-11-18T13:40:00
Description: Watch this Scientific Journal Video about In vitro Digestion of Emulsions in a Single Droplet via Multi Subphase Exchange of Simulated Gastrointestinal Fluids at JoVE.com

This research was funded by projects RTI2018-101309-B-C21 and PID2020-631-116615RAI00, funded by MCIN/AEI/10.13039/501100011033 and by &#34;ERDF A way of making Europe&#34;. This work was (partially) supported by the Biocolloid and Fluid Physics Group (ref. PAI-FQM115) of the University of Granada (Spain).

1. Cleaning sequence for all glassware used in surface science experimentation
<ol>
	<li>Scrub the glassware with a concentrated cleaning solution (see Table of Materials) diluted in water (10%).</li>
	<li>Rinse thoroughly with a sequence of tap water, propanol, distilled water, and ultrapure water. Dry in a cabin and store in a closed cabinet until use.</li>
</ol>
2. Sample preparation
<ol>
	<li>Prepare artificial digestive media according to...

<ol>
	<li>McClements, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biophysics+of+digestion:+Lipids.">The biophysics of digestion: Lipids.</a> Current Opinion in Food Science. 21, 1-6 (2018).</li><li>McClements, D. J., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structured+emulsion-based+delivery+systems:+Controlling+the+digestion+and+release+of+lipophilic+food+components.">Structured emulsion-based delivery systems: Controlling the digestion and release of lipophilic food components.</a> Advances in Colloid and Interface Science. 159 (2), 213-228 (2010).</li><li>Corstens, M. 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M., Carrera S&#225;nchez, C., Rodr&#237;guez Ni&#241;o, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+of+interfacial+characteristics+of+food+foaming+agents+in+foam+formulations.">Implications of interfacial characteristics of food foaming agents in foam formulations.</a> Advances in Colloid and Interface Science. 140 (2), 95-113 (2008).</li><li>Wilde, P. J., Chu, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interfacial+&amp;+colloidal+aspects+of+lipid+digestion.">Interfacial &amp; colloidal aspects of lipid digestion.</a> Advances in Colloid and Interface Science. 165 (1), 14-22 (2011).</li><li>Cabrerizo-V&#237;lchez, M. A., Wege, H. A., Holgado-Terriza, J. A., Neumann, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axisymmetric+drop+shape+analysis+as+penetration+Langmuir+balance.">Axisymmetric drop shape analysis as penetration Langmuir balance.</a> Review of Scientific Instruments. 70 (5), 2438-2444 (1999).</li><li>Maldonado-Valderrama, J., Muros-Cobos, J. L., Holgado-Terriza, J. A., Cabrerizo-V&#237;lchez, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bile+salts+at+the+air-water+interface:+Adsorption+and+desorption.">Bile salts at the air-water interface: Adsorption and desorption.</a> Colloids and surfaces B: Biointerfaces. 120, 176-183 (2014).</li><li>Maldonado-Valderrama, J., Terriza, J. A. H., Torcello-G&#243;mez, A., Cabrerizo-V&#237;lchez, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+digestion+of+interfacial+protein+structures.">In vitro digestion of interfacial protein structures.</a> Soft Matter. 9, 1043-1053 (2013).</li><li>Maldonado-Valderrama, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+in+vitro+digestion+at+oil-water+interfaces.">Probing in vitro digestion at oil-water interfaces.</a> Current Opinion in Colloid and Interface Science. 39, 51-60 (2019).</li><li>Brodkorb, 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=INFOGEST+static+in+vitro+simulation+of+gastrointestinal+food+digestion.">INFOGEST static in vitro simulation of gastrointestinal food digestion.</a> Nature Protocols. 14 (4), 991-1014 (2019).</li><li>del Castillo-Santaella, T., Maldonado-Valderrama, J., Molina-Bolivar, J. A., Galisteo-Gonzalez, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cross-linker+glutaraldehyde+on+gastric+digestion+of+emulsified+albumin.">Effect of cross-linker glutaraldehyde on gastric digestion of emulsified albumin.</a> Colloids and Surfaces B: Biointerfaces. 145, 899-905 (2016).</li><li>Macierzanka, A., Torcello-G&#243;mez, A., Jungnickel, C., Maldonado-Valderrama, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bile+salts+in+digestion+and+transport+of+lipids.">Bile salts in digestion and transport of lipids.</a> Advances in Colloid and Interface Science. 274, 102045 (2019).</li><li>Maldonado-Valderrama, J., Torcello-G&#243;mez, A., del Castillo-Santaella, T., Holgado-Terriza, J. A., Cabrerizo-V&#237;lchez, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subphase+exchange+experiments+with+the+pendant+drop+technique.">Subphase exchange experiments with the pendant drop technique.</a> Advances in Colloid and Interface Science. 222, 488-501 (2015).</li><li>Bellesi, F. A., Ruiz-Henestrosa, V. M. P., Maldonado-Valderrama, J., Del Castillo Santaella, T., Pilosof, A. M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+interfacial+in+vitro+digestion+of+protein+and+polysaccharide+oil/water+films.">Comparative interfacial in vitro digestion of protein and polysaccharide oil/water films.</a> Colloids and Surfaces B: Biointerfaces. 161, 547-554 (2018).</li><li>Del Castillo-Santaella, T., Sanmart&#237;n, E., Cabrerizo-V&#237;lchez, M. A., Arboleya, J. C., Maldonado-Valderrama, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+digestibility+of+&#946;-lactoglobulin+by+pulsed+light+processing:+A+dilatational+and+shear+study.">Improved digestibility of &#946;-lactoglobulin by pulsed light processing: A dilatational and shear study.</a> Soft Matter. 10 (48), 9702-9714 (2014).</li><li>Infantes-Garcia, M. 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=In+vitro+gastric+lipid+digestion+of+emulsions+with+mixed+emulsifiers:+Correlation+between+lipolysis+kinetics+and+interfacial+characteristics.">In vitro gastric lipid digestion of emulsions with mixed emulsifiers: Correlation between lipolysis kinetics and interfacial characteristics.</a> Food Hydrocolloids. 128, 107576 (2022).</li><li>del Castillo-Santaella, T., Cebri&#225;n, R., Maqueda, M., G&#225;lvez-Ruiz, M. J., Maldonado-Valderrama, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+in+vitro+digestibility+of+food+biopreservative+AS-48.">Assessing in vitro digestibility of food biopreservative AS-48.</a> Food Chemistry. 246, 249-257 (2018).</li><li>Torcello-G&#243;mez, A., Maldonado-Valderrama, J., J&#243;dar-Reyes, A. B., Cabrerizo-V&#237;lchez, M. A., Mart&#237;n-Rodr&#237;guez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluronic-covered+oil-water+interfaces+under+simulated+duodenal+conditions.">Pluronic-covered oil-water interfaces under simulated duodenal conditions.</a> Food Hydrocolloids. 34, 54-61 (2014).</li><li>Minekus, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+standardised+static+in+vitro+digestion+method+suitable+for+food+-+an+international+consensus.">A standardised static in vitro digestion method suitable for food - an international consensus.</a> Food &amp; Function. 5 (6), 1113-1124 (2014).</li><li>Wege, H. A., Holgado-Terriza, J. A., Cabrerizo-V&#237;lchez, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+constant+surface+pressure+penetration+langmuir+balance+based+on+axisymmetric+drop+shape+analysis.">Development of a constant surface pressure penetration langmuir balance based on axisymmetric drop shape analysis.</a> Journal of Colloid and Interface Science. 249 (2), 263-273 (2002).</li><li>del Castillo-Santaella, T., 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=Hyaluronic+acid+and+human/bovine+serum+albumin+shelled+nanocapsules:+Interaction+with+mucins+and+in+vitro+digestibility+of+interfacial+films.">Hyaluronic acid and human/bovine serum albumin shelled nanocapsules: Interaction with mucins and in vitro digestibility of interfacial films.</a> Food Chemistry. 383, 132330 (2022).</li><li>Aguilera-Garrido, 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=Applications+of+serum+albumins+in+delivery+systems:+Differences+in+interfacial+behaviour+and+interacting+abilities+with+polysaccharides.">Applications of serum albumins in delivery systems: Differences in interfacial behaviour and interacting abilities with polysaccharides.</a> Advances in Colloid and Interface Science. 290 (5), 102365 (2021).</li></ol>

This article describes a generalized protocol to measure in vitro digestion of interfacial layers by using pendant drop equipment. The protocol can be adjusted to the specific requirements of the experiment by tuning the composition of the digestive buffers, which are based on the INFOGEST11,20 harmonized protocol to facilitate comparison with literature. The digestive enzymes and biosurfactants can be added individually, sequentially, or together. This ...

Understanding how fat is digested, which involves emulsion digestion, is important to rationally design products with tailored functionality1. The substrate for fat digestion is an emulsion since fat is emulsified upon consumption by mechanical action and mixing with biosurfactants in the mouth and stomach. Also, most of the fat consumed by humans is already emulsified (such as milk products), and in the case of infants or some elderly people, this is the only form of consumption. Hence, the design of emulsion-based products with specific digestion profiles is very important in nutrition1. Moreover, emulsions can encapsulate and deliver nutrients, drugs, or lipophilic bioactives2 to tackle different gastrointestinal conditions such as obesity3, nutrient fortification, food allergies, and digestive diseases. In oil-in-water emulsions, lipid droplets are surrounded by interfacial layers of emulsifiers such as proteins, surfactants, polymers, particles, and mixtures4. The role of emulsifiers is twofold: stabilize the emulsion5 and protect/transport the encapsulated compound to a specific site. Achieving a tailored digestibility of emulsions depends on their initial composition but also requires monitoring the continuous evolution of this interface during the transit through the gastrointestinal tract (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64158/64158fig01.jpg" /> 
Figure 1: Applying interfacial engineering of emulsions to tackle some of the main gastrointestinal conditions. <a href="https://www.jove.com/files/ftp_upload/64158/64158fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Lipid digestion is ultimately an interfacial process because it requires the adsorption of lipases (gastric or pancreatic) onto the oil-water interface of emulsified lipid droplets through the interfacial layer to reach and hydrolyze the triglycerides contained in the oil into free fatty acids and monoacylglycerides6. This is schematized in Figure 2. Gastric lipase competes with pepsin and phospholipids in the stomach for the oil-water interface (Figure 2, gastric digestion). Then, pancreatic lipase/colipase compete with trypsin/chymotrypsin, phospholipids, bile salts, and digestive products in the small intestine. Proteases can alter the interfacial coverage, preventing or favoring lipase adsorption, while bile salts are highly surface active and displace most of the remaining emulsifier to promote lipase adsorption (Figure 2, intestinal digestion). Eventually, the rate and extent of lipolysis depend on the interfacial properties of the initial/gastric digested emulsion, such as the thickness, inter/intramolecular connections, and electrostatic and steric interactions. Accordingly, monitoring the evolution of the interfacial layer as it is digested offers an experimental platform to identify interfacial mechanisms and events affecting lipase adsorption and, hence, lipid digestion.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64158/64158fig02.jpg" /> 
Figure 2: Schematic diagram illustrating the role of interfaces in gastrointestinal lipid digestion. Pepsin hydrolysis alters interfacial composition at the gastric phase, while gastric lipase hydrolyzes triglycerides. In the small intestine, trypsin/chymotrypsin further hydrolyze the interfacial film, while lipolysis proceeds by the adsorption of BS/lipases, the hydrolysis of triglycerides, and the desorption of lipolytic products by solubilization in the BS micelles/complex. <a href="https://www.jove.com/files/ftp_upload/64158/64158fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The pendant drop equipment at the University of Granada (UGR) is implemented with a patented technology, the coaxial double capillary, that enables subphase exchange of the bulk solution7. The capillary, which holds the pendant drop, consists of an arrangement of two coaxial capillaries that are independently connected to each channel of a double microinjector. Each microinjector can operate independently, allowing the exchange of the dropped content by through-flow7. Accordingly, the subphase exchange consists of the simultaneous injection of the new solution with the inner capillary and the extraction of the bulk solution with the outer capillary using the same flow rate. This process allows the replacement of the bulk solution with no disturbance of the interfacial area or the volume of the droplet. This procedure was later upgraded to a multi-subphase exchange, which allows up to eight sequential subphase exchanges of the droplet bulk solution8. This enables the simulation of the digestive process in a single aqueous droplet suspended in lipidic media by sequentially exchanging the bulk solution with artificial media mimicking the different compartments (mouth, stomach, small intestine). The whole setup is represented in Figure 3, including the details of the components. The syringes in the microinjector are connected to the eight vias valves, each connecting to a microcentrifuge tube containing the artificial digestive fluid with components described in Figure 2.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64158/64158fig03.jpg" /> 
Figure 3: General view of the OCTOPUS with all components. The CCD camera, microscope, micro-positioner, thermostabilized cell, and double capillary connected independently to a double microinjector with two syringes connected to eight vias valves. Each syringe connects with capillary, four&#160;microcentrifuge tubes with sample and one discharge. <a href="https://www.jove.com/files/ftp_upload/64158/64158fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 4A shows how each of the artificial digestive fluids is injected into the pendant drop by subphase exchange through the double capillary. Each digestive compound detailed in Figure 2 can be applied simultaneously/sequentially, simulating the passage through the gastrointestinal tract. The artificial digestive fluids contain different enzymes and biosurfactants, which alter the interfacial tension of the initial emulsifier, as schematized in Figure 4B. The software DINATEN (see Table of Materials), also developed at the UGR, records the evolution of the interfacial tension in real time as the initial interfacial layer is digested in vitro. Also, after each digestive phase, the dilatational elasticity of the interfacial layer is computed by imposing periodic oscillations of volume/interfacial area onto the stabilized interfacial layer and recording the response of the interfacial tension. The period/frequency and the amplitude of the oscillation can be varied, and image processing with the software CONTACTO provides the dilatational rheological parameters8.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64158/64158fig04.jpg" /> 
Figure 4: Examples of digestion profiles. (A) The initial emulsifier layer is subjected to artificial digestive media placed in the microcentrifuge by sequential subphase exchange of the different solutions into the pendant drop. (B) The general evolution of the interfacial tension (y-axis) of the initial emulsifier as a function of time (x-axis) as it is digested in vitro by the various enzymes/biosurfactants in the artificial media. A final subphase exchange with plain intestinal fluid measures the desorption of digested lipid by solubilization in mixed micelles. <a href="https://www.jove.com/files/ftp_upload/64158/64158fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
This study presents the general protocol designed to measure in vitro digestion of interfacial layers with pendant drop equipment9. The initial interfacial layer is subjected sequentially to conditions mimicking the passage through the gastrointestinal tract, as depicted in Figure 2. These different digestive media are injected into the pendant drop by subphase exchange of the different solutions contained in the microcentrifuge tubes (Figure 4A). The composition of these media can be customized depending on the gastrointestinal conditions that will be evaluated, namely, gastric/intestinal proteolysis/lipolysis, allowing for measuring cumulative effects and sinergies10. The experimental conditions used to mimic the digestion process in each compartment follow the international consensus protocol published by INFOGEST detailing the pH and amounts of electrolytes and enzymes11. The experimental device based on pendant drop allows recording of the interfacial tension in situ throughout the simulated digestion process. The dilatational rheology of the interfacial layer is computed at the end of each digestive step. In this way, each emulsifier offers a digestion profile illustrating the properties of the digested interfaces, as depicted in Figure 4B. This allows the extraction of conclusions regarding its susceptibility or resistance to the different stages of the digestive process. In general, the artificial digestive media contains acid/basic pH, electrolytes, proteases (gastric and intestinal), lipases (gastric and intestinal), bile salts, and phospholipids, which are dissolved in their respective digestive fluids (gastric or intestinal). Figure 4B shows a generic profile of the evolution of an emulsifier&#39;s interfacial tension, first subjected to protease action, followed by lipases. In general, proteolysis of the interfacial layer promotes an increase in the interfacial tension owing to the desorption of hydrolyzed peptides9,12, while lipolysis results in a very steep reduction in the interfacial tension due to the adsorption of bile salts and lipases13. A final subphase exchange with intestinal fluid depletes the bulk solution of unadsorbed/digested material and promotes the desorption of soluble compounds and the solubilization of digested lipids in mixed micelles. This is quantified by the increased interfacial tension recorded (Figure 4B).
In summary, the experimental design implemented in the pendant drop to simulate in vitro digestion in a single droplet allows for measuring cumulative effects and synergies as the digestion process is applied sequentially to the initial interfacial layer10. The composition of each digestive media can be easily tuned to account for the particularities of the digestive conditions, including gastrointestinal pathologies or infant digestive media14. Then, identification of the interfacial mechanisms affecting proteolysis and lipolysis can be used to modulate digestion by the interfacial engineering of emulsions. The obtained results can be applied in designing novel food matrices with tailored functionalities such as low allergenicity, controlled energy intake, and decreased digestibility15,16,17,18,19.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alpha-chymotrypsin from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>C4129</td>
			<td>Enzyme</td>
		</tr>
		<tr>
			<td>Beta-lactoglobulin</td>
			<td>Sigma-Aldrich</td>
			<td>L0130</td>
			<td>Emulsfier</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>9048-46-8</td>
			<td>Emulsfier</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>10043-52-4</td>
			<td>Electrolyte</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Kronton instruments</td>
			<td>Centrikon T-124</td>
			<td>For separating oil and resins</td>
		</tr>
		<tr>
			<td>Citrus pectin</td>
			<td>Sigma-Aldrich</td>
			<td>P9135</td>
			<td>Emulsfier</td>
		</tr>
		<tr>
			<td>co-lipase FROM PORCINE PANCREAS</td>
			<td>Sigma</td>
			<td>C3028</td>
			<td>Enzyme</td>
		</tr>
		<tr>
			<td>CONTACTO</td>
			<td>University of Granada (UGR)</td>
			<td></td>
			<td>https://core.ugr.es/dinaten/, last access: 07/18/2022</td>
		</tr>
		<tr>
			<td>DINATEN</td>
			<td>University of Granada (UGR)</td>
			<td></td>
			<td>https://core.ugr.es/dinaten/, last access: 07/18/2022</td>
		</tr>
		<tr>
			<td>Gastric lipase</td>
			<td>Lipolytech</td>
			<td>RGE15-1G</td>
			<td>Enzyme</td>
		</tr>
		<tr>
			<td>Human Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>70024-90-7</td>
			<td>Emulsifier</td>
		</tr>
		<tr>
			<td>INFOGEST</td>
			<td></td>
			<td></td>
			<td>http://www.proteomics.ch/IVD/</td>
		</tr>
		<tr>
			<td>Lipase from porcine pancreas, type II</td>
			<td>Sigma-Aldrich</td>
			<td>L33126</td>
			<td>Enzyme</td>
		</tr>
		<tr>
			<td>Magnesium metasilicate resins</td>
			<td>Fluka</td>
			<td>1343-88-0</td>
			<td>Resins to purify oil</td>
		</tr>
		<tr>
			<td>Micro 90</td>
			<td>International products</td>
			<td>M-9051-04</td>
			<td>Cleaner</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>7647-14-5</td>
			<td>Electrolyte</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Scharlau</td>
			<td>10049-21-5</td>
			<td>To prepare buffer</td>
		</tr>
		<tr>
			<td>OCTOPUS</td>
			<td>Producciones Cient&#237;ficas y T&#233;cnicas S.L. (G&#243;jar, Spain)</td>
			<td></td>
			<td>Pendandt Drop Equipment implemented with multi subphase exchange</td>
		</tr>
		<tr>
			<td>Olive oil</td>
			<td>Sigma-Aldrich</td>
			<td>1514</td>
			<td>oil</td>
		</tr>
		<tr>
			<td>Pancreatic from porcine pancreas</td>
			<td>Sigma</td>
			<td>P7545-25 g</td>
			<td>Enzyme</td>
		</tr>
		<tr>
			<td>Pepsin</td>
			<td>Sigma-Aldrich</td>
			<td>P6887</td>
			<td>Enzyme</td>
		</tr>
		<tr>
			<td>Pluronic F127</td>
			<td>Sigma</td>
			<td>P2443</td>
			<td>Emulsifier</td>
		</tr>
		<tr>
			<td>Pluronic F68</td>
			<td>Sigma</td>
			<td>P1300</td>
			<td>Emulsfier</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma</td>
			<td></td>
			<td>Bile salts</td>
		</tr>
		<tr>
			<td>Sodium glycodeoxycholate</td>
			<td>Sigma</td>
			<td>C9910</td>
			<td>Bile salts</td>
		</tr>
		<tr>
			<td>Sodium taurocholate</td>
			<td>Sigma</td>
			<td>86339</td>
			<td>Bile salts</td>
		</tr>
		<tr>
			<td>Syringe Filter</td>
			<td>Millex-DP</td>
			<td>SLGP033R</td>
			<td>&#160;Syringe Filter 0.22 &#181;m pore size polyethersulfone</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>T1426</td>
			<td>Enzyme</td>
		</tr>
	</tbody>
</table>

in vitro digestion of emulsions in a single droplet via multi subphase exchange of simulated gastrointestinal fluids

Emulsions are currently being used to encapsulate and deliver nutrients and drugs to tackle different gastrointestinal conditions such as obesity, nutrient fortification, food allergies, and digestive diseases. The ability of an emulsion to provide the desired functionality, namely, reaching a specific site within the gastrointestinal tract, inhibiting/retarding lipolysis, or facilitating digestibility, ultimately depends on its susceptibility to enzymatic degradation in the gastrointestinal tract. In oil-in-water emulsions, lipid droplets are surrounded by interfacial layers, where the emulsifiers stabilize the emulsion and protect the encapsulated compound. Achieving a tailored digestibility of emulsions depends on their initial composition but also requires monitoring the evolution of those interfacial layers as they are subjected to different phases of gastrointestinal digestion. A pendant drop surface film balance implemented with a multi-subphase exchange allows for simulating the in vitro digestion of emulsions in a single aqueous droplet immersed in oil by applying a customized static digestion model. The transit through the gastrointestinal tract is mimicked by the subphase exchange of the original droplet bulk solution with artificial media, mimicking the physiological conditions of each compartment/step of the gastrointestinal tract. The dynamic evolution of the interfacial tension is recorded in situ throughout the whole simulated gastrointestinal digestion. The mechanical properties of digested interfaces, such as interfacial dilatational elasticity and viscosity, are measured after each digestion phase (oral, gastric, small intestine). The composition of each digestive media can be tuned to account for the particularities of the digestive conditions, including gastrointestinal pathologies and infant digestive media. The specific interfacial mechanisms affecting proteolysis and lipolysis are identified, providing tools to modulate digestion by the interfacial engineering of emulsions. The obtained results can be manipulated for designing novel food matrices with tailored functionalities such as low allergenicity, controlled energy intake, and decreased digestibility.

This section shows different examples of digestion profiles measured with the OCTOPUS. The general appearance of the simulated digestion profile matches is shown in Figure 4B. The interfacial tension is usually represented against time in the digestion profile. The different phases/digestion steps considered are represented in different colors. The first phase forms the initial layer and corresponds to the adsorption phase of the emulsifier or protein/surfactant/polymer, depending on each ca...

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In vitro Digestion of Emulsions in a Single Droplet via Multi Subphase Exchange of Simulated Gastrointestinal Fluids

A pendant drop surface film balance implemented with a multi-subphase exchange, nicknamed the OCTOPUS, allows for mimicking digestive conditions by the sequential subphase exchange of the original bulk solution with simulated gastrointestinal fluids. The simulated in vitro digestion is monitored by recording in situ the interfacial tension of the digested interfacial layer.

In vitro Digestion of Emulsions in a Single Droplet via Multi Subphase Exchange of Simulated Gastrointestinal Fluids

Emulsions are currently being used to encapsulate and deliver nutrients and drugs to tackle different gastrointestinal conditions such as obesity, ...

Our method provides model to investigate how emotions are digested by simulating the process of digestion in a single aquas droplet, which is covered by emulsifier and is immersed in oil phase. We can evaluate the impact of different emulsifiers, and the action of different digestive components, and design interfaces that can resist or retard digestion at different locations within the gastrointestinal tract. We can measure interfacial tension in C2 throughout the whole simulated digestion process, and the elasticity and viscosity of the interfacial layer at the end of it digesting step.The concentrations and the conditions of the digestion can be adjusted to the requirements of the experiment, and this can be applied sequentially, or simultaneously allowing to evaluate synergism and cumulative effects. And we work with a single droplet, so we need a very low amount of sample, and we have high precision control of experimental variables. We have applied this technique to design a strategy to take over this.One of those approaches is to design in the fossil layer that retires or receipts the process of lipolysis. Another approach is the use of lipase innovators, and we have investigated the interfacial mechanism of action of lipase. This device was originally developed to study the digestion in vitro.Nevertheless, it can be used for study mono and interaction with lipid, and also can be applied to study automatically the critical micro concentration. The experimental technique is accessible. A manual researcher has been trained to use it in one week.You need to clean up the equipment thoroughly, and purify the samples and oil in order to prevent the presence of surface active contaminants. You need to get familiar with the software computer, and learn to control the lights and position of capability, and get rid of tiny bubbles. To begin, formal water droplet to check the surface tension of water at room temperature.Set the differential density to air water in the left dialogue, and measure the surface tension in real time. for five minutes. Fill the clean cuvet with at least 0.002 liters of clean vegetable oil, and place it in the cuvet holder in the thermostatic cell.Set the differential density to vegetable oil water and inject 40 microliters at a rate of 0.5 microliters per second. Measure the tension in real time every second until the end of the injection. This is a simple dynamic process.Next, save the data and plot the interfacial tension as a function of droplet volume and a data sheet. Check that the droplet volume range provides a value for the interfacial tension independent of the droplet volume for clean water. Plot the interfacial area as a function of the droplet volume.This curve will be used later to match the volume with the corresponding interfacial area. With the left syringe, inject the volume within the range of constant interfacial tension and record the interfacial tension for five minutes. This is to check the absence of surface active components in the system.To perform the initial control, inject around 10 microliters of the emulsifier solution into the capillary for drop formation, and record the absorption at a constant interfacial area of around 20 square millimeter for one hour. The exact values of volume and area can be obtained from the calibration curve plotted previously. Program the dilutional reology by setting the amplitude of oscillation to 1.25 microliters in the period to 10 seconds.Then program the absorption at the selected interfacial area for 10 seconds. Next, to record the gastric digestion program the absorption at the selected interfacial area for 10 seconds. Fill the left syringe with liquid from valve two.Inject 125 microliters from valve two at five microliters per second with the left syringe, and simultaneously extract the same volume at the same rate with the right syringe. This is a visualization of the process of sub phase exchange of water with methylene blue. Unload the right syringe to exit valve eight and load again the left syringe with liquid from valve two.Repeat these two steps 10 times to assure complete sub phase exchange with liquid and valve two and gastric enzymes. Then, record the absorption at the selected interfacial area for one hour and record the dilutional reology as shown earlier. To record the intestinal digestion after recording the absorption at the selected interfacial area, fill the left syringe with liquid from valve three and follow the same steps shown earlier for recording the gastric digestion.Similarly, to record the desorption, after recording the absorption at the selected interfacial area, fill the left syringe with liquid from valve five and repeat the rest of the steps used to record the gastric digestion. Fill the micro centrifuge tubes with the artificial digestion media, and connect each of them to the respective valve by the corresponding tubing. Fill the tubing in valves two to eight by cleaning from valve two, valve three, valve four, and valve five to the external exit that is valve eight.Fill the tubing in valve one by cleaning from valve one to valve six capillary five times. Place the capillary into the oil phase, and load the left syringe with valve one. Start sequentially processing the initial control gastric digestion, intestinal digestion, and desorption, saving the data at the end of each process.Experimental results obtained for the gastric digestion of emulsifiers are shown in these figures. Gastric proteolysis of human serum albumin is presented here. Digestive media are applied by sub phases exchange with solutions at 37 degrees Celsius.Here blue represents the initial buffer with protein and red represents simplified SGF with pepsin. During sub phase exchange with simplified SGF and pepsin, the interfacial tension increases owing to the hydrolysis of the protein, which dilutes the initial protein layer. The graphical image represents the gastric lipolysis of citrus pectin.Here, blue represents the initial buffer with citrus pectin, yellow represents simplified SGF with gastric lipase, and gray represents simplified SGF. Sub phases exchange with gastric lipase decreases the surface tension while sub phases exchange with simplified SGF, provides a null response of the interfacial tension. Shown here is an example of the intestinal digestion profiles.Absorption, desorption, profiles of bio salts, lipase, and lipase plus bile salts in simplified SIF at 37 degrees Celsius are presented here. The graphical image shows the evolution of the interfacial tension upon lipolysis of two variants of plueronic F127 and F68. A steep decrease can be seen in interfacial tension due to the absorption of lipase and bile salts, and the production of free fatty acids onto previously formed interfacial films of F68 and F127 at the oil water interface.The desorption step shows the sub phase exchange with simplified SIF from bile salts, F68, and F127. In vitro digestion profile of AS48 absorbed film at the air water interface in human and bovine serum albumin absorbed films at the olive oil water interface. The representative images show the interfacial tension, the dilation elasticity, and the dilation viscosity of in vitro digestion of beta lactoglobulin absorbed film at the olive oil water interface.The dilation parameters were measured at 1 hertz, 0.1 hertz, and 0.01 hertz after the digested interface was equilibrated in each step. It is important to match the balls when programming the process with the corresponding centrifuge tubes, capillary, or axis, the evolution of the drop site distribution, then, electrophoretic mobility set a potential of droplets, and the amino free fatty acid produces in lipolysis offer complementary information to corresponding with findings from interfacial tension. This technique provides interfacial layers with different digestibility profiles to deliver nutrients and drugs at different locations within gastrointestinal tract, and also to develop new encapsulation systems.

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Research

JoVE Journal

Biochemistry

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for skin-care cosmetics. The goals of the protocol are: (1) to process chicken feathers into KH by alkaline-enzymatic hydrolysis and purify it by dialysis, and (2) to test if adding KH into an ointment base (OB) increases hydration of the skin and improves skin barrier function by diminishing transepidermal water loss (TEWL). During alkaline-enzymatic hydrolysis feathers are first incubated at a higher temperature in an alkaline environment and then, under mild conditions, hydrolyzed with proteolytic enzyme. The solution of KH is dialyzed, vacuum dried, and milled to a fine powder. Cosmetic formulations comprising from oil in water emulsion (O/W) containing 2, 4, and 6 weight% of KH (based on the weight of the OB) are prepared. Testing the moisturizing properties of KH is carried out on 10 men and 10 women at time intervals of 1, 2, 3, 4, 24, and 48 h. Tested formulations are spread at degreased volar forearm sites. The skin hydration of stratum corneum (SC) is assessed by measuring capacitance of the skin, which is one of the most world-wide used and simple methods. TEWL is based on measuring the quantity of water transported per a defined area and period of time from the skin. Both methods are fully non-invasive. KH makes for an excellent occlusive; depending on the addition of KH into OB, it brings about a 30% reduction in TEWL after application. KH also functions as a humectant, as it binds water from the lower layers of the epidermis to the SC; at the optimum KH addition in the OB, up to 19% rise in hydration in men and 22% rise in women occurs.

The goal of the protocol is to prepare keratin hydrolysate from chicken feathers by alkaline-enzymatic hydrolysis and to test whether adding keratin hydrolysate into a cosmetics ointment base improves skin barrier function (heightening hydration and diminishing transepidermal water loss). Tests are conducted on men and woman volunteers.

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

Fluorescent Visualization of Mango-tagged RNA in Polyacrylamide Gels via a Poststaining Method

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, respectively. As many gel-imaging techniques use nonspecific stains or expensive fluorophore probes, sensitive, discriminating, and economical gel-imaging methodologies are highly desirable. RNA Mango core sequences are small (19&#8211;22 nt) sequence motifs that, when closed by an arbitrary RNA stem, can be simply and inexpensively appended to an RNA of interest. These Mango tags bind with high affinity and specificity to a thiazole-orange fluorophore ligand called TO1-Biotin, which becomes thousands of times more fluorescent upon binding. Here we show that Mango I, II, III, and IV can be used to specifically image RNA in gels with high sensitivity. As little as 62.5 fmol of RNA in native gels and 125 fmol of RNA in denaturing gels can be detected by soaking gels in an imaging buffer containing potassium and 20 nM TO1-Biotin for 30 min. We demonstrate the specificity of the Mango-tagged system by imaging a Mango-tagged 6S bacterial RNA in the context of a complex mixture of total bacterial RNA.

Here we present a sensitive, rapid, and discriminating post-gel staining method to image RNAs tagged with RNA Mango aptamers I, II, III, or IV, using either native or denaturing polyacrylamide gel electrophoresis (PAGE) gels. After running standard PAGE gels, Mango-tagged RNA can be easily stained with TO1-Biotin and then analyzed using commonly available fluorescence readers.

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, ...

Ion Exchange Chromatography (IEX) Coupled to Multi-angle Light Scattering (MALS) for Protein Separation and Characterization

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination of the high-specificity separation technique IEX with the accurate molar mass analysis achieved by MALS allows the characterization of heterogeneous protein samples, including mixtures of oligomeric forms or protein populations, even with very similar molar masses. Therefore, IEX-MALS provides an additional level of protein characterization and is complementary to the standard size-exclusion chromatography with multi-angle light scattering (SEC-MALS) technique.
Here we describe a protocol for a basic IEX-MALS experiment and demonstrate this method on bovine serum albumin (BSA). IEX separates BSA to its oligomeric forms allowing a molar mass analysis by MALS of each individual form. Optimization of an IEX-MALS experiment is also presented and demonstrated on BSA, achieving excellent separation between BSA monomers and larger oligomers. IEX-MALS is a valuable technique for protein quality assessment since it provides both fine separation and molar mass determination of multiple protein species that exist in a sample.

Ion Exchange Chromatography IEX Coupled to Multi-angle Light Scattering MALS for Protein Separation and Characterization

This protocol describes the use of high-specificity ion-exchange chromatography with multi-angle light scattering for an accurate molar mass determination of proteins, protein complexes, and peptides in a heterogeneous sample. This method is valuable for quality assessment, as well as for the characterization of native oligomers, charge variants, and mixed-protein samples.

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination ...

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide&#8211;ribosome&#8211;mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and networks often contain subsets of microtubule arrays that differ in their dynamic properties. For example, in dividing cells, stable bundles of crosslinked microtubules coexist in close proximity to dynamic non-crosslinked microtubules. TIRF-microscopy-based in vitro reconstitution studies enable the simultaneous visualization of the dynamics of these different microtubule arrays. In this assay, an imaging chamber is assembled with surface-immobilized microtubules, which are either present as single filaments or organized into crosslinked&#160;bundles. Introduction of tubulin, nucleotides, and protein regulators allows direct visualization of associated proteins and of dynamic properties of single and crosslinked microtubules. Furthermore, changes that occur as dynamic single microtubules organize into bundles can be monitored in real-time. The method described here allows for a systematic evaluation of the activity and localization of individual proteins, as well as synergistic effects of protein regulators on two different microtubule subsets under identical experimental conditions, thereby providing mechanistic insights that are inaccessible by other methods.

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Here, a TIRF microscopy-based in vitro reconstitution assay is presented to simultaneously quantify and compare the dynamics of two microtubule populations. A method is described to simultaneously view the collective activity of multiple microtubule-associated proteins on crosslinked microtubule bundles and single microtubules.

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and ...