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<meta name="robots" content="INDEX,FOLLOW,NOARCHIVE" /><meta name="citation_inbook_title" content="Glycoscience Protocols (GlycoPODv2) [Internet]" /><meta name="citation_title" content="Preparation of membrane rafts" /><meta name="citation_publisher" content="Japan Consortium for Glycobiology and Glycotechnology" /><meta name="citation_date" content="2022/03/17" /><meta name="citation_author" content="Jin-ichi Inokuchi" /><meta name="citation_pmid" content="37590582" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK593831/" /><meta name="citation_keywords" content="detergent-resistant membrane microdomains (DRMs)" /><meta name="citation_keywords" content="microdomain" /><meta name="citation_keywords" content="lipid rafts" /><meta name="citation_keywords" content="glycosphingolipids" /><meta name="citation_keywords" content="cholesterol" /><meta name="citation_keywords" content="sphingomyelin" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Preparation of membrane rafts" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="Japan Consortium for Glycobiology and Glycotechnology" /><meta name="DC.Contributor" content="Jin-ichi Inokuchi" /><meta name="DC.Date" content="2022/03/17" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK593831/" /><meta name="description" content="Two decades ago, the concept that biological membranes contain microdomains of specialized lipid and protein composition (lipid rafts) was proposed (1). 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Initially, the research was focused on the characterization of cholesterol and sphingolipid-rich membrane microdomains that were resistant to solubilization in the cold nonionic detergent Triton X-100. Such detergent-resistant membrane microdomains (DRMs) were of low buoyant density and could be readily purified on sucrose equilibrium density gradients. However, studies with other nonionic detergents, such as Lubrol WX and Brij-98, have revealed the existence of various raft subtypes with different lipid compositions and functions from those reported for Triton X-100 rafts (2). While there remains a large degree of controversy in terms of the purity, the physiological importance, and even the existence of different types of lipid rafts in intact cells, the ability to routinely purify such domains has led to significant progress in understanding the functional architecture of biological membranes. 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<div class="pre-content"><div><div class="bk_prnt"><p class="small">NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.</p><p>Nishihara S, Angata K, Aoki-Kinoshita KF, et al., editors. Glycoscience Protocols (GlycoPODv2) [Internet]. Saitama (JP): Japan Consortium for Glycobiology and Glycotechnology; 2021-. </p></div></div></div>
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<div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><h1 id="_NBK593831_"><span class="title" itemprop="name">Preparation of membrane rafts</span></h1><div class="contrib half_rhythm"><span itemprop="author">Jin-ichi Inokuchi</span>, Ph.D.<div class="affiliation small">Tohoku Medical and Pharmaceutical University<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.upm-ukohot@nij" class="oemail">pj.ca.upm-ukohot@nij</a></div></div><div class="small">Corresponding author.</div></div><p class="small">Created: <span itemprop="datePublished">October 11, 2021</span>; Last Revision: <span itemprop="dateModified">March 17, 2022</span>.</p></div><div class="body-content whole_rhythm" itemprop="text"><div id="g13-prepmembrane.Introduction"><h2 id="_g13-prepmembrane_Introduction_">Introduction</h2><p>Two decades ago, the concept that biological membranes contain microdomains of specialized lipid and protein composition (lipid rafts) was proposed (<a class="bk_pop" href="#g13-prepmembrane.REF.1">1</a>). Lipid rafts provide a means for cell membranes to form dynamic platforms within the bilayer and function in membrane trafficking, signal transduction, and cell polarization (<a class="bk_pop" href="#g13-prepmembrane.REF.1">1</a>). Initially, the research was focused on the characterization of cholesterol and sphingolipid-rich membrane microdomains that were resistant to solubilization in the cold nonionic detergent Triton X-100. Such detergent-resistant membrane microdomains (DRMs) were of low buoyant density and could be readily purified on sucrose equilibrium density gradients. However, studies with other nonionic detergents, such as Lubrol WX and Brij-98, have revealed the existence of various raft subtypes with different lipid compositions and functions from those reported for Triton X-100 rafts (<a class="bk_pop" href="#g13-prepmembrane.REF.2">2</a>). While there remains a large degree of controversy in terms of the purity, the physiological importance, and even the existence of different types of lipid rafts in intact cells, the ability to routinely purify such domains has led to significant progress in understanding the functional architecture of biological membranes. Here, a number of widely used methods to prepare rafts, based on early preparations of caveolae using density gradient ultracentrifugation, are summarized (<a class="bk_pop" href="#g13-prepmembrane.REF.3">3</a>, <a class="bk_pop" href="#g13-prepmembrane.REF.4">4</a>, <a class="bk_pop" href="#g13-prepmembrane.REF.5">5</a>).</p></div><div id="g13-prepmembrane.Protocol"><h2 id="_g13-prepmembrane_Protocol_">Protocol</h2><p>This protocol describes widely-used methods for preparing rafts, based on early preparations of caveolae, using density gradient ultracentrifugation.</p><div id="g13-prepmembrane.Materials"><h3>Materials</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Phosphate-buffered saline (PBS), pH 7.4, precooled on ice</p></dd><dt>2.</dt><dd><p class="no_top_margin">Detergent lysis (DL) buffer: 10 mM of Tris-HCl, 1 mM of ethylenediaminetetraacetic acid (EDTA), 0.5 mM of egtazic acid (EGTA), pH 7.4, and a suitable nonionic detergent, such as 1% (v/v) Triton X-100 (TX-100), 0.5% (v/v) Lubrol WX, 0.5% (v/v) Brij-96, or 0.5% (v/v) Brij-98</p></dd><dt>3.</dt><dd><p class="no_top_margin">Complete<sup>TM</sup> protease inhibitor cocktail tablets (Roche Applied Science, Penzberg, Germany) are added just before the start of the experiments at 1 tablet per 10 mL of the DL or all buffers. We have found that this inhibitor cocktail is effective in blocking proteases.</p></dd><dt>4.</dt><dd><p class="no_top_margin">Sucrose solutions for the density gradient: Sucrose (analytical grade) is added to 10 mM of Tris-HCl, 1 mM of EDTA, and 0.5 mM of EGTA, pH 7.4, to give final concentrations of 5%, 30%, and 80% sucrose (all sucrose concentrations are w/v) required for the density gradient described here.</p></dd><dt>5.</dt><dd><p class="no_top_margin">12-mL ultracentrifuge tubes (Ultra-ClearTM polycarbonate Cat. No. 344059, Beckman Coulter, Indianapolis, IN)</p></dd><dt>6.</dt><dd><p class="no_top_margin">BCA (bicinchoninic acid) protein assay reagent (Pierce Biotechnology, Rockford, IL).</p></dd></dl></div><div id="g13-prepmembrane.Instruments"><h3>Instruments</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">An ultracentrifuge (XPN-100, Beckman Coulter, or equivalent) and a swing-out bucket rotor (SW41 Ti, Beckmann Coulter, Indianapolis, IN) or equivalents.</p></dd></dl></div><div id="g13-prepmembrane.Methods"><h3>Methods</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Cells (5–8 × 10<sup>7</sup>, corresponding to 4–7 mg of proteins) are suspended in 1 mL of DL buffer containing protease inhibitor (<b>Note 1</b>) allowed to stand on ice for 30 min.</p></dd><dt>2.</dt><dd><p class="no_top_margin">The cell homogenate treated with DL buffer is pipetted into the bottom of a 12-mL ultracentrifuge tube. The volume is adjusted to 2 mL and then an equal volume of 80% sucrose is added. The two solutions are then mixed thoroughly by pipetting to give a final concentration of 40% sucrose in 4 mL. This forms the bottom sucrose density layer in the ultracentrifuge tube.</p></dd><dt>3.</dt><dd><p class="no_top_margin">4 mL of 30% sucrose solution is now layered on top using a 1-mL pipette. This must be done slowly to avoid mixing with 40% sucrose layer.</p></dd><dt>4.</dt><dd><p class="no_top_margin">Finally, 4 mL of 5% sucrose is added and the tube is filled with additional 5% sucrose until the level of the sucrose solution reaches within a couple of millimeters from the rim. It is important to do this because even a 5-mm gap can result in the tube collapsing in a swing-out rotor bucket during ultracentrifugation. Performing this step will reduce the chance of spilling or disturbing the layers during and after ultracentrifugation.</p></dd><dt>5.</dt><dd><p class="no_top_margin">The tube is then carefully placed in a swing-out rotor bucket and centrifuged overnight (or for at least 4 h) at 4°C at 175,000 ×<i>g</i>.</p></dd><dt>6.</dt><dd><p class="no_top_margin">After centrifugation, the swing-out buckets are carefully removed from the rotor. At this stage it may be possible to visualize a turbid white or opaque membrane band located between 5% and 30% sucrose layers and contains the detergent-insoluble, low-buoyant density rafts.</p></dd><dt>7.</dt><dd><p class="no_top_margin">To harvest gradient fractions, a 1-mL pipette is used to slowly aspirate 12 1-mL fractions beginning at the top of the sucrose gradient. As the volume of the tube more than 12 mL, the 12th fraction becomes a volume greater than 1 mL.</p></dd><dt>8.</dt><dd><p class="no_top_margin">Protein contents are determined by BCA method.</p></dd><dt>9.</dt><dd><p class="no_top_margin">Western blotting: it is important to confirm that the membrane fraction containing lipid rafts contains markers reported in the literature. Western blotting can be used to confirm the presence of caveolin and flotillin. Conversely, western blotting should also be used to demonstrate the absence of established nonraft markers, such as the transferrin receptor and clathrin.</p></dd><dt>10.</dt><dd><p class="no_top_margin">Discussion is required for data mining (<b>Note 2</b>).</p></dd></dl></div><div id="g13-prepmembrane.Notes"><h3>Notes</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">For performing the lipid raft preparation, we suggest that an initial experiment should be designed to use the most commonly used detergent concentrations reported in the literature. However, for weakly raft-associated proteins, these concentrations may be too high and lead to their exclusion from the isolated lipid raft preparation, misleading conclusion that they are not raft-associated. A typical example is the insulin receptor, which localizes to rafts prepared using lysis conditions at low TX-100 concentrations (0.05%) but is lost at the standard 1% concentration (<a class="bk_pop" href="#g13-prepmembrane.REF.6">6</a>). Therefore, if on the first round of analysis, a protein appears to not be raft-associated, repeating experiments with reduced concentrations of detergents (>critical micelle concentration) may be worthwhile to detect weaker raft interactions.</p></dd><dt>2.</dt><dd><p class="no_top_margin">The basic principle of the technique is to use a specific detergent to selectively solubilize nonraft regions of the membrane and to subsequently isolate the detergent-insoluble, low-buoyant density rafts using sucrose equilibrium density gradient centrifugation. The choice of detergent and concentration to use is a key issue (<a class="bk_pop" href="#g13-prepmembrane.REF.7">7</a>). However, it is usually not possible to predict either of these based solely on information, such as the primary structure of the protein or its subcellular localization as determined by immunofluorescence staining or electron microscopy. Therefore, the idea is to comprehensively test different detergents to establish which raft or raft subtype best defines the membrane domain to which the protein under investigation is targeted. There is no universally ideal single detergent, and results can be misleading: A given protein may be raft-associated in an intact cell (as determined by other techniques such as immunoelectron microscopy, fluorescence perturbation, or chemical cross-linking studies), but selectively solubilized from the raft by a detergent (<a class="bk_pop" href="#g13-prepmembrane.REF.8">8</a>, <a class="bk_pop" href="#g13-prepmembrane.REF.9">9</a>); conversely, a nonraft membrane protein or proteins present in different rafts may be inherently insoluble in a detergent and thereby left apparently co-localized in isolated detergent-insoluble rafts (<a class="bk_pop" href="#g13-prepmembrane.REF.10">10</a>, <a class="bk_pop" href="#g13-prepmembrane.REF.11">11</a>). The most commonly used detergents have been nonionic detergents, such as TX-100, Lubrol WX, Brij-58/97/98, and NP-40, each of which is selective for compositionally distinct cholesterol-rich membrane rafts (<a class="bk_pop" href="#g13-prepmembrane.REF.7">7</a>). As an example, Lubrol WX can be used to isolate cholesterol-sphingolipid lipid rafts that contain a high molar proportion of phosphatidylcholine, which is largely absent from TX-100 rafts (<a class="bk_pop" href="#g13-prepmembrane.REF.12">12</a>).</p></dd></dl></div></div><div id="g13-prepmembrane.References"><h2 id="_g13-prepmembrane_References_">References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="g13-prepmembrane.REF.1">Simons K., Ikonen E. Functional rafts in cell membranes. <span><span class="ref-journal">Nature. </span>1997 June 5;<span class="ref-vol">387</span>:569–72.</span> doiPMID: . [<a href="https://pubmed.ncbi.nlm.nih.gov/9177342" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9177342</span></a>] [<a href="http://dx.crossref.org/10.1038/42408" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="g13-prepmembrane.REF.2">Pike L.J. Lipid rafts: Heterogeneity on the high seas. <span><span class="ref-journal">Biochem J. </span>2004 Mar 1;<span class="ref-vol">378</span>(Pt2):281–92.</span> doiPMID: . [<a href="/pmc/articles/PMC1223991/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC1223991</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/14662007" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 14662007</span></a>] [<a href="http://dx.crossref.org/10.1042/BJ20031672" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="g13-prepmembrane.REF.3">Schuck S., Honsho M., Ekroos K., Shevchenko A., Simons K. Resistance of cell membranes to different detergents. <span><span class="ref-journal">Proc Natl Acad Sci U S A. </span>2003 May 13;<span class="ref-vol">100</span>(10):5795–800.</span> doiPMID: . [<a href="/pmc/articles/PMC156280/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC156280</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/12721375" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12721375</span></a>] [<a href="http://dx.crossref.org/10.1073/pnas.0631579100" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="g13-prepmembrane.REF.4">Inokuchi J. Preparation of membrane rafts. GlycoPOD. <a href="https://jcggdb.jp/GlycoPOD/protocolShow.action?nodeId=t238" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">https://jcggdb<wbr style="display:inline-block"></wbr>.jp/GlycoPOD/protocolShow<wbr style="display:inline-block"></wbr>.action?nodeId=t238</a>.</div></dd><dt>5.</dt><dd><div class="bk_ref" id="g13-prepmembrane.REF.5">Song KS., Li S., Okamoto T., Quilliam LA., Sargiacomo M., Lisanti MP. Co-purification and direct interaction of Ras with caveolin, an integral membrane protein of caveolae microdomains. Detergent-free purification of caveolae microdomains. <span><span class="ref-journal">J Biol Chem. </span>1996 Apr 19;<span class="ref-vol">271</span>(16):9690–7.</span> doiPMID: . [<a href="https://pubmed.ncbi.nlm.nih.gov/8621645" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 8621645</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.271.16.960" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="g13-prepmembrane.REF.6">Kabayama K., Sato T., Kitamura F., Uemura S., Kang BW., Igarashi Y. Inokuchi J. TNF alpha-induced insulin resistance in adipocytes as a membrane microdomain disorder: involvement of ganglioside GM3. <span><span class="ref-journal">Glycobiology. </span>2005 Jan;<span class="ref-vol">15</span>(1):21–9.</span> doiPMID: . [<a href="https://pubmed.ncbi.nlm.nih.gov/15306563" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15306563</span></a>] [<a href="http://dx.crossref.org/10.1093/glycob/cwh135" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>7.</dt><dd><div class="bk_ref" id="g13-prepmembrane.REF.7">Chamberlain LH. Detergents as tools for the purification and classification of lipid rafts. <span><span class="ref-journal">FEBS Lett. </span>2004 Feb 13;<span class="ref-vol">559</span>(1-3):1–5.</span> doiPMID: . [<a href="https://pubmed.ncbi.nlm.nih.gov/14986659" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 14986659</span></a>] [<a href="http://dx.crossref.org/10.1016/s0014-5793(04)00050-x" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>8.</dt><dd><div class="bk_ref" id="g13-prepmembrane.REF.8">Song KS., Li S., Okamoto T., Quilliam LA., Sargiacomo M., Lisanti MP. Co-purification and direct interaction of Ras with caveolin, an integral membrane protein of caveolae microdomains. Detergent-free purification of caveolae microdomains. <span><span class="ref-journal">J Biol Chem. </span>1996 Apr 19;<span class="ref-vol">271</span>(16):9690–7.</span> doiPMID: . [<a href="https://pubmed.ncbi.nlm.nih.gov/8621645" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 8621645</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.271.16.9690" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>9.</dt><dd><div class="bk_ref" id="g13-prepmembrane.REF.9">Stan RV., Roberts WG., Predescu D., Ihida K., Saucan L., Ghitescu L., Palade GE. Immunoisolation and partial characterization of endothelial plasmalemmal vesicles (caveolae). <span><span class="ref-journal">Mol Biol Cell. </span>1997 Apr;<span class="ref-vol">8</span>(4):595–605.</span> doiPMID: . [<a href="/pmc/articles/PMC276112/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC276112</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/9247641" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9247641</span></a>] [<a href="http://dx.crossref.org/10.1091/mbc.8.4.595" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>10.</dt><dd><div class="bk_ref" id="g13-prepmembrane.REF.10">Mayor S., Maxfield FR. Insolubility and redistribution of GPI-anchored proteins at the cell surface after detergent treatment. <span><span class="ref-journal">Mol Biol Cell. </span>1995 Jul;<span class="ref-vol">6</span>(7):929–44.</span> doiPMID: . [<a href="/pmc/articles/PMC301249/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC301249</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/7579703" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 7579703</span></a>] [<a href="http://dx.crossref.org/10.1091/mbc.6.7.929" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>11.</dt><dd><div class="bk_ref" id="g13-prepmembrane.REF.11">Wilson BS., Steinberg SL., Liederman K., Pfeiffer JR., Surviladze Z., Zhang J., Samelson LE., Yang LH., Kotula PG., Oliver JM. Markers for detergent-resistant lipid rafts occupy distinct and dynamic domains in native membranes. <span><span class="ref-journal">Mol Biol Cell. </span>2004 Jun;<span class="ref-vol">15</span>(6):2580–92.</span> doiPMID: . [<a href="/pmc/articles/PMC420084/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC420084</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/15034144" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15034144</span></a>] [<a href="http://dx.crossref.org/10.1091/mbc.e03-08-0574" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>12.</dt><dd><div class="bk_ref" id="g13-prepmembrane.REF.12">Drobnik W. Borsukova H. Bottcher A. Pfeiffer A. Liebisch G. Schutz GJ. Schindler H. Schmitz G. Apo AI/ABCA1-dependent and HDL3-mediated lipid efflux from compositionally distinct cholesterol-based microdomains. <span><span class="ref-journal">Traffic. </span>2002 Apr;<span class="ref-vol">3</span>(4):268–78.</span> doiPMID: . [<a href="https://pubmed.ncbi.nlm.nih.gov/11929608" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11929608</span></a>] [<a href="http://dx.crossref.org/10.1034/j.1600-0854.2002.030404.x" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl></div><h2 id="NBK593831_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt></dt><dd><div id="g13-prepmembrane.FN1"><p class="no_top_margin">The authors declare no competing or financial interests.</p></div></dd></dl><div id="bk_toc_contnr"></div></div></div>
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