Name: the extraction of liver glycogen molecules for glycogen structure determination
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Description: Watch this Scientific Journal Video about The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination at JoVE.com

The authors are grateful to Mr. Gaosheng Wu and Miss Yunwen Zhu for technical assistance with FACE and Mr. Zhenxia Hu and Mr. Dengbin for technical assistance with SEC. MAS is supported by an Advance Queensland Industry Research Fellowship, Mater Foundation, Equity Trustees, and the L G McCallam Est and George Weaber Trusts. This work was supported by the Priority Academic Program of Jiangsu Higher Education Institutions, a Natural Science Foundation of China grant C1304013151101138, and the 2017 Jiangsu Innovation and Entrepreneurship talents program. Figure 1-5 were created using BioRender.

Mouse livers used to optimize this procedure21 were from 12 male BKS-DB/Nju background mice (7.2 weeks old, see the Table of Materials). Animal use was approved by Renmin Hospital of Wuhan University Animal Care and Ethics Committee, IACUC Issue No. WDRM 20181113.
1 Animal tissues
<ol>
	<li>Weigh mouse liver (1-1.8 g of whole liver from each mouse).</li>
	<li>Rapidly freeze the mouse liver in liquid nitrogen and store it at -80 &#176;C.</li>
</ol>
<p ...

<ol>
	<li>Hills, D. M., Heller, H. C., Hacker, S. D., Hall, D. W., Sadava, D., Laskowski, M., Freeeman, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Life: the science of biology. 12th edn. , (2020).</li><li>Calder, P. C., Geddes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+of+high+molecular+weight+from+mammalian+muscle.">Glycogen of high molecular weight from mammalian muscle.</a> Carbohydrate Research. 135 (2), 249-256 (1985).</li><li>Rybicka, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosomes+-+The+organelles+of+glycogen+metabolism.">Glycosomes - The organelles of glycogen metabolism.</a> Tissue and Cell. 28 (3), 253-265 (1996).</li><li>Takeuchi, T., Iwamasa, T., Miyayama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafine+structure+of+glycogen+macromolecules+in+mammalian-tissues.">Ultrafine structure of glycogen macromolecules in mammalian-tissues.</a> Journal of Electron Microscopy. 27 (1), 31-38 (1978).</li><li>Drochmans, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologie+du+glycogene+-+etude+au+microscope+electronique+de+colorations+negatives+du+glycogene+particulaire.">Morphologie du glycogene - etude au microscope electronique de colorations negatives du glycogene particulaire.</a> Journal of Ultrastructure Research. 6, 141-163 (1962).</li><li>Besford, Q. 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=The+structure+of+cardiac+glycogen+in+healthy+mice.">The structure of cardiac glycogen in healthy mice.</a> International Journal of Biological Macromolecules. 51 (5), 887-891 (2012).</li><li>Lumsden, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+structure+of+glycogen+in+some+cyclophyllidean+and+trypanorhynch+cestodes.">Macromolecular structure of glycogen in some cyclophyllidean and trypanorhynch cestodes.</a> Journal of Parasitology. 51 (4), 501-515 (1965).</li><li>Deng, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+structure+of+glycogen+in+diabetic+liver.">Molecular structure of glycogen in diabetic liver.</a> Glycoconjugate Journal. 32 (3-4), 113-118 (2015).</li><li>Deng, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+for+stopping+chain+and+total-molecule+growth+in+complex+branched+polymers,+exemplified+by+glycogen.">The mechanism for stopping chain and total-molecule growth in complex branched polymers, exemplified by glycogen.</a> Biomacromolecules. 16 (6), 1870-1872 (2015).</li><li>Jiang, X., 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=The+molecular-size+dependence+of+glycogen+enzymatic+degradation+rate+and+its+importance+for+diabetes.">The molecular-size dependence of glycogen enzymatic degradation rate and its importance for diabetes.</a> European Polymer Journal. 82 (1), 175-180 (2016).</li><li>Hu, Z., 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=Glycogen+structure+in+type+1+diabetic+mice:+towards+understanding+the+origin+of+diabetic+glycogen+molecular+fragility.">Glycogen structure in type 1 diabetic mice: towards understanding the origin of diabetic glycogen molecular fragility.</a> International Journal of Biological Macromolecules. 128, 665-672 (2019).</li><li>Suzuki, Y., 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=Insulin+control+of+glycogen+metabolism+in+knockout+mice+lacking+the+muscle-specific+protein+phosphatase+PP1G/R-GL.">Insulin control of glycogen metabolism in knockout mice lacking the muscle-specific protein phosphatase PP1G/R-GL.</a> Molecular and Cellular Biology. 21 (8), 2683-2694 (2001).</li><li>Stetten, M. R., Katzen, H. M., Stetten, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+inhomogeneity+of+glycogen+as+a+function+of+molecular+weight.">Metabolic inhomogeneity of glycogen as a function of molecular weight.</a> Journal of Biological Chemistry. 122 (2), 587-599 (1956).</li><li>Nahorski, S. R., Rogers, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzymic+fluorometric+micro+method+for+determination+of+glycogen.">An enzymic fluorometric micro method for determination of glycogen.</a> Analytical Biochemistry. 49 (2), 492-497 (1972).</li><li>Wang, Z., 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=Molecular+structural+features+of+glycogen+in+the+kidneys+of+diabetic+rats.">Molecular structural features of glycogen in the kidneys of diabetic rats.</a> Carbohydrate Polymers. 229, 115526 (2020).</li><li>Wang, L., 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=Molecular+structure+of+glycogen+in+Escherichia+coli.">Molecular structure of glycogen in Escherichia coli.</a> Biomacromolecules. 20 (7), 2821-2829 (2019).</li><li>Parker, G. J., Koay, A., Gilbert-Wilson, R., Waddington, L. J., Stapleton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMP-activated+protein+kinase+does+not+associate+with+glycogen+alpha-particles+from+rat+liver.">AMP-activated protein kinase does not associate with glycogen alpha-particles from rat liver.</a> Biochemical and Biophysical Research Communications. 362, 811-815 (2007).</li><li>Ryu, J. -. H., 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=Comparative+structural+analyses+of+purified+glycogen+particles+from+rat+liver,+human+skeletal+muscle+and+commercial+preparations.">Comparative structural analyses of purified glycogen particles from rat liver, human skeletal muscle and commercial preparations.</a> International Journal of Biological Macromolecules. 45 (5), 478-482 (2009).</li><li>Sullivan, M. 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=Nature+of+alpha+and+beta+particles+in+glycogen+using+molecular+size+distributions.">Nature of alpha and beta particles in glycogen using molecular size distributions.</a> Biomacromolecules. 11 (4), 1094-1100 (2010).</li><li>Tan, X., 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+new+non-degradative+method+to+purify+glycogen.">A new non-degradative method to purify glycogen.</a> Carbohydrate Polymers. 147 (1), 165-170 (2016).</li><li>Wang, Z., Liu, Q., Wang, L., Gilbert, R. G., Sullivan, 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=Optimization+of+liver+glycogen+extraction+when+considering+molecular+fine+structure.">Optimization of liver glycogen extraction when considering molecular fine structure.</a> Carbohydrate Polymers. 261, 117887 (2020).</li><li>Zhao, Y., Tan, X., Wu, G., Gilbert, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+molecular+fine+structure+to+identify+optimal+methods+of+extracting+starch.">Using molecular fine structure to identify optimal methods of extracting starch.</a> Starch - Starke. 72, 1900214 (2020).</li><li>Shokri-Afra, H., Ostovar-Ravari, A., Rasouli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+the+classical+assay+method+for+liver+glycogen+fractions:+ASG+is+the+main+and+metabolic+active+fraction.">Improvement of the classical assay method for liver glycogen fractions: ASG is the main and metabolic active fraction.</a> European Review for Medical and Pharmacological Sciences. 20, 4328-4336 (2016).</li><li>Kerly, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+solubility+of+glycogen.">The solubility of glycogen.</a> The Biochemical Journal. 24, 67-76 (1930).</li><li>Sullivan, M. 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=Improving+size-exclusion+chromatography+for+glycogen.">Improving size-exclusion chromatography for glycogen.</a> Journal of Chromatography A. 1332 (1), 21-29 (2014).</li><li>Sullivan, M. 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=Skeletal+muscle+glycogen+chain+length+correlates+with+insolubility+in+mouse+models+of+polyglucosan-associated+neurodegenerative+diseases.">Skeletal muscle glycogen chain length correlates with insolubility in mouse models of polyglucosan-associated neurodegenerative diseases.</a> Cell Reports. 27 (5), 1334-1344 (2019).</li><li>Orrell, S. A., Bueding, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+products+obtained+by+various+procedures+used+for+the+extraction+of+glycogen.">A comparison of products obtained by various procedures used for the extraction of glycogen.</a> Journal of Biological Chemistry. 239 (12), 4021-4026 (1964).</li><li>Sullivan, M. 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=Molecular+insights+into+glycogen+alpha-particle+formation.">Molecular insights into glycogen alpha-particle formation.</a> Biomacromolecules. 13 (11), 3805-3813 (2012).</li></ol>

Previous studies have shown that the structure of glycogen is important for its properties; for example, the molecular size affects the degradation rate of glycogen10, and the chain length distribution affects its solubility26. To properly understand these relationships, it is important to extract glycogen with a procedure that isolates, as much as possible, a representative and undamaged sample. Traditional methods of extraction utilized either hot alkaline conditions or c...

Glycogen is a complex, hyperbranched polymer of glucose found in animals, fungi, and bacteria1. In mammals, liver glycogen functions as a blood glucose buffer, preserving homeostasis, while muscle glycogen acts as a short-term glucose reservoir to provide energy directly2. The structure of glycogen is often described by three levels (shown in Figure 1): 1. Linear chains are formed by glucose monomers via (1&#8594;4)-&#945; glycosidic bonds, with branch points being connected via (1&#8594;6)-&#945; glycosidic bonds; 2. highly branched &#946; particles (~20 nm in diameter) that, especially in tissues such as skeletal muscle, act as independent glycogen molecules3,4; 3. larger &#945; glycogen particles (up to 300 nm in diameter) that consist of smaller &#946; glycogen units, which are found in the liver5, heart6, and in some non-mammalian species7. Hepatic &#945; particles from diabetic mice are molecularly fragile, with a propensity to degrade to &#946;-particles when dissolved in dimethyl sulfoxide (DMSO), while &#945; particles from non-diabetic controls generally remain unchanged. One hypothesis is that this fragility may exacerbate the poor blood glucose balance seen in diabetes, with the fragile &#945; particles potentially resulting in higher proportions of the more rapidly degraded &#946; particle8,9,10,11.
Traditional glycogen extraction methods utilize the relatively harsh conditions of exposing the liver tissue to hot alkaline solution12 or acid solutions such as trichloroacetic acid (TCA)13 or perchloric acid (PCA)14. While effective at separating the glycogen from other components of the liver tissue, these methods inevitably degrade the glycogen structure to some extent15,16. Although these methods are suitable for quantitative measurement of the glycogen content, they are not ideal for studies focused on obtaining structural information on the glycogen due to this structural damage. Since the development of these methods, a milder extraction procedure has been developed that utilizes cold Tris buffer (shown to inhibit glucosidase degradation) with sucrose density gradient ultracentrifugation17,18,19. With the pH controlled at ~8, this method does not subject the glycogen to the acid or alkaline hydrolysis seen in previous procedures.
Sucrose density gradient ultracentrifugation of homogenized liver tissue can separate glycogen particles from the majority of cell material. If necessary, additional purification can be performed by preparative size exclusion chromatography, resulting in the collection of purified glycogen with attached glycogen-associating proteins20. Although this method, with milder conditions, is more likely to preserve the structure of glycogen, it is difficult to prevent some portion of the glycogen from being lost in the supernatant, especially smaller glycogen particles that are less dense15. Another potential cause of glycogen loss is that the milder conditions allow some enzymatic degradation, resulting in lower glycogen yields compared to harsher extraction methods. Recent research reported optimization of the liver-glycogen extraction method to preserve the structure of glycogen21. Here, various sucrose concentrations (30%,50%, 72.5%) were tested to determine whether lower sucrose concentrations minimized the loss of smaller glycogen particles. The rationale was that the lower density would allow for smaller, less dense particles to penetrate the sucrose layer and aggregate in the pellet with the rest of the glycogen.
In this study, the extraction methods with and without a 10 min boiling step were compared to test whether glycogen degradation enzymes could be denatured, resulting in the extraction of more glycogen that was also free from partial degradation. Whole molecular size distributions and the glycogen chain length distributions were used to determine the structure of the extracted glycogen, similar to a starch extraction optimization published previously22. Size exclusion chromatography (SEC) with differential refractive index (DRI) detection was used to obtain the size distributions of glycogen, which describe the total molecular weight as a function of molecular size. Fluorophore-assisted carbohydrate electrophoresis (FACE) was used to analyze the chain-length distributions, which describe the relative number of glucoside chains of each given size (or degree of polymerization). This paper describes the methodology of extracting glycogen from liver tissues based on the previous optimization study21. The data suggest that the method most suited to preserve glycogen structure is a sucrose concentration of 30% with a 10 min boiling step.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>8-aminopyrene-1,3,6-trisulfonate (APTS)</td>
			<td>SIGMA Aldrich</td>
			<td>9341</td>
			<td>0.1 M solution</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>SIGMA Aldrich</td>
			<td>695092</td>
			<td>0.1 M, pH 3.5 solution</td>
		</tr>
		<tr>
			<td>Agilent 1260 Infinity SEC system</td>
			<td>Agilent, Santa Clara, CA, USA</td>
			<td></td>
			<td>Size-exclusion chromatography (SEC)</td>
		</tr>
		<tr>
			<td>BKS-DB/Nju background mice</td>
			<td>Nanjing Biomedical Research Institution of Nanjing University</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose Assay Kit (GOPOD Format)</td>
			<td>Megazyme</td>
			<td>K-GLUC</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenedinitrilotetraacetic acid (EDTA)</td>
			<td>SIGMA Aldrich</td>
			<td>431788</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer</td>
			<td>IKA</td>
			<td>T 25</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>SIGMA Aldrich</td>
			<td>2104</td>
			<td>0.1 M solution</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>SIGMA Aldrich</td>
			<td>2104</td>
			<td>0.1 M solution</td>
		</tr>
		<tr>
			<td>P/ACE MDQ plus system</td>
			<td>Ab Sciex, US</td>
			<td></td>
			<td>Fluorophore-assisted carbohydrate electrophoresis (FACE)</td>
		</tr>
		<tr>
			<td>Refractive index detector</td>
			<td>Optilab UT-rEX, Wyatt, Santa Barbara, CA, USA)</td>
			<td></td>
			<td>Size-exclusion chromatography (SEC)</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>SIGMA Aldrich</td>
			<td>241245</td>
			<td>1 M, pH 4.5 solution</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>SIGMA Aldrich</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>SIGMA Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium cyanoborohydride</td>
			<td>SIGMA Aldrich</td>
			<td>156159</td>
			<td>1 M solution</td>
		</tr>
		<tr>
			<td>Sodium fluoride</td>
			<td>SIGMA Aldrich</td>
			<td>201154</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>SIGMA Aldrich</td>
			<td>43617</td>
			<td>0.1 M solution</td>
		</tr>
		<tr>
			<td>Sodium nitrate</td>
			<td>SIGMA Aldrich</td>
			<td>NISTRM8569</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyrophosphate</td>
			<td>SIGMA Aldrich</td>
			<td>221368</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>SIGMA Aldrich</td>
			<td>V90016</td>
			<td></td>
		</tr>
		<tr>
			<td>SUPREMA pre-column, 1,000 and 10,000 columns</td>
			<td>Polymer Standards Services, Mainz, Germany</td>
			<td></td>
			<td>Size-exclusion chromatography (SEC)</td>
		</tr>
		<tr>
			<td>Trizma</td>
			<td>SIGMA Aldrich</td>
			<td>T 1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes</td>
			<td>Beckman</td>
			<td></td>
			<td>4 mL, Open-Top Thinwall Ultra-Clear Tube, 11 x 60 mm</td>
		</tr>
	</tbody>
</table>

the extraction of liver glycogen molecules for glycogen structure determination

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are influenced by its structure. Hence, a suitable extraction method that isolates representative samples of glycogen is crucial to the study of this macromolecule. Compared to other extraction methods, a method that employs a sucrose density gradient centrifugation step can minimize molecular damage. Based on this method, a recent publication describes how the density of the sucrose solution used during centrifugation was varied (30%, 50%, 72.5%) to find the most suitable concentration to extract glycogen particles of a wide variety of sizes, limiting the loss of smaller particles. A 10 min boiling step was introduced to test its ability to denature glycogen degrading enzymes, thus preserving glycogen. The lowest sucrose concentration (30%) and the addition of the boiling step were shown to extract the most representative samples of glycogen.

While the procedure described above is for the most optimal method (30% sucrose with the addition of a 10min boiling step), data are provided here for glycogen extracted via three sucrose concentrations (30%, 50%, 72.5%), with and without a boiling step, as previously described21. Following the protocol, the purity, crude yield, and glycogen yield of dry glycogen extracted by different conditions are given in Table 1, reproduced from21. There were no signif...

Watch this Scientific Journal Video about The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination at JoVE.com

The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination

An optimal sucrose concentration was determined for the extraction of liver glycogen using sucrose density gradient centrifugation. The addition of a 10 min boiling step to inhibit glycogen-degrading enzymes proved beneficial.

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are ...

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