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Volumn 61, Issue 4, 2012, Pages 490-497

SiRNA knock down of glutamate dehydrogenase in astrocytes affects glutamate metabolism leading to extensive accumulation of the neuroactive amino acids glutamate and aspartate

Author keywords

15N; Alanine; Aspartate; Astrocyte; Glutamate; Glutamine; Homeostasis; Mass spectrometry; Metabolism; siRNA; TCA cycle

Indexed keywords

2 OXOGLUTARIC ACID; ALANINE; AMMONIUM CHLORIDE; ASPARTIC ACID; GLUTAMATE DEHYDROGENASE; GLUTAMIC ACID; OXALOACETIC ACID; SMALL INTERFERING RNA;

EID: 84865720909     PISSN: 01970186     EISSN: 18729754     Source Type: Journal    
DOI: 10.1016/j.neuint.2012.04.014     Document Type: Article
Times cited : (40)

References (58)
  • 1
    • 77956647974 scopus 로고    scopus 로고
    • Glutamine as a mediator of ammonia neurotoxicity: A critical appraisal
    • J. Albrecht, M. Zielinska, and M.D. Norenberg Glutamine as a mediator of ammonia neurotoxicity: a critical appraisal Biochem. Pharmacol. 80 2010 1303 1308
    • (2010) Biochem. Pharmacol. , vol.80 , pp. 1303-1308
    • Albrecht, J.1    Zielinska, M.2    Norenberg, M.D.3
  • 2
    • 0023572285 scopus 로고
    • Glial glutamate dehydrogenase: Ultrastructural localization and regional distribution in relation to the mitochondrial enzyme, cytochrome oxidase
    • C. Aoki, T.A. Milner, S.B. Berger, K.F. Sheu, J.P. Blass, and V.M. Pickel Glial glutamate dehydrogenase: ultrastructural localization and regional distribution in relation to the mitochondrial enzyme, cytochrome oxidase J. Neurosci. Res. 18 1987 305 318
    • (1987) J. Neurosci. Res. , vol.18 , pp. 305-318
    • Aoki, C.1    Milner, T.A.2    Berger, S.B.3    Sheu, K.F.4    Blass, J.P.5    Pickel, V.M.6
  • 3
    • 33745893228 scopus 로고    scopus 로고
    • The glutamate/GABA-glutamine cycle: Aspects of transport, neurotransmitter homeostasis and ammonia transfer
    • L.K. Bak, A. Schousboe, and H.S. Waagepetersen The glutamate/GABA- glutamine cycle: aspects of transport, neurotransmitter homeostasis and ammonia transfer J. Neurochem. 98 2006 641 653
    • (2006) J. Neurochem. , vol.98 , pp. 641-653
    • Bak, L.K.1    Schousboe, A.2    Waagepetersen, H.S.3
  • 4
    • 84865735219 scopus 로고    scopus 로고
    • The glutamate transporter, GLAST, participates in a macromolecular complex that supports glutamate metabolism
    • in press
    • Bauer, D.E.; Jackson, J.G.; Genda, E.N.; Montoya, M.M.; Yudkoff, M.; Robinson, M.B.; in press. The glutamate transporter, GLAST, participates in a macromolecular complex that supports glutamate metabolism. Neurochem. Int. http://dx.doi.org/10.1016/j.neuint.2012.01.013.
    • Neurochem. Int.
    • Bauer D., .E.1    Jackson J., .G.2    Genda E., .N.3    Montoya M., .M.4    Yudkoff, M.5    Robinson M., .B.6
  • 6
    • 0025696512 scopus 로고
    • Distribution of glutamate-like immunoreactivity in excitatory hippocampal pathways: A semiquantitative electron microscopic study in rats
    • C.R. Bramham, R. Torp, N. Zhang, J. Storm-Mathisen, and O.P. Ottersen Distribution of glutamate-like immunoreactivity in excitatory hippocampal pathways: a semiquantitative electron microscopic study in rats Neuroscience 39 1990 405 417
    • (1990) Neuroscience , vol.39 , pp. 405-417
    • Bramham, C.R.1    Torp, R.2    Zhang, N.3    Storm-Mathisen, J.4    Ottersen, O.P.5
  • 10
    • 0022646352 scopus 로고
    • Purification and characterization of a soluble and a particulate glutamate dehydrogenase from rat brain
    • A.D. Colon, A. Plaitakis, A. Perakis, S. Berl, and D.D. Clarke Purification and characterization of a soluble and a particulate glutamate dehydrogenase from rat brain J. Neurochem. 46 1986 1811 1819
    • (1986) J. Neurochem. , vol.46 , pp. 1811-1819
    • Colon, A.D.1    Plaitakis, A.2    Perakis, A.3    Berl, S.4    Clarke, D.D.5
  • 11
    • 85052778173 scopus 로고
    • L-Glutamate(2-oxoglutarate) aminotransferases
    • E. Kvamme, CRC Press Florida
    • A.J. Cooper l-Glutamate(2-oxoglutarate) aminotransferases E. Kvamme, Glutamine and Glutamate in Mammals 1988 CRC Press Florida 123 152
    • (1988) Glutamine and Glutamate in Mammals , pp. 123-152
    • Cooper, A.J.1
  • 12
    • 80052263625 scopus 로고    scopus 로고
    • 13N as a tracer for studying glutamate metabolism
    • A.J. Cooper 13N as a tracer for studying glutamate metabolism Neurochem. Int. 59 2011 456 464
    • (2011) Neurochem. Int. , vol.59 , pp. 456-464
    • Cooper, A.J.1
  • 13
    • 0023277382 scopus 로고
    • Biochemistry and physiology of brain ammonia
    • A.J. Cooper, and F. Plum Biochemistry and physiology of brain ammonia Physiol. Rev. 67 1987 440 519
    • (1987) Physiol. Rev. , vol.67 , pp. 440-519
    • Cooper, A.J.1    Plum, F.2
  • 14
    • 79955646990 scopus 로고    scopus 로고
    • Inhibition of glutamine synthesis induces glutamate dehydrogenase- dependent ammonia fixation into alanine in co-cultures of astrocytes and neurons
    • S. Dadsetan, L.K. Bak, M. Sorensen, S. Keiding, H. Vilstrup, P. Ott, R. Leke, A. Schousboe, and H.S. Waagepetersen Inhibition of glutamine synthesis induces glutamate dehydrogenase-dependent ammonia fixation into alanine in co-cultures of astrocytes and neurons Neurochem. Int. 59 2011 482 488
    • (2011) Neurochem. Int. , vol.59 , pp. 482-488
    • Dadsetan, S.1    Bak, L.K.2    Sorensen, M.3    Keiding, S.4    Vilstrup, H.5    Ott, P.6    Leke, R.7    Schousboe, A.8    Waagepetersen, H.S.9
  • 16
    • 0021958309 scopus 로고
    • Ontogenetic development of glutamate metabolizing enzymes in cultured cerebellar granule cells and in cerebellum in vivo
    • J. Drejer, O.M. Larsson, E. Kvamme, G. Svenneby, L. Hertz, and A. Schousboe Ontogenetic development of glutamate metabolizing enzymes in cultured cerebellar granule cells and in cerebellum in vivo Neurochem. Res. 10 1985 49 62
    • (1985) Neurochem. Res. , vol.10 , pp. 49-62
    • Drejer, J.1    Larsson, O.M.2    Kvamme, E.3    Svenneby, G.4    Hertz, L.5    Schousboe, A.6
  • 17
    • 0025196959 scopus 로고
    • Metabolism and role of glutamate in mammalian brain
    • M. Erecinska, and I.A. Silver Metabolism and role of glutamate in mammalian brain Prog. Neurobiol. 35 1990 245 296
    • (1990) Prog. Neurobiol. , vol.35 , pp. 245-296
    • Erecinska, M.1    Silver, I.A.2
  • 18
    • 0026668151 scopus 로고
    • Glutamate metabolism in rat cortical astrocyte cultures
    • S.E. Farinelli, and W.J. Nicklas Glutamate metabolism in rat cortical astrocyte cultures J. Neurochem. 58 1992 1905 1915
    • (1992) J. Neurochem. , vol.58 , pp. 1905-1915
    • Farinelli, S.E.1    Nicklas, W.J.2
  • 20
    • 0032529722 scopus 로고    scopus 로고
    • Synaptic vesicular localization and exocytosis of l-aspartate in excitatory nerve terminals: A quantitative immunogold analysis in rat hippocampus
    • V. Gundersen, F.A. Chaudhry, J.G. Bjaalie, F. Fonnum, O.P. Ottersen, and J. Storm-Mathisen Synaptic vesicular localization and exocytosis of l-aspartate in excitatory nerve terminals: a quantitative immunogold analysis in rat hippocampus J. Neurosci. 18 1998 6059 6070
    • (1998) J. Neurosci. , vol.18 , pp. 6059-6070
    • Gundersen, V.1    Chaudhry, F.A.2    Bjaalie, J.G.3    Fonnum, F.4    Ottersen, O.P.5    Storm-Mathisen, J.6
  • 23
    • 33846423878 scopus 로고    scopus 로고
    • Energy metabolism in astrocytes: High rate of oxidative metabolism and spatiotemporal dependence on glycolysis/glycogenolysis
    • L. Hertz, L. Peng, and G.A. Dienel Energy metabolism in astrocytes: high rate of oxidative metabolism and spatiotemporal dependence on glycolysis/glycogenolysis J. Cereb. Blood Flow Metab. 27 2007 219 249
    • (2007) J. Cereb. Blood Flow Metab. , vol.27 , pp. 219-249
    • Hertz, L.1    Peng, L.2    Dienel, G.A.3
  • 24
    • 0016733259 scopus 로고
    • Control of the redox state of the pyridine nucleotides in the rat cerebral cortex. Effect of electroshock-induced seizures
    • D.C. Howse, and T.E. Duffy Control of the redox state of the pyridine nucleotides in the rat cerebral cortex. Effect of electroshock-induced seizures J. Neurochem. 24 1975 935 940
    • (1975) J. Neurochem. , vol.24 , pp. 935-940
    • Howse, D.C.1    Duffy, T.E.2
  • 25
    • 0023653356 scopus 로고
    • Aminooxyacetic acid inhibits the malate-aspartate shuttle in isolated nerve terminals and prevents the mitochondria from utilizing glycolytic substrates
    • R.A. Kauppinen, T.S. Sihra, and D.G. Nicholls Aminooxyacetic acid inhibits the malate-aspartate shuttle in isolated nerve terminals and prevents the mitochondria from utilizing glycolytic substrates Biochim. Biophys. Acta 930 1987 173 178
    • (1987) Biochim. Biophys. Acta , vol.930 , pp. 173-178
    • Kauppinen, R.A.1    Sihra, T.S.2    Nicholls, D.G.3
  • 26
    • 0000027898 scopus 로고
    • Ontogenetic development of glutamate and GABA metabolizing enzymes in cultured cerebral cortex interneurons and in cerebral cortex in vivo
    • O.M. Larsson, J. Drejer, E. Kvamme, G. Svenneby, L. Hertz, and A. Schousboe Ontogenetic development of glutamate and GABA metabolizing enzymes in cultured cerebral cortex interneurons and in cerebral cortex in vivo Int. J. Dev. Neurosci. 3 1985 177 185
    • (1985) Int. J. Dev. Neurosci. , vol.3 , pp. 177-185
    • Larsson, O.M.1    Drejer, J.2    Kvamme, E.3    Svenneby, G.4    Hertz, L.5    Schousboe, A.6
  • 28
    • 0001214769 scopus 로고
    • The tricarboxylic acid cycle
    • D. Greenberg, 3rd edition Academic Press New York
    • J.M. Lowenstein The tricarboxylic acid cycle D. Greenberg, Metabolic Pathways, vol. 1 3rd edition 1967 Academic Press New York 146 270
    • (1967) Metabolic Pathways, Vol. 1 , pp. 146-270
    • Lowenstein, J.M.1
  • 30
    • 0022635525 scopus 로고
    • Analysis of amino acids as their tert.-butyldimethylsilyl derivatives by gas-liquid chromatography and mass spectrometry
    • T.P. Mawhinney, R.S. Robinett, A. Atalay, and M.A. Madson Analysis of amino acids as their tert.-butyldimethylsilyl derivatives by gas-liquid chromatography and mass spectrometry J. Chromatogr. 358 1986 231 242
    • (1986) J. Chromatogr. , vol.358 , pp. 231-242
    • Mawhinney, T.P.1    Robinett, R.S.2    Atalay, A.3    Madson, M.A.4
  • 31
    • 80052290024 scopus 로고    scopus 로고
    • Glutamate dehydrogenase in brain mitochondria: Do lipid modifications and transient metabolon formation influence enzyme activity?
    • M.C. McKenna Glutamate dehydrogenase in brain mitochondria: do lipid modifications and transient metabolon formation influence enzyme activity? Neurochem. Int. 59 2011 525 533
    • (2011) Neurochem. Int. , vol.59 , pp. 525-533
    • McKenna, M.C.1
  • 33
    • 0030061356 scopus 로고    scopus 로고
    • Exogenous glutamate concentration regulates the metabolic fate of glutamate in astrocytes
    • M.C. McKenna, U. Sonnewald, X. Huang, J. Stevenson, and H.R. Zielke Exogenous glutamate concentration regulates the metabolic fate of glutamate in astrocytes J. Neurochem. 66 1996 386 393
    • (1996) J. Neurochem. , vol.66 , pp. 386-393
    • McKenna, M.C.1    Sonnewald, U.2    Huang, X.3    Stevenson, J.4    Zielke, H.R.5
  • 34
    • 0027727915 scopus 로고
    • Regulation of energy metabolism in synaptic terminals and cultured rat brain astrocytes: Differences revealed using aminooxyacetate
    • M.C. McKenna, J.T. Tildon, J.H. Stevenson, R. Boatright, and S. Huang Regulation of energy metabolism in synaptic terminals and cultured rat brain astrocytes: differences revealed using aminooxyacetate Dev. Neurosci. 15 1993 320 329
    • (1993) Dev. Neurosci. , vol.15 , pp. 320-329
    • McKenna, M.C.1    Tildon, J.T.2    Stevenson, J.H.3    Boatright, R.4    Huang, S.5
  • 35
    • 0029972176 scopus 로고    scopus 로고
    • New insights into the compartmentation of glutamate and glutamine in cultured rat brain astrocytes
    • M.C. McKenna, J.T. Tildon, J.H. Stevenson, and X. Huang New insights into the compartmentation of glutamate and glutamine in cultured rat brain astrocytes Dev. Neurosci. 18 1996 380 390
    • (1996) Dev. Neurosci. , vol.18 , pp. 380-390
    • McKenna, M.C.1    Tildon, J.T.2    Stevenson, J.H.3    Huang, X.4
  • 36
    • 0032427875 scopus 로고    scopus 로고
    • Astroglial dysfunction in hepatic encephalopathy
    • M.D. Norenberg Astroglial dysfunction in hepatic encephalopathy Metab. Brain Dis. 13 1998 319 335
    • (1998) Metab. Brain Dis. , vol.13 , pp. 319-335
    • Norenberg, M.D.1
  • 38
    • 0020322196 scopus 로고
    • Abnormal glutamate metabolism in an adult-onset degenerative neurological disorder
    • A. Plaitakis, S. Berl, and M.D. Yahr Abnormal glutamate metabolism in an adult-onset degenerative neurological disorder Science 216 1982 193 196
    • (1982) Science , vol.216 , pp. 193-196
    • Plaitakis, A.1    Berl, S.2    Yahr, M.D.3
  • 40
    • 80052265272 scopus 로고    scopus 로고
    • The human GLUD2 glutamate dehydrogenase and its regulation in health and disease
    • A. Plaitakis, H. Latsoudis, and C. Spanaki The human GLUD2 glutamate dehydrogenase and its regulation in health and disease Neurochem. Int. 59 2011 495 509
    • (2011) Neurochem. Int. , vol.59 , pp. 495-509
    • Plaitakis, A.1    Latsoudis, H.2    Spanaki, C.3
  • 41
    • 0034688312 scopus 로고    scopus 로고
    • Glutamate release in severe brain ischaemia is mainly by reversed uptake
    • D.J. Rossi, T. Oshima, and D. Attwell Glutamate release in severe brain ischaemia is mainly by reversed uptake Nature 403 2000 316 321
    • (2000) Nature , vol.403 , pp. 316-321
    • Rossi, D.J.1    Oshima, T.2    Attwell, D.3
  • 42
    • 0028036505 scopus 로고
    • Quantitative ultrastructural localization of glutamate dehydrogenase in the rat cerebellar cortex
    • F. Rothe, M. Brosz, and J. Storm-Mathisen Quantitative ultrastructural localization of glutamate dehydrogenase in the rat cerebellar cortex Neuroscience 62 1994 1133 1146
    • (1994) Neuroscience , vol.62 , pp. 1133-1146
    • Rothe, F.1    Brosz, M.2    Storm-Mathisen, J.3
  • 43
    • 0025696513 scopus 로고
    • Immunohistochemical demonstration of glutamate dehydrogenase in the postnatally developing rat hippocampal formation and cerebellar cortex: Comparison to activity staining
    • F. Rothe, G. Wolf, and G. Schunzel Immunohistochemical demonstration of glutamate dehydrogenase in the postnatally developing rat hippocampal formation and cerebellar cortex: comparison to activity staining Neuroscience 39 1990 419 429
    • (1990) Neuroscience , vol.39 , pp. 419-429
    • Rothe, F.1    Wolf, G.2    Schunzel, G.3
  • 44
    • 0028341884 scopus 로고
    • Novel human glutamate dehydrogenase expressed in neural and testicular tissues and encoded by an X-linked intronless gene
    • P. Shashidharan, T.M. Michaelidis, N.K. Robakis, A. Kresovali, J. Papamatheakis, and A. Plaitakis Novel human glutamate dehydrogenase expressed in neural and testicular tissues and encoded by an X-linked intronless gene J. Biol. Chem. 269 1994 16971 16976
    • (1994) J. Biol. Chem. , vol.269 , pp. 16971-16976
    • Shashidharan, P.1    Michaelidis, T.M.2    Robakis, N.K.3    Kresovali, A.4    Papamatheakis, J.5    Plaitakis, A.6
  • 46
    • 0024320234 scopus 로고
    • Glutamine and 2-oxoglutarate as metabolic precursors of the transmitter pools of glutamate and GABA: Correlation of regional uptake by rat brain synaptosomes
    • R.P. Shank, W.J. Baldy, and C.W. Ash Glutamine and 2-oxoglutarate as metabolic precursors of the transmitter pools of glutamate and GABA: correlation of regional uptake by rat brain synaptosomes Neurochem. Res. 14 1989 371 376
    • (1989) Neurochem. Res. , vol.14 , pp. 371-376
    • Shank, R.P.1    Baldy, W.J.2    Ash, C.W.3
  • 48
    • 0029662102 scopus 로고    scopus 로고
    • Metabolism of [U-13C5] glutamine in cultured astrocytes studied by NMR spectroscopy: First evidence of astrocytic pyruvate recycling
    • U. Sonnewald, N. Westergaard, P. Jones, A. Taylor, H.S. Bachelard, and A. Schousboe Metabolism of [U-13C5] glutamine in cultured astrocytes studied by NMR spectroscopy: first evidence of astrocytic pyruvate recycling J. Neurochem. 67 1996 2566 2572
    • (1996) J. Neurochem. , vol.67 , pp. 2566-2572
    • Sonnewald, U.1    Westergaard, N.2    Jones, P.3    Taylor, A.4    Bachelard, H.S.5    Schousboe, A.6
  • 49
    • 0031239422 scopus 로고    scopus 로고
    • Glutamate transport and metabolism in astrocytes
    • U. Sonnewald, N. Westergaard, and A. Schousboe Glutamate transport and metabolism in astrocytes Glia 21 1997 56 63
    • (1997) Glia , vol.21 , pp. 56-63
    • Sonnewald, U.1    Westergaard, N.2    Schousboe, A.3
  • 50
    • 84856303217 scopus 로고    scopus 로고
    • The role of glutamate dehydrogenase in mammalian ammonia metabolism
    • C. Spanaki, and A. Plaitakis The role of glutamate dehydrogenase in mammalian ammonia metabolism Neurotox. Res. 21 2012 117 127
    • (2012) Neurotox. Res. , vol.21 , pp. 117-127
    • Spanaki, C.1    Plaitakis, A.2
  • 51
    • 77952778182 scopus 로고    scopus 로고
    • Human GLUD2 glutamate dehydrogenase is expressed in neural and testicular supporting cells
    • C. Spanaki, I. Zaganas, K.A. Kleopa, and A. Plaitakis Human GLUD2 glutamate dehydrogenase is expressed in neural and testicular supporting cells J. Biol. Chem. 285 2010 16748 16756
    • (2010) J. Biol. Chem. , vol.285 , pp. 16748-16756
    • Spanaki, C.1    Zaganas, I.2    Kleopa, K.A.3    Plaitakis, A.4
  • 52
    • 80052259624 scopus 로고    scopus 로고
    • Two genetic forms of hyperinsulinemic hypoglycemia caused by dysregulation of glutamate dehydrogenase
    • C.A. Stanley Two genetic forms of hyperinsulinemic hypoglycemia caused by dysregulation of glutamate dehydrogenase Neurochem. Int. 59 2011 465 472
    • (2011) Neurochem. Int. , vol.59 , pp. 465-472
    • Stanley, C.A.1
  • 53
    • 0036866554 scopus 로고    scopus 로고
    • Demonstration of pyruvate recycling in primary cultures of neocortical astrocytes but not in neurons
    • H.S. Waagepetersen, H. Qu, L. Hertz, U. Sonnewald, and A. Schousboe Demonstration of pyruvate recycling in primary cultures of neocortical astrocytes but not in neurons Neurochem. Res. 27 2002 1431 1437
    • (2002) Neurochem. Res. , vol.27 , pp. 1431-1437
    • Waagepetersen, H.S.1    Qu, H.2    Hertz, L.3    Sonnewald, U.4    Schousboe, A.5
  • 54
    • 0034846461 scopus 로고    scopus 로고
    • Multiple compartments with different metabolic characteristics are involved in biosynthesis of intracellular and released glutamine and citrate in astrocytes
    • H.S. Waagepetersen, U. Sonnewald, O.M. Larsson, and A. Schousboe Multiple compartments with different metabolic characteristics are involved in biosynthesis of intracellular and released glutamine and citrate in astrocytes Glia 35 2001 246 252
    • (2001) Glia , vol.35 , pp. 246-252
    • Waagepetersen, H.S.1    Sonnewald, U.2    Larsson, O.M.3    Schousboe, A.4
  • 55
    • 0030174754 scopus 로고    scopus 로고
    • Evaluation of the importance of transamination versus deamination in astrocytic metabolism of [U-13C]glutamate
    • N. Westergaard, J. Drejer, A. Schousboe, and U. Sonnewald Evaluation of the importance of transamination versus deamination in astrocytic metabolism of [U-13C]glutamate Glia 17 1996 160 168
    • (1996) Glia , vol.17 , pp. 160-168
    • Westergaard, N.1    Drejer, J.2    Schousboe, A.3    Sonnewald, U.4
  • 56
    • 0021037707 scopus 로고
    • Pyruvate carboxylase activity in primary cultures of astrocytes and neurons
    • A.C. Yu, J. Drejer, L. Hertz, and A. Schousboe Pyruvate carboxylase activity in primary cultures of astrocytes and neurons J. Neurochem. 41 1983 1484 1487
    • (1983) J. Neurochem. , vol.41 , pp. 1484-1487
    • Yu, A.C.1    Drejer, J.2    Hertz, L.3    Schousboe, A.4
  • 57
    • 0020335372 scopus 로고
    • Metabolic fate of 14C-labeled glutamate in astrocytes in primary cultures
    • A.C. Yu, A. Schousboe, and L. Hertz Metabolic fate of 14C-labeled glutamate in astrocytes in primary cultures J. Neurochem. 39 1982 954 960
    • (1982) J. Neurochem. , vol.39 , pp. 954-960
    • Yu, A.C.1    Schousboe, A.2    Hertz, L.3
  • 58
    • 0028036798 scopus 로고
    • Tricarboxylic acid cycle in rat brain synaptosomes. Fluxes and interactions with aspartate aminotransferase and malate/aspartate shuttle
    • M. Yudkoff, D. Nelson, Y. Daikhin, and M. Erecinska Tricarboxylic acid cycle in rat brain synaptosomes. Fluxes and interactions with aspartate aminotransferase and malate/aspartate shuttle J. Biol. Chem. 269 1994 27414 27420
    • (1994) J. Biol. Chem. , vol.269 , pp. 27414-27420
    • Yudkoff, M.1    Nelson, D.2    Daikhin, Y.3    Erecinska, M.4


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