Protective effect and mechanism of Krüppel-like factor 7 overexpression on retinal ganglion cells in mice after optic nerve clamp_Chinese Journal of Ocular Fundus Diseases

Authors：

Gu Jingsai , Chen Zhen , Xu Yishuang , Zheng Hongmei ,  Hua Dihao

Eye Center, Renmin Hospital of Wuhan University, Wuhan 430060, China;

Corresponding author：

Hua Dihao, Email: 277058240@qq.com

Keywords：

Kruppel-like factor 7; Optic nerve clamp; Retinal ganglion cells; Nerve growth factor; Tyrosine kinase; B lymphoblastoma-2; B lymphoblastoma-2-associated X protein

DOI：

10.3760/cma.j.cn511434-20230504-00196

Video：

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Abstract Full text Figures/Tables Video References Cited by

Objective To explore the protective effect and mechanism of Krüppel-like factor 7 (KLF7) overexpression on retinal ganglion cells (RGC) in mice after optic nerve crush (ONC). Methods A total of sixty five 10-week-old C57BL/6J mice were randomly divided into five groups: blank control group (group A), intravitreal injection (IVT)-KLF7 group (group B), IVT-phosphate buffer saline-ONC group (group C), IVT-KLF7-ONC group (group D), and IVT-recombinant adeno-associated virus 2-enhanced green fluorescent protein-ONC group (group E), with 13 mice in each group. On the 7 days after the ONC model, the mice in each group were killed. RGC survival rate was counted by whole retina flat mount and immunofluorescence techniques. KLF7, nerve growth factor (NGF), tyrosine kinase A (TrkA), phosphorylated TrkA (pTrkA), tyrosine kinase B (TrkB) and phosphorylated TrkB (pTrk) were detected by western blot, growth associated protein 43 (GAP43), brain-derived neurotrophic factor, B lymphoblastoma-2 (Bcl-2), Bcl-2 associated X protein (BAX), Caspase-3 protein relative expression levels. One-way analysis of variance or Kruskal-Wallis test were used for comparison between groups. Results On the 7 days after the ONC model, the density of RGC in the retina of groups A, B, C, D and E were (3 707.4±12.8), (3 582.4±13.3), (1 396.3±16.1), (1 658.3±22.2) and (1 323.6±16.9)/mm², respectively. Compared with groups C and E, RGC density in group D was significantly increased, and the difference was statistically significant (P=0.028, 0.007). Compared with groups A, B, C and E, the relative expression levels of NGF, pTrkA, pTrkB, GAP43 and Bcl-2 proteins in the retina of mice in group D were increased, while the relative expression levels of BAX and Caspase-3 proteins were decreased, with statistical significance (P＜0.01). Conclusion In mouse ONC model, overexpression of KLF7 can improve RGC survival rate, increase the relative expression levels of NGF, pTrkA, pTrkB, GAP43 and Bcl-2 proteins in retina, and decrease the relative expression levels of BAX and Caspase-3 proteins.

Citation： Gu Jingsai, Chen Zhen, Xu Yishuang, Zheng Hongmei, Hua Dihao. Protective effect and mechanism of Krüppel-like factor 7 overexpression on retinal ganglion cells in mice after optic nerve clamp. Chinese Journal of Ocular Fundus Diseases, 2024, 40(4): 303-310. doi: 10.3760/cma.j.cn511434-20230504-00196 Copy

1.	Laha B, Stafford BK, Huberman AD. Regenerating optic pathways from the eye to the brain[J]. Science, 2017, 356(6342): 1031-1034. DOI: 10.1126/science.aal5060.
2.	Zhang J, Yang D, Huang H, et al. Coordination of necessary and permissive signals by PTEN inhibition for CNS axon regeneration[J]. Front Neurosci, 2018, 12: 558. DOI: 10.3389/fnins.2018.00558.
3.	Pearson R, Fleetwood J, Eaton S, et al. Kruppel-like transcription factors: a functional family[J]. Int J Biochem Cell Biol, 2008, 40(10): 1996-2001. DOI: 10.1016/j.biocel.2007.07.018.
4.	Li ZY, Zhu YX, Chen JR, et al. The role of KLF transcription factor in the regulation of cancer progression[J/OL]. Biomed Pharmacother, 2023, 162: 114661[2023-04-15]. https://doi.org/10.1016/j.biopha.2023.114661. DOI: 10.1016/j.biopha.2023.114661.
5.	Jiang BC, Zhang WW, Yang T, et al. Demethylation of G-protein-coupled receptor 151 promoter facilitates the binding of Kruppel-like factor 5 and enhances neuropathic pain after nerve injury in mice[J]. J Neurosci, 2018, 38(49): 10535-10551. DOI: 10.1523/JNEUROSCI.0702-18.2018.
6.	Dhaliwal NK, Abatti LE, Mitchell JA. KLF4 protein stability regulated by interaction with pluripotency transcription factors overrides transcriptional control[J]. Genes Dev, 2019, 33(15-16): 1069-1082. DOI: 10.1101/gad.324319.119.
7.	Blackmore MG, Wang Z, Lerch JK, et al. Kruppel-like Factor 7 engineered for transcriptional activation promotes axon regeneration in the adult corticospinal tract[J]. Proc Natl Acad Sci USA, 2012, 109(19): 7517-7522. DOI: 10.1073/pnas.1120684109.
8.	李宗媛, 杨宁, 罗晋媛, 等. Krüppel样因子7对视网膜缺血再灌注损伤小鼠视网膜神经节细胞存活及视网膜电图的影响[J]. 中华眼底病杂志, 2020, 36(11): 846-852. DOI: 10.3760/cma.j.cn511434-20200602-00256.Li ZY, Yang N, Luo JY, et al. Effects of Krüppel-like factor 7 on the survival of retinal anglion cells and electroretinogram after retinalischemia-reperfusion injury[J]. Chin J Ocul Fundus Dis, 2020, 36(11): 846-852. DOI: 10.3760/cma.j.cn511434-20200602-00256.
9.	Moore DL, Blackmore MG, Hu Y, et al. KLF family members regulate intrinsic axon regeneration ability[J]. Science, 2009, 326(5950): 298-301. DOI: 10.1126/science.1175737.
10.	欧阳灵艺, 贺涛, 邢怡桥. DJ-1蛋白对视神经钳夹伤后小鼠视网膜神经节细胞及视功能的影响[J]. 中华眼底病杂志, 2021, 37(5): 377-384. DOI: 10.3760/cma.j.cn511434-20201218-00621.Ouyang LY, He T, Xing YQ. Effects of DJ-1 protein on retinal ganglion cells and visual function in mice after optic nerve crush injury[J]. Chin J Ocul Fundus Dis, 2021, 37(5): 377-384. DOI: 10.3760/cma.j.cn511434-20201218-00621.
11.	Lobanovskaya N, Zharkovsky T, Jaako K, et al. PSA modification of NCAM supports the survival of injured retinal ganglion cells in adulthood[J]. Brain Res, 2015, 1625: 9-17. DOI: 10.1016/j.brainres.2015.08.008.
12.	Yin K J, Hamblin M, Fan Y, et al. Krupple-like factors in the central nervous system: novel mediators in stroke[J]. Metab Brain Dis, 2015, 30(2): 401-410. DOI: 10.1007/s11011-013-9468-1.
13.	Wang Z, Winsor K, Nienhaus C, et al. Combined chondroitinase and KLF7 expression reduce net retraction of sensory and CST axons from sites of spinal injury[J]. Neurobiol Dis, 2017, 99: 24-35. DOI: 10.1016/j.nbd.2016.12.010.
14.	Li WY, Wang Y, Zhai FG, et al. AAV-KLF7 promotes descending propriospinal neuron axonal plasticity after spinal cord injury[J/OL]. Neural Plast, 2017, 2017: 1621629[2017-08-13]. https://pubmed.ncbi.nlm.nih.gov/28884027/. DOI: 10.1155/2017/1621629.
15.	Sanchez-Migallon MC, Valiente-Soriano FJ, Nadal-Nicolas FM, et al. Apoptotic retinal ganglion cell death after optic nerve transection or crush in mice: delayed RGC loss with BDNF or a caspase 3 inhibitor[J]. Invest Ophthalmol Vis Sci, 2016, 57(1): 81-93. DOI: 10.1167/iovs.15-17841.
16.	廖良, 徐铭谦, 韦企平, 等. 钳夹法造成大鼠视网膜神经节细胞过量丢失[J]. 眼科新进展, 2011, 31(2): 101-104. DOI: 10.13389/j.cnki.rao.2011.02.003.Liao L, Xu MQ, Wei QP, et al. Excessive retinal ganglion cell loss after optic nerve crushed with forceps in rats[J]. Rec Adv Ophthalmol, 2011, 31(2): 101-104. DOI: 10.13389/j.cnki.rao.2011.02.003.
17.	Li WY, Zhu GY, Yue WJ, et al. KLF7 overexpression in bone marrow stromal stem cells graft transplantation promotes sciatic nerve regeneration[J/OL]. J Neural Eng, 2019, 16(5): 56011[2019-08-12]. https://pubmed.ncbi.nlm.nih.gov/31296795/. DOI: 10.1088/1741-2552/ab3188.
18.	Wang Y, Li WY, Jia H, et al. KLF7-transfected Schwann cell graft transplantation promotes sciatic nerve regeneration[J]. Neuroscience, 2017, 340: 319-332. DOI: 10.1016/j.neuroscience.2016.10.069.
19.	Allen SJ, Watson JJ, Shoemark DK, et al. GDNF, NGF and BDNF as therapeutic options for neurodegeneration[J]. Pharmacol Ther, 2013, 138(2): 155-175. DOI: 10.1016/j.pharmthera.2013.01.004.
20.	Harada T, Harada C, Parada LF. Molecular regulation of visual system development: more than meets the eye[J]. Genes Dev, 2007, 21(4): 367-378. DOI: 10.1101/gad.1504307.
21.	Caiazzo M, Colucci-D'Amato L, Esposito MT, et al. Transcription factor KLF7 regulates differentiation of neuroectodermal and mesodermal cell lineages[J]. Exp Cell Res, 2010, 316(14): 2365-2376. DOI: 10.1016/j.yexcr.2010.05.021.
22.	Kingsbury TJ, Krueger BK. Ca2+, CREB and kruppel: a novel KLF7-binding element conserved in mouse and human TRKB promoters is required for CREB-dependent transcription[J]. Mol Cell Neurosci, 2007, 35(3): 447-455. DOI: 10.1016/j.mcn.2007.04.004.
23.	Mesentier-Louro LA, Rosso P, Carito V, et al. Nerve growth factor role on retinal ganglion cell survival and axon regrowth: effects of ocular administration in experimental model of optic nerve injury[J]. Mol Neurobiol, 2019, 56(2): 1056-1069. DOI: 10.1007/s12035-018-1154-1.
24.	Bai Y, Dergham P, Nedev H, et al. Chronic and acute models of retinal neurodegeneration TrkA activity are neuroprotective whereas p75NTR activity is neurotoxic through a paracrine mechanism[J]. J Biol Chem, 2010, 285(50): 39392-39400. DOI: 10.1074/jbc.M110.147801.
25.	Shi Z, Birman E, Saragovi HU. Neurotrophic rationale in glaucoma: a TrkA agonist, but not NGF or a p75 antagonist, protects retinal ganglion cells in vivo[J]. Dev Neurobiol, 2007, 67(7): 884-894. DOI: 10.1002/dneu.20360.
26.	Brunet A, Datta SR, Greenberg ME. Transcription-dependent and-independent control of neuronal survival by the PI3K-Akt signaling pathway[J]. Curr Opin Neurobiol, 2001, 11(3): 297-305. DOI: 10.1016/s0959-4388(00)00211-7.
27.	Arthur JS, Fong AL, Dwyer JM, et al. Mitogen-and stress-activated protein kinase 1 mediates cAMP response element-binding protein phosphorylation and activation by neurotrophins[J]. J Neurosci, 2004, 24(18): 4324-4332. DOI: 10.1523/JNEUROSCI.5227-03.2004.
28.	Zhan Z, Wu Y, Liu Z, et al. Reduced dendritic spines in the visual cortex contralateral to the optic nerve crush eye in adult mice[J]. Invest Ophthalmol Vis Sci, 2020, 61(10): 55. DOI: 10.1167/iovs.61.10.55.
29.	Kato S, Matsukawa T, Koriyama Y, et al. A molecular mechanism of optic nerve regeneration in fish: the retinoid signaling pathway[J]. Prog Retin Eye Res, 2013, 37: 13-30. DOI: 10.1016/j.preteyeres.2013.07.004.
30.	Vigneswara V, Berry M, Logan A, et al. Pigment epithelium-derived factor is retinal ganglion cell neuroprotective and axogenic after optic nerve crush injury[J]. Invest Ophthalmol Vis Sci, 2013, 54(4): 2624-2633. DOI: 10.1167/iovs.13-11803.
31.	Levkovitch-Verbin H. Retinal ganglion cell apoptotic pathway in glaucoma: initiating and downstream mechanisms[J]. Prog Brain Res, 2015, 220: 37-57. DOI: 10.1016/bs.pbr.2015.05.005.
32.	Bonfanti L, Strettoi E, Chierzi S, et al. Protection of retinal ganglion cells from natural and axotomy-induced cell death in neonatal transgenic mice overexpressing Bcl-2[J]. J Neurosci, 1996, 16(13): 4186-4194. DOI: 10.1523/JNEUROSCI.16-13-04186.1996.
33.	Levkovitch-Verbin H, Waserzoog Y, Vander S, et al. Minocycline upregulates pro-survival genes and downregulates pro-apoptotic genes in experimental glaucoma[J]. Graefe's Arch Clin Exp Ophthalmol, 2014, 252(5): 761-772. DOI: 10.1007/s00417-014-2588-4.
34.	Li B, Barnes GE, Holt WF. The decline of the photopic negative response (PhNR) in the rat after optic nerve transection[J]. Doc Ophthalmol, 2005, 111(1): 23-31. DOI: 10.1007/s10633-005-2629-8.

1. Laha B, Stafford BK, Huberman AD. Regenerating optic pathways from the eye to the brain[J]. Science, 2017, 356(6342): 1031-1034. DOI: 10.1126/science.aal5060.
2. Zhang J, Yang D, Huang H, et al. Coordination of necessary and permissive signals by PTEN inhibition for CNS axon regeneration[J]. Front Neurosci, 2018, 12: 558. DOI: 10.3389/fnins.2018.00558.
3. Pearson R, Fleetwood J, Eaton S, et al. Kruppel-like transcription factors: a functional family[J]. Int J Biochem Cell Biol, 2008, 40(10): 1996-2001. DOI: 10.1016/j.biocel.2007.07.018.
4. Li ZY, Zhu YX, Chen JR, et al. The role of KLF transcription factor in the regulation of cancer progression[J/OL]. Biomed Pharmacother, 2023, 162: 114661[2023-04-15]. https://doi.org/10.1016/j.biopha.2023.114661. DOI: 10.1016/j.biopha.2023.114661.
5. Jiang BC, Zhang WW, Yang T, et al. Demethylation of G-protein-coupled receptor 151 promoter facilitates the binding of Kruppel-like factor 5 and enhances neuropathic pain after nerve injury in mice[J]. J Neurosci, 2018, 38(49): 10535-10551. DOI: 10.1523/JNEUROSCI.0702-18.2018.
6. Dhaliwal NK, Abatti LE, Mitchell JA. KLF4 protein stability regulated by interaction with pluripotency transcription factors overrides transcriptional control[J]. Genes Dev, 2019, 33(15-16): 1069-1082. DOI: 10.1101/gad.324319.119.
7. Blackmore MG, Wang Z, Lerch JK, et al. Kruppel-like Factor 7 engineered for transcriptional activation promotes axon regeneration in the adult corticospinal tract[J]. Proc Natl Acad Sci USA, 2012, 109(19): 7517-7522. DOI: 10.1073/pnas.1120684109.
8. 李宗媛, 杨宁, 罗晋媛, 等. Krüppel样因子7对视网膜缺血再灌注损伤小鼠视网膜神经节细胞存活及视网膜电图的影响[J]. 中华眼底病杂志, 2020, 36(11): 846-852. DOI: 10.3760/cma.j.cn511434-20200602-00256.Li ZY, Yang N, Luo JY, et al. Effects of Krüppel-like factor 7 on the survival of retinal anglion cells and electroretinogram after retinalischemia-reperfusion injury[J]. Chin J Ocul Fundus Dis, 2020, 36(11): 846-852. DOI: 10.3760/cma.j.cn511434-20200602-00256.
9. Moore DL, Blackmore MG, Hu Y, et al. KLF family members regulate intrinsic axon regeneration ability[J]. Science, 2009, 326(5950): 298-301. DOI: 10.1126/science.1175737.
10. 欧阳灵艺, 贺涛, 邢怡桥. DJ-1蛋白对视神经钳夹伤后小鼠视网膜神经节细胞及视功能的影响[J]. 中华眼底病杂志, 2021, 37(5): 377-384. DOI: 10.3760/cma.j.cn511434-20201218-00621.Ouyang LY, He T, Xing YQ. Effects of DJ-1 protein on retinal ganglion cells and visual function in mice after optic nerve crush injury[J]. Chin J Ocul Fundus Dis, 2021, 37(5): 377-384. DOI: 10.3760/cma.j.cn511434-20201218-00621.
11. Lobanovskaya N, Zharkovsky T, Jaako K, et al. PSA modification of NCAM supports the survival of injured retinal ganglion cells in adulthood[J]. Brain Res, 2015, 1625: 9-17. DOI: 10.1016/j.brainres.2015.08.008.
12. Yin K J, Hamblin M, Fan Y, et al. Krupple-like factors in the central nervous system: novel mediators in stroke[J]. Metab Brain Dis, 2015, 30(2): 401-410. DOI: 10.1007/s11011-013-9468-1.
13. Wang Z, Winsor K, Nienhaus C, et al. Combined chondroitinase and KLF7 expression reduce net retraction of sensory and CST axons from sites of spinal injury[J]. Neurobiol Dis, 2017, 99: 24-35. DOI: 10.1016/j.nbd.2016.12.010.
14. Li WY, Wang Y, Zhai FG, et al. AAV-KLF7 promotes descending propriospinal neuron axonal plasticity after spinal cord injury[J/OL]. Neural Plast, 2017, 2017: 1621629[2017-08-13]. https://pubmed.ncbi.nlm.nih.gov/28884027/. DOI: 10.1155/2017/1621629.
15. Sanchez-Migallon MC, Valiente-Soriano FJ, Nadal-Nicolas FM, et al. Apoptotic retinal ganglion cell death after optic nerve transection or crush in mice: delayed RGC loss with BDNF or a caspase 3 inhibitor[J]. Invest Ophthalmol Vis Sci, 2016, 57(1): 81-93. DOI: 10.1167/iovs.15-17841.
16. 廖良, 徐铭谦, 韦企平, 等. 钳夹法造成大鼠视网膜神经节细胞过量丢失[J]. 眼科新进展, 2011, 31(2): 101-104. DOI: 10.13389/j.cnki.rao.2011.02.003.Liao L, Xu MQ, Wei QP, et al. Excessive retinal ganglion cell loss after optic nerve crushed with forceps in rats[J]. Rec Adv Ophthalmol, 2011, 31(2): 101-104. DOI: 10.13389/j.cnki.rao.2011.02.003.
17. Li WY, Zhu GY, Yue WJ, et al. KLF7 overexpression in bone marrow stromal stem cells graft transplantation promotes sciatic nerve regeneration[J/OL]. J Neural Eng, 2019, 16(5): 56011[2019-08-12]. https://pubmed.ncbi.nlm.nih.gov/31296795/. DOI: 10.1088/1741-2552/ab3188.
18. Wang Y, Li WY, Jia H, et al. KLF7-transfected Schwann cell graft transplantation promotes sciatic nerve regeneration[J]. Neuroscience, 2017, 340: 319-332. DOI: 10.1016/j.neuroscience.2016.10.069.
19. Allen SJ, Watson JJ, Shoemark DK, et al. GDNF, NGF and BDNF as therapeutic options for neurodegeneration[J]. Pharmacol Ther, 2013, 138(2): 155-175. DOI: 10.1016/j.pharmthera.2013.01.004.
20. Harada T, Harada C, Parada LF. Molecular regulation of visual system development: more than meets the eye[J]. Genes Dev, 2007, 21(4): 367-378. DOI: 10.1101/gad.1504307.
21. Caiazzo M, Colucci-D'Amato L, Esposito MT, et al. Transcription factor KLF7 regulates differentiation of neuroectodermal and mesodermal cell lineages[J]. Exp Cell Res, 2010, 316(14): 2365-2376. DOI: 10.1016/j.yexcr.2010.05.021.
22. Kingsbury TJ, Krueger BK. Ca2+, CREB and kruppel: a novel KLF7-binding element conserved in mouse and human TRKB promoters is required for CREB-dependent transcription[J]. Mol Cell Neurosci, 2007, 35(3): 447-455. DOI: 10.1016/j.mcn.2007.04.004.
23. Mesentier-Louro LA, Rosso P, Carito V, et al. Nerve growth factor role on retinal ganglion cell survival and axon regrowth: effects of ocular administration in experimental model of optic nerve injury[J]. Mol Neurobiol, 2019, 56(2): 1056-1069. DOI: 10.1007/s12035-018-1154-1.
24. Bai Y, Dergham P, Nedev H, et al. Chronic and acute models of retinal neurodegeneration TrkA activity are neuroprotective whereas p75NTR activity is neurotoxic through a paracrine mechanism[J]. J Biol Chem, 2010, 285(50): 39392-39400. DOI: 10.1074/jbc.M110.147801.
25. Shi Z, Birman E, Saragovi HU. Neurotrophic rationale in glaucoma: a TrkA agonist, but not NGF or a p75 antagonist, protects retinal ganglion cells in vivo[J]. Dev Neurobiol, 2007, 67(7): 884-894. DOI: 10.1002/dneu.20360.
26. Brunet A, Datta SR, Greenberg ME. Transcription-dependent and-independent control of neuronal survival by the PI3K-Akt signaling pathway[J]. Curr Opin Neurobiol, 2001, 11(3): 297-305. DOI: 10.1016/s0959-4388(00)00211-7.
27. Arthur JS, Fong AL, Dwyer JM, et al. Mitogen-and stress-activated protein kinase 1 mediates cAMP response element-binding protein phosphorylation and activation by neurotrophins[J]. J Neurosci, 2004, 24(18): 4324-4332. DOI: 10.1523/JNEUROSCI.5227-03.2004.
28. Zhan Z, Wu Y, Liu Z, et al. Reduced dendritic spines in the visual cortex contralateral to the optic nerve crush eye in adult mice[J]. Invest Ophthalmol Vis Sci, 2020, 61(10): 55. DOI: 10.1167/iovs.61.10.55.
29. Kato S, Matsukawa T, Koriyama Y, et al. A molecular mechanism of optic nerve regeneration in fish: the retinoid signaling pathway[J]. Prog Retin Eye Res, 2013, 37: 13-30. DOI: 10.1016/j.preteyeres.2013.07.004.
30. Vigneswara V, Berry M, Logan A, et al. Pigment epithelium-derived factor is retinal ganglion cell neuroprotective and axogenic after optic nerve crush injury[J]. Invest Ophthalmol Vis Sci, 2013, 54(4): 2624-2633. DOI: 10.1167/iovs.13-11803.
31. Levkovitch-Verbin H. Retinal ganglion cell apoptotic pathway in glaucoma: initiating and downstream mechanisms[J]. Prog Brain Res, 2015, 220: 37-57. DOI: 10.1016/bs.pbr.2015.05.005.
32. Bonfanti L, Strettoi E, Chierzi S, et al. Protection of retinal ganglion cells from natural and axotomy-induced cell death in neonatal transgenic mice overexpressing Bcl-2[J]. J Neurosci, 1996, 16(13): 4186-4194. DOI: 10.1523/JNEUROSCI.16-13-04186.1996.
33. Levkovitch-Verbin H, Waserzoog Y, Vander S, et al. Minocycline upregulates pro-survival genes and downregulates pro-apoptotic genes in experimental glaucoma[J]. Graefe's Arch Clin Exp Ophthalmol, 2014, 252(5): 761-772. DOI: 10.1007/s00417-014-2588-4.
34. Li B, Barnes GE, Holt WF. The decline of the photopic negative response (PhNR) in the rat after optic nerve transection[J]. Doc Ophthalmol, 2005, 111(1): 23-31. DOI: 10.1007/s10633-005-2629-8.

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Protective effect and mechanism of Krüppel-like factor 7 overexpression on retinal ganglion cells in mice after optic nerve clamp

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