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Cellular and Molecular Life Sciences

, Volume 76, Issue 24, pp 5027–5039 | Cite as

Open reading frame mining identifies a TLR4 binding domain in the primary sequence of ECRG4

  • Xitong Dang
  • Raul Coimbra
  • Liang Mao
  • Sonia Podvin
  • Xue Li
  • Hua Yu
  • Todd W. Costantini
  • Xiaorong Zeng
  • Dana Larocca
  • Brian P. Eliceiri
  • Andrew BairdEmail author
Original Article

Abstract

The embedding of small peptide ligands within large inactive pre-pro-precursor proteins encoded by orphan open reading frames (ORFs) makes them difficult to identify and study. To address this problem, we generated oligonucleotide (< 100–400 base pair) combinatorial libraries from either the epidermal growth factor (EGF) ORF that encodes the > 1200 amino acid EGF precursor protein or the orphan ECRG4 ORF, that encodes a 148 amino acid Esophageal Cancer Related Gene 4 (ECRG4), a putative cytokine precursor protein of up to eight ligands. After phage display and 3–4 rounds of biopanning for phage internalization into prostate cancer epithelial cells, sequencing identified the 53-amino acid EGF ligand encoded by the 5′ region of the EGF ORF and three distinct domains within the primary sequence of ECRG4: its membrane targeting hydrophobic signal peptide, an unanticipated amino terminus domain at ECRG437–63 and a C-terminus ECRG4133–148 domain. Using HEK-blue cells transfected with the innate immunity receptor complex, we show that both ECRG437–63 and ECRG4133–148 enter cells by interaction with the TLR4 immune complex but neither stimulate NFkB. Taken together, the results help establish that phage display can be used to identify cryptic domains within ORFs of the human secretome and identify a novel TLR4-targeted internalization domain in the amino terminus of ECRG4 that may contribute to its effects on cell migration, immune cell activation and tumor suppression.

Keywords

Ligand Receptor Peptide targeting Secretome Phage display Cytokine precursor Tumor suppressor gene 

Notes

Acknowledgements

Research originally supported by the National Institutes of Health P20-GM078421 (AB/BPE), EY018479 (AB), HL73396 (BPE), the American Recovery Act (ARRA), and subsequently from GM121530 (TC), UC San Diego Department of Surgery Reinvestment Fund (RC) and completed with funding from grants 81-770-336 and 30-870-903 from the National Natural Science Foundation of China (XD). The authors declare no conflicts of interest and are indebted to Emelie Amburn’s expert laboratory support.

References

  1. 1.
    Lander ES (2011) Initial impact of the sequencing of the human genome. Nature 470(7333):187–197.  https://doi.org/10.1038/nature09792 PubMedCrossRefGoogle Scholar
  2. 2.
    McManus CJ, Graveley BR (2011) RNA structure and the mechanisms of alternative splicing. Curr Opin Genet Dev 21(4):373–379.  https://doi.org/10.1016/j.gde.2011.04.001 PubMedPubMedCentralCrossRefGoogle Scholar
  3. 3.
    Poulos MG, Batra R, Charizanis K, Swanson MS (2011) Developments in RNA splicing and disease. Cold Spring Harb Perspect Biol 3(1):a000778.  https://doi.org/10.1101/cshperspect.a000778 PubMedPubMedCentralCrossRefGoogle Scholar
  4. 4.
    Castro MG, Morrison E (1997) Post-translational processing of proopiomelanocortin in the pituitary and in the brain. Crit Rev Neurobiol 11(1):35–57PubMedCrossRefGoogle Scholar
  5. 5.
    Hanada K, Yang JC (2005) Novel biochemistry: post-translational protein splicing and other lessons from the school of antigen processing. J Mol Med (Berl) 83(6):420–428.  https://doi.org/10.1007/s00109-005-0652-6 CrossRefGoogle Scholar
  6. 6.
    Falth M, Skold K, Svensson M, Nilsson A, Fenyo D, Andren PE (2007) Neuropeptidomics strategies for specific and sensitive identification of endogenous peptides. Mol Cell Proteom 6(7):1188–1197.  https://doi.org/10.1074/mcp.M700016-MCP200 CrossRefGoogle Scholar
  7. 7.
    Svensson M, Skold K, Nilsson A, Falth M, Nydahl K, Svenningsson P, Andren PE (2007) Neuropeptidomics: MS applied to the discovery of novel peptides from the brain. Anal Chem 79(1):15–16PubMedCrossRefGoogle Scholar
  8. 8.
    Bystroff C, Krogh A (2008) Hidden Markov Models for prediction of protein features. Methods Mol Biol 413:173–198PubMedGoogle Scholar
  9. 9.
    Schuster-Bockler B, Bateman A (2007) An introduction to hidden Markov models. Curr Protoc Bioinform Append 3:3.  https://doi.org/10.1002/0471250953.bia03as18 CrossRefGoogle Scholar
  10. 10.
    Wu J, Xie J (2010) Hidden Markov model and its applications in motif findings. Methods Mol Biol 620:405–416.  https://doi.org/10.1007/978-1-60761-580-4_13 CrossRefGoogle Scholar
  11. 11.
    Hathout Y (2007) Approaches to the study of the cell secretome. Expert Rev Proteom 4(2):239–248.  https://doi.org/10.1586/14789450.4.2.239 CrossRefGoogle Scholar
  12. 12.
    Mustafa SA, Hoheisel JD, Alhamdani MS (2011) Secretome profiling with antibody microarrays. Mol BioSyst 7(6):1795–1801.  https://doi.org/10.1039/c1mb05071k PubMedCrossRefGoogle Scholar
  13. 13.
    Bi MX, Han WD, Lu SX (2001) Using Lab On-line to Clone and Identify the Esophageal Cancer Related Gene 4. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Biochem Biophys Bull Shanghai) 33(3):257–261Google Scholar
  14. 14.
    Yue CM, Deng DJ, Bi MX, Guo LP, Lu SH (2003) Expression of ECRG4, a novel esophageal cancer-related gene, downregulated by CpG island hypermethylation in human esophageal squamous cell carcinoma. World J Gastroenterol 9(6):1174–1178PubMedPubMedCentralCrossRefGoogle Scholar
  15. 15.
    Cai Z, Liang P, Xuan J, Wan J, Guo H (2016) ECRG4 as a novel tumor suppressor gene inhibits colorectal cancer cell growth in vitro and in vivo. Tumour Biol 37(7):9111–9120.  https://doi.org/10.1007/s13277-015-4775-2 PubMedCrossRefGoogle Scholar
  16. 16.
    Chen J, Liu C, Yin L, Zhang W (2015) The tumor-promoting function of ECRG4 in papillary thyroid carcinoma and its related mechanism. Tumour Biol 36(2):1081–1089.  https://doi.org/10.1007/s13277-014-2731-1 PubMedCrossRefGoogle Scholar
  17. 17.
    Chen JY, Wu X, Hong CQ, Chen J, Wei XL, Zhou L, Zhang HX, Huang YT, Peng L (2017) Downregulated ECRG4 is correlated with lymph node metastasis and predicts poor outcome for nasopharyngeal carcinoma patients. Clin Transl Oncol 19(1):84–90.  https://doi.org/10.1007/s12094-016-1507-z PubMedCrossRefGoogle Scholar
  18. 18.
    Gotze S, Feldhaus V, Traska T, Wolter M, Reifenberger G, Tannapfel A, Kuhnen C, Martin D, Muller O, Sievers S (2009) ECRG4 is a candidate tumor suppressor gene frequently hypermethylated in colorectal carcinoma and glioma. BMC Cancer 9:447.  https://doi.org/10.1186/1471-2407-9-447 PubMedPubMedCentralCrossRefGoogle Scholar
  19. 19.
    Matsuzaki J, Torigoe T, Hirohashi Y, Tamura Y, Asanuma H, Nakazawa E, Saka E, Yasuda K, Takahashi S, Sato N (2013) Expression of ECRG4 is associated with lower proliferative potential of esophageal cancer cells. Pathol Int 63(8):391–397.  https://doi.org/10.1111/pin.12079 PubMedCrossRefGoogle Scholar
  20. 20.
    Mori Y, Ishiguro H, Kuwabara Y, Kimura M, Mitsui A, Kurehara H, Mori R, Tomoda K, Ogawa R, Katada T, Harata K, Fujii Y (2007) Expression of ECRG4 is an independent prognostic factor for poor survival in patients with esophageal squamous cell carcinoma. Oncol Rep 18(4):981–985PubMedGoogle Scholar
  21. 21.
    Jia J, Dai S, Sun X, Sang Y, Xu Z, Zhang J, Cui X, Song J, Guo X (2015) A preliminary study of the effect of ECRG4 overexpression on the proliferation and apoptosis of human laryngeal cancer cells and the underlying mechanisms. Mol Med Rep 12(4):5058–5064.  https://doi.org/10.3892/mmr.2015.4059 PubMedPubMedCentralCrossRefGoogle Scholar
  22. 22.
    Jiang CP, Wu BH, Wang BQ, Fu MY, Yang M, Zhou Y, Liu F (2013) Overexpression of ECRG4 enhances chemosensitivity to 5-fluorouracil in the human gastric cancer SGC-7901 cell line. Tumour Biol 34(4):2269–2273.  https://doi.org/10.1007/s13277-013-0768-1 PubMedCrossRefGoogle Scholar
  23. 23.
    Deng P, Chang XJ, Gao ZM, Xu XY, Sun AQ, Li K, Dai DQ (2018) Downregulation and DNA methylation of ECRG4 in gastric cancer. Onco Targets Ther 11:4019–4028.  https://doi.org/10.2147/OTT.S161200 PubMedPubMedCentralCrossRefGoogle Scholar
  24. 24.
    Lee J, Dang X, Borboa A, Coimbra R, Baird A, Eliceiri BP (2015) Thrombin-processed Ecrg4 recruits myeloid cells and induces antitumorigenic inflammation. Neuro Oncol 17(5):685–696.  https://doi.org/10.1093/neuonc/nou302 PubMedCrossRefGoogle Scholar
  25. 25.
    Li L, Zhang C, Li X, Lu S, Zhou Y (2010) The candidate tumor suppressor gene ECRG4 inhibits cancer cells migration and invasion in esophageal carcinoma. J Exp Clin Cancer Res 29:133.  https://doi.org/10.1186/1756-9966-29-133 PubMedPubMedCentralCrossRefGoogle Scholar
  26. 26.
    Mirabeau O, Perlas E, Severini C, Audero E, Gascuel O, Possenti R, Birney E, Rosenthal N, Gross C (2007) Identification of novel peptide hormones in the human proteome by hidden Markov model screening. Genome Res 17(3):320–327.  https://doi.org/10.1101/gr.5755407 PubMedPubMedCentralCrossRefGoogle Scholar
  27. 27.
    Gonzalez AM, Podvin S, Lin SY, Miller MC, Botfield H, Leadbeater WE, Roberton A, Dang X, Knowling SE, Cardenas-Galindo E, Donahue JE, Stopa EG, Johanson CE, Coimbra R, Eliceiri BP, Baird A (2011) Ecrg4 expression and its product augurin in the choroid plexus: impact on fetal brain development, cerebrospinal fluid homeostasis and neuroprogenitor cell response to CNS injury. Fluids Barriers CNS 8(1):6.  https://doi.org/10.1186/2045-8118-8-6 PubMedPubMedCentralCrossRefGoogle Scholar
  28. 28.
    Podvin S, Gonzalez AM, Miller MC, Dang X, Botfield H, Donahue JE, Kurabi A, Boissaud-Cooke M, Rossi R, Leadbeater WE, Johanson CE, Coimbra R, Stopa EG, Eliceiri BP, Baird A (2011) Esophageal cancer related gene-4 is a choroid plexus-derived injury response gene: evidence for a biphasic response in early and late brain injury. PLoS One 6(9):e24609.  https://doi.org/10.1371/journal.pone.0024609 PubMedPubMedCentralCrossRefGoogle Scholar
  29. 29.
    Tadross JA, Patterson M, Suzuki K, Beale KE, Boughton CK, Smith KL, Moore S, Ghatei MA, Bloom SR (2010) Augurin stimulates the hypothalamo-pituitary-adrenal axis via the release of corticotrophin-releasing factor in rats. Br J Pharmacol 159(8):1663–1671.  https://doi.org/10.1111/j.1476-5381.2010.00655.x PubMedPubMedCentralCrossRefGoogle Scholar
  30. 30.
    Matsuzaki J, Torigoe T, Hirohashi Y, Kamiguchi K, Tamura Y, Tsukahara T, Kubo T, Takahashi A, Nakazawa E, Saka E, Yasuda K, Takahashi S, Sato N (2012) ECRG4 is a negative regulator of caspase-8-mediated apoptosis in human T-leukemia cells. Carcinogenesis 33(5):996–1003.  https://doi.org/10.1093/carcin/bgs118 PubMedCrossRefGoogle Scholar
  31. 31.
    Huh YH, Ryu JH, Shin S, Lee DU, Yang S, Oh KS, Chun CH, Choi JK, Song WK, Chun JS (2009) Esophageal cancer related gene 4 (ECRG4) is a marker of articular chondrocyte differentiation and cartilage destruction. Gene 448(1):7–15.  https://doi.org/10.1016/j.gene.2009.08.015 PubMedCrossRefGoogle Scholar
  32. 32.
    Kujuro Y, Suzuki N, Kondo T (2010) Esophageal cancer-related gene 4 is a secreted inducer of cell senescence expressed by aged CNS precursor cells. Proc Natl Acad Sci USA 107(18):8259–8264.  https://doi.org/10.1073/pnas.0911446107 PubMedCrossRefGoogle Scholar
  33. 33.
    Kao S, Shaterian A, Cauvi DM, Dang X, Chun HB, De Maio A, Costantini TW, Coimbra R, Eliceiri BP, Baird A (2015) Pulmonary preconditioning, injury, and inflammation modulate expression of the candidate tumor suppressor gene ECRG4 in lung. Exp Lung Res 41(3):162–172.  https://doi.org/10.3109/01902148.2014.983282 PubMedCrossRefGoogle Scholar
  34. 34.
    Shaterian A, Kao S, Chen L, DiPietro LA, Coimbra R, Eliceiri BP, Baird A (2013) The candidate tumor suppressor gene Ecrg4 as a wound terminating factor in cutaneous injury. Arch Dermatol Res 305(2):141–149.  https://doi.org/10.1007/s00403-012-1276-7 PubMedCrossRefGoogle Scholar
  35. 35.
    Kurabi A, Pak K, Dang X, Coimbra R, Eliceiri BP, Ryan AF, Baird A (2013) Ecrg4 attenuates the inflammatory proliferative response of mucosal epithelial cells to infection. PLoS One 8(4):e61394.  https://doi.org/10.1371/journal.pone.0061394 PubMedPubMedCentralCrossRefGoogle Scholar
  36. 36.
    Nakatani Y, Kiyonari H, Kondo T (2019) Ecrg4 deficiency extends the replicative capacity of neural stem cells in a Foxg1-dependent manner. Development.  https://doi.org/10.1242/dev.168120 PubMedCrossRefGoogle Scholar
  37. 37.
    Nishikawa M, Drmanac RT, Lobal I, Tang Y, Lee J, Stache-Crain B (2008) Polypeptide having an activity to support proliferation or survival of hematopoietic stem cell and hematopoietic progenitor cell, and DNA coding for the same. United States Patent Trade Office USA, Nuvelo Inc, San Carlos CaliforniaGoogle Scholar
  38. 38.
    Baird A, Coimbra R, Dang X, Lopez N, Lee J, Krzyzaniak M, Winfield R, Potenza B, Eliceiri BP (2012) Cell surface localization and release of the candidate tumor suppressor Ecrg4 from polymorphonuclear cells and monocytes activate macrophages. J Leukoc Biol 91(5):773–781.  https://doi.org/10.1189/jlb.1011503 PubMedPubMedCentralCrossRefGoogle Scholar
  39. 39.
    Baird A, Lee J, Podvin S, Kurabi A, Dang X, Coimbra R, Costantini T, Bansal V, Eliceiri BP (2014) Esophageal cancer-related gene 4 at the interface of injury, inflammation, infection, and malignancy. Gastrointest Cancer 4:131–142.  https://doi.org/10.2147/GICTT.S49085 CrossRefGoogle Scholar
  40. 40.
    Moriguchi T, Kaneumi S, Takeda S, Enomoto K, Mishra SK, Miki T, Koshimizu U, Kitamura H, Kondo T (2016) Ecrg4 contributes to the anti-glioma immunosurveillance through type-I interferon signaling. Oncoimmunology 5(12):e1242547.  https://doi.org/10.1080/2162402X.2016.1242547 PubMedPubMedCentralCrossRefGoogle Scholar
  41. 41.
    Porzionato A, Rucinski M, Macchi V, Sarasin G, Malendowicz LK, De Caro R (2015) ECRG4 expression in normal rat tissues: expression study and literature review. Eur J Histochem 59(2):2458.  https://doi.org/10.4081/ejh.2015.2458 PubMedPubMedCentralCrossRefGoogle Scholar
  42. 42.
    Qin X, Zhang P (2018) ECRG4: a new potential target in precision medicine. Front Med.  https://doi.org/10.1007/s11684-018-0637-9 PubMedCrossRefGoogle Scholar
  43. 43.
    Ozawa A, Lick AN, Lindberg I (2011) Processing of proaugurin is required to suppress proliferation of tumor cell lines. Mol Endocrinol 25(5):776–784.  https://doi.org/10.1210/me.2010-0389 PubMedPubMedCentralCrossRefGoogle Scholar
  44. 44.
    Dang X, Podvin S, Coimbra R, Eliceiri B, Baird A (2012) Cell-specific processing and release of the hormone-like precursor and candidate tumor suppressor gene product, Ecrg4. Cell Tissue Res 348(3):505–514.  https://doi.org/10.1007/s00441-012-1396-6 PubMedPubMedCentralCrossRefGoogle Scholar
  45. 45.
    Dang X, Zeng X, Coimbra R, Eliceiri BP, Baird A (2017) Counter regulation of ECRG4 gene expression by hypermethylation-dependent inhibition and the Sp1 transcription factor-dependent stimulation of the c2orf40 promoter. Gene 636:103–111.  https://doi.org/10.1016/j.gene.2017.08.041 PubMedCrossRefGoogle Scholar
  46. 46.
    Larocca D, Kassner PD, Witte A, Ladner RC, Pierce GF, Baird A (1999) Gene transfer to mammalian cells using genetically targeted filamentous bacteriophage. FASEB J 13(6):727–734PubMedCrossRefGoogle Scholar
  47. 47.
    Rousselet E, Benjannet S, Marcinkiewicz E, Asselin MC, Lazure C, Seidah NG (2011) Proprotein convertase PC7 enhances the activation of the EGF receptor pathway through processing of the EGF precursor. J Biol Chem 286(11):9185–9195.  https://doi.org/10.1074/jbc.M110.189936 PubMedPubMedCentralCrossRefGoogle Scholar
  48. 48.
    Rall LB, Scott J, Bell GI, Crawford RJ, Penschow JD, Niall HD, Coghlan JP (1985) Mouse prepro-epidermal growth factor synthesis by the kidney and other tissues. Nature 313(5999):228–231PubMedCrossRefGoogle Scholar
  49. 49.
    Xian CJ, Zhou XF (2004) EGF family of growth factors: essential roles and functional redundancy in the nerve system. Front Biosci 9:85–92PubMedCrossRefGoogle Scholar
  50. 50.
    Larocca D, Baird A (2001) Receptor-mediated gene transfer by phage-display vectors: applications in functional genomics and gene therapy. Drug Discov Today 6(15):793–801PubMedCrossRefGoogle Scholar
  51. 51.
    Larocca D, Witte A, Johnson W, Pierce GF, Baird A (1998) Targeting bacteriophage to mammalian cell surface receptors for gene delivery. Hum Gene Ther 9(16):2393–2399.  https://doi.org/10.1089/hum.1998.9.16-2393 PubMedCrossRefGoogle Scholar
  52. 52.
    Larocca D, Jensen-Pergakes K, Burg MA, Baird A (2001) Receptor-targeted gene delivery using multivalent phagemid particles. Mol Ther 3(4):476–484.  https://doi.org/10.1006/mthe.2001.0284 PubMedCrossRefGoogle Scholar
  53. 53.
    Larocca D, Burg MA, Jensen-Pergakes K, Ravey EP, Gonzalez AM, Baird A (2002) Evolving phage vectors for cell targeted gene delivery. Curr Pharm Biotechnol 3(1):45–57PubMedCrossRefGoogle Scholar
  54. 54.
    Burg M, Ravey EP, Gonzales M, Amburn E, Faix PH, Baird A, Larocca D (2004) Selection of internalizing ligand-display phage using rolling circle amplification for phage recovery. DNA Cell Biol 23(7):457–462.  https://doi.org/10.1089/1044549041474760 PubMedCrossRefGoogle Scholar
  55. 55.
    Burg MA, Jensen-Pergakes K, Gonzalez AM, Ravey P, Baird A, Larocca D (2002) Enhanced phagemid particle gene transfer in camptothecin-treated carcinoma cells. Cancer Res 62(4):977–981PubMedGoogle Scholar
  56. 56.
    Faix PH, Burg MA, Gonzales M, Ravey EP, Baird A, Larocca D (2004) Phage display of cDNA libraries: enrichment of cDNA expression using open reading frame selection. Biotechniques 36(6):1018–1022PubMedCrossRefGoogle Scholar
  57. 57.
    Joliot A (2005) Transduction peptides within naturally occurring proteins. Sci STKE.  https://doi.org/10.1126/stke.3132005pe54 PubMedCrossRefGoogle Scholar
  58. 58.
    Peitz M, Pfannkuche K, Rajewsky K, Edenhofer F (2002) Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: a tool for efficient genetic engineering of mammalian genomes. Proc Natl Acad Sci USA 99(7):4489–4494.  https://doi.org/10.1073/pnas.032068699 PubMedCrossRefGoogle Scholar
  59. 59.
    Moriguchi T, Takeda S, Iwashita S, Enomoto K, Sawamura T, Koshimizu U, Kondo T (2018) Ecrg4 peptide is the ligand of multiple scavenger receptors. Sci Rep 8(1):4048.  https://doi.org/10.1038/s41598-018-22440-4 PubMedPubMedCentralCrossRefGoogle Scholar
  60. 60.
    Podvin S, Dang X, Meads M, Kurabi A, Costantini T, Eliceiri BP, Baird A, Coimbra R (2015) Esophageal cancer-related gene-4 (ECRG4) interactions with the innate immunity receptor complex. Inflamm Res 64(2):107–118.  https://doi.org/10.1007/s00011-014-0789-2 PubMedCrossRefGoogle Scholar
  61. 61.
    Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228(4705):1315–1317PubMedCrossRefGoogle Scholar
  62. 62.
    Jespers LS, Messens JH, De Keyser A, Eeckhout D, Van den Brande I, Gansemans YG, Lauwereys MJ, Vlasuk GP, Stanssens PE (1995) Surface expression and ligand-based selection of cDNAs fused to filamentous phage gene VI. Biotechnology (NY) 13(4):378–382Google Scholar
  63. 63.
    Smith GP, Petrenko VA (1997) Phage display. Chem Rev 97(2):391–410PubMedCrossRefGoogle Scholar
  64. 64.
    Thompson J, Pope T, Tung JS, Chan C, Hollis G, Mark G, Johnson KS (1996) Affinity maturation of a high-affinity human monoclonal antibody against the third hypervariable loop of human immunodeficiency virus: use of phage display to improve affinity and broaden strain reactivity. J Mol Biol 256(1):77–88.  https://doi.org/10.1006/jmbi.1996.0069 PubMedCrossRefGoogle Scholar
  65. 65.
    Kramer RA, Cox F, van der Horst M, van der Oudenrijn S, Res PC, Bia J, Logtenberg T, de Kruif J (2003) A novel helper phage that improves phage display selection efficiency by preventing the amplification of phages without recombinant protein. Nucleic Acids Res 31(11):e59PubMedPubMedCentralCrossRefGoogle Scholar
  66. 66.
    Smothers JF, Henikoff S, Carter P (2002) Tech.Sight. Phage display. Affinity selection from biological libraries. Science 298(5593):621–622.  https://doi.org/10.1126/science.298.5593.621 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Department of SurgeryUniversity of California San DiegoSan DiegoUSA
  2. 2.The Key Laboratory of Medical Electrophysiology, Ministry of Education, Institute of Cardiovascular ResearchSouthwest Medical UniversityLuzhouChina
  3. 3.Mandala BiosciencesSan DiegoUSA
  4. 4.Department of SurgeryUniversity of California San DiegoSan DiegoUSA

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