{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T12:47:30Z","timestamp":1790254050239,"version":"4.1.0"},"reference-count":47,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2019,9,18]],"date-time":"2019-09-18T00:00:00Z","timestamp":1568764800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003453","name":"Natural Science Foundation of Guangdong Province","doi-asserted-by":"publisher","award":["2017A030313598"],"award-info":[{"award-number":["2017A030313598"]}],"id":[{"id":"10.13039\/501100003453","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>CXC chemokine receptor 4 (CXCR4) is a co-receptor for HIV-1 entry into target cells. Its natural ligand, the chemokine SDF-1, inhibits viral entry mediated by this receptor. However, the broad expression pattern of CXCR4 and its critical roles in various physiological and pathological processes indicate that the direct application of SDF-1 as an entry inhibitor might have severe consequences. Previously, we constructed an effective SDF-1 mutant, SDF-1\/54, by deleting the \u03b1-helix of the C-terminal functional region of SDF-1. Of note, SDF-1\/54 shows remarkable decreased chemotoxic ability, but maintains a similar binding affinity to CXCR4, suggesting SDF-1\/54 might better serve as a CXCR4 inhibitor. Here, we found that SDF-1\/54 exhibited potent antiviral activity against various X4 HIV-1 strains, including the infectious clone HIV-1 NL4-3, laboratory-adapted strain HIV-1 IIIB, clinical isolates and even drug-resistant strains. By using time-of-addition assay, non-infectious and infectious cell\u2013cell fusion assay and CXCR4 internalization assay, we demonstrated SDF-1\/54 is an HIV-1 entry inhibitor. A combination of SDF-1\/54 with several antiretroviral drugs exhibited potent synergistic anti-HIV-1 activity. Moreover, SDF-1\/54 was stable and its anti-HIV-1 activity was not significantly affected by the presence of seminal fluid, vaginal fluid simulant and human serum albumin. SDF-1\/54 showed limited in vitro cytotoxicity to lymphocytes and vaginal epithelial cells. Based on these findings, SDF-1\/54 could have a therapeutic potential as an HIV-1 entry inhibitor.<\/jats:p>","DOI":"10.3390\/v11090874","type":"journal-article","created":{"date-parts":[[2019,9,18]],"date-time":"2019-09-18T11:01:15Z","timestamp":1568804475000},"page":"874","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["A Novel CXCR4 Targeting Protein SDF-1\/54 as an HIV-1 Entry Inhibitor"],"prefix":"10.3390","volume":"11","author":[{"given":"Suiyi","family":"Tan","sequence":"first","affiliation":[{"name":"Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenjuan","family":"Li","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhaofeng","family":"Li","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yujing","family":"Li","sequence":"additional","affiliation":[{"name":"Department of Microbiology, School of Public Health, Southern Medical University, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiangyan","family":"Luo","sequence":"additional","affiliation":[{"name":"Department of Microbiology, School of Public Health, Southern Medical University, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liangzhentian","family":"Yu","sequence":"additional","affiliation":[{"name":"Department of Microbiology, School of Public Health, Southern Medical University, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jie","family":"Yang","sequence":"additional","affiliation":[{"name":"Department of Microbiology, School of Public Health, Southern Medical University, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mengjie","family":"Qiu","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongyan","family":"Cheng","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wei","family":"Xu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Medical Molecular Virology of Ministries of Education &amp; Health, Shanghai Medical College and Institute of Medical Microbiology, Fudan University, Shanghai 200032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8283-7135","authenticated-orcid":false,"given":"Shibo","family":"Jiang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Medical Molecular Virology of Ministries of Education &amp; Health, Shanghai Medical College and Institute of Medical Microbiology, Fudan University, Shanghai 200032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2255-0391","authenticated-orcid":false,"given":"Lu","family":"Lu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Medical Molecular Virology of Ministries of Education &amp; Health, Shanghai Medical College and Institute of Medical Microbiology, Fudan University, Shanghai 200032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6346-5006","authenticated-orcid":false,"given":"Shuwen","family":"Liu","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weifeng","family":"Ma","sequence":"additional","affiliation":[{"name":"Department of Microbiology, School of Public Health, Southern Medical University, Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2019,9,18]]},"reference":[{"key":"ref_1","unstructured":"Joint United Nations Programme on HIV\/AIDS (UNAIDS) (2018). Unaids Data 2018, UNAIDS. Available online: http:\/\/www.unaids.org\/sites\/default\/files\/media_asset\/unaids-data-2018_en.pdf."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1102","DOI":"10.1086\/378302","article-title":"Enfuvirtide (T-20): a novel human immunodeficiency virus type 1 fusion inhibitor","volume":"37","author":"Cervia","year":"2003","journal-title":"Clin. Infect. Dis."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1504","DOI":"10.2174\/1568026614666140827143745","article-title":"Chemokine receptor CCR5 antagonist maraviroc: medicinal chemistry and clinical applications","volume":"14","author":"Xu","year":"2014","journal-title":"Curr. Top. Med. Chem."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1179","DOI":"10.1126\/science.8356453","article-title":"Genotypic and phenotypic characterization of HIV-1 patients with primary infection","volume":"261","author":"Zhu","year":"1993","journal-title":"Science"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1354","DOI":"10.1128\/jvi.66.3.1354-1360.1992","article-title":"Biological phenotype of human immunodeficiency virus type 1 clones at different stages of infection: progression of disease is associated with a shift from monocytotropic to T\u2013cell-tropic virus population","volume":"66","author":"Schuitemaker","year":"1992","journal-title":"J. Virol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1080\/096876899294751","article-title":"HIV coreceptors, cell tropism and inhibition by chemokine receptor ligands","volume":"16","author":"Clapham","year":"1999","journal-title":"Mol. Membr. Biol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"6996","DOI":"10.1093\/emboj\/16.23.6996","article-title":"Solution structure and basis for functional activity of stromal cell-derived factor-1; dissociation of CXCR4 activation from binding and inhibition of HIV-1","volume":"16","author":"Crump","year":"1997","journal-title":"Embo J."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"309","DOI":"10.3390\/v4020309","article-title":"Novel approaches to inhibit HIV entry","volume":"4","author":"Didigu","year":"2012","journal-title":"Viruses"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"881","DOI":"10.1021\/acschembio.8b00061","article-title":"Inhibition of HIV Fusion by Small Molecule Agonists through Efficacy-Engineering of CXCR4","volume":"13","author":"Berg","year":"2018","journal-title":"ACS Chem. Biol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"829","DOI":"10.1038\/382829a0","article-title":"The lymphocyte chemoattractant SDF-1 is a ligand for LESTR\/fusin and blocks HIV-1 entry","volume":"382","author":"Bleul","year":"1996","journal-title":"Nature"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"299","DOI":"10.1038\/cmi.2017.107","article-title":"The unique structural and functional features of CXCL12","volume":"15","author":"Janssens","year":"2018","journal-title":"Cell. Mol. Immunol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1130","DOI":"10.1016\/j.bmc.2019.02.013","article-title":"Design, synthesis and biological evaluation of low molecular weight CXCR4 ligands","volume":"27","author":"Sakyiamah","year":"2019","journal-title":"Bioorg. Med. Chem."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2024","DOI":"10.1021\/jm0104015","article-title":"C-terminal cyclization of an SDF-1 small peptide analogue dramatically increases receptor affinity and activation of the CXCR4 receptor","volume":"45","author":"Tudan","year":"2002","journal-title":"J. Med. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1385\/ABAB:102-103:1-6:041","article-title":"Synthetic peptides for study of human immunodeficiency virus infection","volume":"102\u2013103","author":"Dettin","year":"2002","journal-title":"Appl. Biochem. Biotechnol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"10298","DOI":"10.1039\/C6OB01484D","article-title":"Design, synthesis and biological evaluation of CXCR4 ligands","volume":"14","author":"Mona","year":"2016","journal-title":"Org. Biomol. Chem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1553","DOI":"10.1016\/j.exphem.2006.07.001","article-title":"Cloning and characterizing mutated human stromal cell-derived factor-1 (SDF-1): C-terminal alpha-helix of SDF-1alpha plays a critical role in CXCR4 activation and signaling, but not in CXCR4 binding affinity","volume":"34","author":"Tan","year":"2006","journal-title":"Exp. Hematol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1557","DOI":"10.1084\/jem.174.6.1557","article-title":"Enhancement of human immunodeficiency virus type 1 infection by antisera to peptides from the envelope glycoproteins gp120\/gp41","volume":"174","author":"Jiang","year":"1991","journal-title":"J. Exp. Med."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"4349","DOI":"10.1128\/AAC.48.11.4349-4359.2004","article-title":"N-substituted pyrrole derivatives as novel human immunodeficiency virus type 1 entry inhibitors that interfere with the gp41 six-helix bundle formation and block virus fusion","volume":"48","author":"Jiang","year":"2004","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"5323","DOI":"10.1074\/jbc.RA118.001729","article-title":"Exceptional potency and structural basis of a T1249-derived lipopeptide fusion inhibitor against HIV-1, HIV-2 and simian immunodeficiency virus","volume":"293","author":"Zhu","year":"2018","journal-title":"J. Biol. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1016\/0065-2571(84)90007-4","article-title":"Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors","volume":"22","author":"Chou","year":"1984","journal-title":"Adv. Enzyme Regul."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1700","DOI":"10.1128\/AAC.01046-09","article-title":"3-hydroxyphthalic anhydride-modified chicken ovalbumin exhibits potent and broad anti-HIV-1 activity: a potential microbicide for preventing sexual transmission of HIV-1","volume":"54","author":"Li","year":"2010","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1455","DOI":"10.1089\/aid.2013.0043","article-title":"3-hydroxyphthalic anhydride-modified human serum albumin as a microbicide candidate against HIV type 1 entry by targeting both viral envelope glycoprotein gp120 and cellular receptor CD4","volume":"29","author":"Li","year":"2013","journal-title":"AIDS Res. Hum. Retrovir."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"859","DOI":"10.3389\/fphar.2019.00859","article-title":"N-Substituted Pyrrole Derivative 12m Inhibits HIV-1 Entry by Targeting Gp41 of HIV-1 Envelope Glycoprotein","volume":"10","author":"Qiu","year":"2019","journal-title":"Front. Pharmacol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1016\/S0166-0934(02)00213-6","article-title":"Automatic quantitation of HIV-1 mediated cell-to\u2013cell fusion with a digital image analysis system (DIAS): application for rapid screening of HIV-1 fusion inhibitors","volume":"107","author":"Lu","year":"2003","journal-title":"J. Virol. Methods"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"7862","DOI":"10.1128\/JVI.00168-09","article-title":"Combinations of the first and next generations of human immunodeficiency virus (HIV) fusion inhibitors exhibit a highly potent synergistic effect against enfuvirtide- sensitive and -resistant HIV type 1 strains","volume":"83","author":"Pan","year":"2009","journal-title":"J. Virol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"621","DOI":"10.1124\/pr.58.3.10","article-title":"Theoretical basis, experimental design and computerized simulation of synergism and antagonism in drug combination studies","volume":"58","author":"Chou","year":"2006","journal-title":"Pharmacol. Rev."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"5123","DOI":"10.1128\/AAC.00385-15","article-title":"ADS-J1 inhibits semen-derived amyloid fibril formation and blocks fibril-mediated enhancement of HIV-1 infection","volume":"59","author":"Xun","year":"2015","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2533","DOI":"10.1093\/jac\/dkt230","article-title":"An engineered HIV-1 gp41 trimeric coiled coil with increased stability and anti-HIV-1 activity: implication for developing anti-HIV microbicides","volume":"68","author":"Tong","year":"2013","journal-title":"J. Antimicrob. Chemother."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"361","DOI":"10.1016\/j.bmcl.2015.12.005","article-title":"Aspernigrins with anti-HIV-1 activities from the marine-derived fungus Aspergillus niger SCSIO Jcsw6F30","volume":"26","author":"Zhou","year":"2016","journal-title":"Bioorg. Med. Chem. Lett."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Neurath, A.R., Strick, N., and Li, Y.Y. (2006). Role of seminal plasma in the anti-HIV-1 activity of candidate microbicides. BMC Infect. Dis., 6.","DOI":"10.1186\/1471-2334-6-150"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"157r","DOI":"10.1126\/scitranslmed.3009634","article-title":"Semen enhances HIV infectivity and impairs the antiviral efficacy of microbicides","volume":"6","author":"Zirafi","year":"2014","journal-title":"Sci. Transl. Med."},{"key":"ref_32","first-page":"152","article-title":"Loss of C-terminal alpha-helix decreased SDF-1alpha-mediated signaling and chemotaxis without influencing CXCR4 internalization","volume":"25","author":"Cai","year":"2004","journal-title":"Acta Pharmacol. Sin."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"863","DOI":"10.1189\/jlb.2MR1115-521RR","article-title":"Proteolytic processing of human serum albumin generates EPI-X4, an endogenous antagonist of CXCR4","volume":"99","author":"Zirafi","year":"2016","journal-title":"J. Leukoc. Biol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"2193","DOI":"10.1002\/art.40230","article-title":"NF-kappaB2 Controls the Migratory Activity of Memory T Cells by Regulating Expression of CXCR4 in a Mouse Model of Sjogren\u2019s Syndrome","volume":"69","author":"Kurosawa","year":"2017","journal-title":"Arthritis Rheumatol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"429","DOI":"10.1182\/blood-2010-01-266833","article-title":"Loss of Cxcl12\/Sdf-1 in adult mice decreases the quiescent state of hematopoietic stem\/progenitor cells and alters the pattern of hematopoietic regeneration after myelosuppression","volume":"117","author":"Tzeng","year":"2011","journal-title":"Blood"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1002\/ijc.29333","article-title":"Role of CXCL12 and CXCR4 in normal cerebellar development and medulloblastoma","volume":"138","author":"Ozawa","year":"2016","journal-title":"Int. J. Cancer"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"388","DOI":"10.1161\/CIRCULATIONAHA.117.027646","article-title":"Vascular CXCR4 Limits Atherosclerosis by Maintaining Arterial Integrity: Evidence From Mouse and Human Studies","volume":"136","author":"Doring","year":"2017","journal-title":"Circulation"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1080\/07357907.2017.1422512","article-title":"Knockdown of CXCR4 Inhibits CXCL12-Induced Angiogenesis in HUVECs through Downregulation of the MAPK\/ERK and PI3K\/AKT and the Wnt\/beta-Catenin Pathways","volume":"36","author":"Song","year":"2018","journal-title":"Cancer Investig."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1186\/s12948-016-0052-1","article-title":"HIV-1 Nef promotes migration and chemokine synthesis of human basophils and mast cells through the interaction with CXCR4","volume":"14","author":"Rossi","year":"2016","journal-title":"Clin. Mol. Allergy"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1016\/j.jneuroim.2015.11.021","article-title":"New insights into the role of stromal cell-derived factor 1 (SDF-1\/CXCL12) in the pathophysiology of multiple sclerosis","volume":"290","author":"Khorramdelazad","year":"2016","journal-title":"J. Neuroimmunol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"487","DOI":"10.1016\/j.tranon.2018.02.002","article-title":"The Chemokine Receptor CXCR4 and c-MET Cooperatively Promote Epithelial-Mesenchymal Transition in Gastric Cancer Cells","volume":"11","author":"Cheng","year":"2018","journal-title":"Transl. Oncol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"17033","DOI":"10.1038\/sigtrans.2017.33","article-title":"Targeting the CXCR4\/CXCL12 axis with the peptide antagonist E5 to inhibit breast tumor progression","volume":"2","author":"Guo","year":"2017","journal-title":"Signal Transduct. Target Ther."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"691","DOI":"10.1051\/medsci\/20173308002","article-title":"The CXCL12\/CXCR4 signaling pathway in the control of human papillomavirus infection: new susceptibility factors in viral pathogenesis","volume":"33","author":"Meuris","year":"2017","journal-title":"Med. Sci. (Paris)"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1016\/j.ejca.2012.05.005","article-title":"A review on CXCR4\/CXCL12 axis in oncology: no place to hide","volume":"49","author":"Domanska","year":"2013","journal-title":"Eur. J. Cancer"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"18111","DOI":"10.1074\/jbc.M200750200","article-title":"CXCR4\/CCR5 down-modulation and chemotaxis are regulated by the proteasome pathway","volume":"277","author":"Fernandis","year":"2002","journal-title":"J. Biol. Chem."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1007\/978-1-4939-7553-2_10","article-title":"Detecting Cell Surface Expression of the G Protein-Coupled Receptor CXCR4","volume":"1722","author":"Nevins","year":"2018","journal-title":"Methods Mol. Biol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"17476","DOI":"10.1074\/jbc.M202063200","article-title":"Exploring the stereochemistry of CXCR4-peptide recognition and inhibiting HIV-1 entry with D-peptides derived from chemokines","volume":"277","author":"Zhou","year":"2002","journal-title":"J. Biol. Chem."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/11\/9\/874\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T13:21:28Z","timestamp":1760188888000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/11\/9\/874"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,9,18]]},"references-count":47,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2019,9]]}},"alternative-id":["v11090874"],"URL":"https:\/\/doi.org\/10.3390\/v11090874","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,9,18]]}}}