{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T11:16:16Z","timestamp":1788434176708,"version":"build-2803163510"},"reference-count":50,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T00:00:00Z","timestamp":1788393600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"JSPS KAKENH","award":["JP21K17323"],"award-info":[{"award-number":["JP21K17323"]}]},{"name":"JSPS KAKENH","award":["JP23K09762"],"award-info":[{"award-number":["JP23K09762"]}]},{"name":"JSPS KAKENH","award":["JP23K18387"],"award-info":[{"award-number":["JP23K18387"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Metabolites"],"abstract":"<jats:p>Background: Cannabis contains \u03949-tetrahydrocannabinol (THC) together with numerous other constituents, whereas HU210 (HU) is a synthetic full agonist at cannabinoid receptors. We applied integrated metabolomics and proteomics to compare Cannabis extract (CS) and HU in GT1-7 neuronal cells. Methods: Cytotoxicity was evaluated using a lactate dehydrogenase assay. Metabolites and proteins were extracted sequentially from single-cell pellets and analyzed using gas chromatography\u2013tandem mass spectrometry and nano-liquid chromatography\u2013quadrupole\u2013Orbitrap mass spectrometry, respectively. Cannabinoids in the exposure medium were quantified using liquid chromatography\u2013tandem mass spectrometry. Results: The lowest CS and HU concentrations that were cytotoxic at 24 h and non-cytotoxic at 3 h were 2000 \u00b5g\/mL and 20 \u00b5M, respectively; CS delivered 166 nM THC, 800 nM tetrahydrocannabinolic acid, and 11.3 nM cannabidiol. After 3 h, HU altered both the metabolome and the proteome, whereas CS altered the metabolome with minimal proteome change (8 of 2720 proteins). Six pathways differed, including the citrate cycle and pentose phosphate pathway. Relative to both the control and CS, HU decreased citrate; increased 2-ketoglutarate, fumarate, malate, gluconate, ribose-5-phosphate and ribose; and increased phosphoenolpyruvate carboxykinase 2, isocitrate dehydrogenase 3 subunit \u03b1, 2-oxoglutarate dehydrogenase complex component E1, succinate dehydrogenase flavoprotein subunit, malate dehydrogenase 1, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase and phosphoglucomutase-1. CS showed the opposite metabolite changes without corresponding enzyme changes. Conclusions: At a pre-lethal time point, CS and HU produced metabolic changes in opposite directions\u2014broad suppression versus coordinated mobilisation\u2014despite comparable cytotoxicity at 24 h.<\/jats:p>","DOI":"10.3390\/metabo16090645","type":"journal-article","created":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T10:10:33Z","timestamp":1788430233000},"page":"645","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Integrated Metabolomic and Proteomic Analyses Reveal Differential Regulation of the Citrate Cycle and Pentose Phosphate Pathway by Cannabis sativa Extract and the Synthetic Cannabinoid HU210 in GT1-7 Neuronal Cells: Sequential Extraction of Metabolites and Proteins from a Single Cell Pellet"],"prefix":"10.3390","volume":"16","author":[{"given":"Yujin","family":"Natori","sequence":"first","affiliation":[{"name":"Department of Legal Medicine and Bioethics, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya-shi 466-8550, Japan"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"software","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4301-829X","authenticated-orcid":false,"given":"Dai","family":"Mizuno","sequence":"additional","affiliation":[{"name":"Department of Forensic Medicine, Faculty of Medicine, Yamagata University, 2-2-2 Iida-Nishi, Yamagata-shi 990-9585, Japan"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"resources","vocabulary":"credit"}]},{"given":"Masaru","family":"Doshi","sequence":"additional","affiliation":[{"name":"Department of Human Physiology and Pathology, Faculty of Pharmaceutical Sciences, Teikyo University, 2-11-1 Kaga, Itabashi-ku 173-8605, Japan"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3334-4235","authenticated-orcid":false,"given":"Masahiro","family":"Kawahara","sequence":"additional","affiliation":[{"name":"Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, 1-1-20 Shin-machi, Nishitokyo-shi 202-8585, Japan"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"resources","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6574-6055","authenticated-orcid":false,"given":"Akira","family":"Ishii","sequence":"additional","affiliation":[{"name":"Department of Legal Medicine and Bioethics, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya-shi 466-8550, Japan"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"funding-acquisition","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,3]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"8332","DOI":"10.1007\/s12035-016-0313-5","article-title":"Endocannabinoid system: The direct and indirect involvement in the memory and learning processes\u2014A short review","volume":"54","author":"Dzik","year":"2017","journal-title":"Mol. Neurobiol."},{"key":"ref_2","first-page":"607","article-title":"Review of the endocannabinoid system","volume":"6","author":"Lu","year":"2021","journal-title":"Biol. Psychiatry Cogn. Neurosci. Neuroimaging"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"605","DOI":"10.1016\/S0026-895X(25)09876-1","article-title":"Determination and characterization of a cannabinoid receptor in rat brain","volume":"34","author":"Devane","year":"1988","journal-title":"Mol. Pharmacol."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Zou, S., and Kumar, U. (2018). Cannabinoid receptors and the endocannabinoid system: Signaling and function in the central nervous system. Int. J. Mol. Sci., 19.","DOI":"10.3390\/ijms19030833"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"91","DOI":"10.4103\/sni.sni_45_18","article-title":"Review of the neurological benefits of phytocannabinoids","volume":"9","author":"Maroon","year":"2018","journal-title":"Surg. Neurol. Int."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"S13","DOI":"10.1038\/sj.ijo.0803272","article-title":"The pharmacology of cannabinoid receptors and their ligands: An overview","volume":"30","author":"Pertwee","year":"2006","journal-title":"Int. J. Obes."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"277","DOI":"10.1016\/j.tips.2015.02.008","article-title":"Endocannabinoid signaling at the periphery: 50 years after THC","volume":"36","author":"Maccarrone","year":"2015","journal-title":"Trends Pharmacol. Sci."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1038\/s41582-019-0284-z","article-title":"Cannabinoids and the expanded endocannabinoid system in neurological disorders","volume":"16","author":"Cristino","year":"2020","journal-title":"Nat. Rev. Neurol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"516","DOI":"10.1016\/j.biopsych.2015.07.028","article-title":"An introduction to the endogenous cannabinoid system","volume":"79","author":"Lu","year":"2016","journal-title":"Biol. Psychiatry"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1344","DOI":"10.1111\/j.1476-5381.2011.01238.x","article-title":"Taming, THC: Potential cannabis synergy and phytocannabinoid\u2013terpenoid entourage effects","volume":"163","author":"Russo","year":"2011","journal-title":"Br. J. Pharmacol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1111\/j.1527-3458.2001.tb00192.x","article-title":"HU 210: A potent tool for investigations of the cannabinoid system","volume":"7","author":"Ottani","year":"2001","journal-title":"CNS Drug Rev."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"327","DOI":"10.2165\/00003088-200342040-00003","article-title":"Pharmacokinetics and pharmacodynamics of cannabinoids","volume":"42","author":"Grotenhermen","year":"2003","journal-title":"Clin. Pharmacokinet."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"16865","DOI":"10.1074\/jbc.273.27.16865","article-title":"Cannabinoid receptor agonist efficacy for stimulating [35S]GTP\u03b3S binding to rat cerebellar membranes correlates with agonist-induced decreases in GDP affinity","volume":"273","author":"Breivogel","year":"1998","journal-title":"J. Biol. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1016\/j.mcn.2004.09.004","article-title":"Neuroprotective effects of the synthetic cannabinoid HU-210 in primary cortical neurons are mediated by phosphatidylinositol 3-kinase\/AKT signaling","volume":"28","author":"Pinteaux","year":"2005","journal-title":"Mol. Cell. Neurosci."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"688","DOI":"10.1038\/sj.bjp.0707273","article-title":"The synthetic cannabinoid HU210 induces spatial memory deficits and suppresses hippocampal firing rate in rats","volume":"151","author":"Robinson","year":"2007","journal-title":"Br. J. Pharmacol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"136953","DOI":"10.1016\/j.neulet.2022.136953","article-title":"Hippocampal CB1 receptor mediates antidepressant-like effect of synthetic cannabinoid HU210 in acute despair reaction model in mice","volume":"792","author":"Fang","year":"2023","journal-title":"Neurosci. Lett."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Eicher, T., Kinnebrew, G., Patt, A., Spencer, K., Ying, K., Ma, Q., Machiraju, R., and Math\u00e9, A.E.A. (2020). Metabolomics and multi-omics integration: A survey of computational methods and resources. Metabolites, 10.","DOI":"10.3390\/metabo10050202"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"170","DOI":"10.1039\/D0MO00041H","article-title":"Multi-omics data integration considerations and study design for biological systems and disease","volume":"17","author":"Graw","year":"2021","journal-title":"Mol. Omics"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"3735","DOI":"10.1016\/j.csbj.2021.06.030","article-title":"Integration strategies of multi-omics data for machine learning analysis","volume":"19","author":"Picard","year":"2021","journal-title":"Comput. Struct. Biotechnol. J."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Sussulini, A., Xia, J., and Ore\u0161i\u010d, M. (2022). Editorial: Multi-omics: Trends and applications in clinical research. Front. Mol. Biosci., 9.","DOI":"10.3389\/fmolb.2022.994239"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"303","DOI":"10.1016\/j.ceca.2009.08.007","article-title":"L-type channel inhibition by CB1 cannabinoid receptors is mediated by PTX-sensitive G proteins and cAMP\/PKA in GT1-7 hypothalamic neurons","volume":"46","author":"Hoddah","year":"2009","journal-title":"Cell Calcium"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"138","DOI":"10.1016\/j.ygcen.2017.01.025","article-title":"How is GnRH regulated in GnRH-producing neurons? Studies using GT1-7 cells as a GnRH-producing cell model","volume":"247","author":"Kanasaki","year":"2017","journal-title":"Gen. Comp. Endocrinol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1093\/bioinformatics\/btn577","article-title":"A novel signaling pathway impact analysis","volume":"25","author":"Tarca","year":"2009","journal-title":"Bioinformatics"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"8268","DOI":"10.1073\/pnas.95.14.8268","article-title":"Cannabidiol and (\u2212)\u03949-tetrahydrocannabinol are neuroprotective antioxidants","volume":"95","author":"Hampson","year":"1998","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"5322","DOI":"10.1523\/JNEUROSCI.18-14-05322.1998","article-title":"Hippocampal neurotoxicity of \u03949-tetrahydrocannabinol","volume":"18","author":"Chan","year":"1998","journal-title":"J. Neurosci."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1016\/j.taap.2013.10.028","article-title":"Cytotoxicity of synthetic cannabinoids on primary neuronal cells of the forebrain: The involvement of cannabinoid CB1 receptors and apoptotic cell death","volume":"274","author":"Tomiyama","year":"2014","journal-title":"Toxicol. Appl. Pharmacol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"102838","DOI":"10.1016\/j.jbc.2022.102838","article-title":"Regulation and function of the mammalian tricarboxylic acid cycle","volume":"299","author":"Arnold","year":"2023","journal-title":"J. Biol. Chem."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Cantu, D., Schaack, J., and Patel, M. (2009). Oxidative inactivation of mitochondrial aconitase results in iron and H2O2-mediated neurotoxicity in rat primary mesencephalic cultures. PLoS ONE, 4.","DOI":"10.1371\/journal.pone.0007095"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Guan, R., Xue, Z., Huang, K., Zhao, Y., He, G., Dai, Y., Liang, M., Wen, Y., Ye, X., and Liu, P. (2025). \u03b1-Ketoglutarate Attenuates Oxidative Stress-Induced Neuronal Aging via Modulation of the mTOR Pathway. Pharmaceuticals, 18.","DOI":"10.3390\/ph18081080"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1007\/s00005-016-0406-x","article-title":"Alpha-ketoglutarate as a molecule with pleiotropic activity: Well-known and novel possibilities of therapeutic use","volume":"65","year":"2017","journal-title":"Arch. Immunol. Ther. Exp."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1247","DOI":"10.1007\/s00439-019-02063-z","article-title":"MDH1 deficiency is a metabolic disorder of the malate\u2013aspartate shuttle associated with early onset severe encephalopathy","volume":"138","author":"Broeks","year":"2019","journal-title":"Hum. Genet."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"399","DOI":"10.1016\/j.bcp.2005.10.011","article-title":"Neuronal and astrocytic shuttle mechanisms for cytosolic\u2013mitochondrial transfer of reducing equivalents: Current evidence and pharmacological tools","volume":"71","author":"McKenna","year":"2006","journal-title":"Biochem. Pharmacol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"323706","DOI":"10.1155\/2015\/323706","article-title":"Tetrahydrocannabinol induces brain mitochondrial respiratory chain dysfunction and increases oxidative stress: A potential mechanism involved in cannabis-related stroke","volume":"2015","author":"Wolff","year":"2015","journal-title":"BioMed Res. Int."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Batsios, G., Taglang, C., Cao, P., Gillespie, A.M., Najac, C., Subramani, E., Wilson, D.M., Flavell, R.R., Larson, P.E.Z., and Ronen, S.M. (2021). Imaging 6-phosphogluconolactonase activity in brain tumors in vivo using hyperpolarized \u03b4-[1\u201313C]gluconolactone. Front. Oncol., 11.","DOI":"10.3389\/fonc.2021.589570"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"857","DOI":"10.1016\/j.molcel.2019.09.007","article-title":"\u03b3-6-Phosphogluconolactone, a Byproduct of the Oxidative Pentose Phosphate Pathway, Contributes to AMPK Activation through Inhibition of PP2A","volume":"76","author":"Gao","year":"2019","journal-title":"Mol. Cell"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"e2100400","DOI":"10.1002\/mnfr.202100400","article-title":"Is galactose a hormetic sugar? An exploratory study of the rat hippocampal redox regulatory network","volume":"65","author":"Homolak","year":"2021","journal-title":"Mol. Nutr. Food Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"112335","DOI":"10.1016\/j.celrep.2023.112335","article-title":"Neurons require glucose uptake and glycolysis in vivo","volume":"42","author":"Li","year":"2023","journal-title":"Cell Rep."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1016\/j.neuint.2017.03.008","article-title":"Catabolism of GABA, succinic semialdehyde or gamma-hydroxybutyrate through the GABA shunt impair mitochondrial substrate-level phosphorylation","volume":"109","author":"Ravasz","year":"2017","journal-title":"Neurochem. Int."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"547","DOI":"10.1016\/S0026-895X(25)15366-2","article-title":"Delta 9-tetrahydrocannabinol inhibits arachidonic acid acylation of phospholipids and triacylglycerols in guinea pig cerebral cortex slices","volume":"40","author":"Reichman","year":"1991","journal-title":"Mol. Pharmacol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"e54812","DOI":"10.7554\/eLife.54812","article-title":"CB1-receptor-mediated inhibitory LTD triggers presynaptic remodeling via protein synthesis and ubiquitination","volume":"9","author":"Monday","year":"2020","journal-title":"eLife"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"22090","DOI":"10.1074\/jbc.M114.566927","article-title":"Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) is a pro-survival, endoplasmic reticulum (ER) stress response gene involved in tumor cell adaptation to nutrient availability","volume":"289","author":"Novellasdemunt","year":"2014","journal-title":"J. Biol. Chem."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1016\/j.freeradbiomed.2021.09.007","article-title":"PCK2 opposes mitochondrial respiration and maintains the redox balance in starved lung cancer cells","volume":"176","author":"Bluemel","year":"2021","journal-title":"Free Radic. Biol. Med."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"577","DOI":"10.1016\/j.redox.2016.12.029","article-title":"Cannabidiol attenuates OGD\/R-induced damage by enhancing mitochondrial bioenergetics and modulating glucose metabolism via pentose-phosphate pathway in hippocampal neurons","volume":"11","author":"Sun","year":"2017","journal-title":"Redox Biol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"274","DOI":"10.1016\/j.cell.2012.03.017","article-title":"MTOR signaling in growth control and disease","volume":"149","author":"Laplante","year":"2012","journal-title":"Cell"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"463","DOI":"10.1083\/jcb.200305129","article-title":"Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain","volume":"163","author":"Bisogno","year":"2003","journal-title":"J. Cell Biol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1186\/s13041-016-0257-8","article-title":"Acute upregulation of neuronal mitochondrial Type-1 cannabinoid receptor and it\u2019s role in metabolic defects and neuronal apoptosis after TBI","volume":"9","author":"Xu","year":"2016","journal-title":"Mol. Brain"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"812","DOI":"10.1212\/01.wnl.0000176753.45410.8b","article-title":"Randomized, controlled trial of cannabis-based medicine in central pain in multiple sclerosis","volume":"65","author":"Rog","year":"2005","journal-title":"Neurology"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1122","DOI":"10.1111\/j.1468-1331.2010.03328.x","article-title":"A randomized, double-blind, placebo-controlled, parallel-group, enriched-design study of nabiximols* (Sativex(\u00ae)), as add-on therapy, in subjects with refractory spasticity caused by multiple sclerosis","volume":"18","author":"Novotna","year":"2011","journal-title":"Eur. J. Neurol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"210","DOI":"10.1016\/j.pain.2007.08.028","article-title":"Sativex successfully treats neuropathic pain characterised by allodynia: A randomised, double-blind, placebo-controlled clinical trial","volume":"133","author":"Nurmikko","year":"2007","journal-title":"Pain"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"861","DOI":"10.1093\/pm\/pnab050","article-title":"Nabiximols in chronic neuropathic pain: A meta-analysis of randomized placebo-controlled trials","volume":"22","author":"Dykukha","year":"2021","journal-title":"Pain Med."}],"container-title":["Metabolites"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-1989\/16\/9\/645\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T10:33:07Z","timestamp":1788431587000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-1989\/16\/9\/645"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,3]]},"references-count":50,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["metabo16090645"],"URL":"https:\/\/doi.org\/10.3390\/metabo16090645","relation":{},"ISSN":["2218-1989"],"issn-type":[{"value":"2218-1989","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,3]]}}}