The transcriptome and miRNome profiling of glioblastoma tissues and peritumoral regions highlights molecular pathways shared by tumors and surrounding areas and reveals differences between short-term and long-term survivors

Oncotarget

Volumn 6, Issue 26, 2015, Pages 22526-22552

  Fazi, Barbara ^a   Felsani, Armando ^b,c   Grassi, Luigi ^d   Moles, Anna ^b,c   D'Andrea, Daniel ^d   Toschi, Nicola ^a,e,f   Sicari, Daria ^a   De Bonis, Pasquale ^g,h   Anile, Carmelo ^g   Guerrisi, Maria Giovanna ^d   Luca, Emilia ⁱ   Farace, Maria Giulia ^a   Maira, Giulio ^g   Ciafré, Silvia Anna ^a   Mangiola, Annunziato ^g  

^a UNIVERSITY OF ROME TOR VERGATA   (Italy)

^b CNR   (Italy)

^c Genomnia   (Italy)

^d SAPIENZA UNIVERSITY OF ROME   (Italy)

^e MASSACHUSETTS GENERAL HOSPITAL   (United States)

^f HARVARD MEDICAL SCHOOL   (United States)

^g CATHOLIC UNIVERSITY   (Italy)

^h UNIVERSITY HOSPITAL OF FERRARA   (Italy)

ⁱ FONDAZIONE POLICLINICO UNIVERSITARIO A GEMELLI IRCCS   (Italy)

Author keywords

Editing; Glioblastoma; MicroRNA; Peritumoral area; TGF

Indexed keywords

EPIDERMAL GROWTH FACTOR RECEPTOR; MESSENGER RNA; MICRORNA; MICRORNA 106B 5P; MICRORNA 10B 5P; MICRORNA 1248; MICRORNA 1260A; MICRORNA 1260B; MICRORNA 148A 3P; MICRORNA 16 2 3P; MICRORNA 182 5P; MICRORNA 183 5P; MICRORNA 18A 5P; MICRORNA 21 3P; MICRORNA 21 5P; MICRORNA 210 3P; MICRORNA 24 2 5P; MICRORNA 3065 5P; MICRORNA 376C 3P; MICRORNA 454 3P; MICRORNA 503 5P; MICRORNA 92B 5P; MICRORNA 93 5P; MITOGEN ACTIVATED PROTEIN KINASE; NEUROTROPHIN; PHOSPHATIDYLINOSITOL 3 KINASE; PROTEIN KINASE B; TRANSFORMING GROWTH FACTOR BETA; UNCLASSIFIED DRUG; UNINDEXED DRUG; WNT PROTEIN; TRANSCRIPTOME;

ADULT; AGED; ARTICLE; ASTROCYTE; CANCER GROWTH; CANCER LOCALIZATION; CANCER SURVIVAL; CANCER SURVIVOR; CLINICAL ARTICLE; CONTROLLED STUDY; EXTRACELLULAR MATRIX; FEMALE; GENE EXPRESSION PROFILING; GENE FUNCTION; GENE SEQUENCE; GENETIC ANALYSIS; GENETIC TRANSCRIPTION; GENETIC VARIABILITY; GLIOBLASTOMA; HUMAN; HUMAN TISSUE; LONG TERM CANCER SURVIVOR; MALE; MICROGLIA; PROTEIN FUNCTION; QUANTITATIVE ANALYSIS; REAL TIME POLYMERASE CHAIN REACTION; SHORT TERM CANCER SURVIVOR; SIGNAL TRANSDUCTION; WHITE MATTER; BRAIN TUMOR; GENE EXPRESSION REGULATION; GENETICS; METABOLISM; MIDDLE AGED; PATHOLOGY;

ADULT; AGED; BRAIN NEOPLASMS; FEMALE; GENE EXPRESSION PROFILING; GENE EXPRESSION REGULATION, NEOPLASTIC; GLIOBLASTOMA; HUMANS; MALE; MIDDLE AGED; SIGNAL TRANSDUCTION; TRANSCRIPTOME;

EID: 84941247973     PISSN: None     EISSN: 19492553     Source Type: Journal    
DOI: 10.18632/oncotarget.4151     Document Type: Article

References (79)

1. Ohgaki H, Dessen P, Jourde B, Horstmann S, Nishikawa T, Di Patre PL, Burkhard C, Schüler D, Probst-Hensch NM, Maiorka PC, Baeza N, Pisani P, Yonekawa Y, et al. Genetic pathways to glioblastoma: a population-based study. Cancer Res. 2004; 64:6892-6899.
2. Smith JS, Jenkins RB. Genetic alterations in adult diffuse glioma: occurrence, significance, and prognostic implications. Front. Biosci. 2000; 5:D213-231.
3. Burton EC, Lamborn KR, Feuerstein BG, Prados M, Scott J, Forsyth P, Passe S, Jenkins RB, Aldape KD. Genetic aberrations defined by comparative genomic hybridization distinguish long-term from typical survivors of glioblastoma. Cancer Res. 2002; 62:6205-6210.
4. Weller M, Felsberg J, Hartmann C, Berger H, Steinbach JP, Schramm J, Westphal M, Schackert G, Simon M, Tonn JC, Heese O, Krex D, Nikkhah G, et al. Molecular predictors of progression-free and overall survival in patients with newly diagnosed glioblastoma: a prospective translational study of the German Glioma Network. J ClinOncol. 2009; 27:5743-5750.
5. Shinawi T, Hill VK, Krex D, Schackert G, Gentle D, Morris MR, Wei W, Cruickshank G, Maher ER, Latif F. DNA methylation profiles of long-and short-term glioblastoma survivors. Epigenetics. 2013; 8:149-156.
6. Reifenberger G, Weber RG, Riehmer V, Kaulich K, Willscher E, Wirth H, Gietzelt J, Hentschel B, Westphal M, Simon M, Schackert G, Schramm J, Matschke J, et al. Molecular characterization of long-term survivors of glioblastoma using genome-and transcriptome-wide profiling. Int J Cancer. 2014; 135:1822-1831.
7. Bai RY V. Staedtke GJ. Riggins Molecular targeting of glioblastoma: Drug discovery and therapies. Trends Mol. Med. 2011; 17:301-312.
8. Mangiola A, de Bonis P, Maira G, Balducci M, Sica G, Lama G, Lauriola L, Anile C. Invasive tumor cells and prognosis in a selected population of patients with glioblastoma multiforme. Cancer. 2008; 113:841-846.
9. Mangiola A, Saulnier N, De Bonis P, Orteschi D, Sica G, Lama G, Pettorini BL, Sabatino G, Zollino M, Lauriola L, Colabianchi A, Proietti G, Kovacs G, et al. Gene expression profile of glioblastoma peritumoral tissue: an ex vivo study. PLoSOne. 2013; 8:e57145.
10. Gill BJ, Pisapia DJ, Malone HR, Goldstein H, Lei L, Sonabend A, Yun J, Samanamud J, Sims JS, Banu M, Dovas A, Teich AF, Sheth SA, et al. MRI-localized biopsies reveal subtype-specific differences in molecular and cellularcompositionat the margins of glioblastoma. Proc. Natl. Acad. Sci. U S A. 2014; 111:12550-12555.
11. Charles NA, Holland EC, Gilbertson R, Glass R, Kettenmann H. The brain tumor microenvironment. Glia. 2012; 60:502-514.
12. Edelman E, Porrello A, Guinney J, Balakumaran B, Bild A, Febbo PG, Mukherjee S. Analysis of sample set enrichment scores: assaying the enrichment of sets of genes for individual samples in genome-wide expression profiles. Bioinformatics. 2006; 22:e108-116.
13. Verhaak RG, Hoadley KA, Purdom E, Wang V, Qi Y, Wilkerson MD, Miller CR, Ding L, Golub T, Mesirov JP, Alexe G, Lawrence M, O'Kelly M, et al. Cancer Genome Atlas Research Network. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell. 2010; 17:98-110.
14. Kononenko I, Simec E, Robnik-Sikonja M. Overcoming the myopia of inductive learning algorithms with RELIEFF. Applied Intelligence. 1997; 7:39-55.
15. Christiansen VJ, Jackson KW, Lee KN, Downs TD, McKee PA. Targeting inhibition of fibroblast activation protein-α and prolyloligopeptidase activities on cells common to metastatic tumor microenvironments. Neoplasia. 2013; 15:348-358.
16. Myöhänen TT, Tenorio-Laranga J, Jokinen B, Vázquez-Sánchez R, Moreno-Baylach MJ, García-Horsman JA, Männistö PT. Prolyloligopeptidase induces angiogenesis both in vitro and in vivo in a novel regulatory manner. Br J Pharmacol. 2011; 163:1666-1678.
17. Brennen WN, Isaacs JT, Denmeade SR. Rationale behind targeting fibroblast activation protein-expressing carcinoma-associated fibroblasts as a novel chemotherapeutic strategy. Mol Cancer Ther. 2012; 11:257-266.
18. Zito A, Cartelli D, Cappelletti G, Cariboni A, Andrews W, Parnavelas J, Poletti A, Galbiati M. Neuritin 1 promotes neuronal migration. Brain Struct Funct. 2014; 219:105-118.
19. Le Jan S, Le Meur N, Cazes A, Philippe J, Le Cunff M, Léger J, Corvol P, Germain S. Characterization of the expression of the hypoxia-induced genes neuritin, TXNIP and IGFBP3 in cancer. FEBSLett. 2006; 580:3395-3400.
20. Cahoy JD, Emery B, Kaushal A, Foo LC, Zamanian JL, Christopherson KS, Xing Y, Lubischer JL, Krieg PA, Krupenko SA, Thompson WJ, Barres BA. A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J Neurosci. 2008; 28:264-278.
21. Mammoto T, Jiang A, Jiang E, Panigrahy D, Kieran MW, Mammoto A. Role of collagen matrix in tumor angiogenesis and glioblastoma multiforme progression. Am J Pathol. 2013; 183:1293-305.
22. Hong Y, Sang M, Shang C, Xue YX, Liu YH. Quantitative analysis of topoisomerase II alpha and evaluation of its effects on cell proliferation and apoptosis in glioblastoma cancer stem cells. Neurosci Lett. 2012; 518:138-143.
23. Futyma K, Miotła P, Róz˙yńska K, Zdunek M, Semczuk A, Rechberger T, Wojcierowski J. Expression of genes encoding extracellular matrix proteins: A macroarray study. Oncol Rep. 2014; 32:2349-2353.
24. Ellert-Miklaszewska A, Dabrowski M, Lipko M, Sliwa M, Maleszewska M, Kaminska B. Molecular definition of the pro-tumorigenic phenotype of glioma-activated microglia. Glia. 2013; 61:1178-1190.
25. Zamanian JL, Xu L, Foo LC, Nouri N, Zhou L, Giffard RG, Barres BA. Genomic analysis of reactive astrogliosis. J Neurosci. 2012; 32:6391-6410.
26. Xu Y, Zhang H, Lit LC, Grothey A, Athanasiadou M, Kiritsi M, Lombardo Y, Frampton AE, Green AR, Ellis IO, Ali S, Lenz HJ, Thanou M, et al. The kinase LMTK3 promotes invasion in breast cancer through GRB2-mediated induction of integrin β1. Sci Signal. 2014; 7:ra58.
27. Bao L, Loda M, Janmey PA, Stewart R, Anand-Apte B, Zetter BR. Thymosin beta 15: a novel regulator of tumor cell motility upregulated in metastatic prostate cancer. Nat Med. 1996; 2:1322-1328.
28. Räisänen SR, Lehenkari P, Tasanen M, Rahkila P, Härkönen PL, Väänänen HK. Carbonic anhydrase III protects cells from hydrogen peroxide-induced apoptosis. FASEB, J. 1999; 13:513-522.
29. Johnson H, Del Rosario AM, Bryson BD, Schroeder MA, Sarkaria JN, White FM. Molecular characterization of EGFR and EGFRvIII signaling networks in human glioblastoma tumor xenografts. Mol Cell Proteomics. 2012; 11:1724-1740.
30. Oh MJ, Yi SJ, Kim HS, Kim JH, Jeong YH, van Agthoven T, Jhun BH. Functional roles of BCAR3 in the signaling pathways of insulin leading to DNA synthesis, membrane ruffling and GLUT4 translocation. Biochem Biophys Res Commun. 2013; 441:911-916.
31. Brézillon S, Pietraszek K, Maquart FX, Wegrowski Y. Lumican effects in the control of tumour progression and their links with metalloproteinases and integrins. FEBS, J. 2013; 280:2369-2381.
32. Atkinson JC, Rühl M, Becker J, Ackermann R, Schuppan D. Collagen VI regulates normal and transformed mesenchymal cell proliferation in vitro. Exp Cell Res. 1996; 228:283-291.
33. Cheon DJ, Tong Y, Sim MS, Dering J, Berel D, Cui X, Lester J, Beach JA, Tighiouart M, Walts AE, Karlan BY, Orsulic S. A collagen-remodeling gene signature regulated by TGF-β signaling is associated with metastasis and poor survival in serous ovarian cancer. Clin Cancer Res. 2014; 20:711-723.
34. Bergsland M, Werme M, Malewicz M, Perlmann T, Muhr J. The establishment of neuronal properties is controlled by Sox4 and Sox11. Genes Dev. 2006; 20:3475-3486.
35. Ikushima H, Todo T, Ino Y, Takahashi M, Miyazawa K, Miyazono K. Autocrine TGF-beta signaling maintains tumorigenicity of glioma-initiating cells through Sry-related HMG-box factors. Cell Stem Cell. 2009; 5:504-514.
36. Xu XL, Lee TC, Offor N, Cheng C, Liu A, Fang Y, Jhanwar SC, Abramson DH, Cobrinik D. Tumor-associated retinal astrocytes promote retinoblastoma cell proliferation through production of IGFBP-5. Am J Pathol. 2010; 177:424-435.
37. Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources. Nature Protoc. 2009; 4:44-57.
38. Huang DW, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. NucleicAcids Res. 2009; 37:1-13.
39. Barbano R, Palumbo O, Pasculli B, Galasso M, Volinia S, D'Angelo V, Icolaro N, Coco M, Dimitri L, Graziano P, Copetti M, Valori VM, Maiello E, et al. MiRNASignature for Defining Aggressive Phenotype and Prognosis in Gliomas. PLoSOne. 2014; 9:e108950.
40. Ma R, Yan W, Zhang G, Lv H, Liu Z, Fang F, Zhang W, Zhang J, Tao T, You Y, Jiang T, Kang X. Upregulation of miR-196b confers a poor prognosis in glioblastoma patients via inducing a proliferative phenotype. PLoS One. 2012; 7:e38096.
41. Xiao S, Yang Z, Lv R, Zhao J, Wu M, Liao Y, Liu Q. miR-135b contributes to the radioresistance by targeting GSK3β in human glioblastoma multiforme cells. PLoS One. 2014; 9:e108810.
42. Tanaka H, Sasayama T, Tanaka K, Nakamizo S, Nishihara M, Mizukawa K, Kohta M, Koyama J, Miyake S, Taniguchi M, Hosoda K. MicroRNA-183 upregulates HIF-1α by targeting isocitrate dehydrogenase 2 (IDH2) in glioma cells. J Neurooncol. 2013; 111:273-283.
43. Zhao X, He X, Han X, Yu Y, Ye F, Chen Y, Hoang T, Xu X, Mi QS, Xin M, Wang F, Appel B, Lu QR. MicroRNA-mediated control of oligodendrocyte differentiation. Neuron. 2010; 65:612-626.
44. Hermeking H. The miR-34 family in cancer and apoptosis. Cell Death Differ. 2010; 17:193-199.
45. Ciafrè SA, Galardi S, Mangiola A, Ferracin M, Liu CG, Sabatino G, Negrini M, Maira G, Croce CM, Farace MG. Extensive modulation of a set of microRNAs in primary glioblastoma. Biochem Biophys Res Commun. 2005; 334:1351-1358.
46. Brower JV, Clark PA, Lyon W, Kuo JS. MicroRNAs in cancer: glioblastoma and glioblastoma cancer stem cells. Neurochem Int. 2014; 77:68-77.
47. Kim J, Zhang Y, Skalski M, Hayes J, Kefas B, Schiff D, Purow B, Parsons S, Lawler S, Abounader R. microRNA-148a is a prognostic oncomiR that targets MIG6 and BIM to regulate EGFR and apoptosis in glioblastoma. Cancer Res. 2014; 74:1541-1553.
48. Song L, Liu L, Wu Z, Li Y, Ying Z, Lin C, Wu J, Hu B, Cheng SY, Li M. Li J.TGF-β induces miR-182 to sustain NF-κB activation in glioma subsets. J Clin Invest. 2012; 122:3563-3578.
49. Karsy M, Arslan E, Moy F. Current Progress on Understanding MicroRNAs in Glioblastoma Multiforme. Genes Cancer. 2012; 3:3-15.
50. Haapa-Paananen S, Chen P, Hellström K, Kohonen P, Hautaniemi S, Kallioniemi O, Perälä M. Functional profiling of precursor MicroRNAs identifies MicroRNAs essential for glioma proliferation. PLoS One. 2013; 8:e60930.
51. Vlachos IS, Kostoulas N, Vergoulis T, Georgakilas G, Reczko M, Maragkakis M, Paraskevopoulou MD, Prionidis K, Dalamagas T, Hatzigeorgiou AG. DIANA miRPath v.2.0: investigating the combinatorial effect of microRNAs in pathways. Nucleic Acids Res. 2012; 40:W498-504.
52. Slotkin W, Nishikura K. Adenosine-to-inosine RNA editing and human disease. Genome Med. 2013; 5:105.
53. Tomaselli S, Bonamassa B, Alisi A, Nobili V, Locatelli F, Gallo A. ADAR enzyme and miRNA story: a nucleotide that can make the difference. Int J Mol Sci. 2013; 14:22796-22816.
54. Bhat KP, Balasubramaniyan V, Vaillant B, Ezhilarasan R, Hummelink K, Hollingsworth F, Wani K, Heathcock L, James JD, Goodman LD, Conroy S, Long L, Lelic N, et al. Mesenchymal differentiation mediated by NF-κB promotes radiation resistance in glioblastoma. Cancer Cell. 2013; 24:331-346.
55. Lin B, Madan A, Yoon JG, Fang X, Yan X, Kim TK, Hwang D, Hood L, Foltz G. Massively parallel signature sequencing and bioinformatics analysis identifies up-regulation of TGFBI and SOX4 in human glioblastoma. PLoS One. 2010; 5:e10210.
56. Codo P, Weller M, Meister G, Szabo E, Steinle A, Wolter M, Reifenberger G, Roth P. MicroRNA-mediated down-regulation of NKG2D ligands contributes to glioma immune escape. Oncotarget. 2014; 5:7651-7662.
57. Donatelli SS, Zhou JM, Gilvary DL, Eksioglu EA, Chen X, Cress WD, Haura EB, Schabath MB, Coppola D, Wei S. TGF-β-inducible microRNA-183 silences tumor-associated natural killer cells. Proc Natl Acad Sci U S A. 2014; 111:4203-4208.
58. Li Q, Zhang D, Wang Y, Sun P, Hou X, Larner J, Xiong W, Mi J. MiR-21/Smad 7 signaling determines TGF-β1-induced CAF formation. Sci Rep. 2013; 3:2038.
59. Smith AL, Iwanaga R, Drasin DJ, Micalizzi DS, Vartuli RL, Tan AC, Ford HL. The miR-106b-25 cluster targets Smad7, activates TGF-β signaling, and induces EMT and tumor initiating cell characteristics downstream of Six1 in human breast cancer. Oncogene. 2012; 31:5162-5171.
60. Han X, Yan S, Weijie Z, Feng W, Liuxing W, Mengquan L, Qingxia F. Critical role of miR-10b in transforming growth factor-β1-induced epithelial-mesenchymal transition in breast cancer. Cancer Gene Ther. 2014; 21:60-67.
61. Yang Z, Zhang Y, Wang L. A feedback inhibition between miRNA-127 and TGFβ/c-Jun cascade in HCC cell migration via MMP13. PLoS One. 2013; 8:e65256.
62. Klegeris A, Li J, Bammler TK, Jin J, Zhu D, Kashima DT, Pan S, Hashioka S, Maguire J, McGeer PL, Zhang J. Prolylendopeptidase is revealed following SILAC analysis to be a novel mediator of human microglial and THP-1 cell neurotoxicity. Glia. 2008; 56:675-685.
63. Orian-Rousseau V, Sleeman J. CD44 is a multidomain signaling platform that integrates extracellular matrix cues with growth factor and cytokine signals. Adv Cancer Res. 2014; 123:231-254.
64. Pietras A, Katz AM, Ekström EJ, Wee B, Halliday JJ, Pitter KL, Werbeck JL, Amankulor NM, Huse JT, Holland EC. Osteopontin-CD44 signaling in the glioma perivascular niche enhances cancer stem cell phenotypes and promotes aggressive tumor growth. Cell Stem Cell. 2014; 14:357-369.
65. Guo J, Canaff L, Rajadurai C, Fils-Aimé N, Tian J, Dai M, Korah J, Villatoro M, Park M, Ali S, Lebrun JJ. Breast Cancer Anti-Estrogen Resistance-3 inhibits transforming growth factor-β/Smad signaling and associates with favorable breast cancer disease outcomes. Breast Cancer Res. 2014; 16:476.
66. Li X, Truty MA, Kang Y, Chopin-Laly X, Zhang R, Roife D, Chatterjee D, Lin E, Thomas RM, Wang H, Katz MH, Fleming JB. Extracellular lumican inhibits pancreatic cancer cell growth and is associated with prolonged survival after surgery. Clin Cancer Res. 2014; 20:6529-6540.
67. Kawahara Y, Zinshteyn B, Sethupathy P, Iizasa H, Hatzigeorgiou AG, Nishikura K. Redirection of silencing targets by adenosine-to-inosine editing of miRNAs. Science. 2007; 315:1137-1140.
68. Chen CX, Cho DS, Wang Q, Lai F, Carter KC, Nishikura K. A third member of the RNA-specific adenosine deaminase gene family, ADAR3, contains both singleand double-stranded RNA binding domains. RNA. 2000; 6:755-767.
69. Choudhury Y, Tay FC, Lam DH, Sandanaraj E, Tang C, Ang BT, Wang S. Attenuated adenosine-to-inosine editing of microRNA-376a* promotes invasiveness of glioblastoma cells. J Clin Invest. 2012; 122:4059-4076.
70. Hoelzinger DB, Mariani L, Weis J, Woyke T, Berens TJ, McDonough WS, Sloan A, Coons SW, Berens ME. Gene expression profile of glioblastoma multiforme invasive phenotype points to new therapeutic targets. Neoplasia. 2005; 7:7-16.
71. Mark Hall, Eibe Frank, Geoffrey Holmes, Bernhard Pfahringer, Peter Reutemann, Ian H. Witten The WEKA Data Mining Software: An Update; SIGKDD Explorations. 2009; 11:.
72. Griffiths-Jones S. Annotating non-coding RNAs with Rfam. CurrProtoc Bioinformatics. 2005; Apr Chapter 12:Unit 12.5.
73. Robinson MD, Oshlack A. A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol. 2010; 11:R25.
74. Brunet JP, Tamayo P, Golub TR, Mesirov JP. Metagenes and molecular pattern discovery using matrix factorization. Proc Natl Acad Sci U S A. 2004; 101:4164-4169.
75. Gaujoux R, Seoighe C. A flexible R package for nonnegative matrix factorization. BMC Bioinformatics. 2010; 11:367.
76. Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010; 26:139-140.
77. Enright AJ, John B, Gaul U, Tuschl T, Sander C, Marks DS. MicroRNA targets in Drosophila. Genome Biol. 2003; 5:R1.
78. Krek A, Grün D, Poy MN, Wolf R, Rosenberg L, Epstein EJ, MacMenamin P, da Piedade I, Gunsalus KC, Stoffel M, Rajewsky N. Combinatorial microRNA target predictions. Nat Genet. 2005; 37:495-500.
79. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001; 25:402-408.