|Molecular Vision 2005;
Received 31 January 2005 | Accepted 27 April 2005 | Published 6 May 2005
Expression of the p53 family of proteins in central and peripheral human corneal endothelial cells
Amanda C. Paull,
David R. Whikehart
Department of Vision Sciences, Vision Science Research Center, School of Optometry, The University of Alabama at Birmingham, Birmingham, AL
Correspondence to: David R. Whikehart, PhD, Department of Vision Sciences, School of Optometry, The University of Alabama at Birmingham, 1716 University Boulevard, Birmingham, AL, 35294; email: firstname.lastname@example.org
Purpose: To determine the protein and mRNA expression of p53, p63, and p73 in central and peripheral human corneal endothelial cells. Since these proteins are known to be involved in the regulation of cell division, this study seeks information about their influence in regulating cell proliferation in the human corneal endothelium.
Methods: Human donor corneas were separated into central and peripheral sections. The endothelial tissue from these samples was dissected and samples were analyzed for mRNA transcription of p53, transactivating p63 (TAp63), delta N p63 (ΔNp63), transactivating p73 (TAp73), and delta N p73 (ΔNp73) via the reverse transcriptase-polymerase chain reaction (RT-PCR). Additional samples were analyzed for p53, p63, and p73 protein expression via SDS-PAGE, western blotting, and immunodetection. Frozen corneal sections were immunostained for p53 and analyzed via fluorescence microscopy.
Results: p53 and TAp63 mRNA and protein expression were detected in central and peripheral human corneal endothelium. p53 and TAp63 protein expression were greater in central than in peripheral tissue. ΔNp63 and all isoforms of p73 were not detected in either central or peripheral corneal endothelium.
Conclusions: p53 is expressed in both peripheral and central human corneal endothelium, although it is more highly expressed in the central endothelium. Similarly, TAp63 is more highly expressed in central rather than in peripheral endothelium. This suggests that the peripheral endothelium may have more potential for cell division than the central endothelium. ΔNp63, a stem cell marker, was not detected in the corneal endothelium. Neither the TAp73 nor the ΔNp73 isoforms were detected in either central or peripheral human corneal endothelium.
At least 40,000 corneal transplants are performed each year in the United States (Eye Bank Association of America). The demand for corneas is so marked that the development of artificial corneas for human transplant is justified. In order to develop functional artificial corneal tissue, a thorough understanding of the human cornea is needed. In the human cornea, the endothelium is the most important layer for the maintenance of osmotic balance and the transparency of corneal tissue . This monolayer of cells appears to be inhibited from dividing in the adult, although these cells have limited proliferation in vitro in response to growth promoting factors and wounding [1-3]. To understand this phenomenon better, the regenerative properties of human endothelial cells have been studied extensively [1,4-6]. In this study, we analyzed the expression of three proteins (the p53 family) involved in cell division in the peripheral and central corneal endothelium. Since stem cells have been found to be isolated at the peripheral/limbal region of the epithelium [7-9], this study also investigates cell division potential in central and peripheral endothelial tissue.
p53 is an important protein involved in the negative regulation of cell division. It is well known that this protein is a common target for genetic mutations in rapidly dividing cancer cells . p53 expression is usually not detected in dividing tissues such as skin, kidney, lung, stomach, or breast [11,12]. However, p53 expression has previously been detected in the cytoplasm of normal human corneal endothelial cells , and these cells have a limited potential for cell division.
In 1997, two other p53 related proteins were identified (p63 and p73) [14,15]. Each of these proteins possess several isoforms with characteristics that either mimic or antagonize the role of p53. Both p63 and p73 contain two classes of isoforms; those containing a transactivating domain (TAp63 and TAp73) and those lacking the transactivating domain (ΔNp63 and ΔNp73). A simplified model attributes transcriptionally active properties to the TA proteins, whereas the ΔN proteins possess dominant negative functions toward themselves and p53 . Therefore, the TA isoforms of p63 and p73 may play a role in negatively regulating cell division like p53, while the ΔN isoforms of p63 and p73 may play a role in positively regulating cell division. The latter may even contribute to immortalization of cell tissue . The interactions between these p53 family members have been thoroughly reviewed by Moll and Slade , and the implications for the results of this study are explained further in the Discussion section.
This investigation showed the expression patterns of p53 protein family members in the reluctantly dividing cells of the human corneal endothelium. Although the corneal endothelium has historically been thought to be nondividing, recent data suggests that endothelial cells may be induced to divide by certain factors [1-3]. Studies have shown that there is a higher cell density in the peripheral corneal endothelium than in the central endothelium . Recent studies have provided evidence which suggests that there may be cell division in the peripheral endothelium . In order to further indicate signs of cell division in the periphery of the corneal endothelium, the relative expressions of the p53 family of proteins in the periphery were compared with those in the central endothelium. Relatively smaller expressions of these proteins in the periphery would be an indicator of cell division in the periphery.
Dissection of human corneas
All research procedures involving human corneal tissues carried out in this project were approved by the Institutional Review Board of the University of Alabama at Birmingham. Human corneas were obtained from the Alabama and Georgia Eye Banks. They were removed from donor buttons within 14 days postmortem after being stored in Optisol (Chiron Opthalmics, Irvine, CA) at 4 °C. Central human corneal endothelium ("central tissue") was collected from the button cut by a 8 mm (diameter) circular trephine. Peripheral endothelium ("peripheral tissue") was collected from the button cut by a 12 mm (diameter) circular trephine after the 8 mm button had been removed. The latter area was considered to have the corneolimbal junction as its outermost border as defined by Hogan et al. . The endothelial tissues within these separate sections were carefully stripped away from their underlying stromas. The tissue was then either frozen at -80 °C or processed immediately for either protein or RNA analysis.
Central and peripheral endothelial tissue from 2-4 corneas were separately incubated in 300 μl of 1X CHAPS buffer (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, pH 7.5) on ice for approximately 2.5 h. For the pooled donor samples, the average age was determined. All samples contained donors whose ages were within 10 years of each other. Samples were spun at 12,000x g for 20 min and the supernatents were frozen at -80 °C. For p53 and p73, the positive control was human embryonal kidney cell line 293 (BD Pharmingen, San Diego, CA). The positive control for p53 and p63 was human cervical cell line ME180 (American Type Culture Connection, Manassas, VA). Positive control cell lines were separated into aliquots and stored at -80 °C.
Protein expression was detected by a standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) western blotting protocol . Each sample contained an equal amount of protein as determined by a dye binding assay (BCA, bicinchroninic acid; Pierce Chemical Co., Rockford, IL). Gels were also stained for proteins to display equal loading per lane using Coomassie blue (CB). Proteins were transferred to a nitrocellulose membrane using a semidry electroblotting method [19,20]. Table 1 lists the primary antibodies that were used. All antibodies were diluted in phosphate buffered saline containing 0.05% Tween (PBS-T).
Proteins were immunodetected using an enhanced chemiluminescence detection system (ECL detection system; Amersham Biosciences, Piscataway, NJ) on Kodak X-ray film. The chemiluminescent material was analyzed for semi-quantitative differences in protein expression with a computerized densitometer (Eagle Eye II; Stratagene, La Jolla, CA).
Samples of central and peripheral human corneal endothelial tissue were obtained as described above. Total RNA from two corneas of the same donor was extracted using the RNABee method  (Tel-Test, Inc., Friendswood, TX). Samples were redissolved in 20 μl of RNase free deionized water, and the concentration of total RNA was determined by UV spectrophotometry. Samples were stored at -80 °C. Subsequently, equal amounts of total RNA (300-500 ng) were reverse transcribed into cDNA using random primers (SuperScript First Strand Synthesis for RT-PCR; Invitrogen, Carlsbad, CA). Primer sequences were obtained from previously published studies as summarized in Table 2. PCR was performed in a 50 μl reaction mixture using Titanium TaqDNA polymerase (BD Biosciences, Palo Alto, CA). The housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was detected to confirm equal loading of RNA. Reactions using water instead of reverse transcriptase or samples were used as controls to confirm the absence of contaminants. The PCR conditions were followed as described previously for p53 and GAPDH , p63 , and p73 .
Amplification products were analyzed by gel electrophoresis. RNA analysis was performed on seven different samples in three different experiments.
The immunofluorescence of p53 was detected in 10 sections of one corneal sample. The cornea was frozen in Tissue-Tek optical cutting temperature medium (Miles Inc., Diagnostic Division, Elkhart, IN). Transverse corneal sections (10 μm) were applied to slides and air dried. Slides were fixed in ethanol (70% ethanol in 50 mM glycine) for 12 h at -20 °C. Immunostaining was performed according to a previously described method . The primary antibody for p53 (see Table 1) was diluted 1:500 in phosphate buffered saline (PBS). The secondary antibody was a mouse anti-goat antibody conjugated to fluorescein (sc-2356; Santa Cruz Biotechnology, Santa Cruz, CA). It was diluted 1:500 in PBS. The samples were preserved in two drops of Vectashield Mounting Medium containing 1.5 μg/ml of 4'6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA), and covered with a coverslip. The slides were viewed using fluorescence microscopy (ApoCam, AxioVision 4.2, Zeiss, Jena, Germany).
The analysis of mRNA from human corneal endothelial cells indicated that p53 and TAp63 transcripts are present in both peripheral and central endothelial cells. However, transcripts for ΔNp63 and all isoforms of p73 were not detected in peripheral or central corneal endothelial cells (Figure 1, Figure 2).
Human corneal endothelial cells expressed both p53 and TAp63; expression was greater in the central endothelium than in the peripheral endothelium (Figure 2). The data for p53 reflect experiments conducted on 16 different samples and the data for p63 reflect experiments conducted on 6 different samples.
Densitometry analysis of the antibody bands was conducted to compare the average density between central and peripheral bands. The central density of each sample was divided by the peripheral density of each sample to obtain a relative ratio of protein expression between the central and peripheral tissue. Thus, a ratio of 2 indicates that the central density was twice the peripheral density. For p53, the central bands were approximately 1.9 times as dense as the peripheral bands. For TAp63, central bands were approximately 2.0 times as dense as the peripheral bands. As shown in Figure 3, the ratio did not vary significantly with age.
The p73 isoforms were not detectable in human endothelial cells in the central and peripheral regions (Figure 1). Both mRNA detection and protein detection was negative for all isoforms of p73. The p73 results were obtained from 13 different samples.
Staining for p53 was found to be higher in the central endothelium than in the peripheral endothelium (Figure 4). Immunofluorescence for p63 and p73 was not performed. Previous findings with western blots showed that the commercially available p63 and p73 antibodies had strong cross-reactivity to p53. Therefore, the results would not be as reliable as the data interpreted from the western blots and PCR experiments.
p53 inhibits cell division primarily through a p21 pathway, involving proteins such as the cyclinD/CDK4 complex; the retinoblastoma protein (Rb), and the E2F protein [25,26]. Rb and E2F remain bound together as a result of cyclin dependent kinase inhibition. This blocks promotion of the cell cycle from the G1 to the S phase. p53 not only down regulates cell division during the normal process of aging and senescence, but it is also involved in a response to DNA damage [27,28] and telomere shortening .
Regarding other members of the p53 family, the p63 gene encodes 6 isoforms which migrate on gels in sizes from 32-90 kDa [21,30]. Two main types of isoforms have been identified; the transactivating (TA) isoforms and the ΔN isoforms. The TA isoforms contain a transactivating domain that is homologous to the transactivating domain of p53 . The ΔN isoforms lack the transactivating domain, but they can still bind to p53 consensus sites and act as dominant inhibitors . It has been suggested that ΔNp63 isoforms are involved in positively regulating cell division, whereas TAp63 isoforms are involved in negatively regulating cell division [16,21]. The ΔN isoforms of p63 may also serve as stem cell markers in a variety of epithelial cell types, including endometrium , cervix , breast , prostate , and in stratified squamous epithelium [21,34]. For example, ΔNp63α is expressed in epithelial stem cells, while TAp63α is expressed in fully differentiated cell types. Intermediate differentiation stages (transient amplifying cells) associate with expression of both types of isoforms . Accordingly, p63 has been shown to play a crucial role in epithelial tissue development [36,37] and in the growth and development of epithelial tumors [30,38-40]. It is also noted that p63 isoform expression is altered during cellular senescence and that the transcription of ΔNp63 is repressed by p53 . All of this suggests that alterations to the isoform expression patterns of p63 positively or negatively regulate cellular growth and division [35,42]. In this study, we found that TAp63 was more upregulated in the central endothelium than in the peripheral endothelium. This would, thereby, contribute to the suppression of cell division in central endothelial tissue.
The p73 gene also encodes six different isoforms which range in size from 47.5 to 80 kDa [22,43]. Like p63, the p73 isoforms consist of two types, the TA isoforms and the ΔN isoforms. The TA isforms can induce apoptosis and even transactivate many p53 target genes . In contrast, the ΔNp73 isoforms act as dominant negative inhibitors and can even promote cell immortalization . However, in this investigation no isoform of p73 was found in endothelial tissues. In fact, p73 expression may not always be related to p53 expression levels as p73 has properties independent of p53 .
Our data show that both p53 and TAp63 were found to be more highly expressed in the central than in the peripheral human corneal endothelium. Since these proteins are involved in negatively regulating cell division, this supports previous findings that there is increased potential for cellular proliferation in the peripheral rather than in the central cornea (unpublished and ).
We have also shown that ΔNp63, a stem cell marker, is not expressed in the human corneal endothelium. Although stem cells have been demonstrated to reside in the peripheral and limbal region of the corneal epithelium [7-9], our experiments were not able to detect stem cells in the periphery of the corneal endothelium based on the absence of this marker. Moreover, other laboratory results  suggest that endothelial stem cells may be present in the posterior limbus.
In summary, the division and survival of corneal endothelial cells may be related to the expression of some p53 family members. Our data support previous studies that there is a greater potential for cell proliferation in the periphery of the corneal endothelium than in the central corneal endothelium. This data should be useful for determining the growth potential and characteristics of human corneal endothelial cells in the development of artificial corneas.
Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology; Ft. Lauderdale, FL; May 2003. Supported by grants from the National Eye Institute: EY13994 and the Eye Sight Foundation of Alabama. Amanda C. Paull was supported by NEI Training Grant: T35-EY07084. The authors thank Marci Wright and Kathy Mishler for their excellent technical assistance in this project.
1. Joyce NC. Proliferative capacity of the corneal endothelium. Prog Retin Eye Res 2003; 22:359-89.
2. Senoo T, Joyce NC. Cell cycle kinetics in corneal endothelium from old and young donors. Invest Ophthalmol Vis Sci 2000; 41:660-7.
3. Zhu C, Joyce NC. Proliferative response of corneal endothelial cells from young and older donors. Invest Ophthalmol Vis Sci 2004; 45:1743-51.
4. Engelmann K, Sobottka Ventura A, Drexler D, Staude HJ. A sensitive method for testing the quality of organ culture media and of individual medium components in a cornea bank. Graefes Arch Clin Exp Ophthalmol 1998; 236:312-9.
5. Bednarz J, Rodokanaki-von Schrenck A, Engelmann K. Different characteristics of endothelial cells from central and peripheral human cornea in primary culture and after subculture. In Vitro Cell Dev Biol Anim 1998; 34:149-53.
6. Griffith M, Osborne R, Munger R, Xiong X, Doillon CJ, Laycock NL, Hakim M, Song Y, Watsky MA. Functional human corneal equivalents constructed from cell lines. Science 1999; 286:2169-72.
7. Schermer A, Galvin S, Sun TT. Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. J Cell Biol 1986; 103:49-62.
8. Cotsarelis G, Cheng SZ, Dong G, Sun TT, Lavker RM. Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells. Cell 1989; 57:201-9.
9. Chen Z, de Paiva CS, Luo L, Kretzer FL, Pflugfelder SC, Li DQ. Characterization of putative stem cell phenotype in human limbal epithelia. Stem Cells 2004; 22:355-66.
10. Ko LJ, Prives C. p53: puzzle and paradigm. Genes Dev 1996; 10:1054-72.
11. Vojtesek B, Bartek J, Midgley CA, Lane DP. An immunochemical analysis of the human nuclear phosphoprotein p53. New monoclonal antibodies and epitope mapping using recombinant p53. J Immunol Methods 1992; 151:237-44.
12. Jacquemier J, Moles JP, Penault-Llorca F, Adelaide J, Torrente M, Viens P, Birnbaum D, Theillet C. p53 immunohistochemical analysis in breast cancer with four monoclonal antibodies: comparison of staining and PCR-SSCP results. Br J Cancer 1994; 69:846-52.
13. Joyce NC, Navon SE, Roy S, Zieske JD. Expression of cell cycle-associated proteins in human and rabbit corneal endothelium in situ. Invest Ophthalmol Vis Sci 1996; 37:1566-75.
14. Yang A, Kaghad M, Caput D, McKeon F. On the shoulders of giants: p63, p73 and the rise of p53. Trends Genet 2002; 18:90-5.
15. Kaghad M, Bonnet H, Yang A, Creancier L, Biscan JC, Valent A, Minty A, Chalon P, Lelias JM, Dumont X, Ferrara P, McKeon F, Caput D. Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers. Cell 1997; 90:809-19.
16. Moll UM, Slade N. p63 and p73: roles in development and tumor formation. Mol Cancer Res 2004; 2:371-86.
17. Amann J, Holley GP, Lee SB, Edelhauser HF. Increased endothelial cell density in the paracentral and peripheral regions of the human cornea. Am J Ophthalmol 2003; 135:584-90.
18. Hogan MJ, Alvarado JA, Weddell JE. Histology of the human eye; an atlas and textbook. Philadelphia: Saunders; 1971.
19. Wu W, Welsh MJ, Kaufman PB, Zhang HH. Methods in Gene Biotechnology. New York: CRC Press; 1997. p. 241-258.
20. Wang MS, Pang JS, Selsted ME. Semidry electroblotting of peptides and proteins from acid-urea polyacrylamide gels. Anal Biochem 1997; 253:225-30.
21. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162:156-9.
22. Kaneko Y, Tsukamoto A, Korokawa K. Co-ordinate expression of c-fos, p53 and cytokeratin genes during the alteration of growth of human hepatoma cells. mRNA levels measured by reverse transcription and polymerase chain reaction. Cancer Lett 1992; 66:155-64.
23. Yang A, Kaghad M, Wang Y, Gillett E, Fleming MD, Dotsch V, Andrews NC, Caput D, McKeon F. p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. Mol Cell 1998; 2:305-16.
24. Nakagawa T, Takahashi M, Ozaki T, Watanabe Ki K, Todo S, Mizuguchi H, Hayakawa T, Nakagawara A. Autoinhibitory regulation of p73 by Delta Np73 to modulate cell survival and death through a p73-specific target element within the Delta Np73 promoter. Mol Cell Biol 2002; 22:2575-85.
25. Schafer KA. The cell cycle: a review. Vet Pathol 1998; 35:461-78.
26. Prives C, Hall PA. The p53 pathway. J Pathol 1999; 187:112-26.
27. Vaziri H, Benchimol S. From telomere loss to p53 induction and activation of a DNA-damage pathway at senescence: the telomere loss/DNA damage model of cell aging. Exp Gerontol 1996; 31:295-301.
28. Brown JP, Wei W, Sedivy JM. Bypass of senescence after disruption of p21CIP1/WAF1 gene in normal diploid human fibroblasts. Science 1997; 277:831-4.
29. Allsopp RC, Vaziri H, Patterson C, Goldstein S, Younglai EV, Futcher AB, Greider CW, Harley CB. Telomere length predicts replicative capacity of human fibroblasts. Proc Natl Acad Sci U S A 1992; 89:10114-8.
30. O'Connell JT, Mutter GL, Cviko A, Nucci M, Quade BJ, Kozakewich HP, Neffen E, Sun D, Yang A, McKeon FD, Crum CP. Identification of a basal/reserve cell immunophenotype in benign and neoplastic endometrium: a study with the p53 homologue p63. Gynecol Oncol 2001; 80:30-6.
31. Quade BJ, Yang A, Wang Y, Sun D, Park J, Sheets EE, Cviko A, Federschneider JM, Peters R, McKeon FD, Crum CP. Expression of the p53 homologue p63 in early cervical neoplasia. Gynecol Oncol 2001; 80:24-9.
32. Barbareschi M, Pecciarini L, Cangi MG, Macri E, Rizzo A, Viale G, Doglioni C. p63, a p53 homologue, is a selective nuclear marker of myoepithelial cells of the human breast. Am J Surg Pathol 2001; 25:1054-60.
33. Signoretti S, Waltregny D, Dilks J, Isaac B, Lin D, Garraway L, Yang A, Montironi R, McKeon F, Loda M. p63 is a prostate basal cell marker and is required for prostate development. Am J Pathol 2000; 157:1769-75.
34. Pellegrini G, Dellambra E, Golisano O, Martinelli E, Fantozzi I, Bondanza S, Ponzin D, McKeon F, De Luca M. p63 identifies keratinocyte stem cells. Proc Natl Acad Sci U S A 2001; 98:3156-61.
35. Nylander K, Vojtesek B, Nenutil R, Lindgren B, Roos G, Zhanxiang W, Sjostrom B, Dahlqvist A, Coates PJ. Differential expression of p63 isoforms in normal tissues and neoplastic cells. J Pathol 2002; 198:417-27.
36. Mills AA, Zheng B, Wang XJ, Vogel H, Roop DR, Bradley A. p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 1999; 398:708-13.
37. Yang A, Schweitzer R, Sun D, Kaghad M, Walker N, Bronson RT, Tabin C, Sharpe A, Caput D, Crum C, McKeon F. p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 1999; 398:714-8.
38. Senoo M, Tsuchiya I, Matsumura Y, Mori T, Saito Y, Kato H, Okamoto T, Habu S. Transcriptional dysregulation of the p73L / p63 / p51 / p40 / KET gene in human squamous cell carcinomas: expression of Delta Np73L, a novel dominant-negative isoform, and loss of expression of the potential tumour suppressor p51. Br J Cancer 2001; 84:1235-41.
39. Parsa R, Yang A, McKeon F, Green H. Association of p63 with proliferative potential in normal and neoplastic human keratinocytes. J Invest Dermatol 1999; 113:1099-105.
40. Hall PA, Woodman AC, Campbell SJ, Shepherd NA. Expression of the p53 homologue p63alpha and DeltaNp63alpha in the neoplastic sequence of Barrett's oesophagus: correlation with morphology and p53 protein. Gut 2001; 49:618-23.
41. Waltermann A, Kartasheva NN, Dobbelstein M. Differential regulation of p63 and p73 expression. Oncogene 2003; 22:5686-93.
42. Djelloul S, Tarunina M, Barnouin K, Mackay A, Jat PS. Differential protein expression, DNA binding and interaction with SV40 large tumour antigen implicate the p63-family of proteins in replicative senescence. Oncogene 2002; 21:981-9.
43. De Laurenzi V, Costanzo A, Barcaroli D, Terrinoni A, Falco M, Annicchiarico-Petruzzelli M, Levrero M, Melino G. Two new p73 splice variants, gamma and delta, with different transcriptional activity. J Exp Med 1998; 188:1763-8.
44. Petrenko O, Zaika A, Moll UM. deltaNp73 facilitates cell immortalization and cooperates with oncogenic Ras in cellular transformation in vivo. Mol Cell Biol 2003; 23:5540-55.
45. Blint E, Phillips AC, Kozlov S, Stewart CL, Vousden KH. Induction of p57(KIP2) expression by p73beta. Proc Natl Acad Sci U S A 2002; 99:3529-34.
46. Whikehart, DR, Edelhauser HF. BrdU fluorescent stainingin a niche of posterior limbal cells spreads to the human corneal endothelium following mechanical wounding. ARVO Annual Meeting; 2004 April 25-29; Fort Lauderdale (FL).