Abstract
An estimate is made of the frequency of occurrence of nexuses ("gap junctions") in a spectrum of human cervical epithelia, ranging from normal to malignant, since a deficiency of nexuses may be important in abnormal cell-to-cell communication in malignant tissues. The normal cervical epithelium has approximately ten nexuses per cell in the basal layer of proliferating cells and 200 nexuses per cell in the more differentiated intermediate zone. Nexuses are rare between invasive malignant epithelial cells (carcinoma cells). In many areas of cell proliferation near the edge of the tumor mass, fewer than one nexus per cell is present. However, up to four nexuses per cell can be found in some well differentiated regions of invasive carcinoma. Preinvasive malignant epithelia (severe dysplasia and carcinoma-in situ) have as few nexuses as invasive carcinoma. In abnormal but benign epithelia (squamous metaplasia and mild dysplasia), nexuses are abundant. The data indicate that a decrease in number of nexuses correlates with the severity of the morphological alteration in the dysplastic epithelium. Also the deficiency of nexuses in groups of carcinoma cells can occur many cell generations before the development of invasion of the malignant epithelium into the connective tissue. The diminution of nexuses before invasion suggests that a deficiency of nexuses may be one of the important factors in eventually permitting the development of the diffusely infiltrating type of invasion which is characteristic of highly malignant tumors such as squamous carcinomas.
Full Text
The Full Text of this article is available as a PDF (2.8 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- ABERCROMBIE M., AMBROSE E. J. Interference microscope studies of cell contacts in tissue culture. Exp Cell Res. 1958 Oct;15(2):332–345. doi: 10.1016/0014-4827(58)90034-x. [DOI] [PubMed] [Google Scholar]
- ABERCROMBIE M., AMBROSE E. J. The surface properties of cancer cells: a review. Cancer Res. 1962 Jun;22:525–548. [PubMed] [Google Scholar]
- ABERCROMBIE M., HEAYSMAN J. E., KARTHAUSER H. M. Social behaviour of cells in tissue culture. III. Mutual influence of sarcoma cells and fibroblasts. Exp Cell Res. 1957 Oct;13(2):276–291. doi: 10.1016/0014-4827(57)90007-1. [DOI] [PubMed] [Google Scholar]
- ABERCROMBIE M., HEAYSMAN J. E. Observations on the social behaviour of cells in tissue culture. II. Monolayering of fibroblasts. Exp Cell Res. 1954 May;6(2):293–306. doi: 10.1016/0014-4827(54)90176-7. [DOI] [PubMed] [Google Scholar]
- ASHWORTH C. T., LUIBEL F. J., SANDERS E. Epithelium of normal cervix uteri studied with electron microscopy and histochemistry. Am J Obstet Gynecol. 1960 Jun;79:1149–1160. doi: 10.1016/0002-9378(60)90528-7. [DOI] [PubMed] [Google Scholar]
- ASHWORTH C. T., LUIBEL F. J., SANDERS E. The fine structure of nuclei in certain human malignant neoplasms. Am J Clin Pathol. 1960 Jul;34:9–20. doi: 10.1093/ajcp/34.1.9. [DOI] [PubMed] [Google Scholar]
- ASHWORTH C. T., STEMBRIDGE V. A., LUIBEL F. J. Symposium on probable or possible malignant cervical lesions - carcinoma in situ. I. Histology of carcinoma in situ. A study of basement membranes of normal epithelium, carcinoma in situ and invasive carcinoma of uterine cervix utilizing electron microscopy and histochemical methods. Acta Cytol. 1961 Sep-Oct;5:369–384. [PubMed] [Google Scholar]
- Anderson H. C., Kim B., Minkowitz S. Calcifying epithelial odontogenic tumor of Pindborg. An electron microscopic study. Cancer. 1969 Sep;24(3):585–596. doi: 10.1002/1097-0142(196909)24:3<585::aid-cncr2820240325>3.0.co;2-v. [DOI] [PubMed] [Google Scholar]
- Atkin N. B. The use of microspectrophotometry. Obstet Gynecol Surv. 1969 Jul;24(7 Pt 2):794–804. doi: 10.1097/00006254-196907001-00011. [DOI] [PubMed] [Google Scholar]
- BARR L., DEWEY M. M., BERGER W. PROPAGATION OF ACTION POTENTIALS AND THE STRUCTURE OF THE NEXUS IN CARDIAC MUSCLE. J Gen Physiol. 1965 May;48:797–823. doi: 10.1085/jgp.48.5.797. [DOI] [PMC free article] [PubMed] [Google Scholar]
- BERNHARD W. SOME PROBLEMS OF FINE STRUCTURE IN TUMOR CELLS. Prog Exp Tumor Res. 1963;3:1–34. doi: 10.1159/000385959. [DOI] [PubMed] [Google Scholar]
- Barr L., Berger W., Dewey M. M. Electrical transmission at the nexus between smooth muscle cells. J Gen Physiol. 1968 Mar;51(3):347–368. doi: 10.1085/jgp.51.3.347. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Benedetti E. L., Emmelot P. Hexagonal array of subunits in tight junctions separated from isolated rat liver plasma membranes. J Cell Biol. 1968 Jul;38(1):15–24. doi: 10.1083/jcb.38.1.15. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bennington J. L. Cellular kinetics of invasive squamous carcinoma of the human cervix. Cancer Res. 1969 May;29(5):1082–1088. [PubMed] [Google Scholar]
- Bergman R. A. Uterine smooth muscle fibers in castrate and estrogen-treated rats. J Cell Biol. 1968 Mar;36(3):639–648. doi: 10.1083/jcb.36.3.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Branton D. Fracture faces of frozen membranes. Proc Natl Acad Sci U S A. 1966 May;55(5):1048–1056. doi: 10.1073/pnas.55.5.1048. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brightman M. W., Reese T. S. Junctions between intimately apposed cell membranes in the vertebrate brain. J Cell Biol. 1969 Mar;40(3):648–677. doi: 10.1083/jcb.40.3.648. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bullivant S., Ames A., 3rd A simple freeze-fracture replication method for electron microscopy. J Cell Biol. 1966 Jun;29(3):435–447. doi: 10.1083/jcb.29.3.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
- COMAN D. R. Adhesiveness and stickiness: two independent properties of the cell surface. Cancer Res. 1961 Nov;21:1436–1438. [PubMed] [Google Scholar]
- Cobb J. L., Bennett T. A study of nexuses in visceral smooth muscle. J Cell Biol. 1969 Apr;41(1):287–297. doi: 10.1083/jcb.41.1.287. [DOI] [PMC free article] [PubMed] [Google Scholar]
- DEFENDI V., GASIC G. SURFACE MUCOPOLYSACCHARIDES OF POLYOMA VIRUS TRANSFORMED CELLS. J Cell Physiol. 1963 Aug;62:23–32. doi: 10.1002/jcp.1030620105. [DOI] [PubMed] [Google Scholar]
- DEWEY M. M., BARR L. A STUDY OF THE STRUCTURE AND DISTRIBUTION OF THE NEXUS. J Cell Biol. 1964 Dec;23:553–585. doi: 10.1083/jcb.23.3.553. [DOI] [PMC free article] [PubMed] [Google Scholar]
- De Terra N. Cytoplasmic control over the nuclear events of cell reproduction. Int Rev Cytol. 1969;25:1–29. doi: 10.1016/s0074-7696(08)60199-0. [DOI] [PubMed] [Google Scholar]
- Deamer D. W., Branton D. Fracture planes in an ice-bilayer model membrane system. Science. 1967 Nov 3;158(3801):655–657. doi: 10.1126/science.158.3801.655. [DOI] [PubMed] [Google Scholar]
- Dewey M. M., Barr L. Intercellular Connection between Smooth Muscle Cells: the Nexus. Science. 1962 Aug 31;137(3531):670–672. doi: 10.1126/science.137.3531.670-a. [DOI] [PubMed] [Google Scholar]
- Dreifuss J. J., Girardier L. Etude de la propagation de l'excitation dans le ventricule de rat au moyen de solutions hypertoniques. Pflugers Arch Gesamte Physiol Menschen Tiere. 1966;292(1):13–33. [PubMed] [Google Scholar]
- FARQUHAR M. G., PALADE G. E. FUNCTIONAL ORGANIZATION OF AMPHIBIAN SKIN. Proc Natl Acad Sci U S A. 1964 Apr;51:569–577. doi: 10.1073/pnas.51.4.569. [DOI] [PMC free article] [PubMed] [Google Scholar]
- FARQUHAR M. G., PALADE G. E. Junctional complexes in various epithelia. J Cell Biol. 1963 May;17:375–412. doi: 10.1083/jcb.17.2.375. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Farquhar M. G., Palade G. E. Cell junctions in amphibian skin. J Cell Biol. 1965 Jul;26(1):263–291. doi: 10.1083/jcb.26.1.263. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fawcett D. W., McNutt N. S. The ultrastructure of the cat myocardium. I. Ventricular papillary muscle. J Cell Biol. 1969 Jul;42(1):1–45. doi: 10.1083/jcb.42.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fox C. H. Biologic behavior of dysplasia and carcinoma in situ. Am J Obstet Gynecol. 1967 Dec 1;99(7):960–974. doi: 10.1016/0002-9378(67)90251-7. [DOI] [PubMed] [Google Scholar]
- Furshpan E. J., Potter D. D. Low-resistance junctions between cells in embryos and tissue culture. Curr Top Dev Biol. 1968;3:95–127. doi: 10.1016/s0070-2153(08)60352-x. [DOI] [PubMed] [Google Scholar]
- GASIC G., GASIC T. Removal and regeneration of the cell coating in tumour cells. Nature. 1962 Oct 13;196:170–170. doi: 10.1038/196170a0. [DOI] [PubMed] [Google Scholar]
- Goodenough D. A., Revel J. P. A fine structural analysis of intercellular junctions in the mouse liver. J Cell Biol. 1970 May;45(2):272–290. doi: 10.1083/jcb.45.2.272. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Govan A. D., Haines R. M., Langley F. A., Taylor C. W., Woodcock A. S. The histology and cytology of changes in the epithelium of the cervix uteri. J Clin Pathol. 1969 Jul;22(4):383–395. doi: 10.1136/jcp.22.4.383. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Grubb C., Janota I. Squamous differentiation in carcinoma in situ of the cervix uteri. A cyto-histological correlation of malignant intraepithelial lesions with invasive carcinoma. J Clin Pathol. 1967 Jan;20(1):7–14. doi: 10.1136/jcp.20.1.7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hackemann M., Grubb C., Hill K. R. The ultrastructure of normal squamous epithelium of the human cervix uteri. J Ultrastruct Res. 1968 Mar;22(5):443–457. doi: 10.1016/s0022-5320(68)90033-6. [DOI] [PubMed] [Google Scholar]
- Hall J. E., Walton L. Dysplasia of the cervix: a prospective study of 206 cases. Am J Obstet Gynecol. 1968 Mar 1;100(5):662–671. doi: 10.1016/s0002-9378(15)33391-3. [DOI] [PubMed] [Google Scholar]
- Harris J. W., Meyskens F., Patt H. M. Biochemical studies of cytokinetic changes during tumor growth. Cancer Res. 1970 Jul;30(7):1937–1946. [PubMed] [Google Scholar]
- Holmquist N. D., McMahan C. A., Williams O. D. Variability in classification of carcinoma in situ of the uterine cervix. Arch Pathol. 1967 Oct;84(4):334–345. [PubMed] [Google Scholar]
- Hyde A., Blondel B., Matter A., Cheneval J. P., Filloux B., Girardier L. Homo- and heterocellular junctions in cell cultures: an electrophysiological and morphological study. Prog Brain Res. 1969;31:283–311. doi: 10.1016/S0079-6123(08)63247-1. [DOI] [PubMed] [Google Scholar]
- JOHNSON L. D., EASTERDAY C. L., GORE H., HERTIG A. T. THE HISTOGENESIS OF CARCINOMA IN SITU OF THE UTERINE CERVIX; A PRELIMINARY REPORT OF THE ORIGIN OF CARCINOMA IN SITU IN SUBCYLINDRICAL CELL ANAPLASIA. Cancer. 1964 Feb;17:213–229. doi: 10.1002/1097-0142(196402)17:2<213::aid-cncr2820170211>3.0.co;2-v. [DOI] [PubMed] [Google Scholar]
- Jamakosmanović A., Loewenstein W. R. Intercellular communication and tissue growth. 3. Thyroid cancer. J Cell Biol. 1968 Sep;38(3):556–561. doi: 10.1083/jcb.38.3.556. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Johnson L. D., Nickerson R. J., Easterday C. L., Stuart R. S., Hertig A. T. Epidemiologic evidence for the spectrum of change from dysplasia through carcinoma in situ to invasive cancer. Cancer. 1968 Nov;22(5):901–914. doi: 10.1002/1097-0142(196811)22:5<901::aid-cncr2820220502>3.0.co;2-j. [DOI] [PubMed] [Google Scholar]
- Jones H. W., Jr, Davis H. J., Frost J. K., Park I. J., Salimi R., Tseng P. Y., Woodruff J. D. The value of the assay of chromosomes in the diagnosis of cervical neoplasia. Am J Obstet Gynecol. 1968 Nov 1;102(5):624–641. doi: 10.1016/0002-9378(68)90377-3. [DOI] [PubMed] [Google Scholar]
- KARRER H. E. Cell interconnections in normal human cervical epithelium. J Biophys Biochem Cytol. 1960 Feb;7:181–184. doi: 10.1083/jcb.7.1.181. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Karnovsky M. J. The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. J Cell Biol. 1967 Oct;35(1):213–236. doi: 10.1083/jcb.35.1.213. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Karnovsky M. J. The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. J Cell Biol. 1967 Oct;35(1):213–236. doi: 10.1083/jcb.35.1.213. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kelly D. E. Fine structure of desmosomes. , hemidesmosomes, and an adepidermal globular layer in developing newt epidermis. J Cell Biol. 1966 Jan;28(1):51–72. doi: 10.1083/jcb.28.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kirkland J. A. The study of chromosomes in cervical neoplasia. Obstet Gynecol Surv. 1969 Jul;24(7 Pt 2):784–794. doi: 10.1097/00006254-196907001-00010. [DOI] [PubMed] [Google Scholar]
- LUFT J. H. Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol. 1961 Feb;9:409–414. doi: 10.1083/jcb.9.2.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Loewenstein W. R., Kanno Y. Intercellular communication and tissue growth. I. Cancerous growth. J Cell Biol. 1967 May;33(2):225–234. doi: 10.1083/jcb.33.2.225. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Loewenstein W. R. Permeability of membrane junctions. Ann N Y Acad Sci. 1966 Jul 14;137(2):441–472. doi: 10.1111/j.1749-6632.1966.tb50175.x. [DOI] [PubMed] [Google Scholar]
- MELLGREN J., BOERYD B., HAGMAN M. Growth potentials of precancer of the cervix uteri in vitro and in cortisone-treated hamsters. Cancer Res. 1962 Feb;22:139–146. [PubMed] [Google Scholar]
- Martinez-Palomo A., Braislovsky C., Bernhard W. Ultrastructural modifications of the cell surface and intercellular contacts of some transformed cell strains. Cancer Res. 1969 Apr;29(4):925–937. [PubMed] [Google Scholar]
- Martínez-Palomo A. Ultrastructural modifications of intercellular junctions in some epithelial tumors. Lab Invest. 1970 Jun;22(6):605–614. [PubMed] [Google Scholar]
- McNutt N. S. Ultrastructure of intercellular junctions in adult and developing cardiac muscle. Am J Cardiol. 1970 Feb;25(2):169–183. doi: 10.1016/0002-9149(70)90577-1. [DOI] [PubMed] [Google Scholar]
- McNutt N. S., Weinstein R. S. Carcinoma of the cervix: deficiency of nexus intercellular junctions. Science. 1969 Aug 8;165(3893):597–599. doi: 10.1126/science.165.3893.597. [DOI] [PubMed] [Google Scholar]
- McNutt N. S., Weinstein R. S. The ultrastructure of the nexus. A correlated thin-section and freeze-cleave study. J Cell Biol. 1970 Dec;47(3):666–688. doi: 10.1083/jcb.47.3.666. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Moscona A. A. STUDIES ON CELL AGGREGATION: DEMONSTRATION OF MATERIALS WITH SELECTIVE CELL-BINDING ACTIVITY. Proc Natl Acad Sci U S A. 1963 May;49(5):742–747. doi: 10.1073/pnas.49.5.742. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nishihara H. Some observations on the fine structure of the guinea-pig taenia coli after incubation in hypertonic solution. J Anat. 1970 Jul;107(Pt 1):101–114. [PMC free article] [PubMed] [Google Scholar]
- PEACHEY L. D. Thin sections. I. A study of section thickness and physical distortion produced during microtomy. J Biophys Biochem Cytol. 1958 May 25;4(3):233–242. doi: 10.1083/jcb.4.3.233. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Payton B. W., Bennett M. V., Pappas G. D. Permeability and structure of junctional membranes at an electrotonic synapse. Science. 1969 Dec 26;166(3913):1641–1643. doi: 10.1126/science.166.3913.1641. [DOI] [PubMed] [Google Scholar]
- Pinto da Silva P., Branton D. Membrane splitting in freeze-ethching. Covalently bound ferritin as a membrane marker. J Cell Biol. 1970 Jun;45(3):598–605. doi: 10.1083/jcb.45.3.598. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Potter D. D., Furshpan E. J., Lennox E. S. Connections between cells of the developing squid as revealed by electrophysiological methods. Proc Natl Acad Sci U S A. 1966 Feb;55(2):328–336. doi: 10.1073/pnas.55.2.328. [DOI] [PMC free article] [PubMed] [Google Scholar]
- RICHART R. M. A RADIOAUTOGRAPHIC ANALYSIS OF CELLULAR PROLIFERATION IN DYSPLASIA AND CARCINOMA IN SITU OF THE UTERINE CERVIX. Am J Obstet Gynecol. 1963 Aug 1;86:925–930. doi: 10.1016/s0002-9378(16)35248-6. [DOI] [PubMed] [Google Scholar]
- RICHART R. M. THE GROWTH CHARACTERISTICS IN VITRO OF NORMAL EPITHELIUM, DYSPLASIA, AND CARCINOMA-IN-SITU OF THE UTERINE CERVIX. Cancer Res. 1964 May;24:662–669. [PubMed] [Google Scholar]
- Redman R. S., Sreebny L. M. Proliferative behavior of differentiating cells in the developing rat parotid gland. J Cell Biol. 1970 Jul;46(1):81–87. doi: 10.1083/jcb.46.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Revel J. P., Karnovsky M. J. Hexagonal array of subunits in intercellular junctions of the mouse heart and liver. J Cell Biol. 1967 Jun;33(3):C7–C12. doi: 10.1083/jcb.33.3.c7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Richart R. M., Barron B. A. A follow-up study of patients with cervical dysplasia. Am J Obstet Gynecol. 1969 Oct 1;105(3):386–393. doi: 10.1016/0002-9378(69)90268-3. [DOI] [PubMed] [Google Scholar]
- STEINBERG M. S. Reconstruction of tissues by dissociated cells. Some morphogenetic tissue movements and the sorting out of embryonic cells may have a common explanation. Science. 1963 Aug 2;141(3579):401–408. doi: 10.1126/science.141.3579.401. [DOI] [PubMed] [Google Scholar]
- Schrodt G. R., Foreman C. D. Methylcholanthrene-induced carcinoma of the mouse cervix: an electron microscope study. Cancer Res. 1965 Jul;25(6):802–811. [PubMed] [Google Scholar]
- Sheridan J. D. Low-resistance junctions between cancer cells in various solid tumors. J Cell Biol. 1970 Apr;45(1):91–99. doi: 10.1083/jcb.45.1.91. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shingleton H. M., Richart R. M., Wiener J., Spiro D. Human cervical intraepithelial neoplasia: fine structure of dysplasia and carcinoma in situ. Cancer Res. 1968 Apr;28(4):695–706. [PubMed] [Google Scholar]
- Staehelin L. A., Mukherjee T. M., Williams A. W. Freeze-etch appearance of the tight junctions in the epithelium of small and large intestine of mice. Protoplasma. 1969;67(2):165–184. doi: 10.1007/BF01248737. [DOI] [PubMed] [Google Scholar]
- Stern E. Epidemiology of dysplasia. Obstet Gynecol Surv. 1969 Jul;24(7 Pt 2):711–723. doi: 10.1097/00006254-196907001-00005. [DOI] [PubMed] [Google Scholar]
- Stoker M. G., Rubin H. Density dependent inhibition of cell growth in culture. Nature. 1967 Jul 8;215(5097):171–172. doi: 10.1038/215171a0. [DOI] [PubMed] [Google Scholar]
- Stoker M. Contact and short-range interactions affecting growth of animal cells in culture. Curr Top Dev Biol. 1967;2:107–128. doi: 10.1016/s0070-2153(08)60285-9. [DOI] [PubMed] [Google Scholar]
- Tillack T. W., Marchesi V. T. Demonstration of the outer surface of freeze-etched red blood cell membranes. J Cell Biol. 1970 Jun;45(3):649–653. doi: 10.1083/jcb.45.3.649. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Trelstad R. L., Revel J. P., Hay E. D. Tight junctions between cells in the early chick embryo as visualized with the electron microscopy. J Cell Biol. 1966 Oct;31(1):C6–10. doi: 10.1083/jcb.31.1.c6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Uehara Y., Burnstock G. Demonstration of "gap junctions" between smooth muscle cells. J Cell Biol. 1970 Jan;44(1):215–217. doi: 10.1083/jcb.44.1.215. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wehrli E., Mühlethaler K., Moor H. Membrane structure as seen with a double replica method for freeze fracturing. Exp Cell Res. 1970 Feb;59(2):336–339. doi: 10.1016/0014-4827(70)90609-9. [DOI] [PubMed] [Google Scholar]
- Weinstein R. S., Clowes A. W., McNutt N. S. Unique cleavage planes in frozen red cell membranes. Proc Soc Exp Biol Med. 1970 Sep;134(4):1195–1198. doi: 10.3181/00379727-134-34973. [DOI] [PubMed] [Google Scholar]
- Wilbanks G. D. Tissue culture in early cervical neoplasia. A review and prospectus. Obstet Gynecol Surv. 1969 Jul;24(7 Pt 2):804–837. doi: 10.1097/00006254-196907001-00012. [DOI] [PubMed] [Google Scholar]
- Younes M. S. Electron microscope observations on carcinoma in situ of the cervix. Obstet Gynecol Surv. 1969 Jul;24(7 Pt 2):768–784. doi: 10.1097/00006254-196907001-00009. [DOI] [PubMed] [Google Scholar]