・Luong MX, Tam J, Lin Q, Hagendoorn J, Moore KJ, Padera TP, Seed B, Fukumura D, Kucherlapati R, Jain RK.
Lack of lymphatic vessel phenotype in LYVE-1/CD44 double knockout mice.
J Cell Physiol. 2009 Jan 23. [Epub ahead of print]
Lymphatic vessels play a key role in maintaining tissue-fluid homeostasis, immune surveillance and metastasis. The hyaluronan receptor, LYVE-1, is widely used as a molecular marker for adult and embryonic lymphatic endothelium, but its physiological functions have not yet been established in vivo. In agreement with a recent report, LYVE-1(-/-) mice, which are healthy and fertile, do not display any defects related to congenital abnormalities of the lymphatic system. One hypothesis for the absence of a phenotype in LYVE-1 null mice is that other hyaluronan receptors, such as CD44, may compensate for LYVE-1. To test this hypothesis, we created LYVE-1/CD44 double knockout mice with appropriate littermate controls. Lymphatic vessel structure and function, as determined by histological methods and intravital microscopy, show that LYVE-1(-/-), CD44(-/-) and LYVE-1(-/-)/CD44(-/-) mice are indistinguishable from wild-type mice under normal conditions. Furthermore, resolution of carrageenan-induced paw edema is comparable in all genotypes. However, LYVE-1(-/-)/CD44(-/-) mice exhibit increased edema formation in a carrageenan-induced paw inflammation model compared to wild-type mice, but not to LYVE(-/-) or CD44(-/-) mice. These data suggest that LYVE-1 and CD44 are not required for the formation or function of lymphatics, but do not rule out a role for LYVE-1 in inflammation.
・Martin-Villar E, Yurrita MM, Fernandez-Munoz B, Quintanilla M, Renart J.
Regulation of podoplanin/PA2.26 antigen expression in tumour cells. Involvement of calpain-mediated proteolysis.
Int J Biochem Cell Biol. 2008 Dec 25. [Epub ahead of print]
Podoplanin/PA2.26 antigen is a small transmembrane mucin expressed in different types of cancer where it is associated with increased cell migration, invasiveness and metastasis. Little is known about the mechanisms that control podoplanin expression. Here, we show that podoplanin synthesis can be controlled at different levels. We analyzed podoplanin expression in a wide panel of tumour cell lines. The podoplanin gene (PDPN) is transcribed in cells derived from sarcomas, embryonal carcinomas, squamous cell carcinomas and endometrial tumours, while cell lines derived from colon, pancreatic, ovarian and ductal breast carcinomas do not express PDPN transcripts. PDPN is expressed as two mRNAs of approximately 2.7 and approximately 0.9kb, both of which contain the coding sequence and arise by alternative polyadenylation. Strikingly, in most of the cell lines where PDPN transcripts were found, no podoplanin or only very low levels of the protein could be detected in Western blot. Treatment of several of these cell lines with the calpain inhibitor calpeptin resulted in podoplanin accumulation, whereas lactacystin, a specific inhibitor of the proteasome, had no effect. In vitro experiments show that podoplanin is a substrate of calpain-1. These results indicate that at least in some tumour cells absence or reduced podoplanin protein levels are due to post-translational calpain-mediated proteolysis. We also report in this article the identification of a novel podoplanin isoform that originates by alternative splicing and differs from the standard form in lacking two cytoplasmic residues (YS). YS dipeptide is highly conserved across species, suggesting that it might be functionally relevant.
・Kuno A, Kato Y, Matsuda A, Kaneko MK, Ito H, Amano K, Chiba Y, Narimatsu H, Hirabayashi J.
Focused differential glycan analysis with the platform antibody-assisted lectin profiling (ALP) for glycan-related biomarker verification.
Molecular & Cellular Proteomics, 8:99-108, 2009.
Protein glycosylation is a critical issue attracting increasing attention in the field of proteomics as it is expected to play a key role in investigation of histological and diagnostic biomarkers. In this context, an enormous number of them have now been nominated as disease-related biomarkers. However, there is no appropriate strategy in the current proteome platform to qualify such marker candidate molecules, which relates their specific expression to particular diseases. Here, we present a new practical system for focused differential glycan analysis in terms of antibody-assisted lectin profiling (ALP). In the developed procedure, i) a target protein is enriched from clinic samples (e.g., tissue extracts, cell supernatants or sera) by immuno-precipitation with a specific antibody recognizing a core protein moiety; ii) the target glycoprotein is quantified by immuno-blotting using the same antibody used in i); and iii) glycosylation difference is analyzed by means of antibody-overlay lectin microarray, an application technique of an emerging glycan profiling microarray. As model glycoproteins having either N-linked or O-linked glycans, prostate-specific antigen or podoplanin, respectively, were subjected to systematic ALP analysis. As a result, specific signals corresponding to the target glycoprotein glycans were obtained at a sub-picomole level with the aid of specific antibodies, whereby disease-specific or tissue specific glycosylation changes could be observed in a rapid, reproducible and high-throughput manner. Thus, the established system should provide a powerful pipeline in support of on-going efforts in glyco-biomarker discovery.
・Sawa Y, Iwasawa K, Ishikawa H.
Expression of podoplanin in the mouse tooth germ and apical bud cells.
Acta Histochem Cytochem. 2008 Oct 29;41(5):121-6. Epub 2008 Sep 9.
This study was designed to investigate the distribution of cells expressing podoplanin in the mouse tooth bud. Podoplanin expression was detected in enamel epithelia of the cervical loop at cell-cell contacts strongly, and weakly on the loosely aggregated stellate reticulum in the center and the neighboring stratum intermedium. Odontoblasts exhibited intense podoplanin expression at the junction with predentin while no expression was detected in the enamel organ containing ameloblasts. These results suggest that proliferating inner and outer enamel epithelia express podoplanin but that the expression is suppressed in the differentiated epithelia containing ameloblasts. On the other hand the podoplanin expression occurs in the differentiating odontoblasts and the expression is sustained in differentiated odontoblasts, indicating that odontoblasts have the strong ability to express podoplanin. In cultured apical bud cells podoplanin was detected at cell-cell contacts. In real-time PCR analysis the amount of podoplanin mRNA of the apical buds was 2-fold compared with the amount of kidney used as a positive control. These findings indicate that apical bud cells have the strong ability to express the podoplanin gene. Podoplanin is a mucin-type glycoprotein negatively charged by extensive O-glycosylation and a high content of sialic acid, which expresses the adhesive property. The podoplanin may contribute to form odontoblastic fiber or function as the anchorage to the tooth development and in proliferating epithelial cells of cervical loop and apical bud.
・Iwasawa K, Kameyama T, Ishikawa H, Sawa Y.
Induction of ICAM-1 and VCAM-1 on the Mouse Lingual Lymphatic Endothelium with TNF-alpha.
Acta Histochem Cytochem. 2008 Oct 29;41(5):115-20. Epub 2008 Sep 9.
This study investigated the TNF-alpha-induced ICAM-1 and VCAM-1 expression on mouse lingual lymphatic vessels. All podoplanin-positive lymphatic vessels expressed PECAM-1. In the lamina propria mucosae of TNF-alpha-treated tongue, almost all initial lymphatics expressed ICAM-1. There were initial lymphatics with the VCAM-1 expression and also the vessels without the expression. In the tunica muscularis of TNF-alpha-treated tongue, collecting lymphatic vessels expressed ICAM-1, but rarely expressed VCAM-1 whereas blood vessels simultaneously expressed ICAM-1 and VCAM-1. The ICAM-1-positive rate increased with TNF-alpha to 75% from 10% on initial lymphatics, and to 40% from 0% on collecting lymphatic vessels while it increased to 90% from 45% on blood vessels. The VCAM-1-positive rate increased with TNF-alpha to 30% from 0% on initial lymphatics, and to 5% from 0% on collecting lymphatic vessels while it increased to 75% from 5% on blood vessels. These findings suggest that the lingual lymphatic endothelium has the ability to express ICAM-1, and VCAM-1 to a lesser extent than the ICAM-1 induction with TNF-alpha, and that the ICAM-1 and VCAM-1 induction predominantly occurs in the initial lymphatics compared with collecting lymphatic vessels.
・Cueni LN, Detmar M.
Galectin-8 interacts with podoplanin and modulates lymphatic endothelial cell functions.
Exp Cell Res. 2009 Mar 4. [Epub ahead of print]
Podoplanin is a small, mucin-like membrane glycoprotein highly expressed by lymphatic but not by blood vascular endothelial cells. Although it was shown to be indispensable for the correct formation and function of the lymphatic vasculature, its precise molecular function has remained unknown. In the present study, we identified the mammalian lectin galectin-8 as a novel, glycosylation-dependent interaction partner of podoplanin. Galectin-8 is a tandem-repeat type galectin, which interacts with cell surface glycoproteins, including certain integrins, as well as with extracellular matrix molecules such as fibronectin. Here we show that, similar to podoplanin, galectin-8 is more highly expressed by lymphatic than by blood vascular endothelial cells, and that it promotes lymphatic endothelial cell adhesion as well as haptotactic migration when immobilized onto a surface, while inhibiting the formation of tube-like structures by lymphatic endothelial cells in a collagen matrix when incorporated into the matrix. Importantly, functions of blood vascular endothelial cells, which lack podoplanin expression, are not affected by galectin-8. These data suggest a role for galectin-8 and podoplanin in supporting the connection of the lymphatic endothelium to the surrounding extracellular matrix, most likely in cooperation with other glycoproteins on the surface of lymphatic endothelial cells.
・AACR annual meeting 2009
今回のAACRミーティングでは、我々の演題を含め、3つの演題があった。
Podoplanin is a novel antibody-based therapeutic target for glioblastoma
Yukinari Kato, Kazuhiko Mishima, Mika Kato Kaneko, Michael Zalutsky, Chien-Tsun Kuan, and Darell D. Bigner
Podoplanin is a mucin-like transmembrane sialoglycoprotein that is known to be expressed on lymphatic endothelial cells and tumor-initiating cells (TICs). Podoplanin is also expressed in several tumors such as squamous cell carcinomas, mesothelioma, and testicular tumors, and is involved in cancer cell migration, invasion, and metastasis. Interaction between podoplanin and C-type lectin-like receptor-2 (CLEC-2) is reported to be critical for podoplanin-induced platelet aggregation and cancer metastasis. Recently, we showed upregulated expression of podoplanin in central nervous system (CNS) germinomas, but not in non-germinomatous germ cell tumors, except for parts of immature teratomas in limited numbers. Furthermore, we investigated the podoplanin expression in CNS astrocytic tumors. Podoplanin mRNA and protein expression were markedly higher in glioblastomas than in anaplastic astrocytomas, and were not detected in diffuse astrocytoma, suggesting that podoplanin expression might be associated with malignancy of astrocytic tumors. We recently produced a novel anti-podoplanin antibody, NZ-1 (rat IgG2a). In this study, the epitope of NZ-1 antibody was investigated using ELISA, Western blot, and flow cytometry with synthesized podoplanin peptides and deletion mutants of recombinant podoplanin. The epitope for NZ-1 is platelet aggregation-stimulating (PLAG) domain-2/3, which is critical for activity of podoplanin. We have shown that two GBM cell lines, LN319 and D397MG, highly express podoplanin and demonstrated the surface binding for NZ-1 in flow cytometry. We then determined the binding affinity of NZ-1 for podoplanin in BIAcore using podoplanin peptide and Scatchard analysis using podoplanin-positive cell lines. NZ-1 was radioiodinated with Iodogen, and immunoreactive fraction (IRF) was 76.4%. Using I125-NZ-1, the KD was determined to be 8.9 x 10-11 M in BIAcore, and 2.6 x 10-9 M for LN319 cells and 9.8 x 10-10 M for D397MG cells in Scatchard analysis. NZ-1 was also internalized into LN319 and D397MG cells. Taken together, we have shown that podoplanin a potential therapeutic target of glioblastoma for antibody-based therapy.
Glioma cancer stem cells expressing podoplanin exhibit a unique gene expression signature, even in the absence of CD133
Lindsey D. Goodman, Thuy T. Le, Patrice Love, Joy Gumin, Frederick F. Lang Jr., Howard Colman, Ken Aldape, Erik P. Sulman. UT M.D. Anderson Cancer Center, Houston, TX
Glioblastoma is a devastating brain tumor, refractory to most treatments and with a uniformly poor prognosis. Glioma cancer stem cells (GCSCs) are a small population of cells within a tumor believed to be responsible for tumor initiation and treatment resistance. Though CD133 has been reported as a putative marker of GCSCs, we have previously identified the transmembrane protein podoplanin (pdpn) in primary glioma cells cultured as neurospheres and have shown that pdpn+ cells exhibit stem-like characteristics, such as neurosphere formation, resistance to radiation and in vivo tumor formation. Moreover, this phenotype was observed even in those cells not expressing CD133. Pdpn-expression in clinical tumor specimens was prognostic for patient survival and response to radiotherapy, which was not the case for CD133. We hypothesized that pdpn+ cells identify a unique cell type within a tumor which most closely resembles the true GCSC. To test this hypothesis, we performed fluorescence activated cell sorting (FACS) using directly-conjugated monocolonal antibodies to either pdpn or CD133 on a panel of primary cell lines (GSCs) obtained from high-grade gliomas and maintained as neurospheres in long-term culture. Single-cell suspension cultures (neurosphere assays) were used to assess neurosphere formation rates and radiation resistance. RNA obtained from subpopulations immediately following sorting was then used for expression profiling on Affymetrix GeneChips (U133A). Expression profiling data was subjected to unsupervised clustering to identify unique molecular signatures that classify the subpopulations. For all GSCs studied, 4 distinct populations were obtained by FACS (pdpn-/CD133-, pdpn-/cd133+, pdpn+/CD133-, and pdpn+/CD133+). Single-cell suspension assays performed on each subpopulation showed higher rates of neurophere formation in the pdpn+ subpopulations compared to the pdpn- (45% vs. 11%), irrespective of the presence of CD133. Similarly, normalized neurosphere formation rates following 2Gy ionizing radiation were higher in the pdpn+ vs. the pdpn- cells (43% vs. 20%) and silencing of pdpn expression led to increased radiation sensitivity. Unsupervised clustering of expression data from subpopulations of multiple cell lines revealed a unique molecular signature in the pdpn+ cells (pdpn+/CD133- and pdpn+/CD133+) not seen in the pdpn- cells (pdpn-/CD133- and pdpn-/CD133+). Interestingly, clustering of pdpn+ subpopulations showed close linkage, while CD133+ subpopulations did not, suggesting that CD133+ cells did not exhibit a unique gene signature. Genes associated with pdpn included members of signal transduction pathways previously reported in glioma tumorigenesis as well as several novel pathways. In conclusion, pdpn-expressing cells exhibit a unique stem-like phenotype and express a unique gene signature not seen in CD133-expressing cells.
Platelet aggregation induced by prostate cancer cells: regulation by tissue factor, podoplanin and a novel secreted protein
Wei-Lian Tseng, Chia-Chun Huang, Chien-Ling Huang, Ju-Chien Cheng, Ching-Ping Tseng. Chang Gung University, Taoyuan, Taiwan, China Medical University, Taichung, Taiwan
Prostate cancer is one of the most common types of cancer that occurs in men in the world. Tumor cell-induced platelet aggregation (TCIPA) can form a platelet barrier surrounding the cancer cells and has been proposed as a major mechanism to protect cancer cells from attack of immune system and from the stress of blood flow leading to rear site metastasis. In this study, we established an analytical platform to explore the molecular basis of prostate cancer cell-platelet interaction. At first, TCIPA analysis of the two prostate cancer lines DU145 and PC3 revealed that, at the same dosage of tumor cells (1X106), DU145 was more aggressive than PC3 to cause platelet aggregation. Western blot analysis revealed that the well-characterized platelet activating proteins podoplanin (PDPN) had similar expression level in DU145 and PC3. In contrast, tissue factor (TF) was relatively abundant in DU145 when compared to the PC3 cells. Accordingly, TF-blocking antibody abolished TCIPA of DU145 and PC3, whereas the PDPN-blocking antibody caused complete inhibition of TCIPA in PC3 but not DU145. Consistent with these observations, no TCIPA was occurred when the TF substrate, coagulation factor VII-deficient plasma was used in the analysis. Under this circumstance, ectopic addition of recombinant FVII can restore TCIPA of DU145. These data indicate that TF and PDPN are the key TCIPA effectors with different potential in various prostate cancer cells. In addition to these findings, we also revealed a novel tumor cell-secreted protein in the promotion of TCIPA. This protein was found to provoke downstream signal by receptor binding and induce tyrosine phosphorylation of specific intracellular proteins. Furthermore, pre-incubation of prostate cancer cells with a specific, receptor-competitive protein abrogated its receptor binding and TCIPA. Taken together, our data suggest that multiple mechanisms are involved in prostate cancer cells-induced platelet aggregation. Profiling of these platelet aggregation-promoting molecules may shed an insight for tumor cell-platelet interaction and the metastatic potential of circulating tumor cells.
・Yamanashi T, Nakanishi Y, Fujii G, Akishima-Fukasawa Y, Moriya Y, Kanai Y, Watanabe M, Hirohashi S.
Podoplanin Expression Identified in Stromal Fibroblasts as a Favorable Prognostic Marker in Patients with Colorectal Carcinoma.
Oncology. 2009 Jun 25;77(1):53-62. [Epub ahead of print]
Objective: The microenvironment of cancer plays a critical role in its progression. However, the molecular features of cancer-associated fibroblasts (CAFs) are less well understood than those of cancer cells. We investigated the clinicopathological significance of podoplanin expression in stromal fibroblasts in patients with colorectal cancer (CRC). Methods: We selected podoplanin as an upregulated marker in CAF from a DNA microarray experiment. Consequently, podoplanin was identified as an upregulated gene. Immunohistochemical podoplanin expression was investigated at the National Cancer Center Hospital, Tokyo, Japan, in 120 patients with advanced CRC, and its clinicopathological significance was examined. The biological function of podoplanin expression was also assessed by a coculture invasion assay with CRC cell lines such as HCT116 and HCT15. Results: Podoplanin expression was exclusively confined to stromal fibroblasts and absent in tumor cells. Podoplanin is absent in normal stroma except for lymphatic vessels. Staining was considered positive when over 30% of the cancer stroma was stained. Positive podoplanin expression was significantly correlated with a more distal tumor localization (p = 0.013) and a shallower depth of tumor invasion (p = 0.011). Univariate analysis revealed that negative podoplanin expression in stromal fibroblasts was significantly associated with reduced disease-specific survival (p = 0.0017) and disease-free survival (p < 0.0001). Multivariate analysis revealed that negative podoplanin expression (p = 0.016) and lymph node metastasis (p = 0.027) were significantly associated with disease-free survival. CRC cell invasion was augmented by co-culture with CAFs that were treated with siRNA for podoplanin. Conclusions: Our results suggest that a positive podoplanin expression in stromal fibroblasts could have a protective role against CRC cell invasion and is a significant indicator of a good prognosis in patients with advanced CRC, supported by biological analysis showing that podoplanin expression in CAFs is associated with decreased CRC cell invasion.
・Chuang WY, Yeh CJ, Wu YC, Chao YK, Liu YH, Tseng CK, Chang HK, Liu HP, Hsueh C.
Tumor cell expression of podoplanin correlates with nodal metastasis in esophageal squamous cell carcinoma.
Histol Histopathol. 2009 Aug;24(8):1021-7. [Epub ahead of print]
Podoplanin is a mucin-like glycoprotein expressed in the lymphatic endothelium. It has been suggested to play a role in lymphangiogenesis, since podoplanin deficient mice were found to have dilated malfunctioning lymphatic vessels and lymphedema. High podoplanin expression in tumor cells was found to correlate with lymph node metastasis and poor clinical outcome in patients with oral squamous cell carcinoma (SCC). However, the prognostic significance of podoplanin expression in esophageal SCC remains unexplored. Herein, we studied podoplanin expression in 59 patients who underwent surgical resection of esophageal SCC, with 43 of them preceded by preoperative concurrent chemoradiotherapy (CCRT). We found that high podoplanin expression strongly correlated with clinical nodal metastasis (cN1; p=0.0063), which was associated with short survival (p=0.012). However, there was no direct association between high podoplanin expression and short survival. We also found that lymphatic vessel invasion in the resected esophagus was strongly associated with pathological nodal metastasis (pN1; p=0.00092). Our results suggest that podoplanin could also play a role in tumor aggressiveness in esophageal SCC, as well as in oral SCC.
・Shimada Y, Ishii G, Nagai K, Atsumi N, Fujii S, Yamada A, Yamane Y, Hishida T, Nishimura M, Yoshida J, Ikeda N, Ochiai A.
Expression of podoplanin, CD44, and p63 in squamous cell carcinoma of the lung.
Cancer Sci. 2009 Jul 21. [Epub ahead of print]
Recent molecular biological studies have identified podoplanin as a candidate cancer stem cell (CSC) marker in squamous cell carcinoma (SqCC). The purpose of this study was to examine the expression pattern of podoplanin, and the other stem cell markers CD44 and p63, and their relationship to clinico-pathological features including survival in pulmonary SqCC. We examined histologically the expression of podoplanin, CD44, and p63 in 162 consecutive SqCC by immunostaining. Podoplanin expression was observed in 107 (66%) tumors, CD44 in 145 (89.5%), and p63 in 151 (93.2%), respectively. In 95.3% of the podoplanin-positive tumors, tumor cells showing strong expression were localized in the periphery of the tumor nests. However, this peripheral localization was observed in only 55.9% of the CD44-positive and 43% of p63-positive tumors. In 88.8% of the podoplanin-positive tumors, positive cells were localized more peripherally in the tumor nests than CD44- or p63-positive cells and when CD44 and p63 expressions were compared in these podoplanin-positive tumors, p63-positive layers in the periphery of the tumor nests were broader compared to CD44-positive layers. These findings suggest tumor cells are aligned in the "hierarchical distribution pattern" according to the expression of these three markers. Patients who had podoplanin-positive tumors with the "hierarchical pattern" resulted in significantly better overall survival than those who had podoplanin-negative tumors (P = 0.043). These results suggest that podoplanin expression would reflect the most immature status in the differentiation process of SqCC, and SqCC with hierarchical expression would be a well-organized tumor group with lower biological aggressiveness based on the CSC concept.
・平成21年度「保健医療分野における基礎研究推進事業」採択研究プロジェクト; 新規血小板凝集促進因子Aggrusを標的とした分子標的治療薬の創製; 藤田直也先生
血小板凝集は、心筋梗塞・脳梗塞などの血栓症、がん転移など様々な疾患の発症に関与している。総括研究代表者らが発見・命名したAggrusは、高転移性がん細胞膜上に発現している糖タンパク質として同定され、血小板凝集促進活性を示す。作製済みの抗マウスAggrus中和抗体投与により血行性がん転移がほぼ完全に抑制されることから、本研究では、Aggrusと血小板上レセプターとの結合を阻害する分子標的治療薬（低分子化合物・ヒト化中和抗体）を創製し、がん、心疾患、脳血管疾患の画期的医薬品として臨床応用することを目指す。
・Ishizawa K, Komori T, Shimada S, Hirose T.
Podoplanin is a potential marker for the diagnosis of ependymoma: a comparative study with epithelial membrane antigen (EMA).
Clin Neuropathol. 2009 Sep-Oct;28(5):373-8.
Podoplanin is a mucin-type transmembrane sialoglycoprotein that is characteristically expressed in lymphatic endothelia. It is also expressed in the ependyma of the central nervous system as well as in ependymomas. Particularly, membrane-bound structures along the luminal surface, ring-like structures, and dot-like structures in the cytoplasm, all of which were originally reported for epithelial membrane antigen (EMA) immunohistochemistry in ependymoma, were also reported for podoplanin immunohistochemistry in ependymoma. This study was undertaken to evaluate podoplanin as compared with EMA as a marker of ependymoma. A total of 16 ependymomas (WHO Grade (G) II, 9 cases; GIII, 4; myxopapillary, 2; GIII clear cell, (1) were immunohistochemically studied using antibodies against podoplanin (clones D2-40 and NZ-1) as well as an antibody against EMA (clone E29). In all cases, D2-40 and NZ-1 excellently labeled linear signals along the luminal surface of ependymal canals/rosettes, dot-like structures, and/or ringlike structures, as did E29. These structures were generally more abundant in GII ependymomas than in GIII ependymomas. A semiquantitative analysis between the immunopositive structures of D2-40 or NZ-1 and E29 was conducted with a focus on the dot-like structures and the ring-like structures in the cases of GII and GIII ependymoma. The result showed that there was no statistical difference between D2-40 or NZ-1 and E29. Our study suggests that podoplanin is a potential marker for the diagnosis of ependymoma that corresponds to EMA. Anti-podoplanin antibodies and anti-EMA antibodies could cooperate with each other for the diagnostic immunohistochemistry of ependymoma.