Role of JNK in a Trp53-Dependent Mouse Model of Breast Cancer

The cJun NH₂-terminal kinase (JNK) signal transduction pathway has been implicated in mammary carcinogenesis. To test the role of JNK, we examined the effect of ablation of the Jnk1 and Jnk2 genes in a Trp53-dependent model of breast cancer using BALB/c mice. We detected no defects in mammary gland development in virgin mice or during lactation and involution in control studies of Jnk1^−/− and Jnk2^−/− mice. In a Trp53^−/+ genetic background, mammary carcinomas were detected in 43% of control mice, 70% of Jnk1^−/− mice, and 53% of Jnk2^−/− mice. These data indicate that JNK1 and JNK2 are not essential for mammary carcinoma development in the Trp53^−/+ BALB/c model of breast cancer. In contrast, this analysis suggests that JNK may partially contribute to tumor suppression. This conclusion is consistent with the finding that tumor-free survival of JNK-deficient Trp53^−/+ mice was significantly reduced compared with control Trp53^−/+ mice. We conclude that JNK1 and JNK2 can act as suppressors of mammary tumor development.     

Distinct Roles for Dectin-1 and TLR4 in the Pathogenesis of Aspergillus fumigatus Keratitis

Aspergillus species are a major worldwide cause of corneal ulcers, resulting in visual impairment and blindness in immunocompetent individuals. To enhance our understanding of the pathogenesis of Aspergillus keratitis, we developed a murine model in which red fluorescent protein (RFP)-expressing A. fumigatus (Af293.1RFP) conidia are injected into the corneal stroma, and disease progression and fungal survival are tracked over time. Using Mafia mice in which c-fms expressing macrophages and dendritic cells can be induced to undergo apoptosis, we demonstrated that the presence of resident corneal macrophages is essential for production of IL-1β and CXCL1/KC, and for recruitment of neutrophils and mononuclear cells into the corneal stroma. We found that β-glucan was highly expressed on germinating conidia and hyphae in the cornea stroma, and that both Dectin-1 and phospho-Syk were up-regulated in infected corneas. Additionally, we show that infected Dectin-1^−/− corneas have impaired IL-1β and CXCL1/KC production, resulting in diminished cellular infiltration and fungal clearance compared with control mice, especially during infection with clinical isolates expressing high β-glucan. In contrast to Dectin 1^−/− mice, cellular infiltration into infected TLR2^−/−, TLR4^−/−, and MD-2^−/− mice corneas was unimpaired, indicating no role for these receptors in cell recruitment; however, fungal killing was significantly reduced in TLR4^−/− mice, but not TLR2^−/− or MD-2^−/− mice. We also found that TRIF^−/− and TIRAP^−/− mice exhibited no fungal-killing defects, but that MyD88^−/− and IL-1R1^−/− mice were unable to regulate fungal growth. In conclusion, these data are consistent with a model in which β-glucan on A.fumigatus germinating conidia activates Dectin-1 on corneal macrophages to produce IL-1β, and CXCL1, which together with IL-1R1/MyD88-dependent activation, results in recruitment of neutrophils to the corneal stroma and TLR4-dependent fungal killing.     

The absence of Ser389 phosphorylation in p53 affects the basal gene expression level of many p53-dependent genes and alters the biphasic response to UV exposure in mouse embryonic fibroblasts

Phosphorylation is important in p53-mediated DNA damage responses. After UV irradiation, p53 is phosphorylated specifically at murine residue Ser389. Phosphorylation mutant p53.S389A cells and mice show reduced apoptosis and compromised tumor suppression after UV irradiation. We investigated the underlying cellular processes by time-series analysis of UV-induced gene expression responses in wild-type, p53.S389A, and p53^−/− mouse embryonic fibroblasts. The absence of p53.S389 phosphorylation already causes small endogenous gene expression changes for 2,253, mostly p53-dependent, genes. These genes showed basal gene expression levels intermediate to the wild type and p53^−/−, possibly to readjust the p53 network. Overall, the p53.S389A mutation lifts p53-dependent gene repression to a level similar to that of p53^−/− but has lesser effect on p53-dependently induced genes. In the wild type, the response of 6,058 genes to UV irradiation was strictly biphasic. The early stress response, from 0 to 3 h, results in the activation of processes to prevent the accumulation of DNA damage in cells, whereas the late response, from 12 to 24 h, relates more to reentering the cell cycle. Although the p53.S389A UV gene response was only subtly changed, many cellular processes were significantly affected. The early response was affected the most, and many cellular processes were phase-specifically lost, gained, or altered, e.g., induction of apoptosis, cell division, and DNA repair, respectively. Altogether, p53.S389 phosphorylation seems essential for many p53 target genes and p53-dependent processes.     

Nuclear Ferritin Protects DNA From UV Damage in Corneal Epithelial Cells

Previously, we identified the heavy chain of ferritin as a developmentally regulated nuclear protein of embryonic chicken corneal epithelial cells. The nuclear ferritin is assembled into a supramolecular form indistinguishable from the cytoplasmic form of ferritin found in other cell types and thus most likely has iron-sequestering capabilities. Free iron, via the Fenton reaction, is known to exacerbate UV-induced and other oxidative damage to cellular components, including DNA. Since corneal epithelial cells are constantly exposed to UV light, we hypothesized that the nuclear ferritin might protect the DNA of these cells from free radical damage. To test this possibility, primary cultures of cells from corneal epithelium and stroma, and from skin epithelium and stroma, were UV irradiated, and DNA strand breaks were detected by an in situ 3′-end labeling method. Corneal epithelial cells without nuclear ferritin were also examined. We observed that the corneal epithelial cells with nuclear ferritin had significantly less DNA breakage than other cell types examined. Furthermore, increasing the iron concentration of the culture medium exacerbated the generation of UV-induced DNA strand breaks in corneal and skin fibroblasts, but not in the corneal epithelial cells. Most convincingly, corneal epithelial cells in which the expression of nuclear ferritin was inhibited became much more susceptible to UV-induced DNA damage. Therefore, it seems that corneal epithelial cells have evolved a novel, nuclear ferritin-based mechanism for protecting their DNA against UV damage.     

Sunless skin tanning with dihydroxyacetone delays broad-spectrum ultraviolet photocarcinogenesis in hairless mice

Sunless tanning with dihydroxyacetone (DHA) is not considered to be a sunscreen although it does absorb parts of the ultraviolet (UV) spectrum. We investigated the protection with topical application of DHA against solar UV-induced skin carcinogenesis in lightly pigmented hairless hr/hr C3H/Tif mice. Broad-spectrum UV radiation, simulating the UV part of the solar spectrum was obtained from one Philips TL12 and five Bellarium-S SA-1-12 tubes. Three groups of mice were UV-exposed four times a week to a dose-equivalent of four times the standard erythema dose (SED), without or with application of 5 or 20% DHA only twice a week. Similarly, three groups of mice were treated with DHA and irradiated with a high UV dose (8 SED), simulating a skin burn. Two groups (controls) were not irradiated, but either left untreated or treated with 20% DHA alone. The UV-induced skin pigmentation by melanogenesis could easily be distinguished from DHA-induced browning and was measured by a non-invasive, semi-quantitative method. Application of 20% DHA reduced by 63% the pigmentation produced by 4 SED, however, only by 28% the pigmentation produced by 8 SED. Furthermore, topical application of 20% DHA significantly delayed the time to appearance of the first tumor ≥1 mm (P=0.0012) and the time to appearance of the third tumor (P=2×10⁻⁶) in mice irradiated with 4 SED. However, 20% DHA did not delay tumor development in mice irradiated with 8 SED. Application of 5% DHA did not influence pigmentation or photocarcinogenesis.In conclusion, this is the first study to show that the superficial skin coloring generated by frequent topical application of DHA in high concentrations may delay skin cancer development in hairless mice irradiated with moderate UV doses.     

Synergistic Interaction of Rnf8 and p53 in the Protection against Genomic Instability and Tumorigenesis

Rnf8 is an E3 ubiquitin ligase that plays a key role in the DNA damage response as well as in the maintenance of telomeres and chromatin remodeling. Rnf8^−/− mice exhibit developmental defects and increased susceptibility to tumorigenesis. We observed that levels of p53, a central regulator of the cellular response to DNA damage, increased in Rnf8^−/− mice in a tissue- and cell type–specific manner. To investigate the role of the p53-pathway inactivation on the phenotype observed in Rnf8^−/− mice, we have generated Rnf8^−/−p53^−/− mice. Double-knockout mice showed similar growth retardation defects and impaired class switch recombination compared to Rnf8^−/− mice. In contrast, loss of p53 fully rescued the increased apoptosis and reduced number of thymocytes and splenocytes in Rnf8^−/− mice. Similarly, the senescence phenotype of Rnf8^−/− mouse embryonic fibroblasts was rescued in p53 null background. Rnf8^−/−p53^−/− cells displayed defective cell cycle checkpoints and DNA double-strand break repair. In addition, Rnf8^−/−p53^−/− mice had increased levels of genomic instability and a remarkably elevated tumor incidence compared to either Rnf8^−/− or p53^−/− mice. Altogether, the data in this study highlight the importance of p53-pathway activation upon loss of Rnf8, suggesting that Rnf8 and p53 functionally interact to protect against genomic instability and tumorigenesis.     

Increased Corneal Epithelial Turnover Contributes to Abnormal Homeostasis in the Pax6+/? Mouse Model of Aniridia

We aimed to test previous predictions that limbal epithelial stem cells (LESCs) are quantitatively deficient or qualitatively defective in Pax6^+/− mice and decline with age in wild-type (WT) mice. Consistent with previous studies, corneal epithelial stripe patterns coarsened with age in WT mosaics. Mosaic patterns were also coarser in Pax6^+/− mosaics than WT at 15 weeks but not at 3 weeks, which excludes a developmental explanation and strengthens the prediction that Pax6^+/− mice have a LESC-deficiency. To investigate how Pax6 genotype and age affected corneal homeostasis, we compared corneal epithelial cell turnover and label-retaining cells (LRCs; putative LESCs) in Pax6^+/− and WT mice at 15 and 30 weeks. Limbal BrdU-LRC numbers were not reduced in the older WT mice, so this analysis failed to support the predicted age-related decline in slow-cycling LESC numbers in WT corneas. Similarly, limbal BrdU-LRC numbers were not reduced in Pax6^+/− heterozygotes but BrdU-LRCs were also present in Pax6^+/− corneas. It seems likely that Pax6^+/− LRCs are not exclusively stem cells and some may be terminally differentiated CD31-positive blood vessel cells, which invade the Pax6^+/− cornea. It was not, therefore, possible to use this approach to test the prediction that Pax6^+/− corneas had fewer LESCs than WT. However, short-term BrdU labelling showed that basal to suprabasal movement (leading to cell loss) occurred more rapidly in Pax6^+/− than WT mice. This implies that epithelial cell loss is higher in Pax6^+/− mice. If increased corneal epithelial cell loss exceeds the cell production capacity it could cause corneal homeostasis to become unstable, resulting in progressive corneal deterioration. Although it remains unclear whether Pax6^+/− mice have LESC-deficiency, we suggest that features of corneal deterioration, that are often taken as evidence of LESC-deficiency, might occur in the absence of stem cell deficiency if corneal homeostasis is destabilised by excessive cell loss.     

CD14 Mediates Toll-like Receptor 4 (TLR4) Endocytosis and Spleen Tyrosine Kinase (Syk) and Interferon Regulatory Transcription Factor 3 (IRF3) Activation in Epithelial Cells and Impairs Neutrophil Infiltration and Pseudomonas aeruginosa Killing in Vivo

In the current study, we examined the role of CD14 in regulating LPS activation of corneal epithelial cells and Pseudomonas aeruginosa corneal infection. Our findings demonstrate that LPS induces Toll-like receptor 4 (TLR4) internalization in corneal epithelial cells and that blocking with anti-CD14 selectively inhibits TLR4 endocytosis, spleen tyrosine kinase (Syk) and IRF3 phosphorylation, and production of CCL5/RANTES and IFN-β, but not IL-8. Using a murine model of P. aeruginosa corneal infection, we show that although infected CD14^−/− corneas produce less CCL5, they exhibit significantly increased CXC chemokine production, neutrophil recruitment to the corneal stroma, and bacterial clearance than C57BL/6 mice. We conclude that CD14 has a critical role in mediating TLR4 signaling through IRF3 in resident corneal epithelial cells and macrophages and thereby modulates TLR4 cell surface activation of the MyD88/NF-κB/AP-1 pathway and production of CXC chemokines and neutrophil infiltration to infected tissues.     

An important role of CD80/CD86-CTLA-4 signaling during photocarcinogenesis in mice

Although previous studies have shown that altered B7 costimulation plays a critical role in UV irradiation-induced regulation of immunity, the individual roles of the B7 receptors (CD28 and CTLA-4) or the B7 family members (CD80 and CD86) have not been explored. Thus, we investigated CTLA-4 signaling during photocarcinogenesis of chronically UV-B-exposed mice using an antagonistic anti-CTLA-4 Ab. Anti-CTLA-4-treated mice developed significantly fewer UV-induced tumors. Moreover, anti-CTLA-4 treatment induced long-lasting protective immunity because progressively growing UV tumors inoculated into anti-CTLA-4- and UV-treated mice that had not developed tumors were rejected. Next, we used mice deficient for CD80, CD86, or both in photocarcinogenesis studies to assess the relative contributions of these CTLA-4 ligands. Double-deficient mice showed significantly reduced UV-induced skin tumor development, whereas CD86(-/-) mice produced skin cancer earlier compared with CD80(-/-) and control mice. The growth of UV-induced tumors appears to be controlled by UV-induced suppressor T cells, because CD80(-/-)/CD86(-/-) mice had strongly reduced numbers of UV-induced CD4(+)CD25(+) suppressor T cells. In vitro, CTLA-4 blockade inhibited the suppressor activity of UV-induced CD4(+)CD25(+) T cells, suggesting that reduced photocarcinogenesis might be due to decreased numbers or function of suppressor T cells. Together, these data indicate that blocking CD80/86-CTLA-4 signaling induced immune protection against the development of UV-induced skin tumors. Furthermore, CD86-mediated costimulation appears to play a more critical role in the protection against photocarcinogenesis than CD80.     

Extracellular Matrix Protein Lumican Promotes Clearance and Resolution of Pseudomonas aeruginosa Keratitis in a Mouse Model

Lumican is an extracellular protein that associates with CD14 on the surface of macrophages and neutrophils, and promotes CD14-TLR4 mediated response to bacterial lipopolysaccharides (LPS). Lumican-deficient (Lum ^−/−) mice and macrophages are impaired in TLR4 signals; raising the possibility that lumican may regulate host response to live bacterial infections. In a recent study we showed that in vitro Lum ^−/− macrophages are impaired in phagocytosis of gram-negative bacteria and in a lung infection model the Lum ^−/− mice showed poor survival. The cornea is an immune privileged barrier tissue that relies primarily on innate immunity to protect against ocular infections. Lumican is a major component of the cornea, yet its role in counteracting live bacteria in the cornea remains poorly understood. Here we investigated Pseudomonas aeruginosa infections of the cornea in Lum ^−/− mice. By flow cytometry we found that 24 hours after infection macrophage and neutrophil counts were lower in the cornea of Lum ^−/− mice compared to wild types. Infected Lum ^−/− corneas showed lower levels of the leukocyte chemoattractant CXCL1 by 24–48 hours of infection, and increased bacterial counts up to 5 days after infection, compared to Lum^+/− mice. The pro-inflammatory cytokine TNF-α was comparably low 24 hours after infection, but significantly higher in the Lum ^−/− compared to Lum ^+/− infected corneas by 2–5 days after infection. Taken together, the results indicate that lumican facilitates development of an innate immune response at the earlier stages of infection and lumican deficiency leads to poor bacterial clearance and resolution of corneal inflammation at a later stage.     

IL-10 controls ultraviolet-induced carcinogenesis in mice

UV radiation-induced immunosuppression contributes significantly to the development of UV-induced skin cancer by inhibiting protective immune responses. IL-10 has been shown to be a key mediator of UV-induced immunosuppression. To investigate the role of IL-10 during photocarcinogenesis, groups of IL-10(+/+), IL-10(+/-), and IL-10(-/-) mice were chronically irradiated with UV. IL-10(+/+) and IL-10(+/-) mice developed skin cancer to similar extents, whereas IL-10(-/-) mice were protected against the induction of skin malignancies by UV. Because UV is able to induce regulatory T cells, which play a role in the suppression of protective immunity, UV-induced regulatory T cell function was analyzed. Splenic regulatory T cells from UV-irradiated IL-10(-/-) mice were unable to confer immunosuppression upon transfer into naive recipients. UV-induced CD4+CD25+ T cells from IL-10(-/-) mice showed impaired suppressor function when cocultured with conventional CD4+CD25- T cells. CD4+CD25- T cells from IL-10(-/-) mice produced increased amounts of IFN-gamma and enhanced numbers of CD4+TIM-3+ T cells were detectable within UV-induced tumors in IL-10(-/-) mice, suggesting strong Th1-driven immunity. Mice treated with CD8+ T cells from UV-irradiated IL-10(-/-) mice rejected a UV tumor challenge significantly faster, and augmented numbers of granzyme A+ cells were detected within injected UV tumors in IL-10(-/-) animals, suggesting marked antitumoral CTL responses. Together, these findings indicate that IL-10 is critically involved in antitumoral immunity during photocarcinogenesis. Moreover, these results point out the crucial role of Th1 responses and UV-induced regulatory T cell function in the protection against UV-induced tumor development.     

Chronic Proliferative Dermatitis in Sharpin Null Mice: Development of an Autoinflammatory Disease in the Absence of B and T Lymphocytes and IL4/IL13 Signaling

SHARPIN is a key regulator of NFKB and integrin signaling. Mice lacking Sharpin develop a phenotype known as chronic proliferative dermatitis (CPDM), typified by progressive epidermal hyperplasia, apoptosis of keratinocytes, cutaneous and systemic eosinophilic inflammation, and hypoplasia of secondary lymphoid organs. Rag1^−/− mice, which lack mature B and T cells, were crossed with Sharpin^−/− mice to examine the role of lymphocytes in CDPM. Although inflammation in the lungs, liver, and joints was reduced in these double mutant mice, dermatitis was not reduced in the absence of functional lymphocytes, suggesting that lymphocytes are not primary drivers of the inflammation in the skin. Type 2 cytokine expression is increased in CPDM. In an attempt to reduce this aspect of the phenotype, Il4ra^−/− mice, unresponsive to both IL4 and IL13, were crossed with Sharpin^−/− mice. Double homozygous Sharpin^−/−, Il4ra^−/− mice developed an exacerbated granulocytic dermatitis, acute system inflammation, as well as hepatic necrosis and mineralization. High expression of CHI3L4, normally seen in CPDM skin, was abolished in Sharpin^−/−, Il4ra^−/− double mutant mice indicating the crucial role of IL4 and IL13 in the expression of this protein. Cutaneous eosinophilia persisted in Sharpin^−/−, Il4ra^−/− mice, although expression of Il5 mRNA was reduced and the expression of Ccl11 and Ccl24 was completely abolished. TSLP and IL33 were both increased in the skin of Sharpin^−/− mice and this was maintained in Sharpin^−/−, Il4ra^−/− mice suggesting a role for TSLP and IL33 in the eosinophilic dermatitis in SHARPIN-deficient mice. These studies indicate that cutaneous inflammation in SHARPIN-deficient mice is autoinflammatory in nature developing independently of B and T lymphocytes, while the systemic inflammation seen in CPDM has a strong lymphocyte-dependent component. Both the cutaneous and systemic inflammation is enhanced by loss of IL4 and IL13 signaling indicating that these cytokines normally play an anti-inflammatory role in SHARPIN-deficient mice.     

Altered Prostanoid Signaling Contributes to Increased Skin Tumorigenesis in Tpl2 Knockout Mice

Squamous cell carcinoma is the second most common form of skin cancer with the incidence expected to double over the next 20 years. Inflammation is believed to be a critical component in skin cancer progression. Therefore, understanding genes involved in the regulation of inflammatory pathways is vital to the design of targeted therapies. Numerous studies show cyclooxygenases (COXs) play an essential role in inflammation-associated cancers. Tpl2 (MAP3K8) is a protein kinase in the MAP Kinase signal transduction cascade. Previous research using a two-stage skin carcinogenesis model revealed that Tpl2 ^−/− mice have significantly higher tumor incidence and inflammatory response than wild-type (WT) controls. The current study investigates whether cyclooxygenase-2 (COX-2) and COX-2- regulated prostaglandins and prostaglandin receptors drive the highly tumorigenic state of Tpl2^−/− mice by investigating the relationship between Tpl2 and COX-2. Keratinocytes from newborn WT or Tpl2 ^−/− mice were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) for various times over 24 hours. Western analysis revealed significant differences in COX-2 and COX-2 dependent prostanoids and prostanoid receptors. Additionally, in vivo experiments confirmed that COX-2 and COX-2 downstream factors were elevated in TPA-treated Tpl2^−/− skin, as well as in papillomas from Tpl2 ^−/− mice. Use of the selective COX-2 inhibitor Celecoxib showed the increased tumorigenesis in the Tpl2^−/− mice to primarily be mediated through COX-2. These experiments illustrate COX-2 induction in the absence of Tpl2 may be responsible for the increased tumorigenesis found in Tpl2 ^−/− mice. Defining the relationship between Tpl2 and COX-2 may lead to new ways to downregulate COX-2 through the modulation of Tpl2.     

Loss of RhoB Expression Enhances the Myelodysplastic Phenotype of Mammalian Diaphanous-Related Formin mDia1 Knockout Mice

Myelodysplastic syndrome (MDS) is characterized by ineffective hematopoiesis and hyperplastic bone marrow. Complete loss or interstitial deletions of the long arm of chromosome 5 occur frequently in MDS. One candidate tumor suppressor on 5q is the mammalian Diaphanous (mDia)-related formin mDia1, encoded by DIAPH1 (5q31.3). mDia-family formins act as effectors for Rho-family small GTP-binding proteins including RhoB, which has also been shown to possess tumor suppressor activity. Mice lacking the Drf1 gene that encodes mDia1 develop age-dependent myelodysplastic features. We crossed mDia1 and RhoB knockout mice to test whether the additional loss of RhoB expression would compound the myelodysplastic phenotype. Drf1 ^−/− RhoB ^−/− mice are fertile and develop normally. Relative to age-matched Drf1 ^−/− RhoB ^+/− mice, the age of myelodysplasia onset was earlier in Drf1 ^−/− RhoB ^−/− animals—including abnormally shaped erythrocytes, splenomegaly, and extramedullary hematopoiesis. In addition, we observed a statistically significant increase in the number of activated monocytes/macrophages in both the spleen and bone marrow of Drf1 ^−/− RhoB ^−/− mice relative to Drf1 ^−/− RhoB ^+/− mice. These data suggest a role for RhoB-regulated mDia1 in the regulation of hematopoietic progenitor cells.     

Chronic Inhibition of Dopamine β-Hydroxylase Facilitates Behavioral Responses to Cocaine in Mice

The anti-alcoholism medication, disulfiram (Antabuse), decreases cocaine use in humans regardless of concurrent alcohol consumption and facilitates cocaine sensitization in rats, but the functional targets are unknown. Disulfiram inhibits dopamine β-hydroxylase (DBH), the enzyme that converts dopamine (DA) to norepinephrine (NE) in noradrenergic neurons. The goal of this study was to test the effects of chronic genetic or pharmacological DBH inhibition on behavioral responses to cocaine using DBH knockout (Dbh −/−) mice, disulfiram, and the selective DBH inhibitor, nepicastat. Locomotor activity was measured in control (Dbh +/−) and Dbh −/− mice during a 5 day regimen of saline+saline, disulfiram+saline, nepicastat+saline, saline+cocaine, disulfiram+cocaine, or nepicastat+cocaine. After a 10 day withdrawal period, all groups were administered cocaine, and locomotor activity and stereotypy were measured. Drug-naïve Dbh −/− mice were hypersensitive to cocaine-induced locomotion and resembled cocaine-sensitized Dbh +/− mice. Chronic disulfiram administration facilitated cocaine-induced locomotion in some mice and induced stereotypy in others during the development of sensitization, while cocaine-induced stereotypy was evident in all nepicastat-treated mice. Cocaine-induced stereotypy was profoundly increased in the disulfiram+cocaine, nepicastat+cocaine, and nepicastat+saline groups upon cocaine challenge after withdrawal in Dbh +/− mice. Disulfiram or nepicastat treatment had no effect on behavioral responses to cocaine in Dbh −/− mice. These results demonstrate that chronic DBH inhibition facilitates behavioral responses to cocaine, although different methods of inhibition (genetic vs. non-selective inhibitor vs. selective inhibitor) enhance qualitatively different cocaine-induced behaviors.