Is a diluted seawater-based solution safe and effective on human nasal epithelium?

PURPOSE: Nasal irrigation is an effective method for alleviating several nasal symptoms and regular seawater-based nasal irrigation is useful for maintaining nasal hygiene which is essential for appropriate functioning of the nose and for preventing airborne particles including some pollutants, pathogens, and allergens from moving further in the respiratory system. However, safety studies on seawater-based nasal irrigation are scarce. In this study, the safety and efficacy of a diluted isotonic seawater solution (Stérimar Nasal Hygiene, SNH) in maintaining nasal homeostasis were evaluated in vitro. METHODS: Safety was assessed by measuring tissue integrity via transepithelial electrical resistance (TEER). Efficacy was measured by mucociliary clearance (MCC), mucin secretion, and tissue re-epithelization (wound repair) assays. All assays were performed using a 3D reconstituted human nasal epithelium model. RESULTS: In SNH-treated tissues, TEER values were statistically significantly lower than the untreated tissues; however, the values were above the tissue integrity limit. SNH treatment significantly increased MCC (88 vs. 36 µm/s, p < 0.001) and mucin secretion (1717 vs. 1280 µg/ml, p < 0.001) as compared to untreated cultures. Faster wound closure profile was noted upon pre-SNH treatment as compared to classical isotonic saline solution pre-treatment (90.5 vs. 50.7% wound closure 22 h after wound generation). CONCLUSION: SNH did not compromise the integrity of the nasal epithelium in vitro. Furthermore, SNH was effective for removal of foreign particles through MCC increase and for enhancing wound repair on nasal mucosa.

In vitro Comparison of Safety and Efficacy of Diluted Isotonic Seawater and Electrodialyzed Seawater for Nasal Hygiene.

BACKGROUND: Nasal irrigation is often used for managing sinonasal conditions and maintaining nasal hygiene, which is critical to overall nasal health and to provide protection against airborne contaminants and pathogens. However, studies comparing efficacies of different solutions are needed. PURPOSE: This in vitro study evaluated the ionic balance of an isotonic diluted seawater solution (Stérimar Nasal Hygiene, SNH) and its safety and efficacy for regular nasal hygiene in comparison to electrodialyzed seawater (EDS). MATERIALS AND METHODS: Ionic balance of SNH, EDS and pure seawater was measured by mass spectrometry and chromatography to be compared to the ionic balance of human plasma as reported in the literature. Safety was measured through cytotoxicity (lactate dehydrogenase release) and pro-inflammation (interleukin-8 secretion) assays using a 3D-reconstituted human nasal epithelium model. For efficacy, adenosine 5'-triphosphate (ATP) release assays, and histological (alcian blue) and immunohistochemical (aquaporin 3) stainings were performed on tissues under hypotonic challenge where saline solution was used as the negative control. RESULTS: Compared to EDS, the ionic balance of SNH was more similar to human plasma and pure seawater. SNH reduced hypotonic stress-associated ATP release and maintained tissue morphology more effectively and lastingly compared to EDS. Both solutions were safe to use on nasal epithelium, as neither of them caused cytotoxicity or induced (pro-) inflammation. CONCLUSION: In comparison to EDS, this study confirms the safety and efficacy of SNH in maintaining good nasal hygiene consistent with its benefits reported in clinical trials.

In vitro safety and performance evaluation of a seawater solution enriched with copper, hyaluronic acid, and eucalyptus for nasal lavage.

BACKGROUND: The common cold is a viral infectious disease with symptoms such as runny nose, sore throat, and mainly, nasal congestion. State-of-the-art therapeutic approaches focus on alleviating the symptoms of this disease by non-invasive and simple-to-use methods. Nasal irrigation is one of the most accepted approaches to ease nasal congestion which, if left untreated, has a negative impact on the quality of life of patients. PURPOSE: In this study, the safety and efficacy of a novel hypertonic seawater solution for nasal lavage enriched with hyaluronic acids, eucalyptus oil, copper, and manganese salts (Stérimar Stop & Protect Cold and Flu; SSPCF) have been investigated in vitro. METHODS: An in vitro 3D reconstituted human nasal epithelium tissue model, MucilAir™, has been used in this study to investigate the safety of SSPCF on nasal epithelium by measuring transepithelial electrical resistance (TEER), lactate dehydrogenase (LDH), and interleukin-8 (IL-8) secretion. The efficacy of SSPCF was measured by mucociliary clearance (MCC), ATP release, Alcian blue and aquaporin (AQP3) stainings. RESULTS: SSPCF treatment respected nasal epithelium tissue integrity and enhanced barrier function without inducing a cytotoxic response. Secreted LDH and IL-8 levels were similar to untreated controls. MCC rate was increased 2.5-fold and ATP release decreased 87% upon SSPCF treatment, indicating improved decongestion activity. SSPCF treatment after hypotonic stress helped recover cellular organization, as shown by Alcian blue and AQP3 staining assays. CONCLUSION: SSPCF appears as a safe and effective nasal irrigation formula that may alleviate the symptoms associated with common cold such as nasal congestion.

Rhinosectan® spray (containing xyloglucan) on the ciliary function of the nasal respiratory epithelium; results of an in vitro study.

BACKGROUND: To assess the effects of Rhinosectan® spray, a medical device containing xyloglucan, on nasal ciliary function (in MucilAir™Nasal cells). METHODS: MucilAir™Nasal, a three-dimensional organotypic airway tissue model (with different cell types), was treated with Rhinosectan® (30 µl) or with a control (saline solution). The effects of Rhinosectan® were evaluated at 15 and 60 min post-exposure by: measurement of the cilia beating frequency (Hz), mucin detection (Enzyme-Linked Lectin Assay-ELLA), mucociliary clearance (µm/s) and phagocytosis assay (fluorescence). RESULTS: Exposure of MucilAir™ to Rhinosectan® did not alter the cilia beating frequency at 15 and 60 min post-exposure (diluted and undiluted). Exposure to Rhinosectan® (undiluted) during 60 min increased mucociliary clearance (93.3 ± 2.1 µm/s vs. 80.9 ± 1.8 µm/s; p < 0.01) and phagocytic activity (1.89-fold increase) in comparison with saline solution. Moreover, a significant decrease in mucin concentration was observed after 15 min of exposure (171.4 ng/ml vs. 306.5 ng/ml; p < 0.01) and at 60 min post-treatment (242.7 ng/ml vs. 339 ng/ml; p < 0.05). CONCLUSIONS: The application of Rhinosectan® to nasal epithelial cells does not impair ciliary movement, enhances mucociliary clearance and facilitates phagocytosis while reducing mucin secretion, which are optimal properties for the management of rhinitis and associated conditions.

Protective barrier properties of Rhinosectan® spray (containing xyloglucan) on an organotypic 3D airway tissue model (MucilAir): results of an in vitro study.

BACKGROUND: To evaluate barrier protective properties of Rhinosectan® spray, a medical device containing xyloglucan, on nasal epithelial cells (MucilAir). METHODS: MucilAir-Nasal, a three-dimensional organotypic (with different cell types) airway tissue model, was treated with the medical device Rhinosectan® (30 µL) or with controls (Rhinocort-budesonide-or saline solution). The protective barrier effects of Rhinosectan® were evaluated by: TEER (trans-epithelial electrical resistance) (preservation of tight junctions), Lucifer Yellow assay (preservation of paracellular flux) and confocal immunofluorescence microscopy (localization of tight junction proteins). RESULTS: Exposure of MucilAir with Rhinosectan® protected cell tight junctions (increases in TEER of 13.1% vs -6.3% with saline solution after 1 h of exposure), and preserved the paracellular flux, even after exposure with pro-inflammatory compounds (TNF-α and LPS from Pseudomonas aeruginosa 10). Results of confocal immunofluorescence microscopy demonstrated that, after treatment with the pro-inflammatory mixture, Rhinosectan® produced a slight relocation of zona occludens-1 in the cytosol compartment (while Rhinocort induced expression of zona-occludens-1), maintaining the localization of occludin (similarly to negative control). CONCLUSIONS: Results of our study indicates that Rhinosectan® creates a protective physical barrier on nasal epithelial cells in vitro, allowing the avoidance of allergens and triggering factors, thus confirming the utility of this medical device in the management of nasal respiratory diseases, as rhinitis or rhinosinusitis.

Efficacy of a New Ocular Surface Modulator in Restoring Epithelial Changes in an In Vitro Model of Dry Eye Syndrome.

PURPOSE: So far tear substitutes have demonstrated a limited role in restoring ocular surface damage in dry eye syndrome (DES). The aim of this study was to assess the efficacy of a new ocular surface modulator in an in vitro model of human corneal epithelium (HCE) damaged by severe osmotic stress mirroring the features of dry eye conditions. METHODS: A reconstructed HCE model challenged by the introduction of sorbitol in the culture medium for 16 h was used to induce an inflammatory pathway and to impair the tight junctions integrity determining a severe modification of the superficial layer ultrastructure. At the end of the overnight stress period in the treated HCE series, 30 µl of the ocular surface modulator (T-LysYal, Sildeha, Switzerland) and of hyaluronic acid (HA) in the control HCE series were applied for 24 h. The following parameters were quantified: scanning electron microscopy (SEM), trans-epithelial electrical resistance (TEER), immunofluorescence analysis of integrin ß1 (ITG-ß1), mRNA expression of Cyclin D-1 (CCND1), and ITG-ß1. RESULTS: In the positive control after the osmotic stress the HCE surface damage was visible at the ultrastructural level with loss of cell-cell interconnections, intercellular matrix destruction, and TEER reduction. After 24 h of treatment with T-LysYal, HCE showed a significant improvement of the ultrastructural morphological organization and increased expression of ITG-ß1 at the tissue level when compared to positive and control series. A significant increase of mRNA expression for ITG-ß1 and CCND1 was shown in the HA-treated cells compared to T-LysYal. TEER measurement showed a significant reduction in all groups after 16 h without modifications after the treatment period. CONCLUSIONS: This study has shown the possibility of a new class of agents denominated ocular surface modulators to restore corneal cells damaged by dry eye conditions. Further in vivo studies are certainly necessary to confirm these results.

Effect of Utipro(®) (containing gelatin-xyloglucan) against Escherichia coli invasion of intestinal epithelial cells: results of an in vitro study.

AIM: To evaluate whether Utipro(®), a natural product approved to prevent urinary tract infections, protects intestinal epithelial cells from Escherichia coli adherence/intracellular invasion in vitro. MATERIALS & METHODS: Caco-2 and CacoGoblet(TM) cells were treated with Utipro (1.5 to 10 mg/ml) or untreated (controls). E. coli adherence/intracellular invasion was evaluated by Trans-Epithelial Electrical Resistance, Lucifer Yellow assay and microbial counts. RESULTS: Utipro was noncytotoxic. Utipro 5 and 10 mg/ml protected cell tight junctions (mean ± SD Trans-Epithelial Electrical Resistance [Ω × cm(2)] 66.83 ± 0.29 and 71.33 ± 0.29, respectively), and protected cells from E. coli intracellular invasion (mean ± SD reductions in total bacteria counts [Log10] 0.9 ± 0.06 and 2.1 ± 0.56, respectively). CONCLUSION: Results indicate that Utipro creates a protective physical barrier on intestinal epithelial cells in vitro which reduces the settling of E. coli reservoirs. These results constitute the first step in the demonstration of the efficacy of Utipro to prevent urinary tract infections. Further research is needed in in vivo models and clinical trials.

Corneal epithelial toxicity of antiglaucoma formulations: in vitro study of repeated applications.

BACKGROUND: By using a biologically relevant and sensitive three-dimensional model of human corneal epithelium and multiple endpoint analysis, assessment of the potential for eye irritation and long-term compatibility of four registered ophthalmological preparations, ie, Timolabak(®), Timoptol(®), Nyogel(®), and Timogel(®), was performed. This approach enables classification of the potential for irritation, discriminating between mildly irritant and non-irritant ocular substances. METHODS: The exposure protocol included two time periods, ie, 24 hours (acute application) and 72 hours (repeated applications twice daily). This approach allows assessment of not only the acute reaction but also possible recovery, as well as mimicking the potential cumulative effects associated with long-term application. Using benzalkonium chloride (BAK) 0.01% as a positive control, the following parameters were quantified: cellular viability by MTT test, histological analysis by hematoxylin and eosin staining, passive release of interleukin-1α by enzyme-linked immunosorbent assay, and OCLN gene expression by quantitative real-time polymerase chain reaction. RESULTS: Cell viability was reduced to under the 50% cutoff value after acute exposure (24 hours) to BAK 0.01%, and after repeated application (72 hours) of Timoptol and Nyogel. Histological analysis after acute exposure showed signs of superficial damage with all formulations, and severe changes after repeated applications of Timoptol, BAK 0.01%, and Nyogel. Timolabak and Timogel did not significantly alter the morphology of the human corneal epithelial cells after the different exposure times. Interleukin-1α release was greater than that for the negative control (>20 pg/mL) and the positive control (BAK 0.01%), Nyogel, and Timoptol treatments and not different after treatment with Timolabak and Timogel. Expression of OCLN, a sign of epithelial barrier impairment, was only significantly upregulated at 24 hours by BAK 0.01%, suggesting a toxic reaction at the ocular surface. OCLN was also overexpressed after repeated application of Nyogel and Timogel. CONCLUSION: OVERALL, THE MULTIPLE ENDPOINT ANALYSIS APPROACH ALLOWS CLASSIFICATION OF THESE PRODUCTS ACCORDING TO DECREASING ORDER OF IRRITATION POTENTIAL AS FOLLOWS: BAK 0.01%, Timoptol, Nyogel, Timogel, and Timolabak.

Molecular mechanism of ocular surface damage: application to an in vitro dry eye model on human corneal epithelium.

PURPOSE: The present study was concerned with the development of a new experimental model of dry eye using human reconstructed in vitro corneal epithelium (HCE). The model is based on the use of adapted culture conditions that induce relevant modifications at the cellular and molecular level thus mimicking dry eye. METHODS: The HCE model was maintained in a controlled environmental setting (relative humidity <40% and 40 °C temperature) for 24 h and up to 72 h to induce dry eye. The evolution of the dry eye condition was assessed by histology, immunohistochemistry staining, scanning electron microscopy, and gene expression by using TaqMan gene assay technology (mucin-4 [MUC4], matrix metallopeptidase-9 [MMP9], tumor necrosis factor-α [TNF-α], and defensin ß-2 [DEFB2). The effects of different commercially available tear substitutes on the induced dry eye condition were tested. RESULTS: This in vitro dry eye HCE model, that was well established within 24 h, has the characteristic features of a dry eye epithelium and could be satisfactorily used for preliminary assessment of the protective activity of some artificial tears. The transcriptional study of selected biomarkers showed an increase in MUC4, MMP9, TNF-α, and hBD-2 (DEFB2) gene expression. CONCLUSIONS: By using a dynamic approach, we were able to define a biomarker gene signature of dry eye-induced effects that could be predictive of corneal damage in vivo and to discriminate the efficacy among different commercial artificial tears.

Molecular modifications of dermal and epidermal biomarkers following UVA exposures on reconstructed full-thickness human skin.

Ultraviolet A (UVA) radiation adversely affects skin health and appearance via multiple molecular pathways. Biologically relevant UVA damage are classified as short-term effects (e.g. formation of reactive oxygen species [ROS], inflammation, photo-oxidation, DNA damage, immunosuppression, photoallergy and cell-mediated contact hypersensitivity) or long-term effects (elastosis, photoageing and photocarcinogenesis). Single and chronic experimental exposure to UVA are limited in humans by ethical concerns, and furthermore it is impossible to quantify long-term endpoints such as photoageing over the life-span of a human volunteer. The aim of the present study was to investigate the biological relevance of the Phenion FT skin model for use in photobiological studies. Biological responses to acute and repeated UVA exposures were investigated by monitoring the kinetics of gene expression during the post-irradiation period. By using a dynamic approach, we were able to define early and stable markers of UVA-induced effects that could be predictive of UVA damage in vivo. The transcriptomic approach applied to 3D human tissues appears to be an encouraging method for gaining a deeper understanding of the UVA effects on skin and for studying the dermal response with non-invasive techniques.

Synthesis of gamma-aminobutyric acid (GABA) by Lactobacillus plantarum DSM19463: functional grape must beverage and dermatological applications.

Agriculture surplus were used as substrates to synthesize gamma-aminobutyric acid (GABA) by Lactobacillus plantarum DSM19463 for the manufacture of a functional beverage or as a novel application for dermatological purposes. Dilution of the grape must to 1 or 4% (w/v) of total carbohydrates favored higher cell yield and synthesis of GABA with respect to whey milk. Optimal conditions for synthesizing GABA in grape must were: initial pH 6.0, initial cell density of Log 7.0 cfu/mL, and addition of 18.4 mM L-glutamate. L. plantarum DSM19463 synthesized 4.83 mM of GABA during fermentation at 30 degrees C for 72 h. The fermented grape must also contain various levels of niacin, free minerals, and polyphenols, and Log 10.0 cfu/g of viable cells of L. plantarum DSM19463. Freeze dried preparation of grape must was applied to the SkinEthic(R) Reconstructed Human Epidermis or multi-layer human skin model (FT-skin tissue). The effect on transcriptional regulation of human beta-defensin-2 (HBD-2), hyaluronan synthase (HAS1), filaggrin (FGR), and involucrin genes was assayed through RT-PCR. Compared to GABA used as pure chemical compound, the up-regulation HBD-2 was similar while the effect on the expression of HAS1 and FGR genes was higher.

Transient dimerization and interaction with ERGIC-53 occur in the fibroblast growth factor receptor 3 early secretory pathway.

The fibroblast growth factor receptor 3 (FGFR3) secretory pathway includes N-linked glycosylation in the endoplasmic reticulum where a stringent quality control system ensures that only correctly folded receptor reaches the cell surface from where mature-functional FGFR3 signals upon ligand-mediated dimerization. We have previously shown that the increased kinase activity associated with FGFR3 bearing the thanatophoric dysplasia type II (TDII) mutation hampers its maturation, enabling the receptor to signal from the endoplasmic reticulum. Here we investigate if this biosynthetic disturbance could be explained by premature dimerization of the receptor. Our observations show that a limited fraction of the immature high-mannose, mutant receptor dimerizes in the early secretory pathway, as does the immature wild type FGFR3. In contrast, the mature fully glycosylated wild type receptor reaches the cell surface as monomer suggesting that dimerization is a transient event. The kinase activity of mutant FGFR3 is not required for dimerization to occur, although it increases dimerization efficiency. Furthermore, mutant FGFR3 trans-phosphorylates the immature wild type receptor indicating that dimerization occurs in the endoplasmic reticulum. Visualization of protein interaction inside the secretory pathway confirms receptor dimerization. In addition, it shows that both wild type and TDII FGFR3 interact with the mannose-specific lectin ERGIC-53. We conclude that transient dimerization is an obligatory step in FGFR3 biosynthesis acting as a pre-assembly quality control mechanism. Furthermore, the TDII/ERGIC-53 complex formation may function as a checkpoint for FGFR3 sorting downstream the endoplasmic reticulum. These findings have implications for understanding the pathogenesis of FGFR3-related disorders.

S100A8 and S100A9 activate MAP kinase and NF-kappaB signaling pathways and trigger translocation of RAGE in human prostate cancer cells.

S100 proteins, a multigenic family of calcium-binding proteins, have been linked to human pathologies in recent years. Deregulated expression of S100 proteins, including S100A8 and S100A9, was reported in association with neoplastic disorders. In a previous study, we identified enhanced expression of S100A8 and S100A9 in human prostate cancer. To investigate potential functional implications of S100A8 and S100A9 in prostate cancer, we examined the influence of over-expressed and of purified recombinant S100A8 and S100A9 proteins in different prostate epithelial cell lines. S100A8 and S100A9 were secreted by prostate cancer cells, a finding which prompted us to analyze a possible function as extracellular ligands. S100A8/A9 induced the activation of NF-kappaB and an increased phosphorylation of p38 and p44/42 MAP kinases. In addition, extracellular S100A8/A9 stimulated migration of benign prostatic cells in vitro. Furthermore, in immunofluorescence experiments, we found a strong speckled co-localization of intracellular S100A8/A9 with RAGE after stimulating cells with recombinant S100A8/A9 protein or by increasing cytosolic Ca2+ levels. In summary, our findings show that S100A8 and S100A9 are linked to the activation of important features of prostate cancer cells.

Glycogen synthase kinase-3 interacts with and phosphorylates estrogen receptor alpha and is involved in the regulation of receptor activity.

Like other steroid hormone receptors, estrogen receptor-alpha (ERalpha) is a substrate for protein kinases, and phosphorylation has profound effects on the function and activity of this receptor. A number of different kinases have been implicated in ERalpha regulation. In this report we show by mutational analysis and in vitro kinase assays that ERalpha is a substrate for glycogen synthase kinase-3 (GSK-3) in vitro and is phosphorylated on two sites, the Ser-102, -104, and -106 motif and Ser-118, both located in the N-terminal transcription activation function (AF-1) domain. GSK-3 forms a complex with ERalpha in vivo as demonstrated by co-immunoprecipitation from cell lysates. The GSK-3 inhibitor lithium chloride was used to determine the role of GSK-3 in phosphorylation of Ser-102, -104, and -106 and Ser-118 in vivo and to explore the role of these serines in the regulation of ERalpha function. Treatment of cells with lithium chloride resulted in dephosphorylation of Ser-104 and -106 and Ser-118, which suggests these serine residues as targets for GSK-3 in vivo. Our results further suggest that ERalpha phosphorylation by GSK-3 stabilizes ERalpha under resting conditions and modulates ERalpha transcriptional activity upon ligand binding. Inhibition and constitutive activation of GSK-3, both, resulted in inhibition of ERalpha transcriptional activity, indicating a function of active as well as inactive GSK-3 in ERalpha regulation. These findings uncover a novel mechanism for the regulation of ERalpha-mediated estrogen signaling controlled by a dual action of GSK-3.

Calcium-binding proteins S100A8 and S100A9 as novel diagnostic markers in human prostate cancer.

PURPOSE: S100 proteins comprise a family of calcium-modulated proteins that have recently been associated with epithelial tumors. We examined the expression of two members of this family, S100A8 and S100A9, together with the S100 receptor RAGE (receptor for advanced glycation end products) in human prostate adenocarcinomas and in prostatic intraepithelial neoplasia. EXPERIMENTAL DESIGN: Tissue specimens of 75 patients with organ-confined prostate cancer of different grades were analyzed by immunohistochemistry for expression of S100A8, S100A9, and RAGE. In addition, in situ hybridization of S100A8 and S100A9 was done for 20 cases. An ELISA was applied to determine serum concentrations of S100A9 in cancer patients compared with healthy controls or to patients with benign prostatic hyperplasia (BPH). RESULTS: S100A8, S100A9, and RAGE were up-regulated in prostatic intraepithelial neoplasia and preferentially in high-grade adenocarcinomas, whereas benign tissue was negative or showed weak expression of the proteins. There was a high degree of overlap of S100A8 and S100A9 expression patterns and of S100A8 or S100A9 and RAGE, respectively. Frequently, a gradient within the tumor tissue with an increased expression toward the invaded stroma of the prostate was observed. S100A9 serum levels were significantly elevated in cancer patients compared with BPH patients or healthy individuals. CONCLUSION: Our data suggest that enhanced expression of S100A8, S100A9, and RAGE is an early event in prostate tumorigenesis and may contribute to development and progression or extension of prostate carcinomas. Furthermore, S100A9 in serum may serve as useful marker to discriminate between prostate cancer and BPH.

Impact of PKCdelta on estrogen receptor localization and activity in breast cancer cells.

Regulation of estrogen receptor (ER) function in breast cancer cells is a complex process involving different signalling mechanisms. One signal transduction component that appears to influence ER signalling is protein kinase C (PKC). PKCdelta is a particular isoenzyme of the novel PKC subfamily that plays a role in growth control, differentiation and apoptosis. The aim of the present study was to investigate the impact of PKCdelta on the regulation of the transcriptional activity of the human ERalpha. By using 12-O-tetradecanoylphorbol-13-acetate (TPA), Bryostatin1 and Rottlerin, we show that active PKCdelta is a proproliferative factor in estrogen-dependent breast cancer cells. Furthermore, activation of PKCdelta by TPA resulted in activation and nuclear translocation of ERalpha and in an increase of ER-dependent reporter gene expression. Transfection and expression of the regulatory domain RDdelta of PKCdelta, which is inhibitory to PKCdelta, inhibited the TPA-induced ERalpha activation and translocation. ERalpha was not phosphorylated by PKCdelta; however, glycogen synthase kinase-3 (GSK3) was identified as a substrate of PKCdelta. The expression of RDdelta resulted in a decrease of TPA-induced GSK3 phosphorylation and translocation into the nucleus. We suggest that GSK3 plays a role in the PKCdelta-related nuclear translocation of ERalpha.