A 3D high resolution MRI method for the visualization of cardiac fibro-fatty infiltrations.

Modifications of the myocardial architecture can cause abnormal electrical activity of the heart. Fibro-fatty infiltrations have been implicated in various cardiac pathologies associated with arrhythmias and sudden cardiac death, such as arrhythmogenic right ventricular cardiomyopathy (ARVC). Here, we report the development of an MRI protocol to observe these modifications at 9.4 T. Two fixed ex vivo human hearts, one healthy and one ARVC, were imaged with an Iterative decomposition with echo asymmetry and least-square estimations (IDEAL) and a magnetization transfer (MT) 3D sequences. The resulting fat fraction and MT ratio (MTR) were analyzed and compared to histological analysis of the three regions ("ARVC triangle") primarily involved in ARVC structural remodeling. In the ARVC heart, high fat content was observed in the "ARVC triangle" and the superimposition of the MTR and fat fraction allowed the identification of fibrotic regions in areas without the presence of fat. The healthy heart exhibited twice less fat than the ARVC heart (31.9%, 28.7% and 1.3% of fat in the same regions, respectively). Localization of fat and fibrosis were confirmed by means of histology. This non-destructive approach allows the investigation of structural remodeling in human pathologies where fibrosis and/or fatty tissue infiltrations are expected to occur.

[Efficacy of visual rehabilitation of patients with pellucid marginal degeneration fitted with SPOT® scleral contact lenses]. / Efficacité de la réhabilitation visuelle de patients atteints de dégénérescence marginale pellucide adaptés en lentilles sclérales SPOT.

PURPOSE: To assess visual improvement in patients with pellucid marginal degeneration (PMD) after fitting with SPOT® scleral contact lenses (Scleral Protection & Ocular Treatment, Laboratoires d'Appareillage Oculaire, Amphion-Les-Bains, France). METHODS: We report a case series of 5 patients with PMD and unsatisfactory refractive correction managed at Clermont-Ferrand university hospital from January to December 2018 fitted with customized SPOT scleral lenses. We assessed the best-corrected visual acuity (BVCA) before and after fitting with SPOT, keratometric data and tolerability of the scleral lenses. RESULTS: Nine eyes of 5 patients aged 51.8±8.47 years were fitted. The BVCA was significantly improved from 0.51 logMAR (±0.39) to 0.04 logMAR (±0.07) (P<0.001). Sixty-six percent of the patients recovered optimal BVCA. No serious adverse event was reported. The presence of whitish deposits and an inordinate amount of manipulation required were the main disadvantages of the lenses. Nevertheless, all the patients considered them to be comfortable. CONCLUSION: Fitting PMD patients who have failed conventional optical devices with SPOT scleral lenses significantly improves BCVA, without serious adverse events, allowing surgery to be deferred even when it appears to be unavoidable.

Efficacy of visual rehabilitation of patients with pellucid marginal degeneration fitted with SPOT® scleral contact lenses.

PURPOSE: To assess visual improvement in patients with pellucid marginal degeneration (PMD) after fitting with SPOT® scleral contact lenses (Scleral Protection & Ocular Treatment, Laboratoires d'Appareillage Oculaire, Amphion-Les-Bains, France). METHODS: We report a case series of 5 patients with PMD and unsatisfactory refractive correction managed at Clermont-Ferrand university hospital from January to December 2018 fitted with customized SPOT scleral lenses. We assessed the best-corrected visual acuity (BCVA) before and after fitting with SPOT, keratometric data and tolerability of the scleral lenses. RESULTS: Nine eyes of 5 patients aged 51.8±8.47 years were fitted. The BCVA was significantly improved from 0.51 logMAR (±0.39) to 0.04 logMAR (±0.07) (P<0.001). Sixty-six percent of the patients recovered optimal BCVA. No serious adverse event was reported. The presence of whitish deposits and an inordinate amount of manipulation required were the main disadvantages of the lenses. Nevertheless, all the patients considered them to be comfortable. CONCLUSION: Fitting PMD patients who have failed conventional optical devices with SPOT scleral lenses significantly improves BCVA, without serious adverse events, allowing surgery to be deferred even when it appears to be unavoidable.

Efficacy of treatments for Demodex blepharitis: A systematic review and meta-analysis.

PURPOSE: We conducted a systematic review and meta-analysis to evaluate the efficacy of different treatment for Demodex blepharitis. Parameters studied were mites count, improvement of symptoms and mites' eradication, stratified on type of treatments and mode of delivery of treatments (local or systemic). METHOD: The PubMed, Cochrane Library, Embase, ClinicalTrials.gov, Google scholar and Science Direct databases were searched for studies reporting an efficacy of treatments for Demodex blepharitis. RESULTS: We included 19 studies (14 observational and 5 randomized clinical trials), for a total of 934 patients, 1741 eyes, and 13 different treatments. For mites count, eradication rate, and symptoms improvement, meta-analysis included fifteen, fourteen and thirteen studies, respectively. The overall effect sizes for efficiency of all treatments, globally, were 1.68 (95CI 1.25 to 2.12), 0.45 (0.26-0.64), and 0.76 (0.59-0.90), respectively. Except usual lid hygiene for mites count, Children's Hospital of Eastern Ontario ointment (CHEO) for both eradication rate and symptoms, and CHEO, 2% metronidazole ointment, and systemic metronidazole for eradication rate, all treatments were efficient. Stratified meta-analysis did not show significant differences between local and systemic treatments (1.22, 0.83 to 1.60 vs 2.24, 1.30 to 3.18 for mites count; 0.37, 0.21 to 0.54 vs 0.56, 0.06 to 0.99 for eradication rate; and 0.77, 0.58 to 0.92 vs 0.67, 0.25 to 0.98 for symptoms improvement). CONCLUSION: We reported the efficiency of the different treatments of Demodex blepharitis. Because of less systemic side effects, local treatments seem promising molecules in the treatment of Demodex blepharitis.

Efficacy of Different Procedures of Intra-Corneal Ring Segment Implantation in Keratoconus: a Systematic Review and Meta-Analysis.

Purpose: To evaluate the efficacy of the functional, keratometric, and refractive postoperative parameters of intracorneal ring segment (ICRS) implantation in keratoconus and its association with collagen cross-linking (CXL), photorefractive keratectomy (PRK), and intraocular lenses (IOLs). Methods: We conducted a systematic review and meta-analysis on case series published between 2007 and 2017. Results: We included 95 case series with a total of 4560 patients. We included 64 studies of the ICRS procedure, 20 studies of ICRS+CXL, 9 studies of ICRS+CXL+PRK, and 5 studies of ICRS+IOL. We demonstrated an overall improvement of all parameters in all procedures. Cylinder was decreased with an overall effect size (ES) of -1.15 (-1.36 to -0.95; I2 = 93.7%). Corrected distance visual acuity was improved with an overall ES of 0.89 (0.78 to 1.00; I2 = 81.9%). Maximal keratometry was decreased with an overall ES of 0.98 (0.85 to 1.11; I2 = 78.9%). ICRS+IOL is the best procedure to improve spherical equivalent and uncorrected distance visual acuity (P < 0.05) compared with other procedures. ICRSs versus ICRS+CXL are similar in all parameters except for corrected distance visual acuity. ICRS+CXL+PRK is better than ICRS alone in all parameters except for the correction of spherical equivalent. Conclusions: Although the quality and strength of the data are questionable, ICRS implantation is an effective strategy to preserve visual function in keratoconic patients. Particularly, ICRS+CXL+PRK could be a low invasive procedure to propose to young keratoconic patients. Translational Relevance: To propose an overview of postoperative parameters on each ICRS procedure on keratoconus.

Short communication: Comparison of 3 rapid methods for analysis of vitamin degradation compounds in fluid skim milk.

Vitamin fortification of dairy products, including fluid milk and fortified whey protein beverages, is an industry standard but can lead to the development of off-flavor compounds that are difficult to extract and detect by instrumental methods. Previous work has identified these compounds and their specific role in off-flavors in skim milk, but efficient extraction and quantification of these compounds remains a challenge. Three rapid methods (stir bar sorptive extraction, solvent-assisted stir bar sorptive extraction, and solid-phase microextraction) were compared for their ability to effectively recover vitamin degradation volatiles from fluid skim milk. The performance of the 3 methods for detecting and quantifying vitamin degradation-related volatile compounds was determined by linear regression of standard curves prepared from spiked standards of 5 vitamin degradation volatiles, the reproducibility on the same day and between days as measured by the average relative standard deviation of each standard curve, and the limits of detection and quantitation. Measurement of vitamin degradation compounds in commercial pasteurized fortified skim milks was also conducted using each method. Detection of selected vitamin degradation volatiles was linear in skim milk (0.005-200 µg/kg). Coefficient of determination values differed between methods and compounds. Within-day and between-day percentage of relative standard deviation also varied with compound and method. Limits of detection and quantitation values for all methods except solid-phase microextraction were lower than concentrations of selected volatile compounds typically found in commercial milk. Solvent-assisted stir bar sorptive extraction with a 10-mL sample volume provided the most consistent detection of selected compounds in commercial milks. Based on linearity, relative standard deviation, and limits of detection and quantitation, cyclohexane solvent-assisted stir bar sorptive extraction with 10-mL sample volume is recommended for the quantitation of vitamin degradation-related volatiles in fluid skim milk.

Technical note: Simultaneous carotenoid and vitamin analysis of milk from total mixed ration-fed cows optimized for xanthophyll detection.

Concentrations of retinol, α-tocopherol, and major carotenoids in dairy products are often determined simultaneously by liquid chromatography. These compounds have different polarity and solubility; thus, extracting them simultaneously can be difficult and inefficient. In milks with low carotenoid concentrations, the xanthophylls lutein and zeaxanthin may not be completely resolved using common extraction techniques. A simplified method was developed to optimize extraction efficiency and the limit of detection and limit of quantification (LoQ) of lutein and zeaxanthin in bovine milk without decreasing sensitivity to other vitamins or carotenoids. The developed method evaluates lutein, zeaxanthin, ß-carotene, retinol, and α-tocopherol simultaneously by ultra-high performance liquid chromatography-photodiode array detection. Common saponification temperatures (40-60°C) and concentrations of KOH in water (10-50% KOH wt/vol) were evaluated. Multiple solvents were evaluated for optimal xanthophyll extraction (diethyl ether, dichloromethane, hexane, and tetrahydrofuran) following saponification. The limit of detection and LoQ were defined as 3:1 and 10:1 signal-to-noise ratio, respectively. All experiments were performed in triplicate. The optimal saponification procedure was a concentration of 25% KOH at either 40 or 50°C. Saponified extracts solubilized in solutions containing diethyl ether had greater concentrations of lutein- than hexane- or tetrahydrofuran-based solutions, with peak areas above LoQ values. The solution containing diethyl ether solubilized similar concentrations of retinol, α-tocopherol, and ß-carotene when compared with other solutions. The proposed optimized method allows for the simultaneous determination of carotenoids from milk with increased lutein and zeaxanthin sensitivity without sacrificing recovery of retinol, α-tocopherol, and ß-carotene.

Flavor and flavor chemistry differences among milks processed by high-temperature, short-time pasteurization or ultra-pasteurization.

Typical high-temperature, short-time (HTST) pasteurization encompasses a lower heat treatment and shorter refrigerated shelf life compared with ultra-pasteurization (UP) achieved by direct steam injection (DSI-UP) or indirect heat (IND-UP). A greater understanding of the effect of different heat treatments on flavor and flavor chemistry of milk is required to characterize, understand, and identify the sources of flavors. The objective of this study was to determine the differences in the flavor and volatile compound profiles of milk subjected to HTST, DSI-UP, or IND-UP using sensory and instrumental techniques. Raw skim and raw standardized 2% fat milks (50 L each) were processed in triplicate and pasteurized at 78°C for 15 s (HTST) or 140°C for 2.3 s by DSI-UP or IND-UP. Milks were cooled and stored at 4°C, then analyzed at d 0, 3, 7, and 14. Sensory attributes were determined using a trained panel, and aroma active compounds were evaluated by solid-phase micro-extraction or stir bar sorptive extraction followed by gas chromatography-mass spectrometry, gas chromatography-olfactometry, and gas chromatography-triple quad mass spectrometry. The UP milks had distinct cooked and sulfur flavors compared with HTST milks. The HTST milks had less diversity in aroma active compounds compared with UP milks. Flavor intensity of all milks decreased by d 14 of storage. Aroma active compound profiles were affected by heat treatment and storage time in both skim and 2% milk. High-impact aroma active compounds were hydrogen sulfide, dimethyl trisulfide, and methional in DSI-UP and 2 and 3-methylbutanal, furfural, 2-heptanone, 2-acetyl-1-pyrroline, 2-aminoacetophenone, benzaldehyde, and dimethyl sulfide in IND-UP. These results provide a foundation knowledge of the effect of heat treatments on flavor development and differences in sensory quality of UP milks.

Sensory and chemical properties of Gouda cheese.

Gouda cheese is a washed-curd cheese that is traditionally produced from bovine milk and brined before ripening for 1 to 20 mo. In response to domestic and international demand, US production of Gouda cheese has more than doubled in recent years. An understanding of the chemical and sensory properties of Gouda cheese can help manufacturers create desirable products. The objective of this study was to determine the chemical and sensory properties of Gouda cheeses. Commercial Gouda cheeses (n = 36; 3 mo to 5 yr; domestic and international) were obtained in duplicate lots. Volatile compounds were extracted by solid-phase microextraction and analyzed by gas chromatography-olfactometry and gas chromatography-mass spectrometry. Composition analyses included pH, proximate analysis, salt content, organic acid analysis by HPLC, and color. Flavor and texture properties were determined by descriptive sensory analysis. Focus groups were conducted to document US consumer perception followed by consumer acceptance testing (n = 149) with selected cheeses. Ninety aroma-active compounds in Gouda cheeses were detected by solid-phase microextraction/gas chromatography-olfactometry. Key aroma-active volatile compounds included diacetyl, 2- and 3-methylbutanal, 2-methylpropanal, methional, ethyl butyrate, acetic acid, butyric acid, homofuraneol, δ-decalactone, and 2-isobutyl-3-methoxypyrazine. Aged cheeses had higher organic acid concentrations, higher fat and salt contents, and lower moisture content than younger cheeses. Younger cheeses were characterized by milky, whey, sour aromatic, and diacetyl flavors, whereas aged cheeses were characterized by fruity, caramel, malty/nutty, and brothy flavors. International cheeses were differentiated by the presence of low intensities of cowy/barny and grassy flavors. Younger cheeses were characterized by higher intensities of smoothness and mouth coating, whereas aged cheeses were characterized by higher intensities of fracture and firmness. American consumers used Gouda cheese in numerous applications and stated that packaging appeal, quality, and age were more important than country of origin or nutrition when purchasing Gouda cheeses. Young and medium US cheeses ≤6 mo were most liked by US consumers. Three distinct consumer segments were identified with distinct preferences for cheese flavor and texture. Findings from this study establish key differences in Gouda cheese regarding age and origin and identify US consumer desires for this cheese category.

[Descemet's membrane endothelial keratoplasty (DMEK): Analysis of a variation in Descemet's endothelial graft preparation]. / Greffes endothéliales par Descemet membrane endothelial keratoplasty (DMEK) : analyse d'une variante de découpe du greffon endothélio-descemétique.

INTRODUCTION: Descemet's membrane endothelial keratoplasty (DMEK) can replace just the corneal endothelium and respect the natural corneal anatomy. Currently, the technique of endothelial graft preparation remains manual and non-standardized. PURPOSE: To report anatomic and functional results after DMEK, and compare two techniques of graft preparation. METHODS: Single-center retrospective study, including 64 eyes of 64 patients undergoing DMEK, from September 2014 to February 2016 at Clermont-Ferrand University Medical Center. The "classic" preparation was used in 44 patients (group 1) and the "variant" preparation was used in 20 patients (group 2). An analysis of functional parameters (visual acuity), anatomy (pachymetry, corneal edema, endothelial cell count) and keratometry (sphere, cylinder, mean keratometry) was performed during the first postoperative year. RESULTS: The mean follow-up was 10.0±2.5 months. The average preparation time was 12.3±8.1minutes, with 14.4±8.8 in group 1 versus 7.8±3.0 in group 2 (P<0.001). At six months, the best corrected distance visual acuity was significantly better in group 1 with an acuity of 0.28±0.28 LogMAR in group 1 and 0.37±0.30 LogMAR in group 2 (P<0.01). The best corrected near visual acuity was also better in group 1 at 6 months, with an acuity of 0.29±0.24 LogMAR (P<0.001) in group 1 and 0.37±0.28 LogMAR in group 2 (P=0.02). Improvement in visual acuity was continuous for the 2 groups. At 6 months, endothelial cell loss was greater in group 1 than in group 2, but the difference was not significant (P=0.44). Central corneal thickness was similar between the 2 groups. Finally, no change in keratometry parameters was found between preoperative data and 6 months in each group or between the two groups. CONCLUSION: DMEK led to an improvement in all parameters as well as rapid visual rehabilitation. The new variation in preparation saved a considerable amount of time without decreasing graft survival or postoperative results.

Oxidative and Anti-Oxidative Stress Markers in Chronic Glaucoma: A Systematic Review and Meta-Analysis.

Chronic glaucoma is a multifactorial disease among which oxidative stress may play a major pathophysiological role. We conducted a systematic review and meta-analysis to evaluate the levels of oxidative and antioxidative stress markers in chronic glaucoma compared with a control group. The PubMed, Cochrane Library, Embase and Science Direct databases were searched for studies reporting oxidative and antioxidative stress markers in chronic glaucoma and in healthy controls using the following keywords: "oxidative stress" or "oxidant stress" or "nitrative stress" or "oxidative damage" or "nitrative damage" or "antioxidative stress" or "antioxidant stress" or "antinitrative stress" and "glaucoma". We stratified our meta-analysis on the type of biomarkers, the type of glaucoma, and the origin of the sample (serum or aqueous humor). We included 22 case-control studies with a total of 2913 patients: 1614 with glaucoma and 1319 healthy controls. We included 12 studies in the meta-analysis on oxidative stress markers and 19 on antioxidative stress markers. We demonstrated an overall increase in oxidative stress markers in glaucoma (effect size = 1.64; 95%CI 1.20-2.09), ranging from an effect size of 1.29 in serum (95%CI 0.84-1.74) to 2.62 in aqueous humor (95%CI 1.60-3.65). Despite a decrease in antioxidative stress marker in serum (effect size = -0.41; 95%CI -0.72 to -0.11), some increased in aqueous humor (superoxide dismutase, effect size = 3.53; 95%CI 1.20-5.85 and glutathione peroxidase, effect size = 6.60; 95%CI 3.88-9.31). The differences in the serum levels of oxidative stress markers between glaucoma patients and controls were significantly higher in primary open angle glaucoma vs primary angle closed glaucoma (effect size = 12.7; 95%CI 8.78-16.6, P < 0.001), and higher in pseudo-exfoliative glaucoma vs primary angle closed glaucoma (effect size = 12.2; 95%CI 8.96-15.5, P < 0.001). In conclusion, oxidative stress increased in glaucoma, both in serum and aqueous humor. Malonyldialdehyde seemed the best biomarkers of oxidative stress in serum. The increase of some antioxidant markers could be a protective response of the eye against oxidative stress.

Antibioprophylaxis in Prevention of Endophthalmitis in Intravitreal Injection: A Systematic Review and Meta-Analysis.

Despite endophthalmitis being the most feared complication, antibioprophylaxis remains controversial in intravitreal injections. Therefore, we conducted a systematic review and meta-analysis on the effects of antibioprophylaxis in intravitreal injections in the prevention of endophthalmitis. The PubMed, Cochrane Library, Embase and Science Direct databases were searched for studies comparing groups with and without antibiotics in intravitreal injection, with the use of the following keywords: "antibiotic*", "endophthalmitis" and "intravitreal injection*". To be included, studies needed to specify number of participants and number of endophthalmitis within each group (with and without antibiotics). We conducted meta-analysis on the prevalence of clinical endophthalmitis including both culture-proven and culture negative samples. Nine studies were included. A total of 88 incidences of endophthalmitis were reported from 174,159 injections (0.051% i.e., one incidence of endophthalmitis for 1979 injections). Specifically, 59 incidences of endophthalmitis were reported from 113,530 injections in the group with antibiotics (0.052% or one incidence of endophthalmitis for 1924 injections) and 29 incidences of endophthalmitis from 60,633 injections in the group without antibiotics (0.048% or one endophthalmitis for 2091 injections). Our meta-analysis did not report a significant difference in the prevalence of clinical endophthalimitis between the two groups with and without topical antibiotics: the odds ratio of clinical endophthalimitis was 0.804 (CI95% 0.384-1.682, p = 0.56) for the antibiotic group compared with the group without antibiotics. In conclusion, we performed the first large meta-analysis demonstrating that antibioprophylaxis is not required in intravitreal injections. Strict rules of asepsis remain the only evidence-based prophylaxis of endophthalmitis. The results support initiatives to reduce the global threat of resistance to antibiotics.

[Vitreous hemorrhage as the initial manifestation of familial exudative vitreoretinopathy in an eight-year-old child]. / Une hémorragie intravitréenne comme manifestation initiale de vitréorétinopathie exsudative familiale chez un enfant de huit ans.

PURPOSE: To report vitreous hemorrhage as the initial manifestation of familial exudative vitreoretinopathy (FEVR). METHODS: Case report. RESULTS: An 8-year-old child presented with vitreous hemorrhage leading to the discovery of large neovascularization. Fundus examination and fluorescein angiography showed the typical appearance of FEVR. A genetic study confirmed the diagnosis. Treatment by laser photocoagulation was performed with good recovery of visual acuity. CONCLUSION: Ophthalmologists should be aware of familial exudative vitreoretinopathy (FEVR) so as not to misdiagnose this sight-threatening disease.

Dependence of collision-induced dissociation energy on molecular degrees of freedom as a means to assess relative binding affinity in multivalent complexes.

In collision-induced dissociation mass spectrometry experiments, the collision energy required for dissociation linearly depends on the degrees of freedom in the precursor ion. The magnitude of the slope of this relationship previously has been shown to qualitatively correlate to the relative binding strength of a noncovalently bound, monovalent complex. The goal of the work presented here is to determine if a similar methodology can be applied for assessing relative binding strengths in multivalent species. We have tested the method on complexes formed from 18-crown-6 and a variety of protonated, primary alkylamines, [C(n)H(2n+1)NH(3)](+) (n=9, 12, 14, 16 and 18) and alkyldiamines, [H(3)NC(n)H(2n)NH(3)](2+) (n=3, 5, 6, 9 and 12), and compared our results with dissociation energies calculated using density functional theory at the B3LYP/6-31G* level. We found that the method correctly assessed the stronger crown ether/headgroup interaction in the two divalent species (1:1 and 2:1 complexes formed from the diaminoalkanes) compared with the weaker interaction in the monovalent species (1:1 complexes formed from mono-aminoalkanes). However, the experimental method could not distinguish between the binding strengths of the two divalent complexes, perhaps because their calculated dissociation energies were quite similar. Our preliminary results suggest that this method could potentially be used for a quick and simple analysis of binding strengths in multivalent species if the binding strengths of the species are significantly different from one another.

Riluzole protects against cardiac ischaemia and reperfusion damage via block of the persistent sodium current.

BACKGROUND AND PURPOSE: Current strategies to ameliorate cardiac ischaemic and reperfusion damage, including block of the sodium-hydrogen exchanger, are therapeutically ineffective. Here we propose a different approach, block of the persistent sodium current (INaP). EXPERIMENTAL APPROACH: Left ventricular pressure was measured as an index of functional deficit in isolated, Langendorff perfused, hearts from adult rats, subjected to 30 min global ischaemia and reperfusion with vehicle only (control) or riluzole (1-10 microM) in the perfusate. Cell shortening and intracellular Ca2+ concentrations [Ca2+](i) were measured in adult rat isolated myocytes subjected to hypoxia and re-oxygenation. The block of transient and persistent sodium currents by concentrations of riluzole between 0.01 and 100 microM were assessed in rat isolated myocytes using patch clamp techniques. KEY RESULTS: In perfused hearts, riluzole produced a concentration-dependent cardioprotective action, with minor protection from 1 microM and produced rapid and almost complete recovery upon reperfusion from 3 and 10 microM. In isolated myocytes, riluzole at 3 and 10 microM greatly attenuated or prevented the hypoxia- and reperfusion-induced rise in [Ca2+](i) and the contractile deficit. In patch clamp experiments, riluzole blocked the persistent sodium current with an IC(50) of 2.7 microM, whereas the block of the transient sodium current was only apparent at concentrations above 30 microM. CONCLUSIONS AND IMPLICATIONS: Riluzole preferentially blocked INaP and was protective in cardiac ischaemia and reperfusion. Thus block of the persistent sodium current would be a viable method of ameliorating cardiac ischaemic and reperfusion damage.