Bicuspid aortic valve hemodynamics induces abnormal medial remodeling in the convexity of porcine ascending aortas

The type-I bicuspid aortic valve (BAV), which differs from the normal tricuspid aortic valve (TAV) most commonly by left-right coronary cusp fusion, is frequently associated with secondary aortopathies. While BAV aortic dilation has been linked to a genetic predisposition, hemodynamics has emerged as a potential alternate etiology. However, the link between BAV hemodynamics and aortic medial degeneration has not been established. The objective of this study was to compare the regional wall shear stresses (WSS) in a TAV and BAV ascending aorta (AA) and to isolate ex vivo their respective impact on aortic wall remodeling. The WSS environments generated in the convex region of a TAV and BAV AA were predicted through fluid–structure interaction (FSI) simulations in an aorta model subjected to both valvular flows. Remodeling of porcine aortic tissue exposed to TAV and BAV AA WSS for 48 h in a cone-and-plate bioreactor was investigated via immunostaining, immunoblotting and zymography. FSI simulations revealed the existence of larger and more unidirectional WSS in the BAV than in the TAV AA convexity. Exposure of normal aortic tissue to BAV AA WSS resulted in increased MMP-2 and MMP-9 expressions and MMP-2 activity but similar fibrillin-1 content and microfibril organization relative to the TAV AA WSS treatment. This study confirms the sensitivity of aortic tissue to WSS abnormalities and demonstrates the susceptibility of BAV hemodynamic stresses to focally mediate aortic medial degradation. The results provide compelling support to the important role of hemodynamics in BAV secondary aortopathy.     

Gene Expression Profiling in Human Lung Development: An Abundant Resource for Lung Adenocarcinoma Prognosis

A tumor can be viewed as a special “organ” that undergoes aberrant and poorly regulated organogenesis. Progress in cancer prognosis and therapy might be facilitated by re-examining distinctive processes that operate during normal development, to elucidate the intrinsic features of cancer that are significantly obscured by its heterogeneity. The global gene expression signatures of 44 human lung tissues at four development stages from Asian descent and 69 lung adenocarcinoma (ADC) tissue samples from ethnic Chinese patients were profiled using microarrays. All of the genes were classified into 27 distinct groups based on their expression patterns (named as PTN1 to PTN27) during the developmental process. In lung ADC, genes whose expression levels decreased steadily during lung development (genes in PTN1) generally had their expression reactivated, while those with uniformly increasing expression levels (genes in PTN27) had their expression suppressed. The genes in PTN1 contain many n-gene signatures that are of prognostic value for lung ADC. The prognostic relevance of a 12-gene demonstrator for patient survival was characterized in five cohorts of healthy and ADC patients [ADC_CICAMS (n = 69, p = 0.007), ADC_PNAS (n = 125, p = 0.0063), ADC_GSE13213 (n = 117, p = 0.0027), ADC_GSE8894 (n = 62, p = 0.01), and ADC_NCI (n = 282, p = 0.045)] and in four groups of stage I patients [ADC_CICAMS (n = 22, p = 0.017), ADC_PNAS (n = 76, p = 0.018), ADC_GSE13213 (n = 79, p = 0.02), and ADC_qPCR (n = 62, p = 0.006)]. In conclusion, by comparison of gene expression profiles during human lung developmental process and lung ADC progression, we revealed that the genes with a uniformly decreasing expression pattern during lung development are of enormous prognostic value for lung ADC.     

Helical flows and asymmetry of blood jet in dilated ascending aorta with normally functioning bicuspid valve

Bicuspid aortic valve (BAV) is associated with aortic dilatation and aneurysm. Several studies evidenced an eccentric systolic flow in ascending aorta associated with increased wall shear stresses (WSS) and the occurrence of an helical systolic flow. This study seeks to elucidate the connections between jet asymmetry and helical flow in patients with normally functioning BAV and dilated ascending aorta. We performed a computational parametric study by varying, for a patient-specific geometry, the valve area and the flow rate entering the aorta and drawing also a tricuspid valve (TAV). We considered also phase-contrast magnetic resonance imaging of four BAV and TAV patients. Measurement of normalized flow asymmetry index, systolic WSS and of a new index (positive helix fraction, PHF) quantifying the presence of a single a single helical flow were performed. In our computation, BAV cases featured higher values of all indices with respect to TAV in both numerical and imaged-based results. Moreover, all indices increased with decreasing valve area and/or with increasing flow rate. This allowed to separate the BAV and TAV cases with respect to the jet asymmetry, WSS localization and helical flow. Interestingly, these results were obtained without modeling the leaflets.     

Unraveling divergent gene expression profiles in bicuspid and tricuspid aortic valve patients with thoracic aortic dilatation: the ASAP study

Thoracic aortic aneurysm (TAA) is a common complication in patients with a bicuspid aortic valve (BAV), the most frequent congenital heart disorder. For unknown reasons TAA occurs at a younger age, with a higher frequency in BAV patients than in patients with a tricuspid aortic valve (TAV), resulting in an increased risk for aortic dissection and rupture. To investigate the increased TAA incidence in BAV patients, we obtained tissue biopsy samples from nondilated and dilated aortas of 131 BAV and TAV patients. Global gene expression profiles were analyzed from controls and from aortic intima-media and adventitia of patients (in total 345 samples). Of the genes found to be differentially expressed with dilation, only a few (<4%) were differentially expressed in both BAV and TAV patients. With the use of gene set enrichment analysis, the cell adhesion and extracellular region gene ontology sets were identified as common features of TAA in both BAV and TAV patients. Immune response genes were observed to be particularly overexpressed in the aortic media of dilated TAV samples. The divergent gene expression profiles indicate that there are fundamental differences in TAA etiology in BAV and TAV patients. Immune response activation solely in the aortic media of TAV patients suggests that inflammation is involved in TAA formation in TAV but not in BAV patients. Conversely, genes were identified that were only differentially expressed with dilation in BAV patients. The result has bearing on future clinical studies in which separate analysis of BAV and TAV patients is recommended.     

Bicuspid aortic valve aortopathies: An hemodynamics characterization in dilated aortas

Bicuspid aortic valve (BAV) aortopathy remains of difficult clinical management due to its heterogeneity and further assessment of related aortic hemodynamics is necessary. The aim of this study was to assess systolic hemodynamic indexes and wall stresses in patients with diverse BAV phenotypes and dilated ascending aortas. The aortic geometry was reconstructed from patient-specific images while the aortic valve was generated based on patient-specific measurements. Physiologic material properties and boundary conditions were applied and fully coupled fluid-structure interaction (FSI) analysis were conducted. Our dilated aortic models were characterized by the presence of abnormal hemodynamics with elevated degrees of flow skewness and eccentricity, regardless of BAV morphotype. Retrograde flow was also present. Both features, predicted by flow angle and flow reversal ratios, were consistently higher than those reported for non-dilated aortas. Right-handed helical flow was present, as well as elevated wall shear stress (WSS) on the outer ascending aortic wall. Our results suggest that the abnormal flow associated with BAV may play a role in aortic enlargement and progress it further on already dilated aortas.     

Gene Expression Profiling in the Type 1 Diabetes Rat Diaphragm

Background

Respiratory muscle contractile performance is impaired by diabetes, mechanisms of which included altered carbohydrate and lipid metabolism, oxidative stress and changes in membrane electrophysiology. The present study examined to what extent these cellular perturbations involve changes in gene expression.

Methodology/Principal Findings

Diaphragm muscle from streptozotocin-diabetic rats was analyzed with Affymetrix gene expression arrays. Diaphragm from diabetic rats had 105 genes with at least ±2-fold significantly changed expression (55 increased, 50 decreased), and these were assigned to gene ontology groups based on over-representation analysis using DAVID software. There was increased expression of genes involved in palmitoyl-CoA hydrolase activity (a component of lipid metabolism) (P = 0.037, n = 2 genes, fold change 4.2 to 27.5) and reduced expression of genes related to carbohydrate metabolism (P = 0.000061, n = 8 genes, fold change −2.0 to −8.5). Other gene ontology groups among upregulated genes were protein ubiquitination (P = 0.0053, n = 4, fold change 2.2 to 3.4), oxidoreductase activity (P = 0.024, n = 8, fold change 2.1 to 6.0), and morphogenesis (P = 0.012, n = 10, fold change 2.1 to 4.3). Other downregulated gene groups were extracellular region (including extracellular matrix and collagen) (P = 0.00032, n = 13, fold change −2.2 to −3.7) and organogenesis (P = 0.032, n = 7, fold change −2.1 to −3.7). Real-time PCR confirmed the directionality of changes in gene expression for 30 of 31 genes tested.

Conclusions/Significance

These data indicate that in diaphragm muscle type 1 diabetes increases expression of genes involved in lipid energetics, oxidative stress and protein ubiquitination, decreases expression of genes involved in carbohydrate metabolism, and has little effect on expression of ion channel genes. Reciprocal changes in expression of genes involved in carbohydrate and lipid metabolism may change the availability of energetic substrates and thereby directly modulate fatigue resistance, an important issue for a muscle like the diaphragm which needs to contract without rest for the entire lifetime of the organism.     

Bicuspid stenotic aortic valves: clinical characteristics and morphological assessment using MRI and echocardiography

Background

Bicuspid aortic valve (BAV) is one of the most common congenital heart defects with a population prevalence of 0.5% to 1.3%. Identifying patients with BAV is clinically relevant because BAV is associated with aortic stenosis, endocarditis and ascending aorta pathology.

Methods and Results

Patients with severe aortic stenosis necessitating aortic valve replacement surgery were included in this study. All dissected aortic valves were stored in the biobank of the University Medical Centre Utrecht. Additionally to the morphological assessment of the aortic valve by the surgeon and pathologist, echocardiographic and magnetic resonance imaging (MRI) images were evaluated. A total of 80 patients were included of whom 32 (40%) were diagnosed with BAV by the surgeon (gold standard). Patients with BAV were significantly younger (55 vs 71 years) and were more frequently male. Notably, a significant difference was found between the surgeon and pathologist in determining valve morphology. MRI was performed in 33% of patients. MRI could assess valve morphology in 96% vs 73% with echocardiography. The sensitivity of MRI for BAV in a population of patients with severe aortic stenosis was higher than echocardiography (75% vs 55%), whereas specificity was better with the latter (91% vs 79%). Typically, the ascending aorta was larger in patients with BAV.

Conclusion

Among unselected patients with severe aortic valve stenosis, a high percentage of patients with BAV were found. Imaging and assessment of the aortic valve morphology when stenotic is challenging.     

Low CD10 mRNA Expression Identifies High-Risk Ductal Carcinoma In Situ (DCIS)

Purpose

Optimal management of breast ductal carcinoma in situ (DCIS) is controversial, and many patients are still overtreated. The local death of myoepithelial cells (MECs) is believed to be a pre-requisite to tumor invasion. We thus hypothesized that loss of CD10 expression, a MEC surface peptidase, would signify basement membrane disruption and confer increased risk of relapse in DCIS. The aim of our study was to retrospectively evaluate the prognostic value of CD10 in DCIS.

Experimental Design

CD10 expression was evaluated by quantitative RT-PCR and immunohistochemistry using paraffin-embedded samples of normal breast tissue (n = 11); of morphologically normal ducts associated with DCIS (n = 10); and of DCIS without an invasive component (n = 154).

Results

CD10 immunostaining was only observed in MECs in normal tissue and in DCIS. Normal tissue showed high mRNA expression levels of CD10, whereas DCIS showed a variable range. After a median follow-up of 6 years, DCIS with CD10 expression below the levels observed in normal tissue (71%) demonstrated a higher risk of local relapse (HR = 1.88; [95CI:1.30–2.70], p = 0.001) in univariate analysis. No relapse was observed in patients expressing high CD10 mRNA levels (29%) similar to the ones observed in normal tissue. In multivariate analysis including known prognostic factors, low CD10 mRNA expression remained significant (HR = 2.25; [95%CI:1.24–4.09], p = 0.008), as did the recently revised Van Nuys Prognostic Index (VNPI) score (HR = 2.03; [95%CI:1.23–3.35], p = 0.006).

Conclusion

The decrease of CD10 expression in MECs is associated with a higher risk of relapse in DCIS; this knowledge has the potential to improve DCIS management.     

Simulations of morphotype-dependent hemodynamics in non-dilated bicuspid aortic valve aortas

Bicuspid aortic valves (BAVs) generate flow abnormalities that may promote aortopathy. While positive helix fraction (PHF) index, flow angle (θ), flow displacement (d) and wall shear stress (WSS) exhibit abnormalities in dilated BAV aortas, it is unclear whether those anomalies stem from the abnormal valve anatomy or the dilated aorta. Therefore, the objective of this study was to quantify the early impact of different BAV morphotypes on aorta hemodynamics prior to dilation. Fluid-structure interaction models were designed to quantify standard peak-systolic flow metrics and temporal WSS characteristics in a realistic non-dilated aorta connected to functional tricuspid aortic valve (TAV) and type-I BAVs. While BAVs generated increased helicity (PHF>0.68) in the middle ascending aorta (AA), larger systolic flow skewness (θ>11.2°) and displacement (d>6.8 mm) relative to the TAV (PHF=0.51; θ<5.5°; d<3.3 mm), no distinct pattern was observed between morphotypes. In contrast, WSS magnitude and directionality abnormalities were BAV morphotype- and site-dependent. Type-I BAVs subjected the AA convexity to peak-systolic WSS overloads (up to 1014% difference vs. TAV). While all BAVs increased WSS unidirectionality on the proximal AA relative to the TAV, the most significant abnormality was achieved by the BAV with left-right-coronary cusp fusion on the wall convexity (up to 0.26 decrease in oscillatory shear index vs. TAV). The results indicate the existence of strong hemodynamic abnormalities in non-dilated type-I BAV AAs, their colocalization with sites vulnerable to dilation and the superior specificity of WSS metrics over global hemodynamic metrics to the valve anatomy.     

Relationship between Circulating and Tissue microRNAs in a Murine Model of Breast Cancer

MiRNAs are key regulators of tumorigenesis that are aberrantly expressed in the circulation and tissue of patients with cancer. The aim of this study was to determine whether miRNA dysregulation in the circulation reflected similar changes in tumour tissue. Athymic nude mice (n = 20) received either a mammary fat pad (n = 8, MFP), or subcutaneous (n = 7, SC) injection of MDA-MB-231 cells. Controls received no tumour cells (n = 5). Tumour volume was monitored weekly and blood sampling performed at weeks 1, 3 and 6 following tumour induction (total n = 60). Animals were sacrificed at week 6 and tumour tissue (n = 15), lungs (n = 20) and enlarged lymph nodes (n = 3) harvested. MicroRNAs were extracted from all samples (n = 98) and relative expression quantified using RQ-PCR. MiR-221 expression was significantly increased in tumour compared to healthy tissue (p<0.001). MiR-10b expression was significantly higher in MFP compared to SC tumours (p<0.05), with the highest levels detected in diseased lymph nodes (p<0.05). MiR-10b was undetectable in the circulation, with no significant change in circulating miR-221 expression detected during disease progression. MiR-195 and miR-497 were significantly decreased in tumour tissue (p<0.05), and also in the circulation of animals 3 weeks following tumour induction (p<0.05). At both tissue and circulating level, a positive correlation was observed between miR-497 and miR-195 (r = 0.61, p<0.001; r = 0.41, p<0.01 respectively). This study highlights the distinct roles of miRNAs in circulation and tissue. It also implicates miRNAs in disease dissemination and progression, which may be important in systemic therapy and biomarker development.     

Fluid Dynamics of Coarctation of the Aorta and Effect of Bicuspid Aortic Valve

Up to 80% of patients with coarctation of the aorta (COA) have a bicuspid aortic valve (BAV). Patients with COA and BAV have elevated risks of aortic complications despite successful surgical repair. The development of such complications involves the interplay between the mechanical forces applied on the artery and the biological processes occurring at the cellular level. The focus of this study is on hemodynamic modifications induced in the aorta in the presence of a COA and a BAV. For this purpose, numerical investigations and magnetic resonance imaging measurements were conducted with different configurations: (1) normal: normal aorta and normal aortic valve; (2) isolated COA: aorta with COA (75% reduction by area) and normal aortic valve; (3) complex COA: aorta with the same severity of COA (75% reduction by area) and BAV. The results show that the coexistence of COA and BAV significantly alters blood flow in the aorta with a significant increase in the maximal velocity, secondary flow, pressure loss, time-averaged wall shear stress and oscillatory shear index downstream of the COA. These findings can contribute to a better understanding of why patients with complex COA have adverse outcome even following a successful surgery.     

Longitudinal Hemodynamic Measurements in Swine Heart Failure Using a Fully Implantable Telemetry System

Chronic monitoring of heart rate, blood pressure, and flow in conscious free-roaming large animals can offer considerable opportunity to understand the progression of cardiovascular diseases and can test new diagnostics and therapeutics. The objective of this study was to demonstrate the feasibility of chronic, simultaneous measurement of several hemodynamic parameters (left ventricular pressure, systemic pressure, blood flow velocity, and heart rate) using a totally implantable multichannel telemetry system in swine heart failure models. Two solid-state blood pressure sensors were inserted in the left ventricle and the descending aorta for pressure measurements. Two Doppler probes were placed around the left anterior descending (LAD) and the brachiocephalic arteries for blood flow velocity measurements. Electrocardiographic (ECG) electrodes were attached to the surface of the left ventricle to monitor heart rate. The telemeter body was implanted in the right side of the abdomen under the skin for approximately 4 to 6 weeks. The animals were subjected to various heart failure models, including volume overload (A-V fistula, n = 3), pressure overload (aortic banding, n = 2) and dilated cardiomyopathy (pacing-induced tachycardia, n = 3). Longitudinal changes in hemodynamics were monitored during the progression of the disease. In the pacing-induced tachycardia animals, the systemic blood pressure progressively decreased within the first 2 weeks and returned to baseline levels thereafter. In the aortic banding animals, the pressure progressively increased during the development of the disease. The pressure in the A-V fistula animals only showed a small increase during the first week and remained stable thereafter. The results demonstrated the ability of this telemetry system of long-term, simultaneous monitoring of blood flow, pressure and heart rate in heart failure models, which may offer significant utility for understanding cardiovascular disease progression and treatment.     

Identification of a DNA Methylome Profile of Esophageal Squamous Cell Carcinoma and Potential Plasma Epigenetic Biomarkers for Early Diagnosis

DNA methylation is a critical epigenetic mechanism involved in key cellular processes. Its deregulation has been linked to many human cancers including esophageal squamous cell carcinoma (ESCC). This study was designed to explore the whole methylation status of ESCC and to identify potential plasma biomarkers for early diagnosis. We used Infinium Methylation 450k array to analyze ESCC tissues (n = 4), paired normal surrounding tissues (n = 4) and normal mucosa from healthy individuals (n = 4), and combined these with gene expression data from the GEO database. One hundred and sixty eight genes had differentially methylated CpG sites in their promoter region and a gene expression pattern inverse to the direction of change in DNA methylation. These genes were involved in several cancer-related pathways. Three genes were validated in additional 42 ESCC tissues and paired normal surrounding tissues. The methylation frequency of EPB41L3, GPX3, and COL14A1 were higher in tumor tissues than in normal surrounding tissues (P<0.017). The higher methylation frequency of EPB41l3 was correlated with large tumor size (P = 0.044) and advanced pT tumor stage (P = 0.001). The higher methylation frequency of GPX3 and COL14A1 were correlated with advanced pN tumor stage (P = 0.001 and P<0.001). The methylation of EPB41L3, GPX3, and COL14A1 genes were only found in ESCC patients'' plasma, but not in normal individuals upon testing 42 ESCC patients and 50 healthy individuals. Diagnostic sensitivity was increased when methylation of any of the 3 genes were counted (64.3% sensitivity and 100% specificity). These differentially methylated genes in plasma may be used as biomarkers for early diagnosis of ESCC.     

The MRC1/CD68 Ratio Is Positively Associated with Adipose Tissue Lipogenesis and with Muscle Mitochondrial Gene Expression in Humans

Background

Alternative macrophages (M2) express the cluster differentiation (CD) 206 (MCR1) at high levels. Decreased M2 in adipose tissue is known to be associated with obesity and inflammation-related metabolic disturbances. Here we aimed to investigate MCR1 relative to CD68 (total macrophages) gene expression in association with adipogenic and mitochondrial genes, which were measured in human visceral [VWAT, n = 147] and subcutaneous adipose tissue [SWAT, n = 76] and in rectus abdominis muscle (n = 23). The effects of surgery-induced weight loss were also longitudinally evaluated (n = 6).

Results

MCR1 and CD68 gene expression levels were similar in VWAT and SWAT. A higher proportion of CD206 relative to total CD68 was present in subjects with less body fat and lower fasting glucose concentrations. The ratio MCR1/CD68was positively associated with IRS1gene expression and with the expression of lipogenic genes such as ACACA, FASN and THRSP, even after adjusting for BMI. The ratio MCR1/CD68 in SWAT increased significantly after the surgery-induced weight loss (+44.7%; p = 0.005) in parallel to the expression of adipogenic genes. In addition, SWAT MCR1/CD68ratio was significantly associated with muscle mitochondrial gene expression (PPARGC1A, TFAM and MT-CO3). AT CD206 was confirmed by immunohistochemistry to be specific of macrophages, especially abundant in crown-like structures.

Conclusion

A decreased ratio MCR1/CD68 is linked to adipose tissue and muscle mitochondrial dysfunction at least at the level of expression of adipogenic and mitochondrial genes.     

Cross-Species Comparison of Genes Related to Nutrient Sensing Mechanisms Expressed along the Intestine

Introduction

Intestinal chemosensory receptors and transporters are able to detect food-derived molecules and are involved in the modulation of gut hormone release. Gut hormones play an important role in the regulation of food intake and the control of gastrointestinal functioning. This mechanism is often referred to as “nutrient sensing”. Knowledge of the distribution of chemosensors along the intestinal tract is important to gain insight in nutrient detection and sensing, both pivotal processes for the regulation of food intake. However, most knowledge is derived from rodents, whereas studies in man and pig are limited, and cross-species comparisons are lacking.

Aim

To characterize and compare intestinal expression patterns of genes related to nutrient sensing in mice, pigs and humans.

Methods

Mucosal biopsy samples taken at six locations in human intestine (n = 40) were analyzed by qPCR. Intestinal scrapings from 14 locations in pigs (n = 6) and from 10 locations in mice (n = 4) were analyzed by qPCR and microarray, respectively. The gene expression of glucagon, cholecystokinin, peptide YY, glucagon-like peptide-1 receptor, taste receptor T1R3, sodium/glucose cotransporter, peptide transporter-1, GPR120, taste receptor T1R1, GPR119 and GPR93 was investigated. Partial least squares (PLS) modeling was used to compare the intestinal expression pattern between the three species.

Results and conclusion

The studied genes were found to display specific expression patterns along the intestinal tract. PLS analysis showed a high similarity between human, pig and mouse in the expression of genes related to nutrient sensing in the distal ileum, and between human and pig in the colon. The gene expression pattern was most deviating between the species in the proximal intestine. Our results give new insights in interspecies similarities and provide new leads for translational research and models aiming to modulate food intake processes in man.     

Spermine and Citrate as Metabolic Biomarkers for Assessing Prostate Cancer Aggressiveness

Separating indolent from aggressive prostate cancer is an important clinical challenge for identifying patients eligible for active surveillance, thereby reducing the risk of overtreatment. The purpose of this study was to assess prostate cancer aggressiveness by metabolic profiling of prostatectomy tissue and to identify specific metabolites as biomarkers for aggressiveness. Prostate tissue samples (n = 158, 48 patients) with a high cancer content (mean: 61.8%) were obtained using a new harvesting method, and metabolic profiles of samples representing different Gleason scores (GS) were acquired by high resolution magic angle spinning magnetic resonance spectroscopy (HR-MAS). Multivariate analysis (PLS, PLS-DA) and absolute quantification (LCModel) were used to examine the ability to predict cancer aggressiveness by comparing low grade (GS = 6, n = 30) and high grade (GS≥7, n = 81) cancer with normal adjacent tissue (n = 47). High grade cancer tissue was distinguished from low grade cancer tissue by decreased concentrations of spermine (p = 0.0044) and citrate (p = 7.73·10⁻⁴), and an increase in the clinically applied (total choline+creatine+polyamines)/citrate (CCP/C) ratio (p = 2.17·10⁻⁴). The metabolic profiles were significantly correlated to the GS obtained from each tissue sample (r = 0.71), and cancer tissue could be distinguished from normal tissue with sensitivity 86.9% and specificity 85.2%. Overall, our findings show that metabolic profiling can separate aggressive from indolent prostate cancer. This holds promise for the benefit of applying in vivo magnetic resonance spectroscopy (MRS) within clinical MR imaging investigations, and HR-MAS analysis of transrectal ultrasound-guided biopsies has a potential as an additional diagnostic tool.