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Objectives: Beta amyloid (beta-A) accumulation in brain is a driving force for Alzheimer's disease (AD) pathogenesis. Apolipoprotein E (ApoE) represents a critical player in beta-A homeostasis, but its role in AD progression is still controversial. We previously reported that Haptoglobin (Hpt), whose level increases during inflammation, binds ApoE and impairs apolipoprotein function in cholesterol homeostasis. Hpt was found associated with beta-A plaques, and shown to inhibit beta-A aggregation. The aim of this study was to evaluate whether the interaction between Hpt and ApoE affects the binding of ApoE to beta-A. Methods: ELISA, immunoprecipitation, and Western blotting, by using purified proteins or cerebrospinal fluid (CSF) were performed in this study. Results: We report here, for the first time, that Hpt forms SDS stable complexes with beta-A, and participates to the interaction between ApoE and beta-A, as shown by immunoprecipitating Hpt- betaA and Hpt- betaA -ApoE complexes from CSF. Furthermore Hpt acts as a positive effector by differently increasing the interaction of beta-A with ApoE2, ApoE3, or ApoE4 (ApoE2>ApoE3>ApoE4). Also, our results showed that the known Hpt activity in preventing oxidative modifications of ApoE is limited in the presence of beta Amyloid1-42. Conclusions: The interaction between Hpt and ApoE might represent a mechanism by which inflammation affects AD pathogenesis. We suggest that Hpt and ApoE functions in brain should be evaluated taking into account their interaction with beta-Amyloid.

Apolipoprotein A-I (ApoA-I), the major protein component of HDL, plays a key role in reverse cholesterol transport, mainly by stimulating the efflux of cholesterol and activating the enzyme LCAT. LCAT converts cholesterol into cholesteryl esters and addresses them to HDL for transport into the circulation. The binding of haptoglobin (Hpt) to ApoA-I is associated with inhibition of LCAT activity[1]. On the basis of the above information, high levels of Hpt, as occurring during the acute phase of inflammation[2], were suggested to be a major cause of both poor cholesterol removal from peripheral cells and low level of HDL cholesterol in the circulation. Previously, we showed that an ApoA-I mimetic peptide (P2a), with amino acid sequence overlapping the stimulatory site for LCAT, was effective in displacing Hpt from ApoA-I, and able to rescue in vitro the stimulatory function of ApoA-I in the presence of high Hpt levels[3]. In this work we characterized the biological activity of peptide P2a in an experimental model of inflammation. In particular, we verified the ability of peptide P2a to rescue LCAT dependent cholesterol esterification in vivo in presence of high level of Hpt. References 1. Balestrieri, M., Cigliano, L., De Simone, M.L., Dale, B., Abrescia, P. Mol. Reprod. Dev. 59, 186 (2001) 2. Langlois MR & Delanghe JR (1996) Biological and clinical significance of haptoglobin polymorphism in humans. Clin Chem 42,1589-1600. 3. Spagnuolo, M.S., Cigliano, L., D’Andrea, L.D., Pedone, C. and Abrescia, P. (2005) Assignment of the binding site for Haptoglobin on Apolipoprotein A-I. J. Biol. Chem. 280, 1193-1198.

Haptoglobin is an acute phase glycoprotein, secreted by hepatocytes and other types of cells including keratinocytes. Haptoglobin has been suggested to impair the immune response, inhibit gelatinases in the extracellular matrix and promote angiogenesis, but its role in psoriasis is obscure to date. Changes in haptoglobin glycan structure were observed in several diseases. The aim of our study was to investigate whether haptoglobin displays glycan variations in psoriasis. We found that the pattern of plasma haptoglobin glycoforms, following two-dimensional electrophoresis, exhibited significant quantitative differences in spot intensities between patients and controls. Quantitative and qualitative differences in glycan mass, between patients and controls, were found by mass spectrometry of glycopeptides from tryptic digests of protein isolated from both patients and controls. The number of distinct fucosylated glycoforms of peptides NLFLNHSENATAK and MVSHHNLTTGATLINEQWLLTTAK was higher in patients than in controls, but no fucosylated glycan was detected on peptide VVLHPNYSQVDIGLIK in either case. The number of peptides with distinct tri- and tetra-antennary glycans was higher in patients than in controls. Abundance or structure of specific glycans, which are present in haptoglobin from patients and different or missing in normal haptoglobin, might be associated to the disease activity.

Alteration of cholesterol (Ch) homeostasis is involved in the pathogenesis of neurodegeneration. Astrocyte-derived lecithin cholesterol acyltransferase (LCAT) was recently shown capable of esterifying Ch on ApoE-lipoproteins. In order to prevent Ch excess in neurons, it can be converted in 24S-hydroxycholesterol (24-OH Ch). Since this cerebrosterol can cross the brain blood barrier, 24-OH Ch is thought to be a major form of Ch for egress from the brain. 24-OH Ch is known to be produced almost exclusively in the brain, and was so far only suggested to be LCAT substrate in order to explain why most of it can be detected in esterified form. Here we report the first experimental evidence that LCAT is capable of esterifying 24-OH Ch in cerebrospinal fluid (CSF). This result comes from experiments of competition between 24-OH Ch and Ch for LCAT and experiments of in vitro production of 24-OH Ch esters. In detail, increasing amounts of 24-OH Ch, in a standard assay of LCAT activity, decreased Ch esterification. Also, purified 24-OH Ch was incubated in a mixture containing CSF as LCAT source, ApoE, and a fluorescent phospholipid (as acyl chain donor). 24-OH Ch esters level, detected by fluorescence, was found increased in time. So, it can be excluded that 24-OH Ch esters found in CSF are produced by ACAT, and released by necrotic cells. Further experiments are required to shed light on how LCAT function on 24-OH Ch may influence brain Ch homeostasis and recovery from neuronal injury.

MHC class II molecules are cell-surface glycoproteins that play a central role in the adaptive immune system because they present peptides, derived mainly from extracellular proteins, to the antigen receptor of CD4+ T cells. MHC class II expression is regulated in a precise cell-type specific manner and restricted to thymic epithelial cells (TECs) and to the antigen-presenting cells (APCs), including B cells, cells of the monocyte–macrophage lineage and dendritic cells (DCs). Other cells such as fibroblasts, astrocytes, endothelial cells and epithelial cells, do not express MHC class II molecules unless they are exposed to specific stimuli, particularly IFN-g, that are produced during infection, inflammation or trauma. MHC class II molecules include different isotypes (HLA-DR, HLA-DP and HLA-DQ) composed of two non-covalently associated polypeptides, alfa and beta chains encoded by separate genes, which expression is co-ordinately controlled at level of transcription. Our results indicated that both 3’UTRs of HLA-DRA and HLA-DQA1 mRNA bind the same cytoplasmic complex. procedure we have identified Ebp1 and DRBP76/NF90 as components of this complex by biochemical purification. Both of them are able to bind dsRNAs, indicating that a secondary structure is shared by MHCII mRNAs. The knockdown of these proteins showed a decrease in the MHCII mRNAs accumulation, demonstrating that they are involved in the stabilization of messengers. Moreover, literature data indicated that both proteins could have a role also in the regulation of translation. Ebp1, found in the nucleoli and associated with polysomes, could represent a link between ribosome biosynthesis and cell proliferation. Its interaction with MHCII mRNAs, resulting in a modulation of the MHCII surface density, could give us clarifications about the antigen presentation in tumor cells. DRBP76/NF90 is also involved in mechanisms of stabilization and protein translation of different messengers but it is especially implicated in the modulation of IL-2 mRNA during T cell activation, occurring during the immune response. We hypothesize, according to the ”RNA operon” model that our mRNAs, encoding functionally related proteins, are co-ordinately regulated by their dsRNA structures in the 3’UTR, through the interaction with RNA-binding proteins. These complexes, immediately after transcription, arrange the mRNAs splicing, export, stability and translation to guarantee an equilibrium in the heterodimer synthesis. Our future aim will be to clarify the link between MHCII post transcriptional regulation and the function of antigen presentation of these molecules, both in professional antigen presenting cells and in tumor cells.

Aims: Apolipoprotein A-I (ApoA-I), the major apolipoprotein of the high density lipoprotein (HDL), plays key roles in the removal of cholesterol excess from peripheral cells, in a recognized anti-atherosclerotic process called “reverse cholesterol transport” (RTC)a. In detail, it stimulates cholesterol (C) efflux and ApoE secretion from macrophages, and enhances Lecithin-Cholesterol Acyl-Transferase (LCAT) activity. The acute phase protein Haptoglobin (Hpt) was found to inhibit RCT, as binding ApoA-I and preventing its stimulation on LCAT. This study aimed to find out whether Hpt influences essential ApoA-I stimulated mechanisms in RCT, such as cholesterol efflux, LCAT activity, and ApoE secretion from macrophages. Methods: Monocytes were isolated from human buffy coats, and kept for 6 days in culture for differentiation into macrophages. After treatment with ³H-C for 24 h, the cells were incubated with ApoA-I (0.04 mg/mL), or ApoA-I plus increasing Hpt amounts (0.1-0.75 mg/mL). After 16 h of incubation, homologous plasma was added as LCAT source. After further incubation for 16 h, supernatants were aliquoted from culture medium to measure their radioactivity content, representing the released C. Supernatant lipids were extracted and separated by TLC to measure the LCAT activity as conversion of unesterified ³H-C into C esters. Other macrophages, not treated with ³H-C, were incubated with ApoA-I (20 µg/mL), or ApoA-I plus Hpt (200 µg/mL). The supernatants of these cultures were collected after 16 h, and analyzed by western blotting to detect the amount of secreted ApoE. Results: Addition of Hpt to macrophages significantly (P

Objective: The aim of this work was to assess whether psoriasis is associated with phenotype prevalence and altered activity of Haptoglobin (Hpt). Background: Hpt is a plasma acute phase glycoprotein, displaying in humans three phenotypes. Phenotype prevalence or structure modification of Hpt was associated with several diseases. The Hpt main function is to bind and carry to the liver free hemoglobin for degradation and iron recycling. Hpt was recently found able to bind the apolipoprotein A-I (ApoA-I), thus impairing its stimulation on the activity of the enzyme lecithin-cholesterol acyl-transferase (LCAT) Study design: Hpt was isolated from patients with psoriasis vulgaris, and its activity in hemoglobin or ApoA-I binding and LCAT inhibition was compared with that of normal protein. Methods: Two affinity chromatography steps, the first using resin-coupled hemoglobin and the second anti-Hpt antibodies, were used to purify Hpt. The protein phenotype was assessed by electrophoresis. Binding experiments were performed by ELISA with stationary hemoglobin or ApoA-I, Hpt in solution, and anti-Hpt antibodies for detection of bound Hpt. Standard LCAT assays were carried out in the presence of Hpt purified from patients or healthy subjects. Results: Phenotype prevalence of Hpt in psoriasis was not found. After affinity chromatography by hemoglobin, albumin and ApoA-I were routinely found heavily contaminating only Hpt from normal subjects. Isolated Hpt from patients had lower activity than normal protein in both hemoglobin binding and LCAT inhibition. Conclusions: In psoriasis, Hpt displays some structure modification(s) which might be associated with the protein function in the disease.

AIMS: Haptoglobin (Hpt) is a plasma acute phase glycoprotein produced mainly in the liver, but also synthesized in extrahepatic tissues including skin. The main function of circulating Hpt is to bind free haemoglobin (Hb) for scavenging iron, and Apolipoprotein A1 (ApoA1) for regulating the cholesterol reverse transport. The function of extrahepatic Hpt is not yet understood. Information on disease-associated isoforms of tissue-specific Hpt is available though poor. The aim of this work was to asses whether Hpt, in psoriasis, displays activity alteration which might be related to structure modification. METHODS: Two affinity chromatography steps, using resin-coupled Hb or anti-Hpt antibodies, were carried out to purify Hpt from human plasma. ELISA with Hb-coated wells was used to assay Hpt binding. After incubation of 0.05, 0.1, 0.2 or 0.3 μM Hpt, bound Hpt was detected by anti-Hpt antibodies. Assays of Lecithin-Cholesterol Acyl Transferase (LCAT) 1, the enzyme which esterifies cholesterol under ApoA1 stimulation, were carried out with Hpt purified from psoriatic patients or healthy subjects. RESULTS: Hpt from patients was found less efficient than control Hpt in Hb binding (30 to 60%). In particular, significant differences were found with 0.1, 0.2 or 0.3 μM Hpt (P

AIMS: Apolipoprotein A1 (ApoA-I) is the major component of HDL and play crucial roles in all steps of RTC. ApoA-I is susceptible to oxidation which contribute to reduce its cardioprotective effects. It has been reported that hypoclorite can affect ApoA-I structure and the ability of this lipoprotein to promote ABCA1-dependent cholesterol efflux. The effects of Superoxide, a potent oxidant produced in blood vessel, on ApoA-I structure and function have never been reported. The aims of this work was to asses if the ApoA-I function to stimulate the cholesterol esterifying enzime (LCAT) was impaired by incubation with superoxide. Furthermore the Apo-A-I funtion to bind Haptoglobin, (Hpt) a plasma glicoprotein, was tested after incubation by superoxide. METHODS: The superoxide production was monitored by measuring the absorbance at 734 nm of a reaction mixture containing several concentration (10 50 100 µM) of KO2 and ABTS 450 µM and kept at 31°C. The binding experiments were performed by ELISA with coated ApoA-I, several concentration of KO2 (10 50 100 µM) was added, after Hpt in solution and anti-Hpt antibodies was used for detection of bound Hpt. Standard LCAT assay were carried out as published 1.ApoA-I-containing liposomes was incubated for 1h at 37°C in the presence and absence of superoxide. RESULTS: After affinity chromatography, in ELISA experiments, Hpt from patients binds less efficiently Hb than Hpt from controls. In particular, significant differences were found for samples with 0.1, 0.2 or 0.3µM Hpt (P

Aim: Haptoglobin (Hpt) was previously found to inhibit reverse cholesterol transport (RCT), as binding ApoA-I and preventing its stimulation of both cholesterol (C) efflux and Lecithin-Cholesterol Acyl-Transferase (LCAT). This study aimed to find out whether Hpt influences RCT from macrophages differentiated from primary culture of human monocytes. Methods: Monocytes were isolated from human buffy coats, and kept for 6 days in culture for differentiation into macrophages. After treatment with ³H-C for 24 h, the cells were incubated with ApoA-I (0.04 mg/mL), or ApoA-I plus increasing Hpt amounts (0.1-0.75 mg/mL). After 16 h of incubation, homologous plasma was added as LCAT source. After further incubation for 16 h, supernatants were aliquoted from culture medium to measure their radioactivity content, representing the released C. Supernatant lipids were extracted and separated by TLC to measure the LCAT activity as conversion of unesterified ³H-C into C esters. Results: Addition of Hpt increasing amounts to macrophages significantly inhibited both ApoA-I dependent stimulation of C efflux (down to 27% of the control value) and LCAT activity (down to 50% of the control value). The changes were significant (P

S-nitrosothiols act as carrier and reservoir of nitric oxide (NO), and release NO under stimulation of Ascorbate (Asc). Erythrocyte can regenerate Asc from its oxidised products, thus saving this powerful antioxidant. In this paper the effect of donors of NO, superoxide and peroxynitrite (SpNONOate, KO2, and SIN-1, respectively) on the erythrocyte production of Asc was investigated. We report here that NO stimulated, while superoxide and peroxynitrite decreased, the Asc recycling. The NO-stimulating effect on the erythrocyte production of Asc was confirmed by using GSNO, a natural occurring S-nitrosothiol, as NO donor. These data highlight a new property of NO, that is the stimulation of erythrocytes for their Asc recycling. Such a property might contribute to regenerate Asc from its oxidised forms, thus preventing its depletion in the circulation. Temperature and pH significantly affected, both in absence and presence of NO, the recycling of Asc by erythrocytes. We propose that a positive feedback, involving the reciprocal stimulation between Asc and S-nitrosothiols, might enhance productions of Asc by erythrocytes and NO release by circulating S-nitrosothiols.

INTRODUCTION: ApoA-1, the major apolipoprotein of the high density lipoprotein (HDL), plays a key role in the removal of cholesterol excess from peripheral cells in a recognized anti-atherosclerotic process called “reverse cholesterol transport (RCT)”. Among known ApoA-1 variants two, with one amino acid change (Zavalla, L159P; Zaragoza, L144R), are associated to poor RCT. Both mutations are located within the domain of ApoA1 (Leu141 – Ala164) binding Haptoglobin (Hpt) 1, a protein of the acute phase of inflammation. Hpt binding to ApoA1 was suggested to impair the role of HDL in RCT1. AIMS: Our aims were to study whether peptides containing the mutations of Zavalla (Pep Zav) or Zaragoza (Pep Zara) are able to bind Hpt or compete with native ApoA1, exposed on HDL, for binding Hpt. METHODS: The binding of biotinylated peptides to Hpt and the experiments of competition of acetylated peptides with HDL for Hpt binding were performed by ELISA, as previously published1. RESULTS: Both Pep-Zav and Pep-Zara bound to Hpt with efficiency similar to that of the peptide harbouring the native sequence of ApoA1 (pep 141–164). Nevertheless, when the same peptides were analysed for their ability to influence Hpt binding to HDL, only Pep Zav effectively displaced (50% at 15μM) Hpt from HDL (fig), as was the case for the pep. 141-164. CONCLUSIONS: We suggest that the L>P conversion, in the Zavalla variant, enhances hydrophilicity in ApoA1, improving the binding to Hpt. Therefore, as ApoA-1 binding to Hpt negatively affects RCT, individuals with Zavalla mutation might be exposed to higher risk of cardiovascular disease. 1.Spagnuolo M.S. et al (2005) J. Biol. Chem. 280, 1193-1198

The present invention relates to methods for identifying agents modulating reverse cholesterol transport or metab. of steroid mols. for treatment of cardiovascular disease, ovarian follicle dysfunction and other metabolic diseases. Haptoglobin (Hpt) was previously found to bind the high d. lipoprotein (HDL) apolipoprotein A-I (ApoA-I) and able to inhibit the ApoA-I-dependent activity of the enzyme lecithin:cholesterol acyltransferase (LCAT), which plays a major role in the reverse cholesterol transport. The ApoA-I sequence from Glu113 to Asn184 harbored the binding site for Hpt. Biotinylated peptides were synthesized overlapping such a sequence, and their Hpt binding activity was detd. by avidin-linked peroxidase. The highest activity was exhibited by the peptide P2a, contg. the ApoA-I sequence from Leu141 to Ala164. Such a sequence contains an ApoA-I domain required for binding cells, promoting cholesterol efflux, and stimulating LCAT. The peptide P2a effectively prevented both binding of Hpt to HDL-coated plastic wells and Hpt-dependent inhibition of LCAT, measured by anti-Hpt antibodies and cholesterol esterification activity, resp. The enzyme activity was not influenced, in the absence of Hpt, by P2a. Differently from ApoA-I or HDL, the peptide did not compete with Hb for Hpt binding in ELISA expts. The results suggest that Hpt might mask the ApoA-I domain required for LCAT stimulation, thus impairing the HDL function. Synthetic peptides, able to displace Hpt from ApoA-I without altering its property of binding Hb, might be used for treatment of diseases assocd. with defective LCAT function.

Aim: Cholesterol supplemented diet, in rabbit, triggers severe hypercholesterolemia which rapidly (less than 2 months) evolves to atherosclerosis. This work aimed to ascertain whether plasma factors playing key roles in the reverse cholesterol transport, such as the activity of the enzyme LCAT and the level of the apolipoprotein (Apo) A-I, are altered during the atherosclerosis rising and progression. Methods: New Zealand rabbits were fed with normal (N=6) or 2% cholesterol enriched (N=6) diet. Serum was collected at 0, 4 and 8 weeks, and analysed for Apo A-I level (by ELISA) and LCAT activity in vitro (assaying free 3H-cholesterol conversion into ester form) or in vivo (measuring by HPLC the amounts of both free and ester forms from isolated HDL). Results: Apo A1 was found decreased in the treated animals (t=4w: 65.5±3.4 vs 95.7±3.6 µg/ml in controls, P

E' stato studiato l'effetto della supplementazione della dieta con silimarina sullo stato redox plasmatico di capre in lattazione. Sono state evidenziate correlazioni positive tra i livelli di antiossidanti plasmatici e lo stato redox del latte.

Glucose-6-phosphate dehydrogenase-deleted embryonic stem (ES) cells (G6pdD) proliferate in vitro without special requirements, but when challenged with oxidants fail to sustain glutathione disulphide reconversion to reduced glutathione (GSH), entering a condition of oxidative stress. Here, we investigate the signalling events downstream of GSH oxidation in G6pdD and wild-type (wt) ES cells. We found that G6pdD ES cells are very sensitive to oxidants, activating an apoptotic pathway at oxidant concentrations otherwise sublethal for wt ES cells. We show that the apoptotic pathway activated by low oxidant concentrations is accompanied by mitochondria dysfunction, and it is therefore blocked by the overexpression of Bcl-XL. Bcl-XL does not inhibit the decrease in cellular GSH and reactive oxygen species formation following oxidant treatment. We also found that oxidant treatment in ES cells is followed by the activation of the MEK/ extracellular signal-regulated kinase (ERK) pathway. Interest¬ingly, ERK activation has opposite outcomes in G6pdD ES cells compared to wt, which has a proapoptotic function in the first and a prosurvival function in the latter. We show that this phenomenon can be regulated by the cellular GSH level.

Haptoglobin (Hp) can be purified by affinity chromatography using hemoglobin(Hb)-linked Sepharose. Elution with 8 M urea is generally performed, resulting in Hp heavy contamination by the apolipoprotein AI (ApoAI), and partial loss of Hb binding activity. Hp, separated from ApoAI, was recovered by elution with glycine-HCl at pH 3. Complexes of the isolated protein with Hb or ApoAI were detected by enzyme-linked immunosorbent assay (ELISA). Competition between the two ligands in their interaction with Hp was observed. Concanavalin A (ConA), which binds the Hp carbohydrate chains, did not influence the Hp binding to ApoAI. The results suggest that changes in the plasma level of ApoAI or Hb affect the Hp role in regulating the cholesterol reverse transport or preventing Hb-dependent oxidative damages.

During exercise reactive oxygen species are produced. The antioxidants prevent or limit tissue damages by these species in physiological conditions. In particular, ascorbate and urate scavenge peroxynitrite, which can alter the function of many molecules, including the lecithin-cholesterol acyltransferase (LCAT) enzyme involved in reverse cholesterol transport. The aims of the present study were to compare the plasma antioxidant response to an ergometric test (ET) in hypertensive and healthy subjects, evaluate the exercise dependent nitrosative stress in plasma, and assess whether the LCAT activity is altered by the exercise. Plasma samples, prepared before and after ET from hypertensive or healthy volunteers, were analysed for their levels of Ascorbate, Urate, -Tocopherol, Retinol, nitrotyrosine, and LCAT activity. The -Tocopherol and Retinol levels did not significantly change in both groups during exercise, while the Ascorbate level changed displaying higher increase in controls (+38.8%) than in hypertensives (+17.2%). In these patients, during ET, the Urate and nitrotyrosine levels changed more than in normotensives (+13.5% and +40.6% versus –3.1% and +25.2% respectively). The antioxidants effectively prevented loss or reduction of LCAT activity, as it was similar in hypertensives and normotensives, and did not change after ET. Conclusions: The results demonstrate that exercise is associated with enhanced protein nitrosation, and suggest that the Ascorbate or Urate levels increase to limit oxidative damage.

Mouse embryonic stem (ES) glucose-6-phosphate (G6P) dehydrogenase-deleted cells (G6pd Delta), obtained by transient Cre recombinase expression in a G6pd-loxed cell line, are unable to produce G6P dehydrogenase (G6PD) protein (EC 1.1.1.42). These G6pd Delta cells proliferate in vitro without special requirements but are extremely sensitive to oxidative stress. Under normal growth conditions, ES G6pd Delta cells show a high ratio of NADPH to NADP+ and a normal intracellular level of GSH. In the presence of the thiol scavenger oxidant, azodicarboxylic acid bis[dimethylamide], at concentrations lethal for G6pd Delta but not for wild-type ES cells, NADPH and GSH in G6pd Delta cells dramatically shift to their oxidized forms. In contrast, wild-type ES cells are able to increase rapidly and intensely the activity of the pentose-phosphate pathway in response to the oxidant. This process, mediated by the [NADPH]/[NADP+] ratio, does not occur in G6pd Delta cells. G6PD has been generally considered essential for providing NADPH-reducing power. We now find that other reactions provide the cell with a large fraction of NADPH under non-stress conditions, whereas G6PD is the only NADPH-producing enzyme activated in response to oxidative stress, which can act as a guardian of the cell redox potential. Moreover, bacterial G6PD can substitute for the human enzyme, strongly suggesting that a relatively simple mechanism of enzyme kinetics underlies this phenomenon.