The adsorption of copper(II) ions on to dehydrated wheat bran (DWB): determination of the equilibrium and thermodynamic parameters

The adsorption of copper(II) ions on to dehydrated wheat bran (DWB), a by-product of the flour process, was investigated as a function of initial pH, temperature, initial metal ion concentration and adsorbent dosage. The optimum adsorption conditions were initial pH 5.0, initial copper concentration 100 mg l⁻¹, temperature 60 °C and adsorbent dosage 0.1 g. The adsorption equilibrium was described well by the Langmuir isotherm model with maximum adsorption capacity of 51.5 mg g⁻¹ of copper(II) ions on DWB. The observation of an increase in adsorption with increasing temperature leads to the result that the adsorption of copper(II) ions on DWB is endothermic in nature. The thermodynamic parameters such as enthalpy, free energy and entropy changes were calculated and these values show that the copper(II)-DWB adsorption process was favoured at high temperatures.     

Genetically encoded red fluorescent copper(I) sensors for cellular copper(I) imaging

Copper ranks among the most important metal ions in living organism, owing to its key catalytic effect in a range of biochemical processes. Dysregulation of in vivo copper(I) metabolism is extremely toxic and would cause serious diseases in human, such as Wilson’s and Menkes. Thus, it would be highly valuable to have a proper approach to monitor the dynamics of copper(I) in vivo, as it is directly related to the onset of human copper(I)-related diseases. Under these circumstance, developing fluorescent protein based copper(I) sensors is highly demanded. However, these established sensors are mostly based on green or yellow FPs. Fluorescent copper(I) sensors with a spectra in the red range are more desirable due to lower phototoxicity, less auto-fluorescent noise and better penetration of red light. In the present work, we grafted a special red FP into three different location of a copper(I) binding protein, and generate a series of red fluorescent copper(I) sensors. Despite their limited in vivo sensitivity toward copper(I), these sensors are viable for cellular copper(I) imaging. Furthermore, these red fluorescent copper(I) sensors are a good starting point to develop superior copper(I) biosensors capable of imaging copper(I) fluctuations within a truly biologically relevant concentration, and further effort to realize this endeavor is under way.     

Mechanism of Copper Uptake from Blood Plasma Ceruloplasmin by Mammalian Cells

Ceruloplasmin, the main copper binding protein in blood plasma, has been of particular interest for its role in efflux of iron from cells, but has additional functions. Here we tested the hypothesis that it releases its copper for cell uptake by interacting with a cell surface reductase and transporters, producing apoceruloplasmin. Uptake and transepithelial transport of copper from ceruloplasmin was demonstrated with mammary epithelial cell monolayers (PMC42) with tight junctions grown in bicameral chambers, and purified human ⁶⁴Cu-labeled ceruloplasmin secreted by HepG2 cells. Monolayers took up virtually all the ⁶⁴Cu over 16h and secreted half into the apical (milk) fluid. This was partly inhibited by Ag(I). The ⁶⁴Cu in ceruloplasmin purified from plasma of ⁶⁴Cu-injected mice accumulated linearly in mouse embryonic fibroblasts (MEFs) over 3-6h. Rates were somewhat higher in Ctr1+/+ versus Ctr1-/- cells, and 3-fold lower at 2°C. The ceruloplasmin-derived ⁶⁴Cu could not be removed by extensive washing or trypsin treatment, and most was recovered in the cytosol. Actual cell copper (determined by furnace atomic absorption) increased markedly upon 24h exposure to holoceruloplasmin. This was accompanied by a conversion of holo to apoceruloplasmin in the culture medium and did not occur during incubation in the absence of cells. Four different endocytosis inhibitors failed to prevent ⁶⁴Cu uptake from ceruloplasmin. High concentrations of non-radioactive Cu(II)- or Fe(III)-NTA (substrates for cell surface reductases), or Cu(I)-NTA (to compete for transporter uptake) almost eliminated uptake of ⁶⁴Cu from ceruloplasmin. MEFs had cell surface reductase activity and expressed Steap 2 (but not Steaps 3 and 4 or dCytB). However, six-day siRNA treatment was insufficient to reduce activity or uptake. We conclude that ceruloplasmin is a circulating copper transport protein that may interact with Steap2 on the cell surface, forming apoceruloplasmin, and Cu(I) that enters cells through CTR1 and an unknown copper uptake transporter.     

The reversible depletion and reconstitution of a copper ion in Coprinus cinereus laccase followed by spectroscopic techniques

The specific activities of crude and purified Coprinus cinereus laccase preparations could be enhanced by a factor of 10-12 by activation with copper ions. The copper to protein contents of purified non-activated laccase were 2.3 ± 0.1 compared to 3.3 ± 0.1 in purified activated laccase indicating that only a fraction of the laccase can be activated. Purified laccase not activated with copper ions shows in isoelectric focusing four bands in order of decreasing pI in a ratio 1/5/3/1 where only bands I and II had laccase activity. Purified activated laccase showed only three bands (I, II and III) in the ratio 5/4/1 all with some laccase activity. The pH profile of the activity for activated and non-activated laccase showed identical behavior indicating that the active forms were the same. The change in UV-Vis around 330 nm following the depletion and reconstitution of the enzyme combined with activity measurements supports the reversibility of the selective removal and insertion of copper ions at the type 2 site. The circular dichroism spectrum of activated purified laccase has characteristic changes around 350 nm relative to non-activated laccase indicative of changes at the type 2/type 3 sites. The difference between the electron paramagnetic resonance spectra of non-activated and activated C. cinereus laccase indicates that a fraction of the non-activated purified laccase contained a copper(II) signal with a coupling constant between a type 1 and a type 2 copper(II). This electron paramagnetic resonance signal could be explained by an induced asymmetry in the type 3 site due to a missing type 2 copper ion.     

A comparative study of the transport dynamics of the peptide moiety of the milk ceruloplasmin molecule in the body of rats with embryonic and adult types of copper metabolism

In chase experiments, we followed the distribution of [125I]-ceruloplasmin prepared from human breast milk orally administered to young rats. Experiments were conducted using six-day-old rat pups (the embryonic type of copper metabolism) or 35-day-old ones (the adult type of copper metabolism). Using the technique of rocket immunoelectrophoresis, we have demonstrated that in six-day-old rats [125I]-ceruloplasmin was transferred from the gastrointestinal tract to the bloodstream and could be detected there over a period of 4 h. In 35-day-old rats, milk ceruloplasmin was digested in the upper part of the intestinal tract. The dynamic aspects of the distribution of labeled milk ceruloplasmin in the body of six-day old rats over a period of 4 h point out that, under the conditions of embryonic copper metabolism, it can serve as a transporter of copper ions to extrahepatic organs. We discuss the role of milk ceruloplasmin in copper metabolism in mammals during the neonatal period.     

A His-tag based immobilization method for the preparation and reconstitution of apoflavoproteins

The NifL PAS domain from Azotobacter vinelandii is a flavoprotein with FAD as the prosthetic group. Here we describe a novel immobilization procedure for the large-scale preparation of apo NifL PAS domain and its efficient reconstitution with either 2,4a-13C-FAD or 2,4a-13C-FMN. In this procedure, the His-tagged holoprotein is bound to an immobilized metal affinity column and the flavin is released by washing the column with buffer containing 2 M KBr and 2 M urea. The apoprotein is reconstituted on-column with the (artificial) flavin cofactor, and then eluted with buffer containing 250 mM imidazole. Alternatively, the immobilized apoprotein can be released from the column matrix before reconstitution.The His-tag based immobilization method of preparing reconstituted (or apo) NifL PAS domain protein has the advantage that it combines a protein affinity chromatography technique with limited protein loss, resulting in a high protein yield with extremely efficient flavin reconstitution. This on-column reconstitution method can also be used in cases where the apoprotein is unstable. Therefore, it may develop as a universal method for replacement of flavin or other cofactors.     

Discrete roles of copper ions in chemical unfolding of human ceruloplasmin

Human ceruloplasmin (CP) is a multicopper oxidase essential for normal iron homeostasis. The protein has six beta-barrel domains with one type 1 copper in each of domains 2, 4, and 6; the remaining copper ions form a catalytic trinuclear cluster, one type 2 and two type 3 coppers, at the interface between domains 1 and 6. We have characterized urea-induced unfolding of holo- and apo-forms of CP by far-UV circular dichroism, intrinsic fluorescence, 8-anilinonaphthalene-1-sulfonic acid binding, visible absorption, copper content, and oxidase activity probes (pH 7, 23 degrees C). We find that holo-CP unfolds in a complex reaction with at least one intermediate. The formation of the intermediate correlates with decreased secondary structure, exposure of aromatics, loss of two coppers, and reduced oxidase activity; this step is reversible, indicating that the trinuclear cluster remains intact. Further additions of urea trigger complete protein unfolding and loss of all coppers. Attempts to refold this species result in an inactive apoprotein with molten-globule characteristics. The apo-form of CP also unfolds in a multistep reaction, albeit the intermediate appears at a slightly lower urea concentration. Again, correct refolding is possible from the intermediate but not the unfolded state. Our study demonstrates that in vitro equilibrium unfolding of CP involves intermediates and that the copper ions are removed in stages. When the catalytic site is finally destroyed, refolding is not possible at neutral pH. This implies a mechanistic role for the trinuclear metal cluster as a nucleation point, aligning domains 1 and 6, during CP folding in vivo.     

Assessment of copper,iron, zinc and manganese status and speciation in patients with Parkinson’s disease: A pilot study

BackgroundThe objective of this pilot study was to assess iron (Fe), copper (Cu), zinc (Zn), and manganese (Mn) status (hair, serum, and urine) and speciation (serum) in Parkinson’s disease (PD) patients.MethodsA pilot study involving a total of 27 subjects (13 PD patients, 14 controls) was performed. Serum, urine, and hair metal content was assessed using ICP-MS. Speciation analysis of Cu, Zn, Fe, and Mn was performed using a hybrid HPLC-ICP-MS system.ResultsGroup comparisons did not reveal any significant group difference in serum Cu, Zn, Fe, and Mn total metal level between PD patients and controls. Speciation analysis revealed a significant decrease in Cu/ceruloplasmin copper in association with elevation of low-molecular weight species (amino acids)-bound copper. It is proposed that in PD, binding of Cu(II) ions to ceruloplasmin is reduced and free copper ions coordinate with low molecular weight ligands. The level of Mn-albumin complexes in PD patients was more than 4-fold higher as compared to the respective value in the control group. The observed difference may be considered as a marker of redistribution between high and low molecular weight ligands.ConclusionsMetal speciation is significantly affected in serum of PD-patients. These findings are indicative of the potential role of metal metabolism and PD pathogenesis, although the exact mechanisms of such associations require further detailed studies.     

The role of metals in the enzymatic and nonenzymatic oxidation of epinephrine

The effects of transition metals on nonenzymatic and ceruloplasmin catalyzed epinephrine oxidation were investigated by studying rates of epinephrine oxidation in purified buffers and in the presence of metal chelating agents. We found that epinephrine does not “autoxidize” in sodium chloride solutions prepared with deionized water that was further purified by chromatography over Chelex 100 resin prior to use. Epinephrine was oxidized rapidly in sodium chloride prepared with tap water (1.20±0.12 nmoles/min) or in deionized water (0.40±0.80 nmoles/min), but this oxidation was prevented by the addition of Desferal, a potent metal chelating agent. Epinephrine oxidation was enhanced upon the addition of ceruloplasmin, and this oxidation rate could be slowed, but not eliminated, by the addition of Desferal. If epinephrine solutions were preincubated for 72 hours with Desferal prior to ceruloplasmin addition, however, no oxidation was observed. Epinephrine was shown to form colored complexes with both iron and copper at pH 7.0. The Fe(III)-epinephrine complex was much more stable than was the Cu(II)-epinephrine complex. Oxygen consumption studies of ceruloplasmin catalyzed epinephrine oxidation showed that copper was a better promoter of epinephrine oxidation than was iron, suggesting that ceruloplasmin-catalyzed epinephrine oxidation results from adventitious copper bound to the purified enzyme. In light of these results, the physiological relevance of ceruloplasmin catalyzed oxidation of biogenic amines may be minor.     

Complexes of copper(II) dihalide with 2,2′-dipyridylamine. X-ray diffraction structures of the [dibromo-bis(dipyam)copper(II)]–water (1:2) and di[chloro-bis(dipyam)copper(II)]diiodide–acetonitrile (1:2) complexes

The formation of complexes between copper(II) halides and 2,2′-dipyridylamine (dipyam) has been studied systematically. Only complexes with a 1:1 and 1:2 metal-to-ligand ratio are formed. Some mixed chloro–iodide and halide–PF₆ compounds have also been isolated. The X-ray diffraction structures of the [Cu(dipyam)₂Br₂] · 2H₂O (I) and the [Cu(dipyam)₂Cl]₂I₂ · 2CH₃CN (II) complexes are reported. I is a rare example of an octahedral coordination among the copper(II) halide complexes of dipyam. The two bromo atoms, which occupy the apical positions, are H-bonded to the water molecules of crystallization. II is a dimer, where each copper forms a cationic chloro-complex of approximately trigonal bipyramidal geometry, the dimerization being due to hydrogen bonds formed by the NH group of one of the two dipyams coordinated to each metal atom with the chlorine atom of the centrosymmetric cationic complex. The iodide anions are hydrogen-bonded to the NH groups of the dipyams not involved in the dimerization.     

Ceruloplasmin and cardiovascular disease

Transition metal ion–mediated oxidation is a commonly used model system for studies of the chemical, structural, and functional modifications of low-density lipoprotein (LDL). The physiological relevance of studies using free metal ions is unclear and has led to an exploration of free metal ion-independent mechanisms of oxidation. We and others have investigated the role of human ceruloplasmin (Cp) in oxidative processes because it the principal copper-containing protein in serum. There is an abundance of epidemiological data that suggests that serum Cp may be an important risk factor predicting myocardial infarction and cardiovascular disease. Biochemical studies have shown that Cp is a potent catalyst of LDL oxidation in vitro. The pro-oxidant activity of Cp requires an intact structure, and a single copper atom at the surface of the protein, near His⁴²⁶, is required for LDL oxidation. Under conditions where inhibitory protein (such as albumin) is present, LDL oxidation by Cp is optimal in the presence of superoxide, which reduces the surface copper atom of Cp. Cultured vascular endothelial and smooth muscle cells also oxidize LDL in the presence of Cp. Superoxide release by these cells is a critical factor regulating the rate of oxidation. Cultured monocytic cells, when activated by zymosan, can oxidize LDL, but these cells are unique in their secretion of Cp. Inhibitor studies using Cp-specific antibodies and antisense oligonucleotides show that Cp is a major contributor to LDL oxidation by these cells. The role of Cp in lipoprotein oxidation and atherosclerotic lesion progression in vivo has not been directly assessed and is an important area for future studies.     

Ab initio simulations of Cu binding sites on the N-terminal region of prion protein

The human prion protein binds Cu²⁺ ions in the octarepeat domain of the N-terminal tail up to full occupancy at pH 7.4. Recent experiments have shown that the HGGG octarepeat subdomain is responsible for holding the metal bound in a square-planar configuration. By using first principle ab initio molecular dynamics simulations of the Car–Parrinello type, the coordination of copper to the binding sites of the prion protein octarepeat region is investigated. Simulations are carried out for a number of structured binding sites. Results for the complexes Cu(HGGGW)(wat), Cu(HGGG), and [Cu(HGGG)]₂ are presented. While the presence of a Trp residue and a water molecule does not seem to affect the nature of the copper coordination, high stability of the bond between copper and the amide nitrogen of deprotonated Gly residues is confirmed in all cases. For the more interesting [Cu(HGGG)]₂ complex, a dynamically entangled arrangement of the two domains with exchange of amide nitrogen bonds between the two copper centers emerges, which is consistent with the short Cu–Cu distance observed in experiments at full copper occupancy.     

Probing copper/tau protein interactions electrochemically

Alzheimer’s disease (AD) is a neurodegenerative disorder that is characterized by peptide and protein misfolding and aggregation, in part due to the presence of excess metal ions such as copper(II) [Cu(II)]. Recently, the brain levels of Cu(II) complexes in vivo were linked to the oxidative stress in neurodegenerative disorders, including AD. Amyloid β-peptide (Aβ), found outside neuronal cells, has been investigated extensively in connection with Cu(II) ion toxicity; however, the effects of metallation on tau are less known. Normal tau protein binds and stabilizes the microtubules in neurons, but in diseased cells tau hyperphosphorylation and aggregation are evident and compromise tau function. There is increasing evidence that the Cu(II) ion may play an important role in tau biochemistry. Here, we present an electrochemical study of the interactions between full-length tau-410 and Cu(II) ions. The coordination of Cu(II) ions to tau immobilized on gold surfaces induces an electrochemical signal at approximately 140 ± 5 mV versus Ag/AgCl due to the Cu(II)/Cu(I) redox couple. Redox potentials and current intensities of Cu(II)-containing nonphosphorylated tau (nTau) and phosphorylated tau (pTau) films were determined at different pH conditions. Greater Cu(II) uptake by pTau over nTau films was observed at low pH. Competitive zinc(II) [Zn(II)] ion binding studies revealed significant Cu(II) ion displacement in pTau films. X-ray photoelectron spectroscopy analysis indicated the presence of Cu 2p and Zn 2p binding energies in protein samples, further supporting metal ion coordination to protein films. The surface-based electrochemical technique requires a minimal protein amount (a few microliters) and allows monitoring the bound Cu(II) ions and the redox activities of the resulting metalloprotein films.     

Circulating ceruloplasmin is an important source of copper for normal and malignant animal cells

The relative uptake of copper from ceruloplasmin and non-ceruloplasmin plasma pools, by normal and malignant cells, was investigated in vivo and in vitro, using ⁶⁴Cu and ⁶⁷Cu. 1. Most of the copper administered intravenously to normal and tumor-bearing rats was removed within 1 h, a substantial portion entering the liver. There were differences in the apparent avidity of individual tissues for ceruplasmin vs. ionic copper, but when calculated on the basis of actual μg absorbed, all showed a preference for ceruplasmin. 2. Appreciable amounts of copper from either source were also absorbed by the tumors, and cultured Ehrlich ascites tumor cells showed a rapid uptake and marked preference for ceruplasmin over non-ceruplasmin copper, as did primary rat muscle cell cultures. 3. Ceruplasmin protein was also absorbed by normal and neoplastic rat tissues, but less rapidly than ceruplasmin copper, as determined by administration of pure [³H]leucine- or [¹²⁵I]ceruloplasmin. Copper deficiency did not accelerate this process. 4. It is concluded that, at least in rat, ceruloplasmin is the preferred plasma source of copper for normal and malignant cells, and that the copper on ceruplasmin turns over more rapidly than the protein moiety, a finding consistent with its role as a copper transport protein.     

The reactions of zirconium(IV) bromide and zirconium(III) bromide and chloride with ammonia and zirconium(IV) bromide with ammonium cyanide

Unlike ZrCl₄, ZrBr₄ is not ammonolysed in liquid ammonia at temperatures up to −33 °C. The existence of ammoniates ZrBr₄nH₃ (n = 17, 12 and 9) at −36 °C has been established; at room temperature, the hexammine ZrBr₄ · 6NH₃ is the stable species which becomes ZrBr₄ · 2NH₃ at 200 °C. When treated with an excess of NH₄CN in liquid ammonia, complete replacement of bromide ions by cyanide occurs to give an inseparable mixture of Zr(CN)₄ · 2NH₃ and NH₄Br. The chloride and bromide of zirconium(III) also undergo no ammonolysis in liquid ammonia; the ammoniates stable at room temperature are ZrCl₃ · 2.5NH₃ and ZrBr₃ · 6NH₃.     

Iron and copper homeostasis and intestinal absorption using the Caco2 cell model

Whole body homeostasis can be viewed as the balance between absorption and excretion, which can be regulated independently. Present evidence suggests that for iron, intestinal absorption is the main site for homeostatic regulation, while for copper it is biliary excretion. There are connections between iron and copper in intestinal absorption and transport. The blue copper plasma protein, ceruloplasmin, and its intracellular homologue, hephaestin, play a role in cellular iron release. The studies reviewed here compare effects of Fe(II) and Cu(II) on their uptake and overall transport by monolayers of polarized Caco2 cells, which model intestinal mucosa. In the physiological range of concentrations, depletion of cellular iron or copper (by half) increased uptake of both metal ions. Depletion of iron or copper also enhanced overall transport of iron from the apical to the basal chamber. Copper depletion enhanced overall copper transport, but iron depletion did not. Pretreatment with excess copper also stimulated copper absorption. Plasma ceruloplasmin (added to the basal chamber) failed to enhance basolateral iron release, and Zn(II) failed to compete with Cu(II) for uptake. Neither copper nor iron deficiency altered expression of IREG1 or DMT1 (-IRE form) at the mRNA level. Thus, in the low-normal range of iron and copper availability, intestinal absorption of both metals appears to be positively related to the need for these elements by the whole organism. The two metal ions also influenced each other's transport; but with copper excess, other mechanisms come into play.     

Copper chaperones: function, structure and copper-binding properties

 Copper is an absolute requirement for living systems and the intracellular trafficking of this metal to copper-dependent proteins is fundamental to normal cellular metabolism. The copper chaperones perform the dual functions of trafficking and the prevention of cytoplasmic exposure to copper ions in transit. Only a small number of copper chaperones have been identified at this time but their conservation across plant, bacterial and animal species suggests that the majority of living systems utilise these proteins for copper routing. The available data suggest that each copper-dependent protein in the cell is served by a specific copper chaperone. Although copper chaperones cannot be substituted for one another in a given cell type, copper chaperones that deliver to the same protein in different cell types appear to be functionally equivalent. The majority of the copper chaperones identified thus far have an "open-faced β-sandwich" global fold with a conserved MXCXXC metal-binding motif. Specificity for a given copper-dependent protein appears to be mediated by the residues surrounding the copper-binding motif. Copper binds to such proteins as Cu(I) in a trigonal complex with three sulfur ligands. Only the copper chaperone specific for cytochrome-c-oxidase, Cox17, deviates from this design. Received: 12 October 1998 / Accepted: 7 December 1998     

Quenching of bathocuproine disulfonate fluorescence by Cu(I) as a basis for copper quantification

In this paper we report the up to now ignored fluorescence properties of the specific Cu(I)-chelator bathocuproine disulfonate and their application in assays of total copper and Cu(I). The method is based on the linear quenching of the bathocuproine disulfonate emission at 770 nm (lambda(ex)580 nm) by increasing concentrations of Cu(I), at pH 7.5. Copper concentrations as low as 0.1 microM can be determined. Other metal ions (iron, manganese, zinc, cadmium, cobalt, nickel) do not interfere. The procedure for total copper determination in proteins includes HCl treatment to release the copper, neutralization to pH 7.5 in the presence of citrate to stabilize the copper, and reduction of the copper to Cu(I) by ascorbate in the presence of the chelator. This assay gave results coincident with the analysis by atomic absorption spectroscopy in two selected proteins. In addition, conditions are described (omitting HCl treatment and reduction by ascorbate) for direct measurement of Cu(I) in native proteins, as illustrated for the Escherichia coli NADH dehydrogenase-2. Data show that the fluorometric assays described in this paper are simple and convenient procedures for total copper and direct Cu(I) quantification in determined biological samples.     

Thermally induced denaturation and aggregation of BLG-A: effect of the Cu<Superscript>2+</Superscript> and Zn<Superscript>2+</Superscript> metal ions

There is growing evidence that metal ions can accelerate the aggregation process of several proteins. This process, associated with several neuro-degenerative diseases, has been reported also for non-pathological proteins. In the present work, the effects of copper and zinc ions on the denaturation and aggregation processes of β-lactoglobulin A (BLG-A) are investigated by differential scanning calorimetry (DSC), fluorescence, electron paramagnetic resonance (EPR) and optical density. The DSC profiles reveal that the thermal behaviour of BLG-A is a complex process, strongly dependent on the protein concentration. For concentrations ≤0.13 mM, the thermogram shows an endothermic peak at 84.3°C, corresponding to denaturation; for concentrations >0.13 mM an exothermic peak also appears, above 90°C, related to the aggregation of the denaturated BLG-A molecules. The thioflavin T fluorescence indicates that the thermally induced aggregates show fibrillar features. The presence of either equimolar Cu²⁺ or Zn²⁺ ions in the protein solution has different effects. In particular, copper binds to the protein in the native state, as evidenced by EPR experiments, and destabilizes BLG-A by decreasing the denaturation temperature by about 10°C, whereas zinc ions probably perturb the partially denaturated state of the protein. The kinetics of BLG-A aggregation shows that both metal ions abolish the lag phase before the aggregation starts. Moreover, the rate of the process is 4.6-fold higher in the presence of copper, whereas the effect of zinc is negligible. The increase of the aggregation rate, induced by copper, may be due to a site-specific binding of the metal ion on the protein.