The VATO project: Development and validation of a dynamic transfer model of tritium in grassland ecosystem.

In this paper, a dynamic compartment model with a high temporal resolution has been investigated to describe tritium transfer in grassland ecosystems exposed to atmospheric 3H releases from nuclear facilities under normal operating or accidental conditions. TOCATTA-χ model belongs to the larger framework of the SYMBIOSE modelling and simulation platform that aims to assess the fate and transport of a wide range of radionuclides in various environmental systems. In this context, the conceptual and mathematical models of TOCATTA-χ have been designed to be relatively simple, minimizing the number of compartments and input parameters required. In the same time, the model achieves a good compromise between easy-to-use (as it is to be used in an operational mode), explicative power and predictive accuracy in various experimental conditions. In the framework of the VATO project, the model has been tested against two-year-long in situ measurements of 3H activity concentration monitored by IRSN in air, groundwater and grass, together with meteorological parameters, on a grass field plot located 2 km downwind of the AREVA NC La Hague nuclear reprocessing plant, as was done in the past for the evaluation of transfer of 14C in grass. By considering fast exchanges at the vegetation-air canopy interface, the model correctly reproduces the observed variability in TFWT activity concentration in grass, which evolves in accordance with spikes in atmospheric HTO activity concentration over the previous 24 h. The average OBT activity concentration in grass is also correctly reproduced. However, the model has to be improved in order to reproduce punctual high concentration of OBT activity, as observed in December 2013. The introduction of another compartment with a fast kinetic (like TFWT) - although outside the model scope - improves the predictions by increasing the correlation coefficient from 0.29 up to 0.56 when it includes this particular point. Further experimental investigation will be undertaken by IRSN and EDF next year to better evaluate (and properly model) other aspects of tritium transfer where knowledge gaps have been identified in both experimental and modelling areas.

The VATO project: An original methodology to study the transfer of tritium as HT and HTO in grassland ecosystem.

Tritium (3H) is mainly released into the environment by nuclear power plants, military nuclear facilities and nuclear reprocessing plants. The construction of new nuclear facilities in the world as well as the evolution of nuclear fuel management might lead to an increase of 3H discharges from the nuclear industry. The VATO project was set up by IRSN (Institut de Radioprotection et de Sûreté Nucléaire) and EDF (Electricité de France) to reduce the uncertainties in the knowledge about transfers of 3H from an atmospheric source (currently releasing HT and HTO) to a grassland ecosystem. A fully instrumented technical platform with specifically designed materials was set up downwind of the AREVA NC La Hague reprocessing plant (Northwest of the France). This study, started in 2013, was conducted in four main steps to provide an hourly data set of 3H concentrations in the environment, adequate to develop and/or validate transfer models. It consisted first in characterizing the physico-chemical forms of 3H present in the air around the plant. Then, 3H transfer kinetics to grass were quantified regarding contributions from various compartments of the environment. For this purpose, an original experimental procedure was provided to take account for biases due to rehydration of freeze-dried samples for the determination of OBT activity concentrations in biological samples. In a third step, the 3H concentrations measured in the air and in rainwater were reconstructed at hourly intervals. Finally, a data processing technique was used to determine the biological half-lives of OBT in grass.

Near-field krypton-85 measurements in stable meteorological conditions around the AREVA NC La Hague reprocessing plant: estimation of atmospheric transfer coefficients.

The aim of this work was to study the near-field dispersion of (85)Kr around the nuclear fuel reprocessing plant at La Hague (AREVA NC La Hague - France) under stable meteorological conditions. Twenty-two (85)Kr night-time experimental campaigns were carried out at distances of up to 4 km from the release source. Although the operational Gaussian models predict for these meteorological conditions a distance to plume touchdown of several kilometers, we almost systematically observed a marked ground signal at distances of 0.5-4 km. The calculated atmospheric transfer coefficients (ATC) show values (1) higher than those observed under neutral conditions, (2) much higher than those proposed by the operational models, and (3) higher than those used in the impact assessments.

GUG is an efficient initiation codon to translate the human mitochondrial ATP6 gene.

A maternally inherited and practically homoplasmic mitochondrial (mtDNA) mutation, 8527A>G, changing the initiation codon AUG into GUG, normally coding for a valine, was observed in the ATP6 gene encoding the ATPase subunit a. No alternate Met codon could replace the normal translational initiator. The patient harboring this mutation exhibited clinical symptoms suggesting a mitochondrial disease but his mother who carried the same mtDNA mutation was healthy. The mutation was absent from 100 controls and occurred once amongst 44 patients suspected of Leber Hereditary Optic Neuropathy (LHON) but devoid of typical LHON mutations. In patient fibroblasts, no effect of 8527A>G mutation could be demonstrated on the biosynthesis of mtDNA-encoded proteins, on size and the content of ATPase subunit a, on ATP hydrolysis and on mitochondrial membrane potential. In addition, ATP synthesis was barely decreased. Therefore, GUG is a functional initiation codon for the human ATP6 gene.

Pathogenicity of missense mutations in SURF1 deficiency inducing the Leigh syndrome. Importance in diagnosis.

Leigh syndrome with cytochrome oxidase (COX) deficiency has been associated with SURF1 mutations. For patient diagnosis, distinction between neutral polymorphisms and pathogenic missense SURF1 mutations in Leigh syndrome is essential. We show that several missense SURF1 mutations did not allow a stable protein to be expressed. Absence of immunologically reactive SURF1 is, therefore, helpful to demonstrate their pathogenicity. In addition, we show that out of two previously described missense mutations housed by the same allele, only one, the T737 C was pathogenic. Indeed, transfection of T737 C mutated SURF1 in SURF1-deficient cells did not restore normal SURF1 stability and COX activity. On the contrary, the G604 C-mutated SURF1 did it and, hence, is a neutral variant.

[A new missense mutation of 574C>T in the SURF1 gene--biochemical and molecular genetic study in seven children with Leigh syndrome]. / Nová missense mutace 574C > T v genu SURF1--biochemická a molekulárne geneticka studie u sedmi detí s leighovým syndromem.

BACKGROUND: Leigh disease, subacute necrotizing encephalopathy, is a serious mitochondrial disorder of energy-providing metabolism. Clinical presentation usually starts in infancy as a progressive neurodegenerative disorder with retardation and regression of psychomotor development. The most common form of the disease is associated with deficiency of the cytochrome c oxidase (COX) due to SURF1 gene mutations. SURF1 encodes an inner mitochondrial membrane protein involved in the biogenesis and assembly of COX complex. METHODS AND RESULTS: The activities of mitochondrial respiratory chain complexes were determined spectrophotometrically in isolated lymphocytes, platelets, muscle mitochondria and cultured fibroblasts. Generalised decrease of COX activity was found in 7 children with typical symptoms of Leigh disease. Two-dimensional electrophoresis of mitochondrial proteins showed altered assembly pattern of COX. As demonstrated by Western blot analysis of mitochondria or mitoplasts with anti-hSurf1 antibodies (gift from Dr. E. A. Shoubridge), the Surf1 protein was absent in all 5 investigated patients. Molecular analyses in the 7 patients revealed the presence of mutations in the SURF1 gene--six patients harboured previously described SURF1 mutations, a new mutation 574C > T was found in one patient. CONCLUSIONS: The co-operation among the patient's families, clinicians and specialised laboratories is essential for the diagnostic of mitochondrial disorders. The treatment of Leigh syndrome is only symptomatic and the prognosis of the disease is unfavourable. The diagnostics on biochemical and molecular level is necessary for genetic counselling and prenatal diagnosis in affected families.

Mitochondrial ATPase and high-energy phosphates in failing hearts.

This study examined high-energy phosphates (HEP) and mitochondrial ATPase protein expression in hearts in which myocardial infarction resulted in either compensated left ventricular remodeling (LVR) or congestive heart failure (CHF). The response of HEP (measured via (31)P magnetic resonance spectroscopy) to a modest increase in the cardiac work state produced by dobutamine-dopamine infusion and pacing (if needed) was examined in 17 pigs after left circumflex coronary artery ligation (9 with LVR and 8 with CHF) and compared with 7 normal pigs. In hearts with LVR, the baseline phosphocreatine (PCr)-to-ATP ratio decreased, and calculated ADP increased; these changes were most severe in hearts with CHF. HEP levels did not change in normal or LVR hearts during dobutamine-dopamine infusion. However, in hearts with CHF, the PCr-to-ATP ratio decreased further, and free ADP increased. The mitochondrial protein levels of the F(0)F(1)-ATPase subunits were normal in hearts with compensated LVR. However, in failing hearts, the alpha-subunit decreased by 36%, the beta-subunit decreased by 16%, the oligomycin sensitivity-conferring protein subunit decreased by 40%, and the initiation factor 1 subunit decreased by 41%. Thus in failing hearts, reductions in mitochondrial F(0)F(1)-ATPase protein expression are associated with increased myocardial free ADP.

Functional characterization of novel mutations in the human cytochrome b gene.

The great variability of the human mitochondrial DNA (mtDNA) sequence induces many difficulties in the search for its deleterious mutations. We illustrate these pitfalls by the analysis of the cytochrome b gene of 21 patients affected with a mitochondrial disease. Eighteen different sequence variations were found, five of which were new mutations. Extensive analysis of the cytochrome b gene of 146 controls found 20 supplementary mutations, thus further demonstrating the high variability of the cytochrome b sequence. We fully evaluated the functional relevance of 36 of these 38 mutations using indirect criteria such as the nature of the mutation, its frequency in controls, or the phylogenetic conservation of the mutated amino acid. When appropriate, the mtDNA haplotype, the heteroplasmic state of the mutation, its tissue distribution or its familial transmission were also assessed. The molecular consequences of the mutations, which appeared possibly deleterious in that first step of evaluation, were evaluated on the complex III enzymological properties and protein composition using specific antibodies that we have generated against four of its subunits. Two original deleterious mutations were found in the group of seven patients with overt complex III defect. Both mutations (G15150A (W135X) and T15197C (S151P)) were heteroplasmic and restricted to muscle. They had significant consequences on the complex III structure. In contrast, only two homoplasmic missense mutations with dubious clinical relevance were found in the patients without overt complex III defect.

Mutations in the SURF1 gene associated with Leigh syndrome and cytochrome C oxidase deficiency.

Cytochrome c oxidase (COX) deficiency is one of the major causes of Leigh Syndrome (LS), a fatal encephalopathy of infancy or childhood, characterized by symmetrical lesions in the basal ganglia and brainstem. Mutations in the nuclear genes encoding COX subunits have not been found in patients with LS and COX deficiency, but mutations have been identified in SURF1. SURF1 encodes a factor involved in COX biogenesis. To date, 30 different mutations have been reported in 40 unrelated patients. We aim to provide an overview of all known mutations in SURF1, and to propose a common nomenclature. Twelve of the mutations were insertion/deletion mutations in exons 1, 4, 6, 8, and 9; 10 were missense/nonsense mutations in exons 2, 4, 5, 7, and 8; and eight were detected at splicing sites in introns 3 to 7. The most frequent mutation was 312_321del 311_312insAT which was found in 12 patients out of 40. Twenty mutations have been described only once. We also list all polymorphisms discovered to date.

[Mitochondrial diabetes complicated by or associated with "MELAS" syndrome?]. / Diabète mitochondrial compliqué ou associé à un syndrome MELAS?

We report the case of fifty-two year-old mentally deficient female who presented with diabetes mellitus, deafness, stroke-like episodes, cardiomyopathy, and macular pattern dystrophy of the retina. Her brain exhibited calcification within basal ganglia, lactacidaemia was not increased. Although her skeletal muscles had never been clinically impaired, a quadriceps biopsy led to the diagnosis of mitochondrial disease because it exhibited ragged red fibers and heteroplasmic point-mutation at position 3243 of the mitochondrial DNA, although not any detectable respiratory chain complex deficiency was found. The mutant percentage in muscle was 70 p.100 and 5 to 10 p.100 in leukocytes. The question of whether a diabetic microangiopathy may be responsible stroke-like episodes is discussed. We suggest it was rather a complicated form of diabetes-deafness than a incomplete MELAS syndrome associated with mitochondrial diabetes.

Missense mutations in SURF1 associated with deficient cytochrome c oxidase assembly in Leigh syndrome patients.

We have studied the fibroblasts of three patients suffering from Leigh syndrome associated with cytochrome c oxidase deficiency (LS-COX-). Their mitochondrial DNA was functional and all nuclear COX subunits had a normal sequence. The expression of transcripts encoding mitochondrial and nuclear COX subunits was normal or slightly increased. Similarly, the OXA1 transcript coding for a protein involved in COX assembly was increased. However, several COX-protein subunits were severely depressed, indicating deficient COX assembly. Surf1, a factor involved in COX biogenesis, was recently reported as mutated in LS-COX- patients, all mutations predicting a truncated protein. Sequence analysis of SURF1 gene in our three patients revealed seven heterozygous mutations, six of which were new : an insertion, a nonsense mutation, a splicing mutation of intron 7 in addition to three missense mutations. The mutation G385 A (Gly124-->Glu) changes a Gly that is strictly conserved in Surfl homologs of 12 species. The substitution G618 C (Asp202-->His), changing an Asp that is conserved only in mammals, appears to be a polymorphism. The mutation T751 C changes Ile246 to Thr, a position at which a hydrophobic amino acid is conserved in all eukaryotic and some bacterial species. Replacing Ile246 by Thr disrupts a predicted beta sheet structure present in all higher eukaryotes. COX activity could be restored in fibroblasts of the three patients by complementation with a retroviral vector containing normal SURF1 cDNA. These mutations identify domains essential to Surf1 protein structure and/or function.

Immunolabelling of mitochondrial superoxide dismutase and of Hsp60 in muscles harbouring a respiratory chain deficiency.

In mitochondrial encephalomyopathies, impairment of the electron transfer chain may lead to overproduction of reduced oxygen species because oxygen consumption is decreased. Whether heat shock proteins (Hsp) are induced or not in mitochondria against oxidative stress is questionable. Muscle ragged-red fibres are the histological hallmark of most respiratory chain deficiencies in humans. They exhibit abnormal mitochondria which accumulate mainly under their sarcolemma. Within these fibres, immunolabelling demonstrated strong expression of mitochondrial manganese-dependent superoxide dismutase and a lack of expression of mitochondrial Hsp60 within the subsarcolemmal spaces. In contrast, Hsp60 was overexpressed within the intermyofibrillar mitochondria. These findings suggest enhanced generation and dismutation of superoxide anions and that processing and integration of imported precursor proteins is impaired within the subsarcolemmal mitochondrial aggregates of ragged-red fibres, whereas protein import and assembly may still be efficient in the intermyofibrillar mitochondria of these fibres.

Sequence conservation from human to prokaryotes of Surf1, a protein involved in cytochrome c oxidase assembly, deficient in Leigh syndrome.

The human SURF1 gene encoding a protein involved in cytochrome c oxidase (COX) assembly, is mutated in most patients presenting Leigh syndrome associated with COX deficiency. Proteins homologous to the human Surf1 have been identified in nine eukaryotes and six prokaryotes using database alignment tools, structure prediction and/or cDNA sequencing. Their sequence comparison revealed a remarkable Surf1 conservation during evolution and put forward at least four highly conserved domains that should be essential for Surf1 function. In Paracoccus denitrificans, the Surf1 homologue is found in the quinol oxidase operon, suggesting that Surf1 is associated with a primitive quinol oxidase which belongs to the same superfamily as cytochrome oxidase.

Functional F1-ATPase essential in maintaining growth and membrane potential of human mitochondrial DNA-depleted rho degrees cells.

F1-ATPase assembly has been studied in human rho degrees cells devoid of mitochondrial DNA (mtDNA). Since, in these cells, oxidative phosphorylation cannot provide ATP, their growth relies on glycolysis. Despite the absence of the mtDNA-coded F0 subunits 6 and 8, rho degrees cells possessed normal levels of F1-ATPase alpha and beta subunits. This F1-ATPase was functional and azide- or aurovertin-sensitive but oligomycin-insensitive. In addition, aurovertin decreased cell growth in rho degrees cells and also reduced their mitochondrial membrane potential, as measured by rhodamine 123 fluorescence. Therefore, a functional F1-ATPase was important to maintain the mitochondrial membrane potential and the growth of these rho degrees cells. Bongkrekic acid, a specific adenine nucleotide translocator (ANT) inhibitor, also reduced rho degrees cell growth and mitochondrial membrane potential. In conclusion, rho degrees cells need both a functional F1-ATPase and a functional ANT to maintain their mitochondrial membrane potential, which is necessary for their growth. ATP hydrolysis catalyzed by F1 must provide ADP3- at a sufficient rate to maintain a rapid exchange with the glycolytic ATP4- by ANT, this electrogenic exchange inducing a mitochondrial membrane potential efficient enough to sustain cell growth. However, since the effects of bongkrekic acid and of aurovertin were additive, other electrogenic pumps should cooperate with this pathway.

Co-existence of high levels of a cytochrome b mutation and of a tandem 200 bp duplication in the D-loop of muscle human mitochondrial DNA.

Previous studies have suggested that some patients with large-scale mitochondrial DNA (mtDNA) deletions also presented a heteroplasmic 260 bp tandem duplication in the mtDNA D-loop region. Such duplications were observed not only in patients with mitochondrial pathology but also in aged subjects. However, the percentage of duplicated mtDNA did not exceed a few per cent of the total mtDNA, except in one example where it reached 30%. We report here another type of 200 bp duplication in the mtDNA D-loop region that, instead of being associated with a large-scale deletion, is correlated to the presence of a point mutation in the cytochrome b gene. The 200 bp duplication concerned up to 95% of the total mtDNA of some muscle mitochondria and was absent from the patient lymphocyte DNA. The percentages of the 200 bp duplication and that of the cytochrome b mutation were relatively close in whole muscle as well as in single muscle fibres, suggesting a correlation between the mutation and the duplication. This duplication could also be detected by PCR in two other patients with mitochondrial disorders but without known deletion or mtDNA mutation. These data suggest that the accumulation of these small duplications in the mtDNA D-loop could be indicative of the presence of other defects of the mtDNA which would damage the respiratory chain function. These deficiencies would induce the generation of small duplications in the D-loop.

Identification of a large-scale mitochondrial deoxyribonucleic acid deletion in endocrinopathies and deafness: report of two unrelated cases with diabetes mellitus and adrenal insufficiency, respectively.

In recent years, a broad variety of chronic diseases have been related to different mitochondrial DNA (mtDNA) rearrangements. We have investigated two 16-yr-old unrelated girls with unexplained endocrine disorders for a mtDNA mutation. One initially presented with an adrenal crisis at the age of 4 yr. Complete adrenal insufficiency for nearly 15 yr was the main clinical manifestation, along with insiduous growth retardation and sensorineural hearing loss since age 6. The other girl presented with ketoacidosis at the age of 15 yr. She exhibited incomplete deafness since age 6 and poor growth. In both patients, brain magnetic resonance imaging abnormalities and raised cerebrospinal fluid protein concentration indicated mild leucodystrophy. Biopsy of skeletal muscle showed a mitochondrial dysfunction; molecular analysis using a PCR screening procedure revealed a 7.4 kb deletion of the mtDNA in skeletal muscle but not in leucocytes. Direct sequence analysis of the junctional regions showed that the deletion spanned 7.436 kb (nucleotide 8649 to nucleotide 16084). The relative amount of deleted mtDNA estimated by Southern blot analysis was 25 and 15%, respectively. No deletion was present in leukocytes obtained from the asymptomatic mothers. The presence of the same mutation in different patients with various endocrine conditions supports the view that the 7.4 kb mtDNA deletion should be considered as one of the candidate causes for phenotypically uncommon cases of endocrinopathies, specially in children with deafness. This is the first report of a mitochondrial disease with primary adrenocortical insufficiency as the clinical onset.

Inhibition of ubiquitous mitochondrial creatine kinase expression in HeLa cells by an antisense oligodeoxynucleotide.

Antisense strategy has been used to inhibit the synthesis of the human ubiquitous mitochondrial creatine kinase (Mi-CK) in HeLa cells. Indeed, elevated levels of Mi-CK in the serum of some cancer patients seem to be an adverse pronostic indicator (for refs see Wallimann T and Hemmer W, Mol Cell Biochem 133/134: 193-220, 1994). A phosphorothioate oligonucleotide, complementary to the second intron-exon splice junction site of the human ubiquitous Mi-CK pre-mRNA was shown to inhibit Mi-CK synthesis by 80% without modifying F1-ATPase beta subunit expression or hampering HeLa cell growth. This inhibition was correlated to a decrease of the Mi-CK mRNA level that could be determined quantitatively after amplification of reverse transcription products (RT) in the presence of varying concentrations of internal standard competitors. This study also demonstrated that the Mi-CK mRNA copy number was much lower in HeLa cells than that of the cytosolic creatine kinase isoform, B-CK. The antisense-induced decrease in Mi-CK mRNA and protein level influenced neither the expression of B-CK which uses up the phosphocreatine produced by Mi-CK during the phosphocreatine shuttle, nor that of another nuclear encoded mitochondrial gene, the F1-ATPase subunit which provides ATP to Mi-CK. In conclusion, an elevated Mi-CK expression is not required for cancer cell growth and therefore, Mi-CK is not a significant limiting factor for the growth of the cancer cells which contain it. In addition, a decrease in Mi-CK synthesis does not induce a change in the expression of mitochondrial F1-ATPase which provides ATP to Mi-CK or in the expression of cytosolic B-CK which is involved together with Mi-CK in the phosphocreatine shuttle. Therefore, the use of the phosphocreatine shuttle as a process mandatory for the active growth of some cancer cells is questioned.

[Leber "plus" disease: optic neuropathy, parkinsonian syndrome and supranuclear ophthalmoplegia]. / Maladie de Leber "plus": neuropathie optique, syndrome parkinsonien et ophtalmoplégie supranucléaire.

A 34-year-old right handed man presented with a bilateral subacute optical neuropathy associated with cervical dystonia, parkinsonism and supranuclear ophtalmoplegia. Magnetic resonance imaging showed in T2 increased intensity of signal in the dorsal mesencephalum and pons as well as in dorsal part of striata. The 3,460 mutation of mitochondrial DNA was found in a blood sample. This observation adds to the variability of presentation of Leber's "plus".

Antimycin resistance and ubiquinol cytochrome c reductase instability associated with a human cytochrome b mutation.

Progressive exercise intolerance was associated with a decreased maximal rate of ubiquinol cytochrome c reductase (complex III) activity in the muscle mitochondria of the studied patient and with a thirty five-fold increase in the I50 for antimycin A. In contrast, myxothiazol sensitivity was not altered. Complex III activity was stable at 37 degrees C, but progressively decreased at 4 degrees C. An heteroplasmic G to A mutation at position 15615 of the mitochondrial DNA, resulting in the replacement of the highly conserved Gly290 in cytochrome b by Asp, was identified. Histochemical studies showed increased cytochrome oxidase and succinate dehydrogenase activities under the sarcolemma of type I fibres. After partial extraction of mitochondria from the muscle, the residual pellet contained a lower percentage of the mutation than did whole muscle, suggesting that the percentage of mutation is higher in the most readily extracted mitochondria, most probably present under the sarcolemma. In the current 8 transmembrane helix model of cytochrome b, Gly290 lies at the end of the sixth transmembrane helix, facing the intermembrane space and close to the presumed sites of interaction between cytochrome b, the iron-sulfur protein and the 9.5 kDa protein. Since immunoblotting experiments showed a relative decrease in the proportions of these three subunits in the patient's mitochondria compared with the other complex III subunits, it is probable that the complex III instability and the relative decrease in these subunits are related to the mutation. The relationship between the decrease in the apparent affinity for antimycin A and the instability of complex III are discussed.

Variations of muscle mitochondrial creatine kinase activity in mitochondrial diseases.

Mitochondrial creatine kinase (mtCK) activity has been measured in the mitochondria isolated from the muscle of 69 patients suspected of mitochondrial diseases. The isolated mitochondria did not contain significant amounts of the muscle isoform of creatine kinase, as checked by an immunoassay performed after electrophoretic separation of the various isoforms. Hence, the enzyme assay reliably represented the mtCK activity. Therefore, a simple measurement of CK activity in isolated mitochondria permitted the measurement of mtCK activity. An absence of mtCK activity in muscle was never observed. The lowest activities were not associated to defined mitochondrial diseases linked to defects of respiratory chain complexes or to defects of citric cycle enzymes. On the contrary, mtCK activity was significantly increased in the muscle of patients exhibiting ragged red fibers. This increase was generally associated to an increase of citrate synthase activity. Since ragged-red fibers and elevated mtCK activities were generally not found in children younger than 3 years, even in cases of characteristic oxidative phosphorylation deficiency, it is suggested that the increase in mtCK activity as well as the appearance of ragged-red fibers are not the first events which occur during the evolution of mitochondrial diseases but would rather be long-term secondary processes which slowly develop in deficient mitochondria.