Imidazo[1,2-a]quinoxalin-4-amines: A novel class of nonxanthine A1-adenosine receptor antagonists

The syntheses and A₁ adenosine receptor affinities of a number of imidazo[1,2-a]quinoxalin-4-amines are reported. Structure—activity relationships within the series and in comparison with other similar tricyclic nonxanthine adenosine antagonists are discussed, leading to a putative common binding mode of these nitrogen-containing heterocycles to A₁ adenosine receptors. Secondary amino compounds displayed the best affinities toward A₁ receptors, while the tertiary amines were almost devoid of activity, thus suggesting a crucial role for the hydrogen bond-forming 4-NH group. Remarkably higher potencies for 1-methyl and N-cyclopentyl derivatives were also found. 4-Cyclopentylamino-1-methylimidazo[1,2-a]quinoxaline (TRFI 165) is the most potent compound in this series, having K_i(A₁) = 7.9 nM. It is also provided with a good A₁ selectivity both versus A_2a and A₃ subtypes and was selected for further pharmacological studies.     

Studies on histaminergic compounds VIII. A new series of compounds showing H1-antagonistic and H2-agonistic properties; synthesis, in vitro pharmacology and structure—activity relationships

To study the structure—activity relationships (SAR) of impromidine-like H₂-agonists, we prepared some analogues of the potent agonist VUF 8405 (an analogue of impromidine in which the 5-methylimidazole moiety was replaced by a phenyl group) by replacing the benzyl group of VUF 8405 with substituted diphenylmethyl moieties and a number of related groups. The compounds have been tested for H₁-activity on guinea pig ileum and trachea. To determine the H₂-activity, the compounds have been investigated on both the right atrium and gastric fundus of the guinea pig. The SAR for H₁-antagonistic activity of this series of compounds on the ileum was found to be analogous to that on the trachea. A significant difference was found between the H₂-SAR on the right atrium and gastric fundus.Because of this combination of H₁-antagonism and H₂-agonism, some of these compounds might be clinically useful, for instance in the treatment of certain allergic disturbances and some kinds of heart failure.     

Experimental studies on the protective efficacy of a Pseudomonas aeruginosa vaccine

Pseudomonas aeruginosa vaccine (PV) containing cell proteins with molecular weight (M_r) 20 000–100 000 and up to 0.08% ( ) admixture of lipopolysaccharide was obtained by water—salt extraction and subsequent ultrafiltration. PV protects mice against experimental P. aeruginosa infection, stimulates production of specific protective antibodies in rabbit and does not provoke obvious toxicity in laboratory animals.     

Simultaneous quantification of bergenin, catechin, and gallic acid from Bergenia ciliata and Bergenia ligulata by using thin-layer chromatography

Bergenia ciliata Sternb. and Bergenia ligulata Wall. (Saxifragaceae) are reputed drugs of Ayurveda, commonly known as Paashaanbhed. A simple TLC method has been developed for the simultaneous quantification of bergenin, catechin, and gallic acid from different parts of B. ciliata and B. ligulata using HPTLC plate precoated with silica gel 60 F₂₅₄. The method was developed in toluene:ethyl acetate:formic acid (4:6:1, v/v) and validated in terms of precision, repeatability, and accuracy. The linearity range for bergenin, catechin and gallic acid were found to be 160–800, 160–480 and 160–560 ng/spot respectively with correlation coefficients of 0.999, 0.999 and 0.999, respectively, which were indicative of good linear dependence of peak area on concentration. The method permits reliable quantification and showed good resolution and separation from other constituents of extract. Accuracy of the method was checked by conducting recovery studies at three different levels for all the three marker compounds and the average percentage recoveries were found to 99.29%, 98.66%, and 99.23%, respectively. The rhizomes were found to contain higher concentration of bergenin, catechin, and gallic acid than other parts of the plants. The proposed method was found to be simple, precise, specific, sensitive, and accurate. It can be used for routine quality control of herbal material and formulations containing bergenia species.     

Gene–environment interactions and obesity—Further aspects of genomewide association studies

Advances in genotyping technologies have facilitated the advent of the genomewide association studies in large study populations and thereby led to the identification of an impressive—and still increasing—number of genetic variants with significant impact on the risk of widespread lifestyle health problems such as obesity, diabetes, and cardiovascular disease. Yet, the scientific community is a long way from reaching a comprehensive picture of the heritable components of these diseases and advancing from plain statistical significance into a biological understanding where the true contribution to a trait is recognized. Increasingly large study populations, denser single-nucleotide polymorphism mapping, deep sequencing, and raised awareness of the importance of structural variants may add to the known genetic variance underlying common complex disorders; however, genetic variance alone probably cannot account for disease susceptibility without the addition of pre- and postnatal environmental and/or behavioral factors. Moreover, an interaction between genetic and environmental factors may hinder the detection of genetic effects if not accounted for, e.g., in genomewide association studies, and prospective cohort studies have hence been proposed to surpass the classic case–control design. With a focus on obesity we describe some of the recently reported gene–environment interactions for polymorphisms identified in the FTO and INSIG2 genes. Ultimately, a thorough understanding of the gene–environment interactions underlying a common complex condition such as obesity may suggest novel treatment or intervention strategies to complement the harmful effect of detrimental genetic variation and thus may assist in improving the quality of life for affected individuals.     

Ecological conditions of natural foci of tularaemia in the Czech Republic

Tularaemia, a zoonosis of veterinary and public health importance, commonly occurs in the Czech Republic as well as other countries of Northern Hemisphere. The objective of this study was to analyse the environmental conditions of distribution of natural foci of tularaemia and their long-term persistence in the Czech Republic. A geographic information system has been used for this purpose. A new variable (x _t), the mean number of natural foci in a specific area, has been suggested for the evaluation of diseases occurring in natural foci. Comparing two 15-year periods, a close correlation between the geographic distribution and numbers of natural foci of tularaemia in the Czech Republic in 1971–1985 and 1986–2000 (r = 0.91, n = 1814, t = 92.50, p = 0.01) was found. Natural foci of tularaemia have been persistent, but not stationary, over the period of 30 years and the geographic area of their occurrence has not been considerably growing or diminishing in the Czech Republic. The highest numbers of natural foci of tularaemia were in habitats of alluvial forests (x _t = 7.20), geographic areas of up to 200 m of elevation above sea (x _t = 9.18), 8.1–10.0 °C of mean annual air temperature (x _t = 6.24), 450–700 mm of mean annual precipitation (x _t = 2.84), and 2001–2200 hour of mean annual sunshine duration (x _t = 8.77). It was proved that tularaemia persists in specific areas of natural foci, the general environmental conditions of which can be defined and make it possible to predict the occurrence of tularaemia in other areas of suitable conditions.     

Accumulation of carbon disulphide metabolites

Summary Biological monitoring for carbon disulphide (CS₂) exposure performed using the iodine-azide test (IAT) and 2-thiothiazolidine-4-carboxylic acid (TTCA) test in urinalysis of workers with high exposure to CS₂ (112–142 mg/m³, n = 34), workers with low exposure (4–7 mg/m³, n = 16), and non-exposed university workers (n =10). Pre-shift and post-shift urine specimens were collected on three consecutive days in the exposed and for only one day in the non-exposed. According to the findings the specificity and the sensitivity seem to be low for the IAT and high for the TTCA test. Contrary to a previous report all pre-shift urine samples showed negative IATs. The TTCA test was positive in pre-shift urine even after 32 to 63.5 h without exposure, and values tended to increase during consecutive days of exposure in highly exposed workers.The possible health implications of these findings should be further investigated.     

Effects of a mixture of vegetable and essential oils and fatty acid potassium salts on Tetranychus urticae and Phytoseiulus persimilis

Laboratory trials were carried out to evaluate the toxicity and the influence of a commercial mixture of vegetal, essential oils, and potassium salts of fatty acids (Acaridoil 13SL^®) on the population growth rate (r_i—instantaneous rate of increase) of two mite species, the phytophagous Tetranychus urticae Koch and the predator Phytoseiulus persimilis Athias-Henriot. A residue of 1.3 mg/cm² of pesticide solution was harmless for Ph. persimilis eggs, while a moderate mortality of eggs and of larvae from treated eggs of T. urticae, was observed (53.8%). The pesticide also caused a delay in the postembryonic development of the tetranychid. Moreover, 83.4% mortality was reported for treated females tetranychids and only 24.0% for Ph. persimilis females. The pesticide influenced negatively the population growth of T. urticae which showed a very low rate of increase (r_i=0.07), compared to that obtained in the control (r_i=0.68). The pesticide did not affect negatively the reproductive potential of Ph. persimilis (r_i=0.54 and r_i=0.57 for test and control, respectively). These results suggest a considerable acaricidal activity of potassium salts of fatty acids and caraway oil on T. urticae and a good selectivity on Ph. persimilis.     

Butyrate and trichostatin A attenuate nuclear factor κB activation and tumor necrosis factor α secretion and increase prostaglandin E2 secretion in human peripheral blood mononuclear cells

The effects of short-chain fatty acids (butyrate, propionate, and acetate) and trichostatin A (TSA), a typical histone deacetylase inhibitor, on tumor necrosis factor (TNF)-α secretion and nuclear factor κB (NF-κB) activation in peripheral blood mononuclear cells induced with lipopolysaccharide were evaluated in relation to prostaglandin E₂ (PGE₂) secretion. Treatment of cells with butyrate; tributyrin, a prodrug of butyrate; propionate; acetate; and TSA down-regulated TNF-α secretion but all up-regulated PGE₂ secretion. Butyrate, propionate, and TSA inhibited NF-κB activation. The effects of the cyclooxygenase-nonspecific inhibitor, indomethacin; the cyclooxygenase-2 selective inhibitor, N-[2-(cyclohexyloxy)-4-nitro-phenyl] methanesulfonamide; and the general lipoxygenase inhibitor, nordihydroguaiaretic acid, varied in cells treated with each short-chain fatty acids. N-[2-(cyclohexyloxy)-4-nitro-phenyl] methanesulfonamide inhibited the effect of propionate on TNF-α secretion, and nordihydroguaiaretic acid inhibited that of acetate. The results showed that butyrate, propionate, and TSA inhibited TNF-α production via PGE₂ secretion and down-regulated NF-κB activation by lipopolysaccharide. These data suggest that the mechanism of butyrate and propionate action is through histone deacetylation and acetate through lipoxygenase activation in the regulation of proinflammatory responses in cells.     

Effect of multivitamin supplementation on the homocysteine and methylmalonic acid blood concentrations in women over the age of 60 years

Summary Background Deficiency of folic acid, vitamin B₆ and/or vitamin B₁₂ can result in elevated total plasma homocysteine concentrations (tHcy), which are considered to be a risk factor for vascular disease. Studies have shown that supplementation of the three vitamins can lower tHcy even in subjects with tHcy in the normal range. Aim of the study The aim of this study was to evaluate the effect of a 6 month supplementation with vitamin B₆, B₁₂ and folate on the concentrations of total plasma homocysteine and serum methylmalonic acid (MMA) of elderly women. Methods The study was designed as a randomized placebo controlled doubleblind trial, and 220 healthy women (aged 60–91 years) were involved. The vitamin and mineral capsule contained pyridoxine (3.4 mg), folic acid (400 µg) and cobalamin (9 µg) in addition to other micronutrients. Blood concentrations of folate, cobalamin, tHcy, MMA and the activity coefficient of erythrocyte alpha-aspartic aminotransferase (alpha-EAST) were measured at baseline and after 6 months of supplementation. Dietary intake was evaluated at the beginning and the end of the intervention by two 3–day diet records. Results Median concentrations of serum cobalamin, serum folate and erythrocyte folate increased significantly and tHcy and alpha–EAST activity (indicative of improved status of vitamin B₆) coefficient decreased significantly in the supplemented group. Median MMA concentration of the supplemented group was significantly lower than that of the placebo group after the intervention. The vitamin supplementation had a greater decreasing effect on the tHcy concentration of volunteers with lower vitamin and higher tHcy initial concentrations. In a linear regression model, baseline tHcy, serum folate, age and alpha–EAST activity coefficient were significantly correlated with the change in tHcy. The change in MMA in the supplement group was significantly associated to the baseline MMA values. Conclusions Our results show that a 6 month supplementation including physiological dosages of B vitamins improves the status of these nutrients and reduces tHcy in presumed healthy elderly women.     

A review of the Salmonellosis surveillance systems in Italy: Evolution during the course of time within the international framework

This paper focuses on the history of the two systems that have been adopted in Italy for the surveillance of Salmonellosis and describes their respective characteristics. Both systems have been subsequently modified: (1) The National Laboratory-based Surveillance System (NLSS) which was created in 1967 for Enteropathogenic Bacteria and subsequently, in 1992, became part of the European computerised Laboratory-based Surveillance System of Salmonellae isolates, the SALM-NET (Salmonella network); (2) The National Infectious Disease Reporting System (NIDRS) which was set up in the 1930s, revised in 1990 and has been used, since 1994, along with the Infectious Disease Informative System (IDIS). The results obtained with the different surveillance systems are presented: (1) The number of isolates from the laboratory surveillance from 1973 to 1997 are described. Total Salmonellae isolates have a slope with an increasing trend from 4372 isolates in 1973 to 15,041 isolates in 1988 drastically dropping to 5479 isolates in 1990 and increasing again to 13,596 isolates in 1993. Attention is given particularly to the epidemiology of S. enteritidis in Italy which increased progressively since 1982 (225 isolates) to 5435 isolates in 1994. S. typhimurium showed a slightly increasing trend in the period 1973–1988 (from 1694 to 3383 isolates) then decreased for reaching again previous levels. S. typhi showed a marked reduction from 573 isolates in 1973 to 33 isolates in 1996. On the contrary, other less frequent serotypes increased. (2) The number of cases of Salmonellosis reported during 1971–1997 are also presented. Other Infections by Salmonellae increased from 12,516 cases in 1976 (renamed Non Typhoidal Salmonellosis in 1990) to more than 20,000 cases in 1992. The number of cases of Typhoid Fever and Infections by S. paratyphi are also described. Particular attention has to be paid to the parallel trends of Salmonellosis using both surveillance systems: number of isolates and number of cases, particularly comparing Other Infections by Salmonellae and total Salmonellae isolates: after the 1992–1993 peak, an initial decrease was observed.     

Role of protein processing,intracellular trafficking and endocytosis in production of and immunity to yeast killer toxin

Yeast strains harboring M₁-dsRNA and its packaging virus ScV-L secrete a disulfide-linked, heterodimeric toxin which kills sensitive yeast cells by disrupting plasma membrane function. The mature toxin is derived from a precursor (preprotoxin) which undergoes post-translational processing steps during export via the established yeast secretory pathway. Cleavage by both the KEX1 and KEX2 endopeptidases is required for expression of killing activity. The same 1.0 kb open reading frame on Ml-dsRNA directs the expression of immunity to toxin. Differentially processed derivatives of protoxin, as well as protoxin itself, have been proposed to serve as mediators of immunity.To understand the mechanisms by which the killing and immunity phenotypes can be derived from a common precursor, we have: 1) studied cellular processes implicated in expression of the phenotypes; and 2) developed a system to produce mutants defective in immunity, killing, or both. In the first approach, the role played by both endocytosis and vesicular trafliking in expression of killing and immunity was examined. Strains defective in endocytosis (end1, end2) or vacuolar protein localization (vnp13, vp16) were transformed with a plasmid encoding killer toxin under control of the pho5 promoter. When induced by phosphate starvation, both end mutants and all vpl mutants expressed killing activity. Immunity to exogenous toxin, however, was significantly decreased in strains carrying both vpl mutant alleles and in one of the endocytosis mutants (end1)). This suicidal phenotype (rex for resistance expression) has been described previously in M₁-containing strains as a leaky phenocopy. The distinct selective disadvantage of the rex phenotype can be overcome in this system by phosphate-mediated repression of killer toxin expression. In the second approach, a strain carrying the phosphate-repressible copy of the toxin gene was mutagenized with EMS, and survivors were scored for the rex phenotype. A large percentage of the rex mutants isolated also exhibited a vpl phenotype, implying that efficacious sorting of vacuolar proteins may be important for expression of immunity. In contrast, the rex mutants and the wild type REX ⁺ strain displayed an END ⁺ phenotype which was distinct from that of the end1 and end2 mutant. At the level of stringency of this fluid phase uptake assay, it appears that defective endocytosis is not a common phenotype obtained among independently isolated and viable rex mutants.Presented at the XXII National Congress of the Italian Society for Microbiology (Salsomaggiore, 1–4 May 1988).     

Physiological Stress of High NH<Stack><Subscript>4</Subscript><Superscript>+</Superscript></Stack> Concentration in Water Column on the Submersed Macrophyte <Emphasis Type="Italic">Vallisneria Natans</Emphasis> L.

The submersed macrophyte, Vallisneria natans L., was cultured in laboratory with NH₄⁺-enriched tap water (1 mg L⁻¹ NH₄-N) for 2 months and the stressful effects of high ammonium (NH₄⁺) concentrations in the water column on this species was evaluated. The plant growth was severely inhibited by the NH₄⁺ supplement in the water column. The plant carbon and nitrogen metabolisms were disturbed by the NH₄⁺ supplement as indicated by the accumulation of free amino acids and the depletion of soluble carbohydrates in the plant tissues. The results suggested that high NH₄⁺ concentrations in the water column may hamper the restoration of submersed vegetation in eutrophic lakes.     

Structure-activity relationship studies of CNS agents. Part 29. N-Methylpiperazino-substituted derivatives of quinazoline, phthalazine and quinoline as novel α1, 5-HT1A and 5-HT2A receptor ligands

New N-methylpiperazino-substituted quinazolines 8 and 9, phthalazine 13, and quinoline 19 have been synthesized. The receptor binding profiles (α₁, 5-HT_1A, 5-HT_2A) of these compounds and their analogs (7–22) have been determined. It has been demonstrated that orientation of a local dipole moment of the heteroaromatic ring system affects both the α₁ and 5-HT_2A affinity of the investigated class of ligands. Distortion of the coplanar unfused heteroaromatic ring system results in a decreased 5-HT_2A affinity. 4-(4-Methylpiperazino)-2-(2-thienyl)quinoline 18 is the most active and selective α₁ ligand (K_i = 4.9 nM) with a much lower affinity for 5-HT_1A (K_i = 3420 nM) and 5-HT_2A (K_i = 211 nM) receptors.     

Induction of microsomal enzymes in the rat liver by inhalation of hydrocarbon solvents

Summary The possibility of increase of microsomal enzyme activities in the liver by inhaled hydrocarbon solvents has been tested in preliminary investigations with male Wistar rats. The animals were exposed 5 hrs per day to an atmosphere containing various solvents, in a test period of 10 or 28 days. Dichloromethane, carbon tetrachloride, trichloroethylene, and benzene in a concentration of 450–500 ppm caused slight increases of cytochrome P₄₅₀ and/or of aminopyrine demethylase activity after 10 days (P>0.02). At a test period of 28 days already 50 ppm of carbon tetrachloride led to an increase of cytochrome P ₄₅₀ in the liver cell (P>0.02). It is reasonable to assume that with prolonged exposure even small solvent concentrations in the respiratory air may lead to undesirable alterations in biotransformation of foreign substances and endogenous compounds in the liver.     

Tolerance of the lichen Xanthoria parietina (L.) Th. Fr. to metal stress

Comparisons were made between the content of Al, Cd, Cu, Fe, Mn, Pb, Sb and Zn in thalli of the lichen Xanthoria parietina and selected physiological processes. Assimilation pigments, activity of photosystem II, parietin, reactive substances produced using thiobarbituric acid (TBA) and CO₂ gas exchange were measured in order to assess tolerance to atmospheric sources of pollution. As expected, lichen thalli accumulated high amounts of the measured elements in relation to distance from pollution sources in Košice, Slovak Republic (US Steel factory and vehicular traffic in the city center). However, except for TBA reactive substances production and to some extent CO₂ gas exchange at the most polluted station, none of the tested physiological parameters showed a clear correlation between accumulation of elements and physiological damage. This reflected a high degree of pollution tolerance in this lichen and corresponded with its high abundance in Košice.     

Approaches to the molecular epidemiology of rickettsioses

This review deals with the developments of molecular approaches to the investigation of rickettsial disease epidemiology. The data presented include changes in the incidence and geographic distribution of endemic rickettsioses.Use of the DNA restriction enzyme technique, in combination with DNA probe analysis, for the molecular genetic differentiation of tick spotted fever — and typhus fever — group rickettsiae and correlation between with the analysis of polypeptide composition of the above group of rickettsiae are discussed.The data are presented on progress in the identification of various Coxiella burnetii strains as a result of restriction analysis of plasmid DNA as well as chromosomal DNA in combination with DNA probe.New and detailed characteristics of classified and newly isolated strains of rickettsiae and Coxiella burnettii revealed by molecular genetic differentiation techniques are discussed.New identification techniques using DNA probes in combination with restriction analysis of chromosomal from rickettsiae and both plasmid and chromosomal DNA from Coxiella burnettii are considered to have good prospects for future use in epidemiological assesment. The establishment of reference file banks containing restriction endonuclease data on the available typical and atypical strains of rickettsiae and Coxiella burnettii is suggested.Presented at the 4th European Congress of Clinical Microbiology, Nice, 17–20 April, 1989.     

Synthesis and preliminary pharmacological evaluation of imidazo[2,1-f]purine-2,4-dione derivatives

A series of N-8-arylpiperazinylpropyl derivatives of 1,3-dimethyl-(1H,8H)-imidazo[2,1-f]purine-2,4-dione (2–10) and amide derivatives of 1,3-dimethyl-6,7-dihydroimidazo[2,1-f]purine-2,4-(1H,3H)-dione-7-carboxylic acid (11–13) were synthesized. Compounds (2–10) evaluated in vitro were potent 5-HT_1A receptor ligands. Preclinical studies indicated that 8-[3-(N4-phenyl)-piperazin-N1-yl-propyl]-1,3-dimethyl-(1H,8H)-imidazo[2,1-f]purine-2,4-dione (2) exerts anxiolytic-like activity in the four-plate test in mice; however its effect was weaker, than that produced by Diazepam. This compound and 8-[3-(N4-2′-metoxyphenyl)-piperazin-N1-yl-propyl]-1,3-dimethyl-(1H,8H)-imidazo[2,1-f]purine-2,4-dione (3) behaved like antidepressants in the forced swimming test in mice; and their activity in that model was comparable with the effect of Imipramine. The obtained results suggested that the long-chain arylpiperazines (LCAPs) linked to tricyclic derivatives of the theophylline remain a worthy of future research for obtaining new derivatives with potential anxiolytic/antidepressant activity.     

Factors Associated with Serum Vitamin D Metabolites and Vitamin D Metabolite Ratios in Premenopausal Women

The most representative indicator of vitamin D status in clinical practice is 25(OH)D₃, but new biomarkers could improve the assessment of vitamin D status and metabolism. The objective of this study is to investigate the association of serum vitamin D metabolites and vitamin D metabolite ratios (VMRs) with potentially influential factors in premenopausal women. This is a cross-sectional study based on 1422 women, aged 39–50, recruited from a Madrid Medical Diagnostic Center. Participants answered an epidemiological and a food frequency questionnaire. Serum vitamin D metabolites were determined using an SPE–LC–MS/MS platform. The association between participant’s characteristics, vitamin D metabolites, and VMRs was quantified by multiple linear regression models. Mean 25(OH)D₃ concentration was 49.2 + 18.9 nmol/L, with greater deficits among obese, nulliparous, dark-skinned women, and with less sun exposure. A lower R2 ratio (1,25(OH)₂D₃/25(OH)D₃) and a higher R4 (24,25(OH)₂D₃/1,25(OH)₂D₃) were observed in nulliparous women, with high sun exposure, and those with low caloric intake or high consumption of calcium, vitamin D supplements, or alcohol. Nulliparous women had lower R1 (25(OH)D₃/Vit D₃) and R3 (24,25(OH)₂D₃/25(OH)D₃), and older women showed lower R3 and R4. Vitamin D status modified the association of the VMRs with seasons. VMRs can be complementary indicators of vitamin D status and its endogenous metabolism, and reveal the influence of certain individual characteristics on the expression of hydroxylase enzymes.     

Stereoselective synthesis of (RP)-8-substituted-N⁶-acylated and N⁶-alkylated adenosine-3',5'-cyclic phosphorothioic acids as cAMP antagonists

N⁶-Monoalkylated, N⁶-dialkylated and N⁶-acylated (R_P)-adenosine 3′,5′-cyclic phosphorothioic acids have been prepared by stereoselective syntheses from cAMP for a study of protein kinase A antagonist activity. The antagonist activity of the parent primary 6-amino cAMP derivative was reduced after N-monoalkylation. No significant activity was detected in the N,N-dialkylated derivative. Mono N-acylation had little effect on the activity. Hydrogen bonding involving the 6-amino group in cAMPS seems necessary for activity.