The present study was designed to evaluate the effect of olive oil and its main polyphenol (oleuropein) in ovariectomised rats with or without inflammation. Rats (6 months old) were ovariectomised or sham-operated as control. Ovariectomised rats were separated into three groups receiving different diets for 3 months: a control diet with 25 g peanut oil and 25 g rapeseed oil/kg (OVX), the control diet with 50 g olive oil/kg or the control diet with 0·15 g oleuropein/kg. The sham-operated group was given the same control diet as OVX. Inflammation was induced 3 weeks before the end of the experiment by subcutaneous injections of talc (magnesium silicate) in one-half of each group. The sucess of ovariectomy was verified at necropsy by the atrophy of uterine horns. Inflammation, oleuropein or olive oil intakes did not have any uterotrophic activity, as they had had no effect on uterus weight. The plasma concentration of α-1-acid glycoprotein (an indicator of inflammation) was increased in OVX rats with inflammation. With regard to bone variables, osteopenia in OVX was exacerbated by inflammation, as shown by a decrease in metaphyseal and total femoral mineral density. Both oleuropein and olive oil prevented this bone loss in OVX rats with inflammation. At necropsy, oleuropein and olive oil consumption had had no effect on plasma osteocalcin concentrations (marker of bone formation) or on urinary deoxypyridinoline excretion (marker of bone resorption). In conclusion, oleuropein and olive-oil feeding can prevent inflammation-induced osteopenia in OVX rats.

The objective of the present study was to evaluate the effect of a nutritional intervention promoting the Mediterranean food pattern in free-living conditions on LDL electrophoretic characteristics in a group of seventy-one healthy women, aged between 30 and 65 years. The 12-week nutritional intervention consisted of two courses on nutrition and seven individual sessions with a dietitian. The first course provided information on the Mediterranean food pattern and the second was a cooking lesson. LDL peak particle diameter (LDL-PPD) and cholesterol levels in small (LDL-cholesterol<255?Å) and large LDL fractions (LDL-cholesterol>260?Å) were obtained by 2–16% polyacrylamide gel electrophoresis of whole plasma. The sample was divided on the basis of baseline LDL-PPD using tertiles of the distribution (258·4 Å and 260·0 Å). Among the total sample of women, no significant change in LDL-PPD was observed in response to the nutritional intervention. However, subjects who at baseline were in the first tertile of the LDL-PPD distribution (>258·4 Å) showed a significant increase in LDL-PPD and in the proportion of LDL% >260?%uest;Å in response to the 12-week nutritional intervention (P>0·05). In contrast, LDL-PPD decreased significantly (P=0·007) among women with large LDL particles at baseline (LDL-PPD >260 Å) while the proportion of LDL%<255 Å and of LDL%>260 Å remained unchanged. To conclude, changes in the food pattern, in response to a nutritional intervention promoting the Mediterranean food pattern, were accompanied by beneficial modifications in LDL electrophoretic characteristics in women who were characterised at baseline by smaller LDL particles.

We have developed a thirty-nine-item semi-quantitative food-frequency questionnaire (FFQ) to assess the dietary consumption of 11 746 men and women in a prospective cohort study that evaluates the health effects of As from drinking water in Bangladesh. In order to validate the FFQ, two 7 d food diaries (FD) were completed for 189 randomly selected cohort participants in two different seasons of the year. Nutrient values were converted based on both the United States Department of Agriculture's National Nutrient Database and a food composition table for the Indian subcontinent. Pearson product-moment and Spearman non-parametric rank correlation coefficients comparing food and nutrient consumptions estimated from FFQ and 7 d FD were calculated based on log-transformed consumption values with or without adjustment for total energy and correction for within-individual variation. Correlations of macronutrients and common micronutrients including total fat, monounsaturated fat, polyunsaturated fat, saturated fat, protein, carbohydrate, dietary fibre, Na, K, vitamin B6, vitamin B12, riboflavin, Mn, thiamin and Fe were moderately good, ranging from 0·30 to 0·76. However, correlations of other micronutrients were weak (<0·30). Large seasonal variations in intakes of retinol equivalents and vitamin C were observed. This analysis documents the degree of validity of the FFQ in measuring specific nutrient intakes in the study population. To our knowledge, the present study is the first to document the validity of a FFQ with the use of 7 d FD in a Bangladeshi population.

The ActiReg® (PreMed AS, Oslo, Norway) system is unique in using combined recordings of body position and motion alone or combined with heart rate (HR) to calculate energy expenditure (EE) and express physical activity (PA). The ActiReg® has two pairs of position and motion sensors connected by cables to a battery-operated storage unit fixed to a waist belt. Each pair of sensors was attached by medical tape to the chest and to the front of the right thigh respectively. The collected data were transferred to a personal computer and processed by a dedicated program ActiCalc®. Calculation models for EE with and without HR are presented. The models were based on literature values for the energy costs of different activities and therefore require no calibration experiments. The ActiReg® system was validated against doubly labelled water (DLW) and indirect calorimetry. The DLW validation demonstrated that neither EE calculated from ActiReg® data alone (EEAR) nor from combined ActiReg® and HR data (EEAR–HR) were statistically different from DLW results. The EEAR procedure causes some underestimation of EE >11 MJ corresponding to a PA level >2·0. This underestimation is reduced by the EEAR–HR procedure. The objective recording of the time spent in different body positions and at different levels of PA may be useful in studies of PA in different groups and in studies of whether recommendations for PA are being met. The comparative ease of data collection and calculation should make ActiReg® a useful instrument to measure habitual PA level and EE.

Thiamin deficiency remains an important public health problem in some populations. The aim of the present investigation was to study thiamin status during the third trimester of pregnancy and its influence on the concentration of this vitamin in transition (days 13–14 of lactation) and mature breast milk (day 40 of lactation) in a group of Spanish women. The pregnancies and lactation periods of fifty-one healthy women 18–35 (mean 26·7 (sd 3·7)) years old were monitored. Vitamin intake during the third trimester was determined by recording the consumption of foods over 5 d and of the quantities provided by dietary supplements. Thiamin status during this stage of pregnancy was determined by measuring the activation coefficient of erythrocyte transketolase (α-ETK). Milk thiamin content was estimated (in 41% of the subjects) by oxidizing thiamin to thiocrome and measuring fluorescence. Subjects with thiamin intakes above that recommended (group H) had more satisfactory serum α-ETK coefficients (1·01 (sd 0·19)) than did those with lower intakes (group L) (1·21 (sd 0·30); P>0·05). Mature milk thiamin concentrations were significantly higher in group H subjects (0·59 (sd 0·44) μmol/l) than group L subjects (0·25 (sd 0·07) μmol/l). Subjects with α-ETK coefficients >1·25 in the third trimester had significantly lower mature milk thiamin concentration (0·31 (sd 0·10) μmol/l) than did subjects with more satisfactory α-ETK levels at this time (0·55 (sd 0·42) μmol/l; P>0·05). The thiamin status of women can be improved since 25·5% of subjects took less than that recommended and 13·7% showed signs of severe deficiency (α-ETK >1·25). The influence of maternal thiamin intake on α-ETK coefficients and on mature breast milk thiamin concentration is confirmed.

The prevalence of oral candidiasis and its association with malnutrition in terms of protein–energy malnutrition and mineral and vitamin depletion were evaluated in ninety-seven hospitalised older adults aged 82·1 (SD 8·6) years. Patients underwent a complete oral examination with microbiological investigation on admission to our geriatric rehabilitation unit. Patients were assessed nutritionally by evaluation of dietary intake and measurement of anthropometric variables, serum nutritional proteins, ferritin, Zn, folate, vitamins B12 and C. The prevalence of oral candidiasis was 37% (n 36); the proportion of patients with BMI <20 kg/m2 was 32% (n 31). The nutritional status of the population was studied by comparing two groups defined according to the absence (group I; n 61) or presence (group II; n 36) of oral candidiasis. The two groups did not differ on the basis of BMI and mid-arm circumference. However, group II had a smaller leg circumference, lower daily energy and protein intakes, lower albumin and transthyretin levels. Patients successfully treated with fluconazole increased their intake on day 30. The proportion of patients with hypozincaemia (<12·5 μmol/l) and vitamin C deficiency (<0·7 mg/l) was higher in group II. Treatment with antibiotics, poor oral hygiene, denture wearing, and vitamin C deficiency appeared as the most significant independent risk factors associated with oral candidiasis. The present findings show that oral candidiasis appears to be related to malnutrition and results in mucosal lesions that have a negative impact on energy intake, which may subsequently worsen nutritional status.

High doses of n–3 PUFA found in fish oils can reduce the circulating concentration of triacylglycerol (TG), which may contribute to the positive impact of these fatty acids on the risk of CVD. The present study aimed to establish the differential impact of EPA and docosahexaenoic (DHA) on plasma lipids and apo in adults. Forty-two normolipidaemic adult subjects completed a double-blind placebo controlled parallel study, receiving an EPA-rich oil (4·8 g EPA/d), DHA-rich oil (4·9 g DHA/d) or olive oil as control, for a period of 4 weeks. No effects of treatment on total cholesterol, LDL-cholesterol or HDL-cholesterol were evident. There was a significant 22 % reduction in TG level relative to the control value following the DHA treatment (P=0·032), with the 15 % decrease in the EPA group failing to reach significance (P=0·258). There were no significant inter-group differences in response to treatment for plasma apoA1, -C3 or -E levels, although a significant 15 % within-group increase in apoE was evident in the EPA (P=0·006) and DHA (P=0·003) groups. In addition, a within-group decrease in the apoA1:HDL-cholesterol ratio was observed in the DHA group, suggesting a positive impact of DHA on HDL particle size. The DHA intervention resulted in a significant increase in the proportion of EPA P=0·000 and DHA P=0·000 in plasma phospholipids, whilst significant increases in EPA P=0·000 and docosapentaenoic acid P=0·002, but not DHA P=0·193, were evident following EPA supplementation (P>0·05). Our present results indicate that DHA may be more efficacious than EPA in improving the plasma lipid profile.

The Finnish national food composition database Fineli® was updated with recent analytical values for plant sterols (PS) (sitosterol, campesterol, stigmasterol, avenasterol, brassicasterols and stanols) and cholesterol. The quality of the new analytical data was assessed. The aims of the present study were: (1) to compare the effect of old and new database values on PS and cholesterol intakes based on average per capita food consumption data; (2) to estimate the current intake and major sources of these compounds in various population groups according to the national FINDIET 1997 survey data. The intake of total PS was 305 mg/d for men and 237 mg/d for women. The respective intakes for cholesterol were 284 mg/d and 201 mg/d. Women had a higher density of PS in their diets than men, whereas the cholesterol density in the diets did not differ between genders. Cereals, margarine, vegetables and vegetable oils were the main food sources of PS. Meat, meat products and eggs were the main sources of cholesterol. A 9 % greater PS intake estimate was obtained with the new PS database compared with the old PS database, probably due to minor methodological differences between the new and old analyses. Notable changes in analytical methods suggest a lower value (−19 %) for cholesterol intake calculated from the new database compared with the old one. We conclude that researchers can have confidence in the new values for PS and cholesterol, because systematic evaluation of the new analytical values showed them to be of high quality.

The effect of supplementation with different levels of all-rac-α-tocopheryl acetate and the inclusion of different dietary contents of PUFA on the deposition of α-tocopherol stereoisomers in liver and thigh of chickens was evaluated. Ninety-six 1-d-old Ross female broiler chickens were randomly distributed into eight experimental treatments (three replicates each) resulting from four levels of α-tocopheryl acetate without supplementation and supplemented with 100, 200 and 400 mg α-tocopheryl acetate/kg and two levels of dietary PUFA (15 and 61 g/kg). The feeds supplemented with α-tocopheryl acetate contained a similar proportion of each stereoisomer. The diets without α-tocopheryl acetate had the following α-tocopherol stereoisomers (%): RRR 35·1, RRS 24·5, RSR 25·3, RSS 13·9 and total 2S forms 1·3. Consumption of different levels of α-tocopheryl acetate did not lead to statistical differences in α-tocopherol stereoisomer proportion in the liver and thigh. In general, the stereoisomer profiles in the tissues studied were similar, responding to the stereoisomer profile of the diet. Both tissues preferentially accumulated 2R stereoisomer (69–100%). However, when α-tocopheryl acetate was used the discrimination was not specific for the RRR α-tocopherol form. Furthermore, the 2R:2S ratio had a tendency to increase as the polyunsaturation level of the diet increased.

Linoleic acid-rich sunflower-seed supplements (SSS) were used in two experiments (experiment 1, high-concentrate diets; experiment 2, high-forage diets) to study effects on rumen protozoa and the growth of lambs. Both experiments consisted of four treatments, two with a low-protein diet (120 g/kg) and two with a high-protein diet (160 g/kg). For both diets, one treatment was without (control) and one with the SSS (140 g/kg dietary DM). The lambs were fed ad libitum for 70 and 140 d in experiments 1 and 2, respectively. Thereafter, the digestibility of organic matter (OM), acid-detergent fibre and neutral-detergent fibre were determined for each diet with four lambs, and then all lambs were slaughtered and rumen fluid samples were collected and analysed. The results showed substantial decreases (P<0·001) or total elimination of protozoa in the rumen fluid of the SSS-receiving lambs. In the first experiment the SSS also decreased (P<0·05) feed intake, but an increase in average daily gain (P<0·06) resulted in an improved (P<0·05) feed:gain ratio. Also, the SSS increased (P<0·05) the digestibility of fibre. In the second experiment the SSS decreased (P<0·05) the OM digestibility, feed intake and growth of lambs. It was concluded that the use of sunflower-seed supplementation in high-concentrate diets of ruminants reduces rumen fauna, resulting in savings on dietary protein supplements and an increased digestion of feed.

The objectives of the present study were to study the correlation between the change in serum Fe and Fe absorption when administering 100 mg Fe (as FeSO4) orally, and to study the correlation between the absorption from a 3 mg and a 100 mg Fe (as FeSO4) dose. The study was conducted in a group of eleven male blood donors, without any evident infection, who had given blood 8 weeks before the study. On three consecutive mornings the subjects were served a wheat roll fortified with Fe. On the first 2 d the roll was fortified with 3 mg Fe labelled with 59Fe; on day 3 the roll was fortified with 100 mg Fe labelled with 55Fe. The serum Fe response to the 100 mg dose was followed for 6 h. Fe absorption was measured by whole-body counting. High correlations were seen between the absorption of Fe and the change in serum Fe after 100 mg Fe (r2 0·94, P>0·001), between the absorption from 3 mg and 100 mg Fe (r2 0·88, P>0·001), and between the absorption from 3 mg Fe and change in serum Fe after 100 mg Fe (r2 0·90, P>0·001). This strengthens the evidence that it is possible to use the change in serum Fe as a measure of Fe absorption, e.g. when establishing the relative bioavailability for Fe powders. The results also imply that the induced serum Fe increase following 100 mg Fe added to a food could predict the Fe absorption of a small dose of Fe added to the same meal.

The present study investigated trends in reported energy intake, macronutrient intake, physical activity level (PAL) and body weight and effects of excluding under-reporters (UR). Dietary intake and time spent in sixteen activity categories were recorded by 887 female university students (median age 29 years) from 1988 to 2003. Energy expenditure (EE) and PAL were measured using a factorial method. All data collected were self-reported. Individuals with reported EI:EE<0·76 were classified as UR. The remainder were classified as non-under-reporters (NUR). Trends were determined from simple linear regression of median data for each year for the entire cohort (ALL) and for NUR and UR separately, and from multiple regression analysis with the subgroups (NUR and UR) as an additional predictor (BOTH). Prevalence of under-reporting and overweight increased between 1988 and 2003. In ALL and BOTH there were trends to increased body mass, protein intake (g/d and % energy) and carbohydrate intake (% energy only) and decreased fat and alcohol intakes (g/d and % energy). In BOTH there were also increases in reported EI and carbohydrate intake (g/d). None of the trends in NUR was significantly different from those in UR, but some trends in ALL and/or BOTH were not significant when UR were excluded. Trends remaining significant in NUR were increased reported energy intake, protein (g/d) and carbohydrate (g/d) intakes, and decreased fat (% energy only) intake. There were no significant trends in PAL. We conclude that some, but not all, dietary trends were affected by exclusion of UR.

Relationships between vitamin E status (α and γ-tocopherol and their ratio in plasma), anthropometric and biochemical indices, and food and nutrient intakes, were studied in four British National Diet and Nutrition Surveys: children aged 1·5–4·5 years, young people aged 4·0–18·0 years, adults 19·0–64·0 years and adults aged ≥65·0 years. γ-Tocopherol:α-tocopherol ratio declined with age. In older women γ-tocopherol and γ-tocopherol:α-tocopherol ratios were directly related to indices of obesity. In young men α- and γ-tocopherols were directly correlated with obesity, but γ-tocopherol:α-tocopherol ratio was not. For young people and toddlers, fewer obesity indices were available and relationships were weaker. Other fat- and water-soluble vitamin indices correlated directly with α-tocopherol and inversely with γ-tocopherol and γ-tocopherol:α-tocopherol ratio. Whereas α-tocopherol correlated directly with ‘healthy’ nutrient choices (such as intrinsic sugars, dietary fibre, vitamins and potassium) and inversely with ‘unhealthy’ choices (extrinsic sugars and monounsaturated fats, i.e. avoidance of polyunsaturated fat), γ-tocopherol and the γ-tocopherol:α-tocopherol ratio related inversely with ‘healthy’ choices. Food groups had analogous relationships; thus, α-tocopherol related directly to use of polyunsaturated fats, fresh fruits and fruit juices, and inversely to non-polyunsaturated fats and extrinsic sugar. The reverse was true for γ-tocopherol and γ-tocopherol:α-tocopherol ratio. Although the mechanisms underlying these relationships are obscure, the γ-tocopherol:α-tocopherol ratio may reveal poor dietary choices, status predictors and a propensity for obesity in later life, especially in women.

There is evidence that a diet rich in fruit and vegetables reduces blood pressure (BP). Characteristically, the Mediterranean diet is rich in plant-derived foods and also in fat, but studies conducted in Mediterranean countries to relate diet to BP are scarce. We studied the association between fruit and vegetable consumption and BP in a cross-sectional analysis of 4393 participants in the Seguimiento Universidad de Navarra (SUN) Study, an ongoing dynamic cohort study in Spain. Diet was measured using a food-frequency questionnaire previously validated in Spain. Fat represented more than 37% total energy intake. Subjects were considered to have undiagnosed hypertension if they reported systolic BP ≥140 mmHg or diastolic BP ≥90 mmHg, and not a medical diagnosis of hypertension. The adjusted prevalence odds ratio of undiagnosed hypertension (upper v. lowest quintile) was 0·58 (95% CI 0·36, 0·91; P for trend 0·01) for vegetable consumption and 0·68 (95% CI 0·43, 1·09; P for trend 0·10) for fruit consumption. Comparing those in the highest quintile of both fruit and vegetable consumption with those in the lowest quintile of both food groups, the prevalence odds ratio was 0·23 (95% CI 0·10, 0·55; P=0·001), after adjusting for risk factors for hypertension and other dietary exposures. In a Mediterranean population with an elevated fat consumption, a high fruit and vegetable intake is inversely associated with BP levels.

Dietary vitamin K1 (phylloquinone) levels that are sufficient to maintain normal blood coagulation may be sub-optimal for bone, and habitual low dietary intakes of vitamin K may have an adverse effect on bone health. The objective of the present study was to measure the intake and adequacy of phylloquinone intake and the contribution of foods to phylloquinone intake in a nationally representative sample of Irish adults. The North/South Ireland Food Consumption Survey database was used, which contains data collected using a 7 d food diary in a randomly selected sample of Irish adults aged 18–64 years (n 1379; 662 men and 717 women). Phylloquinone intakes were estimated using recently compiled food composition data for phylloquinone. The mean daily intake of phylloquinone from food sources was 79 (sd 44) μg. Intakes were significantly higher (P>0·001) in men than in women at levels of 84 and 75quest;μgsol;d. The main contributors to phylloquinone intakes were vegetables (48%), particularly green vegetables (26%). Potatoes (including chipped and fried potatoes), dairy products and fat spreads contributed 10% each and meat contributed 8%. In men, social class and smoking status influenced phylloquinone intakes. Of the population, 52% had phylloquinone intakes below 1 μg/kg body weight and only 17% of men and 27% of women met the US adequate intakes of 120 and 90 μg/d, respectively. The present study shows that habitual phylloquinone intakes in Irish adults are low, which may have implications for bone health.

Folate deficiency is often discussed as a potential risk factor for CVD and some cancers. Reliable assessment of folate status in large-scale epidemiological studies is therefore of major importance. The present study assessed the value of plasma folate (PF) compared with erythrocyte folate (EF) as a marker of folate status in 363 participants in the European Investigation into Cancer and Nutrition (EPIC)-Potsdam cohort. EF and PF, total homocysteine (tHcy), pyridoxine, cobalamin, creatinine, total protein and packed cell volume were determined; glutamate carboxypeptidase (GCP) C1561T, reduced folate carrier (RFC) G80A and methylenetetrahydrofolate (MTHFR) C677T polymorphisms were analysed. Anthropometric measurements were taken and dietary intake was assessed with the EPIC-Potsdam food-frequency questionnaire. Comparison of EF and PF with factors that may modulate their concentrations was performed. Cross-classification of blood folates in quintile categories resulted in correct classification into the same or adjacent category of 75·5 % of all subjects. Age, BMI, pyridoxine and cobalamin, fruit and vegetable intake, and vitamin supplementation 24 h before blood draw were positively associated with EF and with PF. For tHcy an inverse association was found. Participants with the MTHFR 677TT genotype showed significantly elevated EF concentrations compared with those with 677CT genotype; EF and PF were more strongly correlated (r 0·78, P>0·0001) for participants with MTHFR 677TT genotype than for those with the 677CC or 677CT genotype. In summary, our present results indicate that plasma folate seems to be a suitable marker for assessment of folate status for use in large-scale epidemiological studies.

The interactions of lauric (C12) and myristic acid (C14) in suppressing ruminal methanogenesis and methanogens were investigated with the rumen simulation technique (Rusitec) using bovine ruminal fluid. The fatty acids were added to basal substrates (grass hay:concentrate, 1:1.5) at a level of 48 g/kg DM, provided in C12:C14 ratios of 5:0, 4:1, 3:2, 2·5:2.5, 2:3, 1:4 and 0:5. Additionally, an unsupplemented control consisting of the basal substrates only was employed. Incubation periods lasted for 15 (n 4) and 25 (n 2) d. CH4 formation was depressed by any fatty acid mixture containing at least 40 % C12, and effects persisted over the complete incubation periods. The greatest depression (70 % relative to control) occurred with a C12:C14 ratio of 4:1, whereas the second most effective treatment in suppressing CH4 production (60 % relative to control) was found with a ratio of 3:2. Total methanogenic counts were decreased by those mixtures of C12 and C14 also successful in suppressing methanogenesis, the 4:1 treatment being most efficient (60 % decline). With this treatment in particular, the composition of the methanogenic population was altered in such a way that the proportion of Methanococcales increased and Methanobacteriales decreased. Initially, CH4 suppression was associated with a decreased fibre degradation, which, however, was reversed after 10 d of incubation. The present study demonstrated a clear synergistic effect of mixtures of C12 and C14 in suppressing methanogenesis, mediated probably by direct inhibitory effects of the fatty acids on the methanogens.

Monitoring adolescent diets over time enables the assessment of the effectiveness of public health messages which are particularly important in vulnerable groups such as adolescents. In 2000, 424 children aged 11–12 years old completed two 3 d estimated dietary records. On the fourth day one nutritionist interviewed each child to clarify the information in the diary and foods were quantified with the aid of food models. Nutrient intake was calculated using computerised food tables. These children attended the same seven schools in the same Northumberland area as the 11- to 12-year-old children who recorded their diet using the same method in 1980 (n 405) and 1990 (n 379), respectively. Height and weight, and parental occupation were recorded in all three surveys for each child. Height and weight were used to calculate BMI, weight was used to estimate BMR and parental occupation was used to determine social class. Comparing the macronutrient intakes in 2000 with 1980 and 1990, energy intakes (EI) fell in boys (to 8·45 MJ) and girls (to 7·60 MJ). This fall may, at least in part, be due to an increase in low energy reporting. For 1980, 1990 and 2000 the percentage of boys with EI:BMR below 1·1 was 6, 15 and 23%, respectively; for girls, 3, 14 and 18%, respectively. Percentage energy from fat was unchanged between 1980 and 1990 but fell to 35% (about 76 g/d) in 2000, alongside a 3% increase in percentage energy from starch (30%). Percentage energy from non-milk extrinsic sugars remained above recommendations (16%; about 82 g/d). The number of overweight and obese children increased from 11% to 30% between 1980 and 2000. Positive changes have occurred in the Northumbrian adolescent diet but social inequalities, reported in previous surveys, remain.