Novel immunoadjuvants based on cationic lipid: Preparation, characterization and activity in vivo

The interactions between three different protein antigens and dioctadecyldimethylammonium bromide (DODAB) dispersed in aqueous solutions from probe sonication or adsorbed its one bilayer onto particles was comparatively investigated. The three model proteins were bovine serum albumin (BSA), purified 18 kDa/14 kDa antigens from Taenia crassiceps (18/14-Tcra) and a recombinant, heat-shock protein hsp-18 kDa from Mycobacterium leprae. Protein-DODAB complexes in water solution were characterized by dynamic light scattering for sizing and zeta-potential analysis. Cationic complexes (80-100 nm of mean hydrodynamic diameter) displayed sizes similar to those of DODAB bilayer fragments (BF) in aqueous solution and good colloid stability over a range of DODAB and protein concentrations. The amount of cationic lipid required for attaining zero of zeta-potential at a given protein amount depended on protein nature being smaller for 18 kDa/14 kDa antigens than for BSA. Mean diameters for DODAB/protein complexes increased, whereas zeta-potentials decreased with NaCl or protein concentration. In mice, weak IgG production but significant cellular immune responses were induced by the complexes in comparison to antigens alone or carried by aluminum hydroxide as shown from IgG in serum determined by ELISA, delayed type hypersensitivity reaction from footpad swelling tests and cytokines analysis. The novel cationic adjuvant/protein complexes revealed good colloid stability and potential for vaccine design at a reduced DODAB concentration. (C) 2009 Elsevier Ltd. All rights reserved.

The relative absorption of fatty acids in brown trout (Salmo trutta) fed a commercial extruded pellet coated with different lipid sources

The objective of the present study was to investigate the fatty acid absorption capabilities of brown trout (Salmo trutta) fed commercial extruded diets. Five commercial extruded pellets, different only in the lipid sources used for fat coating, were tested on juvenile brown trout for 45 days. The trout were reared in fresh water at 14.6 ± 0.4° C and 7.7 ±
0.3 mg/l, temperature and dissolved oxygen, respectively. The tested lipid sources were fish oil, canola oil, oleine oil, swine fat and poultry fat. After the adaptation period faeces were collected by gently stripping from naesthetized fish. Fatty acid analysis was performed on experimental diets and on collected faeces to evaluate the relative absorption capabilities of the trout digestive system with respect to each detected fatty acid. The use of the relative absorption efficiency (rAE) was opted to evaluate the intrinsic capability of each fatty acid to be absorbed. Brown trout showed a
specific preferential order of absorption of the fatty acids, preferring shorter over longer chain fatty acids and preferring the more unsaturated to the more saturated fatty acids. The fatty acid that showed the best relative absorbability was the C18:4n-3 (rAE = 5.14 ± 0.72), which has a fairly short carbon chain, but at the same time a high unsaturation level, followed by the C18:3n-3 (rAE = 3.38 ± 0.30). The fatty acid that showed the worst relative absorbability (rAE = 0.21 ± 0.02) was C24:1n-9.

Effect of elevated lipid concentrations on human skeletal muscle gene expression

Dietary fatty acids regulate the abundance and activity of various proteins involved in the regulation of fat oxidation by functioning as regulators of gene transcription. To determine whether the transcription of key lipid metabolic proteins necessary for fat metabolism within human skeletal muscle are regulated by acute elevations in circulating free fatty acid (FFA) concentrations, 7 healthy men underwent 3 randomized resting infusions of Intralipid (20%) with heparin sodium, saline and heparin sodium, or saline only for 5 hours. These infusions significantly elevated plasma FFA concentrations by 15-fold (to 1.67 ± 0.13 mmol/L) in the Intralipid infusion trial, with modest elevations observed in the saline and heparin sodium and saline alone infusion groups (0.67 ± 0.09 and 0.49 ± 0.087 mmol/L, P < .01 both vs Intralipid infusion). Analysis of messenger RNA (mRNA) concentration demonstrated that pyruvate dehydrogenase kinase isoform 4 (PDK4) mRNA, a key negative regulator of glucose oxidation, was increased in all trials with a 24-fold response after Intralipid infusion, 15-fold after saline and heparin infusion, and 9-fold after saline alone. The PDK4 increases were not significantly different between the 3 trials. The mRNA concentration of the major uncoupling protein within skeletal muscle, uncoupling protein 3, was not elevated in parallel to the increased plasma FFA as similar (not, vert, similar2-fold) increases were evident in all trials. Additional genes involved in lipid transport (fatty acid translocase/CD36), oxidation (carnitine palmitoyltransferase I), and metabolism (1-acylglycerol-3-phosphate O-acyltransferase 1, hormone-sensitive lipase, and peroxisomal proliferator-activated receptor-γ coactivator-1α) were not altered by increased circulating FFA concentrations. The present data demonstrate that of the genes analyzed that encode proteins that are key regulators of lipid homeostasis within skeletal muscle, only the PDK4 gene is uniquely sensitive to increasing FFA concentrations after increased plasma FFA achieved by intravenous lipid infusion.

Effects of alternative dietary lipid sources on performance, tissues chemical composition, mitochondrial fatty acid oxidation capabilities, and sensory characteristics in brown trout (Salmo trutta L.)

The efficiency of five dietary lipid sources (fish oil as control—C; canola oil—CO; poultry fat—PF; pork lard—PL; and oleine oil—OO) were evaluated in juvenile brown trout (58.4±0.7 g) in an experiment conducted over 70 days at 14.6±0.4 °C. The best growth was observed in fish fed the C diet whereas the PL diet fed fish had the best feed utilization. Significant differences in carcass and muscle proximate composition, but not in liver, were noted among fish fed the different dietary treatments. The fatty acid composition of muscle largely reflected that of the diets, while total cholesterol was not affected. The atherogenicity and the thrombogenicity qualities of the trout flesh were modified by the lipid sources. Sensory analysis did not show any significant differences among the cooked fillets with respect to dietary treatments, while in uncooked products, some significant differences were observed. The carnitine palmitoyltransferase I and II (CPT-I and CPT-II) activities of liver and white muscle were assayed for a better understanding of the potential β-oxidation capability of the different dietary lipid sources. The hepatic, but not white muscle CPT-I and CPT-II activities were affected by dietary treatments. This study showed that alternative lipid sources could be used effectively for oil coating extruded diets for brown trout.

Effects of dietary lipid source on fillet chemical composition, flavour volatile compounds and sensory characteristics in the freshwater fish tench (Tinca tinca L.)

In this study the effects of soybean and linseed oils on chemical and sensory characteristics of fillets were evaluated in the freshwater fish tench (Tinca tinca L.). Five experimental diets, differing only in the relative amount of soybean and linseed oil, were formulated and the experiment was conducted on 360 sub-adult tench for 12 weeks. The fatty acid composition of muscle reflected that of the diets and significant correlations were observed. Diets containing higher amounts of n − 6 fatty acids were responsible for an increased level of n − 6 fatty acids in the fish flesh. Consequently, an increase in the relative amount of n − 6-derived volatile aldehydes was also observed. These latter compounds are generally reported to contribute negatively to the general aroma of fish muscle and, consistently, the results of the sensory analysis showed a high value for the “off-flavour” attribute for fish fed the diet containing only n − 6-rich soybean oil.

17beta-estradiol upregulates the expression of peroxisome proliferator-activated receptor alpha and lipid oxidative genes in skeletal muscle

This study examined the actions of 17β-estradiol (E2) and progesterone on the regulation of the peroxisome proliferator-activated receptors (PPARα and PPARγ) family of nuclear transcription factors and the mRNA abundance of key enzymes involved in fat oxidation, in skeletal muscle. Specifically,
carnitine palmitoyltransferase I (CPT I), β-3-hydroxyacyl CoA dehydrogenase (β-HAD), and pyruvate dehydrogenase kinase 4 (PDK4) were examined. Sprague–Dawley rats were ovariectomized and treated with placebo (Ovx), E2, progesterone, or both hormones in combination (E+P). Additionally,
sham-operated rats were treated with placebo (Sham) to serve as controls. Hormone (or vehicle only) delivery was via time release pellets inserted at the time of surgery, 15 days prior to analysis. E2 treatment increased PPARα mRNA expression and protein content (P<0·05), compared with Ovx treatment. E2 also resulted in upregulated mRNA of CPT I and PDK4 (P<0·05). PPARγ mRNA expression was also increased (P<0·05) by E2 treatment, although protein content remained unaltered. These data
demonstrate the novel regulation of E2 on PPARα and genes encoding key proteins that are pivotal in regulating skeletal muscle lipid oxidative flux.

Effect of dietary modificatio of muscle long-chainN-3 fatty acid on plasma insulin and lipid metabolite, carcass traits, and fat deposition in lambs

In a previous study we showed that feeding fish meal significantly increased muscle long chain n-3 fatty acids (FA) and hot carcass weight. In this study we compared the effect of fish meal and fish oil on increasing muscle long-chain FA. We also investigated whether the increase in carcass weight was due to the effect of dietary enrichment of muscle long-chain n-3 FA on muscle membrane phospholipids and(or) to rumen by-pass protein provided by fish meal. Forty crossbred ([Merino x Border Leicester] x Poll Dorset) wether lambs between 26 and 33 kg BW were randomly assigned to one of five treatments: 1) basal diet of oaten:lucerne chaff (Basal); 2) Basal + fish meal (9% DM) = FM; 3) Basal + fish oil (1.5% DM) with protected sunflower meal (9% DM ) = FOSMP; 4) Basal + fish oil (1.5% DM) = FO; or 5) Basal + protected sunflower meal (10.5% DM) = SMP. Daily intake of ME (9.60 - 10.5 MJ ME/d) and CP (150 to 168 g/d) in all treatments was kept similar by varying the ratio of oaten:lucerne chaff and by feeding the animals at 90% ad libitum intake. Blood samples were collected at the start of the experiment and on the day (d 42) prior to slaughter. Lambs were then slaughtered at a commercial abattoir. At 24 h postmortem carcass traits were measured and longis-simus thoracis muscle taken for analysis of FA of phospholipid and triglyceride fractions. Lambs fed FO and FOSMP showed a marked increase in muscle longchain n-3 FA (P < 0.001) and a reduction in magnitude of the rise in insulin concentration (P < 0.001) after feeding compared with lambs fed Basal and SMP diets. Lambs in FM had a moderate increase (P < 0.001) in muscle long-chain n-3 FA content. Compared with Basal diet, both plasma total cholesterol (P < 0.02) and high-density lipoprotein cholesterol (P < 0.001) levels were greater in SMP and less in FO and FOSMP treat- ments. The i.m. fat content was reduced (P < 0.05) in FM and FO treatments, but carcass weight was increased only with fish meal (P < 0.03). Adding SMP to FO produced muscle with an intermediate level of i.m. fat, whereas muscle long-chain n-3 FA, i.m. fat, and insulin concentration were unchanged with SMP treatment. These results indicate that an increase in carcass weight in FM may be due to the supply of ruminally undegraded protein. They also suggest that fish oil along with fish meal can increase long-chain n-3 FA content in phospholipid of muscle membrane. This may be associated with reduced i.m. fat content and altered insulin action and lipoprotein metabolism.

Lipids and FA analysis of canine prostate tissue

It is widely reported that an association exists between dietary fat intake and the incidence of prostate cancer in humans. To study this association, there is a need for an animal model where prostate carcinogenesis occurs spontaneously. The canine prostate is considered a suitable experimental model for prostate cancer in humans since it is morphologically similar to the human prostate and both humans and dogs have a predisposition to benign and malignant prostate disease. In this study, the FA and lipids profiles of the normal canine prostate tissue from nine dogs were examined. The total lipid content of the canine prostate tissue was 1.7±0.5% (wet weight). The lipid composition analysis using TLC-FID showed that the two major lipid classes were phospholipids and TAG. Total FA, phospholipid, and TAG FA analysis showed that the major FA were palmitic acid (16∶0), stearic acid (18∶0), oleic acid (18∶1), linoleic acid (18∶2n−6), and arachidonic acid (20∶4n−6), The n−3 FA were present at <3% of total FA and included α-linolenic acid (18∶3n−3) (in total and TAG tissue FA), EPA (20∶5n−3) (not in TAG), and DHA (22∶6n−3) (not in TAG). The n−3/n−6 ratio was 1∶11, 1∶13, and 1∶8 in total, phospholipid, and TAG FA, respectively. This study shows the canine prostate has a low level of n−3 FA and a low n−3/n−6 ratio. This is perhaps due to low n−3 content of the diet of the dogs. FA analysis of dogfoods available in Australia showed that the n−3 content in both supermarket and premium bran dogfoods was <3% (wet weight), and the n−3/n−6 ratio was low.

Growth and product quality of European eel (Anguilla anguilla) as affected by dietary protein and lipid sources

A 12-week feeding trial was undertaken to evaluate the effects of partial replacement of marine raw materials in the diet with soybean meal and beef tallow on growth and product quality of European eel (Anguilla anguilla ). Based on growth, feed efficiency and chemical composition, soybean meal was found to be an unsuitable ingredient for eel diets as a substitute for fishmeal (25% on a protein basis), probably because of the low digestibility of its carbohydrate content. However, beef tallow can be used to replace 50% of fish oil without reduction in growth, provided that digestible carbohydrates are present in the diet. No major effects of protein and lipid sources in the diet were found on fillet chemical composition. Sensory analysis revealed no significant differences between fish fed the control and the experimental diets, with the exception of salty taste which was significantly higher in fish fed combined soybean meal and beef tallow compared with fish fed the control diet.

Parametric conditional frailty models for recurrent cardiovascular events in the lipid study

Background Analysis of recurrent event data is frequently needed in clinical and epidemiological studies. An important issue in such analysis is how to account for the dependence of the events in an individual and any unobserved heterogeneity of the event propensity across individuals.Methods We applied a number of conditional frailty and nonfrailty models in an analysis involving recurrent myocardial infarction events in the Long-Term Intervention with Pravastatin in Ischaemic Disease study. A multiple variable risk prediction model was developed for both males and females. Results A Weibull model with a gamma frailty term fitted the data better than other frailty models for each gender. Among nonfrailty models the stratified survival model fitted the data best for each gender. The relative risk estimated by the elapsed time model was close to that estimated by the gap time model. We found that a cholesterol-lowering drug, pravastatin (the intervention being tested in the trial) had significant protective effect against the occurrence of myocardial infarction in men (HR¼0.71, 95% CI0.60–0.83). However, the treatment effect was not significant in women due to smaller sample size (HR¼0.75, 95% CI 0.51–1.10). There were no significant interactions between the treatment effect and each recurrent MI event (p¼0.24 for men and p¼0.55 for women). The risk of developing an MI event for a male who had an MI event during follow-up was about 3.4 (95% CI 2.6–4.4) times the risk compared with those who did not have an MI event. The corresponding relative risk for a female was about 7.8 (95% CI 4.4–13.6). Limitations The number of female patients was relatively small compared with their male counterparts, which may result in low statistical power to find real differences in the effect of treatment and other potential risk factors.Conclusions The conditional frailty model suggested that after accounting for all the risk factors in the model, there was still unmeasured heterogeneity of the risk for myocardial infarction, indicating the effect of subject-specific risk factors. These risk prediction models can be used to classify cardiovascular disease patients into different risk categories and may be useful for the most effective targeting of preventive therapies for cardiovascular disease.

A protocol for the combined sub-fractionation and delipidation of lipid-binding proteins by hydrophobic interaction chromatography

Cellular lipids frequently co-purify with lipid binding proteins isolated from tissue extracts or heterologous host systems and as such hinder in vitro ligand binding approaches for which the apo-protein is a prerequisite. Here we present a technique for the complete removal of unesterified fatty acids, phospholipids, steroids and other lipophilic ligands bound to soluble proteins, without protein denaturation. Peroxisome proliferator activated receptor gamma ligand binding domain and intracellular fatty acid binding proteins were expressed in an Escherichia coli host and completely delipidated by hydrophobic interaction chromatography using phenyl sepharose. The delipidation procedure operates at room temperature with complete removal of bound lipids in a single step, as ascertained by mass spectrometry analysis of organic solvent extracts from purified protein samples. The speed and capacity of this method makes it amenable to scale-up and high-throughput applications. The method can also easily be adapted for other lipid binding proteins that require delipidation under native conditions.

Effect of hydrogen/air flow rates and scan rate on the flame ionisation detection response of phopholipids, and their quantitative and qualitative analysis by Iatroscan (Mark‐6s) TLC‐FID

The combined effect of scan speed, hydrogen and air flow rates on the flame ionization detection (FID) peak response of phospholipid classes has been studied to determine the optimum levels of these parameters. The phospholipid composition of different types of commercial lecithins, as well as lecithins combined with fish oils, has been analyzed by Iatroscan TLC‐FID Mark‐6s under optimized conditions. An air flow rate of 2 L/min, a hydrogen flow rate of 150–160 mL/min, and a scan speed of 30 s/rod seem to be the ideal conditions for scanning phospholipids with complete pyrolysis in the flame in the Mark‐6 model. Increasing the scan speed rapidly decreased the FID response. A hydrogen flow rate as high as 170 mL/min could be used at relatively low air flow rates (&#x003C2 L/min) and the response declined when both air flow rate and hydrogen flow rate increased simultaneously. Both linear and curvilinear relationships had highly significant correlations (p&#x003C0.01) with the sample load. Time course reactions, including the hydrolysis of phosphatidylserine using enzymes, can be successfully monitored by the Iatroscan TLC‐FID Chromarod system.

Long-term effect of calcium-vitamin D3 fortified milk on blood pressure and serum lipid concenstrations in healthy older men

Background/Objectives:
Some epidemiological and clinical studies have shown that increased dairy consumption or calcium and/or vitamin D supplementation can have a beneficial effect on blood pressure, and lipid and lipoprotein concentrations. The aim of this study was to assess the long-term effects of calcium-vitamin D3 fortified milk on blood pressure and lipid-lipoprotein concentrations in community-dwelling older men.

Subjects/Methods:
This is a substudy of a 2-year randomized controlled trial in which 167 men aged >50 years were assigned to receive either 400 ml per day of reduced fat (approx1%) milk fortified with approximately 1000 mg of calcium and 800 IU of vitamin D3 or to a control group receiving no additional fortified milk. Weight, blood pressure, lipid and lipoprotein concentrations were measured every 6 months. Participants on lipid-lowering (n=32) or antihypertensive medication (n=39) were included, but those who commenced, increased or decreased their medication throughout the intervention were excluded (n=27).

Results:
In the 140 men included in this study (milk, n=73; control, n=67), there were no significant effects of the calcium-vitamin D3 fortified milk on weight, systolic or diastolic blood pressure, total cholesterol, high-density lipoprotein or low-density lipoprotein cholesterol or triglyceride concentrations at any time throughout the intervention. Similar results were observed after excluding men taking antihypertensive or lipid-lowering medication or limiting the analysis to those with baseline calcium intakes <1000 mg per day and/or with hypovitaminosis D (25(OH)D <75 nmol/l).

Conclusions:
Supplementation with reduced-fat calcium-vitamin D3 fortified milk did not have a beneficial (nor detrimental) effect on blood pressure, lipid or lipoprotein concentrations in healthy community-dwelling older men.

Lipid composition of cashmere goat fibres

This study examined the differences in the chemical composition, particularly fatty acids, of the lipid extracted from the fibre of bucks, does and castrated goats. The study provides a more detailed understanding of the chemical composition of buck fibre lipid and how it varies throughout the year, and also details the effect of body region and nutrition on the production and chemical composition of lipid from buck fibre. Lipid was extracted with either petroleum ether (non-polar) or chloroform/methanol azeotrope (polar) and analysed by gas chromatography and gas chromatography-mass spectrometry. The more polar solvent system extracted larger amounts of lipid and more of each individual fatty acid. The following buck specific ethyl branched fatty acids were identified: 2-ethylhexanoic, 4-ethylhexanoic, 2-ethyloctanoic, 4-ethyloctanoic, 6-ethyloctanoic, 2-ethyldecanoic, 4-ethyldecanoic, 2-ethyldodecanoic, 6-ethyldodecanoic, 4-ethyldodecanoic, 2-ethyltetradecanoic, 6-ethyltetradecanoic, 4-ethyltetradecanoic, 2-ethylhexadecanoic and 4-ethyloctadecanoic acids. Of these buck specific fatty acids only 4-ethylhexanoic (T), 4-ethyloctanoic, 4-ethyldecanoic, 4-ethyldodecanoic, 6-ethyldodecanoic (T), 4-ethyltetradecanoic, 2-ethylhexadecanoic (T) and 4-ethylhexadecanoic acids have been previously identified or tentatively identified (T) in buck fibre extracts. This shows that the chemical composition of buck fibre lipid is more complex than previously reported, and that it may be more difficult than previously thought to artificially duplicate the odour of the buck. Buck fibre samples had lower average concentrations of 2-methylpropanoic, 2-methylbutanoic, iso-pentadecanoic, anteiso-pentadecanoic, iso-hexadecanoic, anteiso-heptadecanoic, iso-octadecanoic and anteiso-nonadecanoic acids as compared with fibre samples from does, spayed does, or wethers that were castrated at one month of age. The reduced concentrations of these fatty acids in buck fibre extracts were likely to be due to the synthesis of ethyl branched derivatives of iso and anteiso fatty acids. Buck fibre samples had higher concentrations of benzoic acid as compared with fibre samples from does, spayed does, or wethers that were castrated at one month of age. The significance of these results is that non buck specific fatty acids may also make a contribution to the odour of bucks. When fibre samples were collected at various times throughout the year, it was found that the bucks had increased amounts of lipid and ethyl branched fatty acids in fibre samples shorn from March to September, as compared with fibre samples shorn in November and January. The increase in the amount of lipid and ethyl branched fatty acids corresponded with both the rutting period of the buck and the period when the buck odour was increased. This suggests that ethyl branched fatty acids could be pheromones. The variation in lipid content and fatty acid composition was also examined between fibre samples collected from different body regions of the buck during April, as alterations in sebaceous gland activity around the neck during rutting have been reported. It was found that the average amount of lipid in the neck region of the bucks was not statistically higher than the average amounts in the midside and hind regions. However, the ethyl branched fatty acid concentrations were statistically higher in the fibre from around the neck as compared with the fibre from the other body regions, which is consistent with the odour of the buck being most pronounced around the head and neck region. The lipid content and composition of fibre samples from bucks fed high and low quality diets (lucerne and pangola grass, respectively) was examined to determine the effect of nutrition on buck specific components. The high quality diet increased the amount of lipid and ethyl branched fatty acids in fibre samples collected in April from the neck, midside and hind regions, as compared with fibre samples from the corresponding body regions from bucks fed the low quality diet. Thus it may be possible for the pheromone levels of bucks to be increased by simply providing them with good nutrition. The lipid content and ethyl branched fatty acid concentrations of fibre samples increased earlier in the year for the lucerne fed bucks as compared with the pangola grass fed bucks. The lucerne fed bucks had increased concentrations of ethyl branched fatty acids in fibre samples shorn during December to June (6 months) whereas the pangola grass fed bucks had increased concentrations of ethyl branched fatty acids in fibre samples shorn during April to August (4 months). These observations show that good nutrition can result in both the earlier production of ethyl branched fatty acids and an extended period when ethyl branched fatty acids are produced. This suggests that nutrition can be used to manipulate pheromone levels in the buck. The period when the ethyl branched fatty acids were increased corresponded with the period when the plasma luteinizing hormone (LH) and testosterone concentrations, odour and sebaceous gland volume of the bucks were increased, which supports the assumption that ethyl branched fatty acids are involved in odour production and act as pheromones.

Effects of rosiglitazone on intramyocellular lipid accumulation in Psammomys obesus

Objective: To examine the effects of rosiglitazone in intramyocellular lipid (IMCL) content in diabetic Psammomys obesus using novel electron microscopy technologies.

Background: P. obesus is an unique polygenic model of obesity and type 2 diabetes. Male diabetic P. obesus were treated daily with 5 mg/Kg Rosiglitazone by oral gavage for 14 days. Data were compared with a group of age-matched diabetic P. obesus treated with saline vehicle.

Methods: Assessment of insulin resistance and adiposity were determine before and after the treatment period by oral glucose tolerance test (oGTT) and dual energy X-ray absorptiometry (DEXA) analysis. We used a new scanning electron microscopy technology, (WETSEM) to investigate the effects of rosiglitazone administration on IMCL content, size and distribution in red gastrocnemius muscle.

Results: Rosiglitazone treatment improved glucose tolerance in P. obesus with no difference in the overall body fat content although a significant reduction in subscapular fat mass was observed. Rosiglitazone changed the distribution of lipid droplet size in skeletal muscle. Treated animals tended to have smaller lipid droplets compared with saline-treated controls.

Conclusions: Since smaller IMCL droplets are associated with improvements in insulin sensitivity, we propose that this may be an important mechanism by which rosiglitazone affects glucose tolerance.