HIV and people over 50 years old in Europe.

OBJECTIVE: The aim of the study was to report on HIV and older people in the European Region, including new data stratified by subregion and year. METHODS: Data were collected from the 2008 World Health Organization Regional Office for Europe, Communicable Diseases Unit survey on HIV/AIDS and health systems. RESULTS: It was found that 12.9% of newly reported cases of HIV infection in Western Europe in 2007 were in people aged 50 years or older. In Central Europe, almost one-in-10 newly reported cases of HIV infection were in older people, while the proportion in Eastern Europe was 3.7% in 2007. CONCLUSIONS: The issue of HIV infection among older people is of increasing concern as more people age with HIV infection as a result of the availability of combination antiretroviral therapy.

Direct measurements of the magnetic entropy change.

An experimental device that can accurately measure the magnetic entropy change, Δs, as a function of temperature, T, and magnetic field, H, is presented. The magnetic field source is in this case a set of counter-rotating concentric Halbach-type magnets, which produce a highly homogeneous applied field with constant orientation. The field may be varied from 0 to 1.5 T in a continuous way. The temperature stability of the system is controlled to within ±10 mK and the standard range for the current setup is from 230 K to 330 K. The device is under high vacuum and we show that thermal losses to the ambient are negligible in terms of the calorimetric determination of the magnetic entropy change, while the losses cannot be ignored when correcting for the actual sample temperature. We apply the device to two different types of samples; one is commercial grade Gd, i.e., a pure second-order phase transition material, while the other is Gd5Si2Ge2, a first order magnetic phase transition material. We demonstrate the device's ability to fully capture the thermal hysteresis of the latter sample by following appropriate thermal resetting scheme and magnetic resetting scheme.

An acidic class III chitinase in sugar beet: induction by Cercospora beticola, characterization, and expression in transgenic tobacco plants.

An acidic chitinase (SE) was found to accumulate in leaves of sugar beet (Beta vulgaris) during infection with Cercospora beticola. Two isoforms, SE1 and SE2, with MW of 29 kDa and pI of approximately 3.0 were purified to homogeneity. SE2 is an endochitinase that also exhibits exochitinase activity, i.e., it is capable of hydrolyzing chito-oligosaccharides, including chitobiose, into N-acetyl-glucosamine. Partial amino acid sequence data for SE2 were used to obtain a cDNA clone by polymerase chain reaction. The clone was used to isolate a cDNA clone encoding SE2. The deduced amino acid sequence for SE2 is 58-67% identical to the class III chitinases from cucumber, Arabidopsis, and tobacco. A transient induction of SE2 mRNA during the early stages of infection with C. beticola is much stronger in tolerant plants than in susceptible plants. Transgenic tobacco (Nicotiana benthamiana) plants constitutively accumulate SE2 protein in the intercellular space of their leaves. In a preliminary infection experiment, the transgenic plants did not show increase in resistance against C. nicotianae.

Characterization and localization of new antifungal cysteine-rich proteins from Beta vulgaris.

Two novel antifungal proteins, AX1 and AX2, were isolated from leaves of sugar beet infected with Cercospora beticola. AX1 (MW = 5078 +/- 3D) and AX2 (MW = 5193 +/- 3D) were N-terminally sequenced and identified as monomeric, basic proteins consisting of 46 amino acid residues, of which eight are cysteines. Both AX proteins strongly inhibit growth of C. beticola and other filamentous fungi, but have little or no effect against bacteria. Based on primary sequence homology (24 to 46% identity), they are related to the superfamily of gamma-thionins, which have been isolated recently from seeds of monocotyledons and Brassicaceae. Specific antibodies were raised against the AX proteins after conjugation to diphtheria toxoid. Using immunoblotting and immunohistology, we detected high concentrations of AX proteins extracellularly in cell walls and in globular bodies around necrotic lesions in sugar beet leaves infected with C. beticola, suggesting that AX proteins are involved in antifungal defense. Furthermore, AX proteins or serologically related proteins were detected in xylem, stomata, and stomatal cells as well as in sugar beet styles.

Single-dose kinetics of clomipramine: relationship to the sparteine and S-mephenytoin oxidation polymorphisms.

The influence of the sparteine and the S-mephenytoin oxidation polymorphisms on the kinetics of clomipramine were investigated in 25 healthy volunteers: 10 extensive metabolizers of sparteine and mephenytoin (EMs/EMm), nine poor metabolizers of sparteine and extensive metabolizers of mephenytoin (PMs/EMm), five extensive metabolizers of sparteine and poor metabolizers of mephenytoin (EMs/PMm), and one poor metabolizer of sparteine and mephenytoin (PMs/PMm). A single oral dose of 100 mg clomipramine hydrochloride was given to each subject after an overnight fast. Serum and urine levels of clomipramine and its metabolites were monitored after 1, 2, 3, 4, 6, 8, 11, 14, 24, 36, 48, and 96 hours. Additional serum was monitored after 6, 9, 12, and 15 days in the poor metabolizers. 2-Hydroxyclomipramine was undetectable in most subjects before enzymatic hydrolysis of serum and urine. The total median clearance of clomipramine was 99 L.hr-1 (range, 68 to 210) in the EMs/EMm subjects, 56 L.hr-1 (range, 37 to 183) in the PMs/EMm subjects, 66 L.hr-1 (range, 37 to 89) in the EMs/PMm subjects, and 43 L.hr-1 in the PMs/PMm subject. It was significantly lower in PMs/EMm and EMs/PMm subjects compared with EMs/EMm subjects (p = 0.006 and 0.028, respectively; Mann-Whitney). In addition, the formation clearance of 2-hydroxyclomipramine and the hydroxylation indexes were significantly lower in PMs/EMm subjects, as was the demethylation index in EMs/PMm subjects compared with EMs/EMm subjects. Our data thus provide evidence that the 2- and 8-hydroxylation of clomipramine are catalyzed by CYP2D6 and that the N-demethylation is catalyzed in part by CYP2C.

Chloroguanide metabolism in relation to the efficacy in malaria prophylaxis and the S-mephenytoin oxidation in Tanzanians.

S-Mephenytoin and chloroguanide (proguanil) oxidation was studied in 216 tanzanians. The mephenytoin S/R ratio in urine ranged from <0.1 to 1.16. The distribution was skewed to the right, without evidence of a bimodal distribution. Ten subjects (4.6%, 2.2% to 8.3%, 95% CI) with an S/R mephenytoin ratio >0.9, were arbitrarily defined as poor metabolizers of mephenytoin. The chloroguanide/cycloguanil ratio ranged from 0.82 to 249. There was a significant correlation between the mephenytoin S/R ratio and the chloroguanide/cycloguanil ratios (rs = 0.73; p<0.00001). This indicates that cytochrome P4502C19 or CYP2C19 is a major enzyme that catalyzes the bioactivation of chloroguanide to cycloguanil. Chloroguanide is a pro-drug, and hence a low CYP2C19 activity may lead to prophylactic failure caused by inadequate formation of cycloguanil. Fifty-eight women who previously took either 200 mg chloroguanide daily (n = 26) or 200 mg chloroguanide daily plus 300 mg chloroquine weekly (n = 32) in a malaria chemoprophylaxis study showed that there was significant correlation between the number of earlier breakthrough parasitemia episodes and the chloroguanide/cycloguanil ratio (rs = 0.30; p = 0.02). The breakthrough rate did not correlate with the S/R mephenytoin ratio. However, other factors, such as exposure to mosquitoes and sensitivity of the plasmodium to cycloguanil, are probably more important.

Pharmacokinetics of repaglinide in subjects with renal impairment.

OBJECTIVE: To evaluate the effect of renal impairment and renal failure on the pharmacokinetics and safety of repaglinide. METHODS: We conducted a phase I, multicenter, parallel-group, pharmacokinetic comparison trial with single and multiple doses of repaglinide in subjects with various degrees of renal impairment. Subjects with normal renal function (n = 6) and subjects with renal impairment (mild to moderate, n = 6; severe, n = 6) received treatment with 2 mg repaglinide for 7 days. Subjects in the hemodialysis group (n = 6) received two single doses of 2 mg repaglinide separated by a 7- to 14-day washout period. All subjects had repaglinide serum pharmacokinetic profiles measured for the first and last doses administered. Serum steady-state levels, urine levels, and dialysate levels were also measured. RESULTS: Pharmacokinetic parameters did not show significant changes after single or multiple doses of repaglinide, although the elimination rate constant in the group with severe renal impairment decreased after 1 week of treatment. Subjects with severe impairment had significantly higher area under the curve values after single and multiple doses of repaglinide than subjects with normal renal function. No significant differences in values for maximum serum concentration or time to reach maximum concentration were detected between subjects with renal impairment and those with normal renal function. Hemodialysis did not significantly affect repaglinide clearance. CONCLUSIONS: Repaglinide was safe and well tolerated in subjects with varying degrees of renal impairment. Although adjustment of starting doses of repaglinide is not necessary for renal impairment or renal failure, severe impairment may require more care when upward adjustments of dosage are made.

The hypoalgesic effect of imipramine in different human experimental pain models.

In a randomized, placebo-controlled, double-blind, cross-over study, the hypoalgesic effect of a single oral dose of 100 mg imipramine was investigated in 12 healthy volunteers. Test procedures performed before, 3, 6, and 9 h after medication included determination of (1) pain detection and tolerance thresholds to heat and pressure; (2) the thresholds of quadriceps femoris muscle withdrawal reflex to single and repeated electric stimulation of the sural nerve; (3) amplitude of the reflex evoked by 1.5 times the premedication reflex threshold; and (4) continuous pain rating during the cold pressor test. Imipramine significantly increased pain tolerance thresholds to heat (P = 0.03) and pressure (P = 0.01), and both the psychophysical pain tolerance threshold and the reflex threshold to single electric stimulation (P = 0.02 and P = 0.03, respectively). On the repeated stimuli, which consisted of 4 pulses given at 3 Hz, imipramine induced a significant increase in the threshold at which the pain summated through the stimulation series (P = 0.03), whereas the increase in the threshold at which the reflex summated was not significant (P = 0.09). Pain detection thresholds to heat and pressure, the amplitude of the reflex to single suprathreshold stimulation, and pain ratings during the cold pressor test were unaltered by imipramine. It is concluded that imipramine has a differential hypoalgesic effect on different human experimental pain tests. This provides new possibilities of assessing the differential effect of different tricyclic antidepressants on different pain modalities and intensities.

A new series of highly potent growth hormone-releasing peptides derived from ipamorelin.

A new series of GH secretagogues derived from ipamorelin is described. In an attempt to obtain oral bioavailability, by reducing the size and the number of potential hydrogen-bonding sites of the compounds, a strategy using the peptidomimetic fragment 3-(aminomethyl)benzoic acid and sequential backbone N-methylations was applied. Several compounds from this series release GH with high in vitro potency and efficacy in a rat pituitary cell assay and high in vivo potency and efficacy in anesthetized rats. The tetrapeptide NNC 26-0235 (3-(aminomethyl)benzoyl-D-2Nal-N-Me-D-Phe-Lys-NH2) shows, following iv administration, comparable in vivo potency to ipamorelin, GHRP-2, and GHRP-6 with an ED50 in swine at 2 nmol/kg. NNC 26-0235 demonstrated a 10% oral bioavailability in dogs, and NNC 26-0235 and ipamorelin were able to increase basal GH level by more than 10-fold after oral administration of a dose of 1.8 and 2.7 mg/kg, respectively. The tripeptide NNC 26-0323 (3-(aminomethyl)benzoic acid-N-Me-D-2Nal-N-Me-D-Phe-ol) which showed moderate in vitro potency but lacked in vivo potency demonstrated a 20% oral bioavailability in rats.

Novel orally active growth hormone secretagogues.

A novel class of growth hormone-releasing compounds with a molecular weight in the range from 500 to 650 has been discovered. The aim of this study was to obtain growth hormone secretagogues with oral bioavailability. By a rational approach we were able to reduce the size of the lead compound ipamorelin (4) and simultaneously to reduce hydrogen-bonding potential by incorporation of backbone isosters while retaining in vivo potency in swine. A rat pituitary assay was used for screening of all compounds and to evaluate which compounds should be tested further for in vivo potency in swine and oral bioavailability, fpo, in dogs. Most of the tested compounds had fpo in the range of 10-55%. In vivo potency in swine after iv dosing is reported, and ED50 was found to be 30 nmol/kg of body weight for the most potent compound.

Pharmacological characterisation of a new oral GH secretagogue, NN703.

NN703 is a novel orally active GH secretagogue (GHS) derived from ipamorelin. NN703 stimulates GH release from rat pituitary cells in a dose-dependent manner with a potency and efficacy similar to that of GHRP-6. The effect is inhibited by known GHS antagonists, but not by a GH-releasing hormone antagonist. Binding of (35)S-MK677 to the human type 1A GHS receptor (GHS-R 1A) stably expressed on BHK cells was inhibited by GHRP-6 and MK677 as expected. NN703 was also able to inhibit the binding of (35)S-MK677. However, the observed K(i) value was lower than expected, as based on the observed potencies regarding GH release from rat pituitary cells. Similarly, the effect of NN703 on the GHS-R 1A-induced inositol phosphate turnover in these cells showed a lower potency, when compared with GHRP-6 and MK677, than that observed in rat pituitary cells. The effect of i.v. administration of NN703 on GH and cortisol release was studied in swine. The potency and efficacy of NN703 on GH release were determined to be 155+/-23 nmol/kg and 91+/-7 ng GH/ml plasma respectively. A 50% increase of cortisol, compared with basal levels, was observed for all the tested doses of NN703, but no dose-dependency was shown. The effect of NN703 on GH release after i. v. and oral dosing in beagle dogs was studied. NN703 dose-dependently increased the GH release after oral administration. At the highest dose (20 micromol/kg), a 35-fold increase in peak GH concentration was observed (49.5+/-17.8 ng/ml, mean+/-s.e.m.). After a single i.v. dose of 1 micromol/kg the peak GH plasma concentration was elevated to 38.5+/-19.6 ng/ml (mean+/-s.e.m.) approximately 30 min after dosing and returned to basal level after 360 min. The oral bioavailability was 30%. The plasma half-life of NN703 was 4.1+/-0.4 h. A long-term biological effect of NN703 was demonstrated in a rat study, where the body weight gain was measured during a 14-day once daily oral challenge with 100 micromol/kg. The body weight gain was significantly increased after 14 days as compared with a vehicle-treated group. In summary, we here describe an orally active and GH specific secretagogue, NN703. This compound acts through a similar mechanism as GHRP-6, but has a different receptor pharmacology. NN703 induced GH release in both swine and dogs after i.v. and/or p.o. administration, had a high degree of GH specificity in swine and significantly increased the body weight gain in rats.

Pharmacokinetics of repaglinide in healthy caucasian and Japanese subjects.

The objective of this study was to investigate the pharmacokinetics of three different single doses (0.5, 1.0, and 2.0 mg) of repaglinide in healthy Caucasian and Japanese subjects. In this single-center, open-label, randomized, three-period crossover study, 27 healthy male subjects (15 Caucasian and 12 Japanese) each received three different single doses of repaglinide (0.5, 1.0, and 2.0 mg) at consecutive 24-hour intervals. Pharmacokinetic profiles, including area under the curve (AUC0-t), maximum serum concentration (Cmax), time to Cmax (tmax), and half-life (t1/2), were determined for each dose of repaglinide. The relative change in blood glucose level (RC1h) and area under the blood glucose curve (AUGC0-1) at 1 hour after dose were also measured. After oral dosing, both Cmax and AUC0-t increased linearly with dose within the 0.5- to 2.0-mg dose range, regardless of ethnic group. Both Cmax and AUC0-t were significantly higher in Japanese subjects than in Caucasian subjects. At each dose of repaglinide, Cmax and AUC were statistically significantly higher in Japanese than in Caucasian subjects (p = 0.0038 and 0.023, respectively). Discrepancies in body weight and body mass index (BMI) between Caucasian and Japanese subjects could not explain the between-group differences in Cmax or AUC0-t. Statistically significant differences in pharmacodynamic parameters (RC1h and AUGC0-1) were found between ethnic groups (p < 0.0001), the difference being more pronounced for RC1h than AUGC0-1. At a dose of 2.0 mg, the mean decrease in RC1h was 41% for Japanese subjects and 24% for Caucasian subjects. Hypoglycemic reactions were more common at the highest dose (2.0 mg), where they were observed more frequently in Japanese (7 cases) than in Caucasian subjects (4 cases). It was concluded that higher serum levels of repaglinide and greater reductions in blood glucose levels are found in Japanese than in Caucasian subjects following a single oral dose of repaglinide within the 0.5- to 2.0-mg dose range. Repaglinide is well tolerated in both ethnic groups. The results indicate that glycemic control targets may be achieved at lower doses within the recommended range (0.5-4.0 mg/meal) when repaglinide is used to treat Japanese patients in comparison to Caucasian patients.

Ragaglitazar: the pharmacokinetics, pharmacodynamics, and tolerability of a novel dual PPAR alpha and gamma agonist in healthy subjects and patients with type 2 diabetes.

Ragaglitazar is a novel dual peroxisome proliferator-activated receptor (PPAR) alpha and gamma agonist intended to restore insulin sensitivity and correct diabetic dyslipidemia. These studies assessed single-dose pharmacokinetics and tolerability of ragaglitazar in healthy subjects, as well as multiple-dose pharmacokinetics, pharmacodynamics, and tolerability of ragaglitazar in healthy subjects and in patients with type 2 diabetes. Healthy subjects received a single oral dose (1-120 mg), and healthy subjects and type 2 diabetic patients received a loading dose and thereafter once-daily doses (0.5-16 mg) of ragaglitazar for 6 and 20 days, respectively. Ragaglitazar was rapidly absorbed (tmax: 1.5-1.7 h), with mean AUC0-24 h and Cmax proportional to dose after single and multiple dosing; t1/2 was 80 hours following a single dose and 104 hours in healthy subjects and 122 hours in patients after multiple dosing. Administration of 4 mg ragaglitazar to patients (n = 4) for 21 days resulted in mean decreases from baseline in fasting levels of plasma glucose (18%), C-peptide (18%), fructosamine (6%), triglycerides (36%), free fatty acids (49%), total cholesterol (11%), low-density lipoprotein (LDL) cholesterol (21%), and very low-density lipoprotein (VLDL) cholesterol (15%), as well as an increase in high-density lipoprotein (HDL) cholesterol (33%). Overall, ragaglitazar was well tolerated; with multiple dosing, there was a higher incidence of adverse events for patients that, at the highest dose level (16 mg), included peripheral edema and anemia.

Urethral stricture following transurethral prostatectomy.

Urethral stricture is the most common late complication of transurethral prostatectomy. Uroflowmetry is recommended as the routine screening procedure for strictures postoperatively. If maximal urinary flow rate (Qmax) is below 10 mL/second the patients should be investigated further. The etiology of urethral stricture is still unclear. Further studies are necessary to evaluate the possible etiologic role of infected urine pre- and/or postoperatively, urethral catheterization pre- and postoperatively, catheter material, and the type and size of the resectoscope. A narrow urethra is probably a predisposing factor for stricture formation, but this is not definitively clarified. Only few randomized studies have been performed to evaluate the different prophylactic methods against development of strictures postresection. Resection via perineal urethrotomy, perhaps preceded by urethral calibration, seems to be a way to avoid anterior urethral strictures. The effects of internal urethrotomy preoperatively on stricture formation are conflicting. Further randomized studies are necessary.

New highly potent dipeptidic growth hormone secretagogues with low molecular weight.

Based on NN703, low molecular weight growth hormone secretagouges (GHSs) with a reduced number of hydrogen binding sites were designed by removal of the C-terminal amide group. The compounds were highly potent in combination with high efficacy in a rat pituitary cell assay, being characterized with EC(50) values down to 0.8 nM. Selected compounds were tested in in vivo animal models. The oral bioavailability in dogs was 16-44%. Also, the ED(50) values of the compounds were determined both in dog and swine.

Population pharmacokinetics of tiagabine in epileptic patients on monotherapy.

A retrospective study of the population pharmacokinetics of tiagabine was performed from sparse data collected in a multicentre clinical trial in patients with newly diagnosed partial seizures. The purpose was to estimate the inter patient variability and to study the influence of various demographic, environmental and pathophysiological parameters on the pharmacokinetics of tiagabine in patients on monotherapy. A total of 593 plasma concentrations from 130 patients dosed with 2.5, 5, 7.5 or 10 mg tiagabine twice daily were used for modelling. A one-compartment open model with first-order absorption and elimination was fitted to the concentration-time data using the NONMEM program. Selection of covariates was initially performed using stepwise linear regression analyses. The selected covariates were incorporated in the population model and the importance of each covariate was investigated by means of backwards elimination. A one-compartment model with first-order absorption and elimination adequately described the tiagabine concentration-time profile. The apparent clearance as well as the apparent volume of distribution were both significantly correlated to body height in a nonlinear relationship. No other demographic, environmental or clinical chemical parameters were identified as covariates although only a few pathological values of the latter were present in the data. The mean values of CL/f was 6.10 l/h, of V/f was 62.0 l and of k(a) was 1.25 h(-1) for a subject of 170-cm height. The population half-life was 5.72 h. The apparent clearance and volume of distribution of tiagabine in epilepsy patients on monotherapy were both dependent on body height. Prospective studies are required in order to reveal if dose adjustments based on body height will result in improved therapeutic outcome.

Blood loss in transurethral prostatectomy: epidural versus general anaesthesia.

The peroperative and postoperative blood loss was determined in 36 patients undergoing transurethral prostatectomy (TUR-P) using a photometric method. Seventeen patients were randomly allocated to epidural and 19 patients to general anaesthesia. No significant differences were found between the two groups in the total blood loss peroperatively, the corrected blood loss peroperatively (ml/g/min) or the blood loss postoperatively. The systolic blood pressures were equal in the two groups and no correlation between blood loss and blood pressure was demonstrated. The blood loss per g resected tissue was fairly constant and independent of prostatic weight, but bleeding per minute operating time increased significantly with prostatic size. As the total peroperative blood loss increases with operating time, rapid surgery is a possibility of reducing blood loss in TUR-P. Visual estimation of blood loss during TUR-P of larger glands was unreliable with underestimates of about 100 per cent in one third of the patients, when bleeding exceeded 400 ml. Therefore we recommend an accurate determination of blood loss during resection of larger glands.

[Unusual clinical picture of a rotator cuff lesion]. / Usaedvanligt klinisk billede af rotatorcuff-laesion.

A case of a 76 year-old male admitted to hospital because of a fast-growing tumour over his right shoulder is described. Malignity was initially suspected, but examination showed that the tumour was a cyst overlying, and arising from, the acromioclavicular joint. Furthermore we found a complete lesion of the rotator cuff. Acromioclavicular joint cysts are a known but unusual presentation of complete rotator cuff lesions, and their treatment includes anterior acromioplasty, acromioclavicular arthroplasty, and repair of the rotator cuff. Isolated surgical cystectomy is without purpose, as the cyst is bound to reappear. Surgical treatment is only indicated if there is severe pain, and/or unacceptable cosmetic problems.

[Bottle injuries in Denmark 1991-1995]. / Flaskelaesioner i Danmark 1991-1995.

In the period from 1991-95, 1843 injuries caused by bottles were reported to the Danish EHLASS register, the latter representing 14.2% of the total number of patients seen in the Emergency Room in Denmark. The reports were classified as sharp or blunt injuries; age, sex, and body localisation were registered, and the injuries were grouped into eight body localisations. Hand injuries made up largest group (62%), and of these 92% were sharp injuries. The rest of the injuries (38%) were spread over the other zones, each representing 3-11%. Among the injuries there was a significant overrepresentation of males and young people between 10-24 years of age. Nearly 30% of the injuries were associated with a fall. We estimate that the total number of injuries caused by bottles was 2596/year, and that the incidence rate was 4.98/10,000 person years.

[The urethral plug--an alternative treatment of women with urinary stress incontinence]. / Uretralprop--en alternativ behandling til kvinder med stress-urinikontinens.

A developed urethral plug was evaluated for the treatment of women with genuine urinary stress incontinence. The plug consists of an oval meatal plate, a soft stalk and one or two spheres along the stalk with fixed distances between the meatal plate and the spheres. Inside the stalk is a removable semi-rigid guide pin to ease insertion. Forty women were randomly allocated to treatment with either the two-sphere or the one-sphere plug during period one (two weeks). In period two (two weeks) the patients used the other plug. They then continued with what they judged to be the better plug in period three (two months). Eighteen patients (45%) completed period three with the "preference" plug and 17 were subjectively and objectively continent or improved. Fourteen of these women preferred the two-sphere device. The plugs were equally effective in patients with mild or severe incontinence. Six women developed urinary tract infections and two of these had a plug in the bladder. The urethral plug is an effective treatment in a group of women with stress incontinence. Removal by hand is advisable in order to avoid retention of plugs in the bladder.