Impact of educational level on incident chronic kidney disease during 13 years of follow-up: a prospective cohort study.

OBJECTIVES: To examine the association between educational level and chronic kidney disease (CKD) among the Iranian population. STUDY DESIGN: This is a prospective cohort study conducted in the framework of the Tehran Lipid and Glucose Study. METHODS: A total of 8173 Iranians (men = 3659) aged ≥20 years were included in the study. CKD was defined as estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m2. The association between educational status and CKD was explored using multivariate Cox proportional regression analyses, adjusted for age, gender, current smoking, marital status, body mass index, waist circumference, baseline eGFR, diabetes, hypertension, physical activity, history of cardiovascular diseases and dyslipidaemia. RESULTS: During a median follow-up of 13.14 years, 2609 cases of incident CKD were identified; the corresponding incidence rate was 26.35 (range 25.39-27.34) per 1000 person-years. Compared to low educational level, middle and high educational levels showed lower risks for incident CKD in the crude model [hazard ratio (HR) 0.37 (95% confidence interval {CI} 0.34-0.40) and HR 0.40 (95% CI 0.35-0.45), respectively]; however, these HRs changed direction after further adjustment for age and gender [HR 1.26 (95% CI 1.14-1.39) and HR 1.40 (95% CI 1.22-1.61), respectively]. The increased risk of incident CKD for those at higher educational levels remained significant in the fully adjusted model. In addition, results from the gender stratified analyses were in the same direction as those found among the whole study population (P-value for interaction of gender and education >0.8). CONCLUSIONS: Higher educational levels were associated with incident CKD during more than a decade of follow-up; this finding may be attributed to unhealthy lifestyle behaviours among this population group.

Characterization of virulence genes, serogroups and antimicrobial susceptibility of Shiga toxin producing Escherichia coli isolated from bovine mastitic milk in Tehran, Iran.

Clinical mastitis caused by Enterobacteriaceae accounts for significant economic loss in dairy herds. One of the important pathogens that causes mastitis is Shiga toxinproducing Escherichia coli (STEC). Moreover, mastitis caused by STEC can be considered as a source of transmission of STEC strains to humans through unpasteurized milk. The aim of the current study was to determine of the prevalence, the identification of serogroups, the molecular characterization of virulence factors, and the antibiotic resistance properties of STEC isolates from bovine mastitic milk in dairy cattle in Tehran. A total of 325 milk samples from dairy cattle with clinical signs of mastitis were collected. All E. coli isolates (n: 87, 26.7%) were subjected to multiplex PCR for the detection of stx1, stx2, eaeA, and ehly genes and serogroups. Antibiotic susceptibility testing was carried out by the disc diffusion method for all the STEC isolates. Eighty-seven (26.8%) E. coli and 9 (2.8%) STEC strains were isolated from the bovine mastitic milk samples. Shiga-like toxin genes (stx1 and stx2 or one of them), eaeA and ehly were detected in 100%, 66.6%, and 33.3% of STEC isolates, respectively. O26 (22.2%) and O111 (22.2%) were the most commonly detected STEC serogroups. Other serogroups included O145, O121, O128, O157 and O113. High resistance rate to ampicillin and tetracycline (100%) was observed, followed by trimethoprim/sulfamethoxazole (66.6%) and chloramphenicol (55.5%). STEC isolates were found in bovine mastitic milk in Tehran and most of the STEC isolates in our study were non-O157 strains.

Electric-field-driven magnetization switching and nonlinear magnetoelasticity in Au/FeCo/MgO heterostructures.

Voltage-induced switching of magnetization, as opposed to current-driven spin transfer torque switching, can lead to a new paradigm enabling ultralow-power and high density instant-on nonvolatile magnetoelectric random access memory (MeRAM). To date, however, a major bottleneck in optimizing the performance of MeRAM devices is the low voltage-controlled magnetic anisotropy (VCMA) efficiency (change of interfacial magnetic anisotropy energy per unit electric field) leading in turn to high switching energy and write voltage. In this work, employing ab initio electronic structure calculations, we show that epitaxial strain, which is ubiquitous in MeRAM heterostructures, gives rise to a rich variety of VCMA behavior with giant VCMA coefficient (~1800 fJ V(-1)m(-1)) in Au/FeCo/MgO junction. The heterostructure also exhibits a strain-induced spin-reorientation induced by a nonlinear magnetoelastic coupling. The results demonstrate that the VCMA behavior is universal and robust in magnetic junctions with heavy metal caps across the 5d transition metals and that an electric-field-driven magnetic switching at low voltage is achievable by design. These findings open interesting prospects for exploiting strain engineering to harvest higher efficiency VCMA for the next generation MeRAM devices.

Obesity is associated with prolonged activity of the quadriceps and gastrocnemii during gait.

PURPOSE: To examine the effect of obesity and its potential interaction with knee OA presence on the electromyography patterns of the major knee joint periarticular muscles during walking. SCOPE: One hundred and eighteen asymptomatic adults and 177 adults with moderate knee osteoarthritis were subdivided into categories of healthy weight (n = 77; 20 kg/m(2) < BMI < 25 kg/m(2)), overweight (n = 117; 25 kg/m(2) ⩽ BMI < 30 kg/m(2)), and obese (n = 101; BMI ⩾ 30 kg/m(2) based on their body mass index (BMI). All individuals underwent a three-dimensional gait analysis. Surface electromyograms from the lateral and medial gastrocnemii, lateral and medial hamstrings, vastus lateralis, vastus medialis, and rectus femoris were recorded during self-selected speed walking. Principal component analysis was used to extract major features of amplitude and temporal pattern variability from the electromyograms of each muscle group (gastrocnemii, quadriceps, hamstrings separately). Analysis of variance models tested for main BMI category effects and interaction effects for these features (α = 0.05). Statistically significant BMI category (i.e. obesity) effects were found for features that described more prolonged activations of the gastrocnemii and quadriceps muscles during the stance phase of gait with obesity (P < 0.05). CONCLUSIONS: Obesity was associated with prolonged activation of quadriceps and gastrocnemii, which can result in prolonged knee joint contact loading, and thereby may contribute to the predisposition of knee OA development and progression in obese individuals.

Diagnostic values of different definitions of metabolic syndrome to detect poor health status in Iranian adults without diabetes.

AIMS: This study aimed to compare the diagnostic impact of four definitions of the metabolic syndrome for detection of poor health status in adults without diabetes living in Tehran. METHODS: A representative sample of 950 individuals (64% women), aged ≥ 20 years, participants of the Tehran Lipid and Glucose Study in 2005-2007, were recruited for the study. Health status was assessed using the Iranian version of the 36-item Short Form Health Survey. We assessed the detectability of poor health status by definitions of the National Cholesterol Education Program Adult Treatment Panel III, the International Diabetes Federation, the American Heart Association/National Heart, Lung, and the Blood Institute and the Joint Interim Statement. RESULTS: Compared with other definitions, the Joint Interim Statement identified more participants (46.9%) having the metabolic syndrome. Using the National Cholesterol Education Program Adult Treatment Panel III, the International Diabetes Federation and the Joint Interim Statement, the metabolic syndrome was significantly related to poor physical health status, even after adjustment for confounding variables, in women, but not in men. None of the four definitions of the metabolic syndrome was related to the mental health status in either gender. The receiver operating characteristic curves showed no significant difference in the discriminative power of the metabolic syndrome definitions in detecting poor health status in either gender. However, women showed a higher area under the curve for all definitions, in comparison with men. CONCLUSION: There was no difference in the four different definitions of the metabolic syndrome in detecting poor health status among Iranian adults.

Body mass index (BMI)-for-age and health-related quality of life (HRQOL) among high school students in Tehran.

BACKGROUND: The study aimed to determine the association between body mass index (BMI)-for-age and health-related quality of life (HRQOL) among high school students in Tehran. METHODS: A total of 465 high school students (227 girls and 238 boys) and their parents were participated in this cross-sectional study. Body weight and height of the students were measured. For assessing HRQOL, both adolescent self-report and parent proxy-report of the Persian version of Pediatric Quality of Life Inventory (PedsQL™4.0) questionnaire were completed by adolescents and their parents respectively. RESULTS: The prevalence of overweight and obesity (38.5%) was higher than severe thinness and thinness (2.8%). Mean of adolescent self-reported and parent proxy-reported HRQOL total score were 80.26 ± 12.07 and 81.30 ± 14.08 respectively. In terms of HRQOL subscale scores, the highest subscale score of HRQOL was reported in social functioning (87.27 ± 14.40) and the lowest score was reported in emotional functioning (69.83 ± 18.69). Based on adolescent self-report, adolescent boys had significantly higher mean score for total and all subscale scores of HRQOL compared with girls (P < 0.05). BMI-for-age was inversely correlated to adolescent self-reported HRQOL total score (r = -0.25, P < 0.05). Based on adolescents self-report, HRQOL total score was significantly different by body weight status (F = 16.16, P < 0.05). Normal weight adolescents had significantly higher HRQOL total score compared with overweight (mean difference: 7.32; P < 0.05) and obese adolescents (mean difference: 9.10, P < 0.05). The HRQOL total score was not significantly different between normal weight and underweight adolescents (mean difference: 1.65, P = 0.96). However, based on parent proxy-reports, HRQOL total score was not significantly different by body weight status (F = 2.64, P = 0.059). CONCLUSION: More than one-third of adolescents were overweight and obese. BMI-for-age was inversely correlated to adolescent self-reported HRQOL. Based on adolescents' perspective, overweight and obese adolescents had poorer HRQOL compared with normal weight adolescents. Intervention studies are needed to improve the HRQOL of overweight and obese adolescents in Tehran.

Association between MIF gene variation and Meniere's disease.

Several pieces of evidence support the involvement of immune system in Menière's disease (MD). Macrophage migration inhibitory factor (MIF) plays a key role in immune-mediated reactions. Several studies have shown an association between MIF gene polymorphisms and susceptibility to various inflammatory and autoimmune disorders. The aim of this study was to explore the association between MIF-173 G/C polymorphism and MD in an Iranian population. In this case-control association study, MD cases (N = 72) were recruited and were comprised of definitive MD (N = 58) and probable MD (N = 14) subjects. Normal healthy subjects (N = 100) were also included. Genotyping for MIF-173 G/C polymorphism was carried out using PCR-RFLP technique. There was a significant increase in genotype GG in patients with MD compared with the control group. (GG vs. GC + CC, P = 0.02, OR = 2.08, 95% CI: 1.02-4.3). This was more significant when definitive MD was stratified and compared with the controls (GG vs. GC + CC, P = 0.009, OR = 2.6, 95% CI = 1.19-6.18). This study's result indicates the potential role of MIF in MD of which further evaluation is required. Also, the more significant association between MIF gene polymorphism and definitive MD designates the involvement of specific pathogenic mechanisms which may be considered as a marker for diagnosis.

Apolipoprotein E gene polymorphism and total serum cholesterol level in Iranian population.

BACKGROUND: Apolipoprotein E (APOE) is known as a major regulator of blood lipid levels in humans. A number of APOE gene allelic variants have been reported including E2, E3 and E4. Recent studies suggested a role for APOE in obesity and increased Body Mass Index (BMI) and plasma lipid levels in obese children. AIM: The aim of this study was to examine the association between APOE genetic variants and the BMI and lipid profile in an Iranian cohort. SETTING AND DESIGN: Samples were obtained from subjects who participated in a study based on the WHO-designed MONICA (multinational monitoring of trends and determinants in cardiovascular disease) study for coronary artery disease risk assessment in Zone 17 of Tehran. The study was approved by the local ethical committee. Informed consent was obtained from all subjects included in this study. MATERIALS AND METHODS: Subjects (n=320) were recruited. The level of triglyceride (TG) and total serum cholesterol was tested for all subjects in this study. Genotyping for APOE was carried using polymerase chain reaction-Restriction fragment length polymorphism (PCR-RFLP)technique. STATISTICAL ANALYSIS: Levels of significance were determined using contingency tables by either Chi-square or Fisher exact analysis using the STATA (v8) software. The analysis of regression and significance of differences for level of cholesterol and TG was established by one-way analysis of variance followed by Dunnett post hoc multiple comparison tests using SPSS software Version 11.5. RESULTS: The frequency of allele E2 was significantly higher in patients with total serum cholesterol level <200 mg/dl (P 0.01 OR 2.1 95% CI 1.1-4.2). CONCLUSION: The association found in this study between allele E2 and lower total cholesterol level had been reported in previous studies. We have also observed that the frequency of genotype E2/E3 and E2/E4 was significantly higher in patients with normal total serum cholesterol level compared to patients with abnormal cholesterol (P=0.003 OR 2.4 95% CI; 1.3-4.6). Our data needs to be repeated in a larger population with more information for serum LDL and HDL levels and their subgroups.

Early activation of c-Jun N-terminal kinase and p38 kinase regulate cell survival in response to tumor necrosis factor alpha.

Fas ligand and tumor necrosis factor alpha (TNF) bind to members of the TNF receptor superfamily. Stimulation by Fas ligand results in apoptosis, whereas TNF induces multiple effects including proliferation, differentiation, and apoptosis. Activation of the c-Jun N-terminal kinase (JNK) and p38 kinase pathways is common to Fas and TNF signaling; however, their role in apoptosis is controversial. Fas receptor cross-linking induces apoptosis in the absence of actinomycin D and activates JNK in a caspase-dependent manner. In contrast, TNF requires actinomycin D for apoptosis and activates JNK and p38 kinase with biphasic kinetics. The first phase is transient, precedes apoptosis, and is caspase-independent, whereas the second phase is coincident with apoptosis and is caspase-dependent. Inhibition of early TNF-induced JNK and p38 kinases using MKK4/MKK6 mutants or the p38 inhibitor SB203580 increases TNF-induced apoptosis, whereas expression of wild type MKK4/MKK6 enhances survival. In contrast, the Mek inhibitor PD098059 has no effect on survival. These results demonstrate that early activation of p38 kinase (but not Mek) are necessary to protect cells from TNF-mediated cytotoxicity. Thus, early stress kinase activation initiated by TNF plays a key role in regulating apoptosis.

Requirement of an ICE-like protease for induction of apoptosis and ceramide generation by REAPER.

Genetic studies indicated that the Drosophila melanogaster protein REAPER (RPR) controls apoptosis during embryo development. Induction of RPR expression in Drosophila Schneider cells rapidly stimulated apoptosis. RPR-mediated apoptosis was blocked by N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), which suggests that an interleukin-1 beta converting enzyme (ICE)-like protease is required for RPR function. RPR-induced apoptosis was associated with increased ceramide production that was also blocked by Z-VAD-fmk, which suggests that ceramide generation requires an ICE-like protease as well. Thus, the intracellular RPR protein uses cell death signaling pathways similar to those used by the vertebrate transmembrane receptors Fas (CD95) and tumor necrosis factor receptor type 1.

Anti-immunoglobulin E treatment decreases worm burden and egg production in Schistosoma mansoni-infected normal and interferon gamma knockout mice.

The immunoglobulin E (IgE) response is generally considered an essential component of the host defense against parasitic helminths such as Schistosoma mansoni. In contrast, work on antischistosome vaccines suggests that interferon gamma (IFN-gamma) is the critical immune mediator for vaccine-induced immunity to the parasite. In this study, the total IgE response to a primary S. mansoni infection was suppressed by anti-IgE treatment in both normal mice and in mice with defective IFN genes (gene knockout [GKO]). Reduction of the IgE response resulted in decreased worm burden and a decrease in the number of eggs produced per worm in both normal and GKO mice. Whereas anti-IgE treatment also resulted in reduced hepatosplenomegaly, granulomas around existing schistosome eggs showed normal cellularity. Serum interleukin 4 levels fell in response to the reduction in serum IgE as well. The data suggest that IgE plays a detrimental, rather than beneficial, role for the host in schistosomiasis. Furthermore, the absence of IFN-gamma was found to be of little consequence to the host-response to adults or eggs in a primary schistosome infection.

Ca(2+)-regulated serine protease associated with the nuclear scaffold.

The nuclear scaffold (NS) is a proteinaceous network of orthogonally arrayed intermediate filament proteins, termed lamins, which is responsible for nuclear structure. Recent work has demonstrated that a subset of lamins A/C is proteolytically cleaved to produce an ATP-binding protein. This proteolytic cleavage is accomplished by a NS protease activity, which shows a considerable selectivity for lamins A/C and is stringently regulated by Ca2+ in vitro, suggesting that it might also participate in control of NS breakdown in various scenarios. Here, we identify the major NS protease as a novel serine protease with a predominantly chymotryptic-like substrate preference, and we show that even transient perturbations in cytosolic Ca2+ have significant effects on the NS protease activity. This NS protease activity shows extensive similarities to the multicatalytic proteinase complex. In addition to a potential role in control of NS breakdown at mitosis and/or under pathological conditions, this NS protease is also strategically located for other functions, such as inactivation of various oncogenic proteins or maturation-promoting factor.

Schistosoma mansoni: cell-specific expression and secretion of a serine protease during development of cercariae.

Eukaryotic serine proteases are an important family of enzymes whose functions include fertilization, tissue degradation by neutrophils, and host invasion by parasites. To avoid damaging the cells or organisms that produced them, serine proteases must be tightly regulated and sequestered. This study elucidates how the parasitic blood fluke Schistosoma mansoni synthesizes, stores, and releases a serine protease during differentiation of its invasive larvae. In situ hybridization with a cDNA probe localized the protease mRNA to acetabular cells, the first morphologically distinguishable parasite cells that differentiate from the embryonic cell masses present in the intermediate host snail. The acetabular cells contained vimentin but not cytokeratins, consistent with a mesenchymal, not epithelial, origin. Antiprotease antibodies, localized by immunoperoxidase, showed that the protease progressively accumulated in these cells and was packaged in vesicles of three morphologic types. Extension of cytoplasmic processes containing protease vesicles formed "ducts" which reached the anterior end of fully differentiated larvae. During invasion of human skin, groups of intact vesicles were released through the acetabular cytoplasmic processes and ruptured within the host tissue. Ruptured protease vesicles were noted adjacent to degraded epidermal cells and dermal-epidermal basement membrane, as well as along the surface of the penetrating larvae themselves. These observations are consistent with the proposed dual role for the enzyme in facilitating invasion of host skin by larvae and helping to release the larval surface glycocalyx during metamorphosis to the next stage of the parasite.

Tumour necrosis factor alpha restores granulomas and induces parasite egg-laying in schistosome-infected SCID mice.

Schistosomiasis (bilharzia) is a parasitic disease caused by several species of schistosome worms (blood flukes). The key pathogenic event in this disease is the formation of granulomas around schistosome eggs trapped in portal venules of the liver. Granulomas are a distinctive form of chronic inflammation characterized by localized aggregation of activated macrophages around an inciting stimulus. Each granuloma evolves to form a fibrous scar; in schistosomiasis, the result is widespread hepatic fibrosis and portal hypertension. To identify the specific immune signal molecules necessary for granuloma formation, we studied schistosome infections in severe combined immunodeficient (SCID) mice, which have normal macrophages but lack functional B or T lymphocytes. Here we report that the immunoregulatory cytokine tumour necrosis factor alpha is necessary and sufficient to reconstitute granuloma formation in schistosome-infected SCID mice. Moreover, we find that the parasitic worms require tumour necrosis factor alpha for egg-laying and for excretion of eggs from the host. The implication of this latter result is that the parasite has adapted so successfully to its host that it uses a host-derived immunoregulatory protein as a signal for replication and transmission.

Betaine:homocysteine methyltransferase from rat liver: purification and inhibition by a boronic acid substrate analog.

Betaine:homocysteine methyltransferase (BHMT) from rat liver has been highly purified by an efficient procedure requiring only two chromatographic steps: Sephadex G-100 chromatography and fast protein liquid chromatography chromatofocusing. A 170-fold purification and 7.5% overall yield were achieved. Chromatofocusing yielded three active forms of BHMT with pI values near 8.0, 7.6, and 7.0. The subunit molecular weight of each active form is 45,000 Da as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the native enzyme has a molecular weight of 270,000 as determined by exclusion chromatography. The stability of the purified enzyme was found to be potentiated by the presence of 1 mM dimethylglycine and 1 mM homocysteine. Boronate analogs of betaine (pinanyl N,N,N-trimethylaminomethaneboronate) (4) and dimethylglycine (pinanyl N,N-dimethylaminomethaneboronate) were synthesized from pinanyl iodomethaneboronate (3) and trimethylamine or dimethylamine, respectively. The free acid of the betaine analog (5) was reversibly generated from (4). The inhibition of BHMT by (5) appears competitive with a Ki = 45 microM. Since the Km for betaine measured with the purified enzyme is near 0.1 mM, the boronic acid analog of betaine appears to function effectively as a substrate analog inhibitor of BHMT. The analog does not appear to act as a methyl donor to homocysteine when (5) is substituted for betaine in the enzyme reaction. In addition, an enzyme assay based upon C3-cyano reverse phase HPLC detection of the o-phthalaldehyde derivative of methionine was developed as an alternative to the standard radiochemical assay. Betaine:homocysteine methyltransferase in the picomole range can be quantitated using this assay as indicated by a linear response of enzyme activity to protein concentration.

Arresting tissue invasion of a parasite by protease inhibitors chosen with the aid of computer modeling.

Computer modeling of the three-dimensional structure of an enzyme, based upon its primary sequence alone, is a potentially powerful tool to elucidate the function of enzymes as well as design specific inhibitors. The cercarial (larval) protease from the blood fluke Schistosoma mansoni is a serine protease hypothesized to assist the schistosome parasite in invading the human circulatory system via the skin. A three-dimensional model of the protease was built, taking advantage of the similarity of the sequence of the cercarial enzyme to the trypsin-like class of serine proteases. A large hydrophobic S-1 binding pocket, suspected from previous kinetic studies, was located in the model and confirmed by new kinetic studies with both synthetic peptide substrates and inhibitors. Unexpected structural characteristics of the enzyme were also predicted by the model, including a large S-4 binding pocket, again confirmed by assays with synthetic peptides. The model was then used to design a peptide inhibitor with 4-fold increased solubility, and a series of synthetic inhibitors were tested against live cercariae invading human skin to confirm that predictions of the model were also applicable in a biologic assay.

The Schistosomatium douthitti cercarial elastase is biochemically and structurally distinct from that of Schistosoma mansoni.

The cercarial acetabular gland proteinase of Schistosomatium douthitti, an agent of 'swimmer's itch', has been identified and characterized. Like the corresponding proteinase of Schistosoma mansoni, it has significant elastase activity and can degrade a model of dermal extracellular matrix. However, unlike the S. mansoni enzyme, it has a higher molecular weight (50,000 versus 30,000), is of a different proteinase class (metallo versus serine), and has no significant primary structure homology to the S. mansoni proteinase. While these findings indicate that the failure of S. douthitti to produce chronic schistosomiasis in humans is not due to its lacking, or having a less potent 'penetration proteinase' than S. mansoni, the proteolytic enzymes are sufficiently different to support the hypothesis that the Schistosomatium line diverged quite early from the main branch of Schistosoma evolution.

Benzamidomethaneboronic acid: synthesis and inhibition of chymotrypsin.

Benzamidomethaneboronic acid (2) has been synthesized unambiguously from the reaction of dibutyl iodomethaneboronate and N-lithiohexamethyldisilazane to form dibutyl [bis(trimethylsilyl)amino]methaneboronate (4), which was desilylated, benzoylated, and hydrolyzed to 2. It has been shown that 2 is a strong competitive inhibitor of alpha-chymotrypsin (Ki = 8.1 X 10(-6) M, pH 7.5). The reaction product from dibutyl iodomethaneboronate and sodiobenzamide, previously shown to be a potent inhibitor of chymotrypsin, was shown by this work to be O-linked isomer, benzimidoxy-methaneboronic acid (3). The pH-Ki profile over the pH range 6.5-9.5 was consistent with the formation of an enzyme-inhibitor complex which resembled the metastable tetrahedral reaction intermediates occurring during acylation and deacylation of chymotrypsin-catalyzed hydrolysis.

Experimental induction of caprylate-dependent albumin antibodies.

To provide evidence for the immune nature of the albumin agglutination phenomenon or caprylate dependent albumin agglutinins (CDAA), rabbits were injected with native serum that had been incubated in a solution of sodium caprylate. Two of three rabbits responded with the production of CDAA, which in vitro behaved identical to human antisera. Human cells were agglutinated only when caprylate stabilized albumin was added to the cell/serum mixture or when caprylate free albumin plus sodium caprylate were used. The CDAA failed to agglutinate rabbit cells, although, both human adult and cord cells were agglutinated. These experiments indicate that the CDAA represent an immune response to native albumin that had been altered by caprylate.