Sperm functional aspects and enriched proteomic pathways of seminal plasma of adult men with obesity.

BACKGROUND: Previous studies have demonstrated an association between obesity and the decreased male fertility. OBJECTIVE: to observe the mechanisms by which obesity affects semen quality. MATERIALS AND METHODS: A prospective study was performed including 47 male volunteers, of which 27 were obese group (body mass index >30 kg/m2 ) and 20 were eutrophic (body mass index between 18.5 and 25 kg/m2 ) controls. Sperm functional analysis was performed. The remaining seminal plasma was pooled-four pools per group- and submitted to proteomic analysis by liquid chromatography coupled to tandem mass spectrometry. Groups were compared by an unpaired Student's t-test. Differentially expressed proteins were submitted to functional enrichment analysis using the online platform PantherDB. RESULTS: Obese men presented decreased non-progressive motility, morphology, acrosome integrity, mitochondrial activity, and increased sperm DNA fragmentation. In proteomics analysis, 69 proteins were differentially expressed between the two groups. Among them, one protein was absent, 19 were down-regulated, 49 were up-regulated, and one was exclusive in the study group. The main functions enriched were as follows: negative regulation of the intrinsic pathway of apoptosis, activation of immune and inflammatory, antioxidant activity, among others. CONCLUSION: molecular pathways suggest there is a causative link, and that the effector mechanisms alter sperm metabolic status and defective testicular selection 5 mechanisms.

Organic solvent exposure and contrast sensitivity: comparing men and women.

The goal of this study was to compare the visual contrast sensitivity (CS) of men and women exposed and not exposed to organic solvents. Forty-six volunteers of both genders aged between 18 and 41 years (mean±SD=27.72±6.28) participated. Gas station attendants were exposed to gas containing 46.30 ppm of solvents at a temperature of 304±274.39 K, humidity of 62.25±7.59% and ventilation of 0.69±0.46 m/s (a passive gas chromatography-based sampling method was used considering the microclimate variables). Visual CS was measured via the psychophysical method of two-alternative forced choice using vertical sinusoidal gratings with spatial frequencies of 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, and 16.0 cpd (cycles per degree) and an average luminance of 34.4 cd/m2. The results showed that visual CS was significantly lower (P<0.05) in the following groups: i) exposed men compared to unexposed men at frequencies of 0.2, 0.5, 1.0, and 2.0 cpd; ii) exposed women compared to unexposed women at a frequency of 5.0 cpd; and iii) exposed women compared to exposed men at a frequency of 0.5 cpd, even at exposures below the tolerance limit (300 ppm). These results suggest that the visual CS of exposed men was impaired over a wider range of spatial frequencies than that of exposed women. This difference may have been due to the higher body fat content of women compared to that of men, suggesting that body fat in women can serve as a protective factor against neurotoxic effects.

Taller height as a risk factor for venous thromboembolism: a Mendelian randomization meta-analysis.

Essentials Observational data suggest taller people have a higher risk of venous thromboembolism (VTE). We used Mendelian randomization techniques to further explore this association in three studies. Risk of VTE increased by 30-40% for each 10 cm increment in height. Height was more strongly associated with deep vein thrombosis than with pulmonary embolism. SUMMARY: Background Taller height is associated with a greater risk of venous thromboembolism (VTE). Objectives To use instrumental variable (IV) techniques (Mendelian randomization) to further explore this relationship. Methods Participants of European ancestry were included from two cohort studies (Atherosclerosis Risk in Communities [ARIC] study and Cardiovascular Health Study [CHS]) and one case-control study (Mayo Clinic VTE Study [Mayo]). We created two weighted genetic risk scores (GRSs) for height; the full GRS included 668 single-nucleotide polymorphisms (SNPs) from a previously published meta-analysis, and the restricted GRS included a subset of 362 SNPs not associated with weight independently of height. Standard logistic regression and IV models were used to estimate odds ratios (ORs) for VTE per 10-cm increment in height. ORs were pooled across the three studies by the use of inverse variance-weighted random effects meta-analysis. Results Among 9143 ARIC and 3180 CHS participants free of VTE at baseline, there were 367 and 109 incident VTE events. There were 1143 VTE cases and 1292 controls included from Mayo. The pooled ORs from non-IV models and models using the full and restricted GRSs as IVs were 1.27 (95% confidence interval [CI] 1.11-1.46), 1.34 (95% CI 1.04-1.73) and 1.45 (95% CI 1.04-2.01) per 10-cm greater height, respectively. Conclusions Taller height is associated with an increased risk of VTE in adults of European ancestry. Possible explanations for this association, including taller people having a greater venous surface area, a higher number of venous valves, or greater hydrostatic pressure, need to be explored further.

Is there still room for additional common susceptibility alleles for venous thromboembolism?

UNLABELLED: Essentials Genetic architecture of venous thromboembolism (VTE) remains to be fully disentangled. 11 newly discovered candidate polymorphisms were genotyped in 3019 VTE cases and 2605 controls. None of the 11 polymorphisms were significantly associated with VTE risk. Additional major efforts are needed to identify VTE-associated genetic variants. SUMMARY: Background Through a meta-analysis of 12 genome-wide association studies, the International Network against VENous Thrombosis (INVENT) consortium identified two novel susceptibility loci for venous thromboembolism (VTE). This project has also generated other candidates that need to be confirmed. Objectives To assess the association with VTE of common single-nucleotide polymorphisms (SNPs) that demonstrated strong statistical, but not genome-wide, significance in the INVENT cohorts. Patients/methods Eleven SNPs were genotyped and tested for association with VTE in three case-control studies totaling 3019 patients and 2605 healthy individuals. Results and conclusions None of the tested SNPs showed evidence for association with VTE. Different strategies are needed to decipher the whole spectrum of common and rare genetic variations associated with VTE risk.

Possible evidence of systemic lupus erythematosus and periodontal disease association mediated by Toll-like receptors 2 and 4.

Toll-like receptors (TLRs) participate in the innate immune response and trigger the immune responses of the body. Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown aetiology, characterized by an excessive autoimmune response in the body affecting the connective tissues. The disease is possibly triggered by both environmental aetiological factors and pathological organic processes such as exposure to sunlight, chronic infectious processes and genetic factors. Conversely, periodontal disease is an infectious disease caused by microorganisms in the oral cavity, resulting in a chronic inflammatory process which continuously stimulates the immune response, thus causing damage to the periodontal tissues. The expression of both TLR-2 and TLR-4 receptors are increased in both SLE and periodontal disease. Periodontitis might trigger excessive activation of immune response occurring in SLE by maintaining a high expression of TLRs, leading in turn to the acceleration of the onset and progression of autoimmune reactions. In addition, periodontal treatment is able to reduce the expression of these receptors and therefore the symptoms of SLE. Here we discuss the possible interaction between SLE and periodontitis, and suggest further studies evaluating common features in both factors that could explored, due to morbidity and mortality of SLE and the high incidence of periodontal infections around the world.

A comprehensive assessment of environmental exposures among 1000 North American patients with primary sclerosing cholangitis, with and without inflammatory bowel disease.

BACKGROUND: The relationships between primary sclerosing cholangitis (PSC) and the environment are largely unknown. AIM: To validate associations reported in previous studies and to identify novel environmental exposures among PSC patients. METHODS: We performed a multicenter, case-control analysis utilising self-administered questionnaires. Responses between cases (n = 1000) and controls (n = 663) were compared using multivariable logistic regression adjusted for age and gender. The model was further stratified based on inflammatory bowel disease (IBD) status (with IBD n = 741 without IBD n = 259). RESULTS: Smoking was associated with PSC only when IBD was present (OR, 0.5; 95% CI 0.4-0.7) but not among those PSC patients without IBD (OR, 0.9; 95% CI 0.7-1.2). Compared to controls, women with PSC (irrespective of the presence of IBD) were less likely to have received hormone replacement therapy (HRT; OR, 0.5; 95% CI 0.4-0.7) and were more likely to have recurrent urinary tract infections (OR, 1.6; 95% CI 1.2-2.3). PSC patients regardless of gender or IBD status were less likely to eat fish (OR, 0.4; 95% CI 0.3-0.6) and grilled/barbecued meat (OR, 0.8; 95% CI 0.7-0.9). In contrast, PSC patients with and without IBD were more likely to consume steak/burgers that were more well done (OR, 1.3; 95% CI 1.2-1.5). CONCLUSIONS: IBD (rather than PSC) is associated with smoking. Women with PSC are more likely to have recurrent urinary tract infections and less likely to receive HRT. Dietary intake and methods of food preparation differ in PSC patients when compared to controls.

Longitudinal results of a 10-year clinical trial of repair of amalgam restorations.

The aim of this prospective, blind, and randomized clinical trial was to assess the effectiveness of repair of localized clinical defects in amalgam restorations that were initially scheduled for replacement. A cohort of 20 patients with 40 (Class I and Class II) amalgam restorations that presented one or more clinical features that deviated from the ideal (Bravo or Charlie) according to US Public Health Service criteria, were randomly assigned to either the repair or the replacement group-A: repair, n = 19; and B: replacement, n = 21. Two examiners who had calibration expertise evaluated the restorations at baseline and 10 years after according to seven parameters: marginal occlusal adaptation, anatomic form, surface roughness, marginal staining, contact, secondary caries, and luster. After 10 years, 30 restorations (75%) were evaluated (Group A: n = 17; Group B: n = 13). Repaired and replaced amalgam restorations showed similar survival outcomes regarding marginal defects and secondary caries in patients with low and medium caries risk, and most of the restorations were considered clinically acceptable after 10 years. Repair treatment increased the potential for tooth longevity, using a minimally interventional procedure. All restorations trend to downgrade over time.

The temperature of water ingested ad libitum does not influence performance during a 40-km self-paced cycling trial in the heat.

AIM: The aim of the present study was to evaluate the effects of the temperature of ingested water on performance during a 40-km self-paced cycling trial in the heat (35º C and 60% relative humidity). METHODS: The study was randomized, counterbalanced, crossover and single-blinded. Ten well-trained male cycling athletes (cyclists, mountain bikers or triathletes) who were non-acclimatized to heat were subjected to four experimental situations divided into two sets. In the first set, the participants performed two trials, during which they were given either cold (10º C) or warm water (37º C) ad libitum. In these situations, the volume and timing of the water ingestion (when each bolus was ingested) were recorded and replicated in the second set, but the water temperature was reversed. RESULTS: The performance times were unaffected by the water intake volume (P=0.425), but the water at a temperature of 37º C tended to induce lower performance times (P=0.078) during the trials (AL10=93.0±3.5 min; AL37=94.4±4.1 min; SC10=93.4±4.0; SC37=97.4±4.3 min). The water intake was greater when the water was cold (P<0.05), but the temperature did not affect the heat storage rate, rectal temperature, mean skin temperature, heart rate, blood glucose level, sweat loss, sweat rate, perceived exertion rate or plasma volume changes. However, a significant reduction in the plasma volume change from pre- to postexercise was observed (P<0.01). CONCLUSION: The performance, thermoregulatory, cardiovascular and metabolic responses during a 40-km self-paced cycling trial in the heat were unaffected by different water temperatures.

Genetic variation in the HLA region is associated with susceptibility to herpes zoster.

Herpes zoster, commonly referred to as shingles, is caused by the varicella zoster virus (VZV). VZV initially manifests as chicken pox, most commonly in childhood, can remain asymptomatically latent in nerve tissues for many years and often re-emerges as shingles. Although reactivation may be related to immune suppression, aging and female sex, most inter-individual variability in re-emergence risk has not been explained to date. We performed a genome-wide association analyses in 22,981 participants (2280 shingles cases) from the electronic Medical Records and Genomics Network. Using Cox survival and logistic regression, we identified a genomic region in the combined and European ancestry groups that has an age of onset effect reaching genome-wide significance (P>1.0 × 10(-8)). This region tags the non-coding gene HCP5 (HLA Complex P5) in the major histocompatibility complex. This gene is an endogenous retrovirus and likely influences viral activity through regulatory functions. Variants in this genetic region are known to be associated with delay in development of AIDS in people infected by HIV. Our study provides further suggestion that this region may have a critical role in viral suppression and could potentially harbor a clinically actionable variant for the shingles vaccine.

Can repair increase the longevity of composite resins? Results of a 10-year clinical trial.

OBJECTIVES: The aim of this double-blind clinical trial was to assess the longevity of repairs to localized clinical defects in composite resin restorations that were initially planned to be treated with a restoration replacement. METHODS: Twenty-eight patients aged 18-80 years old with 50 composite resin restorations (CR) were recruited. The restorations with localized, marginal, anatomical deficiencies and/or secondary caries adjacent to CR that were "clinically judged" to be suitable for repair or replacement according to the USPHS criteria were randomly assigned to Repair (n=25) or Replacement (n=25) groups, and the quality of the restorations was scored according to the modified USPHS criteria. The restorations were blind and two examiners scored them at baseline (Cohen Kappa agreement score 0.74) and at ten years (Cohen Kappa agreement score 0.87) restorations. Wilcoxon tests were performed for comparisons within the same group (95% CI), and Friedman tests were utilized for multiple comparisons between the different years within each group. RESULTS: Over the decade, the two groups behaved similarly on the parameters of marginal adaptation (MA) (p>0.05), secondary caries (SC) (p>0.05), anatomy (A) (p<0.05), and colour (C) (p>0.05). CONCLUSIONS: Given that the MA, SC, A and C parameters behaved similarly in both groups, the repair of composite resins should be elected when clinically indicated, because it is a minimally invasive treatment that can consistently increase the longevity of restorations. CLINICAL SIGNIFICANCE: The repair of defective composite resins as an alternative treatment to increase their longevity proved to be a safe and effective treatment in the long term.

Critical properties of the Ziff, Gulari, and Barshad (ZGB) model with inert sites.

We study the model proposed by Ziff, Gulari, and Barshad to mimic the oxidation of carbon monoxide (CO) in the presence of fixed impurities distributed over the catalytic surface. Our focus is on the continuous phase transition between the active phase, where occurs the production of carbon dioxide (CO2), and the inactive phase, where all the non inert sites become filled with oxygen molecules. We employ Monte Carlo simulations to calculate the different ratios between moments of the order parameter at the critical point, as well as, we determine the critical exponents ß and ν⊥ as a function of the concentration of impurities. We show that the presence of impurities over the catalytic surface changes the critical behavior of the system. The critical exponents depend on the concentration of impurities and the model does not belong to the directed percolation universality class.

African ancestry protects against Alzheimer's disease-related neuropathology.

Previous studies in dementia epidemiology have reported higher Alzheimer's disease rates in African-Americans when compared with White Americans. To determine whether genetically determined African ancestry is associated with neuropathological changes commonly associated with dementia, we analyzed a population-based brain bank in the highly admixed city of São Paulo, Brazil. African ancestry was estimated through the use of previously described ancestry-informative markers. Risk of presence of neuritic plaques, neurofibrillary tangles, small vessel disease, brain infarcts and Lewy bodies in subjects with significant African ancestry versus those without was determined. Results were adjusted for multiple environmental risk factors, demographic variables and apolipoprotein E genotype. African ancestry was inversely correlated with neuritic plaques (P=0.03). Subjects with significant African ancestry (n=112, 55.4%) showed lower prevalence of neuritic plaques in the univariate analysis (odds ratio (OR) 0.72, 95% confidence interval (CI) 0.55-0.95, P=0.01) and when adjusted for age, sex, APOE genotype and environmental risk factors (OR 0.43, 95% CI 0.21-0.89, P=0.02). There were no significant differences for the presence of other neuropathological alterations. We show for the first time, using genetically determined ancestry, that African ancestry may be highly protective of Alzheimer's disease neuropathology, functioning through either genetic variants or unknown environmental factors. Epidemiological studies correlating African-American race/ethnicity with increased Alzheimer's disease rates should not be interpreted as surrogates of genetic ancestry or considered to represent African-derived populations from the developing nations such as Brazil.

A genome-wide association study of venous thromboembolism identifies risk variants in chromosomes 1q24.2 and 9q.

OBJECTIVES: To identify venous thromboembolism (VTE) disease-susceptibility genes. PATIENTS AND METHODS: We performed in silico genome wide association scan (GWAS) analyses using genotype data imputed to approximately 2.5 million single-nucleotide polymorphisms (SNPs) from adults with objectively-diagnosed VTE (n=1503), and controls frequency matched on age and gender (n=1459; discovery population). Single-nucleotide polymorphisms exceeding genome-wide significance were replicated in a separate population (VTE cases, n=1407; controls, n=1418). Genes associated with VTE were re-sequenced. RESULTS: Seven SNPs exceeded genome-wide significance (P<5×10(-8)): four on chromosome 1q24.2 (F5 rs6025 [factor V Leiden], BLZF1 rs7538157, NME7 rs16861990 and SLC19A2 rs2038024) and three on chromosome 9q34.2 (ABO rs2519093 [ABO intron 1], rs495828, rs8176719 [ABO blood type O allele]). The replication study confirmed a significant association of F5, NME7 and ABO with VTE. However, F5 was the main signal on 1q24.2 as only ABO SNPs remained significantly associated with VTE after adjusting for F5 rs6025. This 1q24.2 region was shown to be inherited as a haplotype block. ABO re-sequencing identified 15 novel single nucleotide variations (SNV) in ABO intron 6 and the ABO 3' UTR that were strongly associated with VTE (P<10(-4)) and belonged to three distinct linkage disequilibrium (LD) blocks; none were in LD with ABO rs8176719 or rs2519093. Our sample size provided 80% power to detect odds ratios (ORs)=2.0 and 1.51 for minor allele frequencies=0.05 and 0.5, respectively (α=1×10(-8); 1% VTE prevalence). CONCLUSIONS: Apart from F5 rs6025, ABO rs8176719, rs2519093 and F2 rs1799963, additional common and high VTE-risk SNPs among whites are unlikely.

Absorbing states in a catalysis model with anti-Arrhenius behavior.

We study a model of heterogeneous catalysis with competitive reactions between two monomers A and B. We assume that reactions are dependent on temperature and follow an anti-Arrhenius mechanism. In this model, a monomer A can react with a nearest neighbor monomer A or B, however, reactions between monomers of type B are not allowed. We assume attractive interactions between nearest neighbor monomers as well as between monomers and the catalyst. Through mean-field calculations, at the level of site and pair approximations, and extensive Monte Carlo simulations, we determine the phase diagram of the model in the plane y(A) versus temperature, where y(A) is the probability that a monomer A reaches the catalyst. The model exhibits absorbing and active phases separated by lines of continuous phase transitions. We calculate the static, dynamic, and spreading exponents of the model, and despite the absorbing state be represented by many different microscopic configurations, the model belongs to the directed percolation universality class in two dimensions. Both reaction mechanisms, Arrhenius and anti-Arrhenius, give the same set of critical exponents and do not change the nature of the universality class of the catalytic models.

Moment ratios and dynamic critical behavior of a reactive system with several absorbing configurations.

We determine the critical behavior of a reactive model with many absorbing configurations. Monomers A and B land on the sites of a linear lattice and can react depending on the state of their nearest-neighbor sites. The probability of a reaction depends on temperature of the catalyst as well as on the energy coupling between pairs of nearest-neighbor monomers. We employ Monte Carlo simulations to calculate the moments of the order parameter of the model as a function of temperature. Some ratios between pairs of moments are independent of temperature and are in the same universality class of the contact process. We also find the dynamical critical exponents of the model and we show that they are in the directed percolation universality class whatever the values of temperature.

Genetic variation within the anticoagulant, procoagulant, fibrinolytic and innate immunity pathways as risk factors for venous thromboembolism.

BACKGROUND: Venous thromboembolism (VTE) is highly heritable (estimated heritability [h(2)]=0.62) and likely to be a result of multigenic action. OBJECTIVE: To systematically test variation within genes encoding for important components of the anticoagulant, procoagulant, fibrinolytic and innate immunity pathways for an independent association with VTE. METHODS: Non-Hispanic adults of European ancestry with objectively-diagnosed VTE, and age- and sex- matched controls, were genotyped for 13 031 single nucleotide polymorphisms (SNPs) within 764 genes. Analyses (n=12296 SNPs) were performed with plink using an additive genetic model and adjusted for age, sex, state of residence, and myocardial infarction or stroke. RESULTS: Among 2927 individuals, one or more SNPs within ABO, F2, F5, F11, KLKB1, SELP and SCUBE1 were significantly associated with VTE, including factor (F) V Leiden, prothrombin G20210A, ABO non-O blood type, and a novel association with ABO rs2519093 (OR=1.68, P-value=8.08×10(-16) ) that was independent of blood type. In stratified analyses, SNPs in the following genes were significantly associated with VTE: F5 and ABO among both genders and LY86 among women; F2, ABO and KLKB1 among FV Leiden non-carriers; F5, F11, KLKB1 and GFRA1 in those with ABO non-O blood type; and ABO, F5, F11, KLKB1, SCUBE1 and SELP among prothrombin G20210A non-carriers. The ABO rs2519093 population-attributable risk (PAR) exceeded that of FV Leiden and prothrombin G20210A, and the joint PAR of FV Leiden, prothrombin G20210A, ABO non-O and ABO rs2519093 was 0.40. CONCLUSIONS: Anticoagulant, procoagulant, fibrinolytic and innate immunity pathway genetic variation accounts for a large proportion of VTE among non-Hispanic adults of European ancestry.

Drug interactions during periodontal therapy in HIV-infected subjects.

Despite the beneficial effect of the HAART, adverse reactions and drug interaction have been observed. Abnormalities in lipid and glucose metabolism make HIV-positive patients to high risk for the development of coronary heart disease and diabetes, respectively. Besides adverse reactions, drug interaction with other medication can also be observed. In fact, drug interaction may interfere in the periodontal therapy in HIV-infected individuals. For instance, fluconazole, ketoconazole, itraconazole, metronidazole, ciprofloxacin, midazolam and triazolam can interact with some antiretroviral medications, such as zidovudine, nevirapine and ritonavir. The aim of the current study was to show to periodontists and general dental practitioners the importance of understanding the drug interaction in HIV-infection in order to establish a better control during periodontal treatment.

Competing reaction model with many absorbing configurations.

We study a competitive reaction model between two monomers A and B on a linear lattice. We assume that monomer A can react with a nearest-neighbor monomer A or B , but reactions between monomers of type B are prohibited. We include in our model lateral interactions between monomers as well as the effects of temperature of the catalyst. The model is considered in the adsorption controlled limit, where the reaction rate is infinitely larger than the adsorption rate of the monomers. We employ site and pair mean-field approximations as well as static Monte Carlo simulations. We determine the phase diagram of the model in the plane y_{A} versus temperature, where y_{A} is the probability that a monomer of the type A arrives at the surface. This phase diagram shows regions of active and absorbing states separated by a line of continuous phase transitions. Despite the absorbing state of the model to be strongly dependent on temperature, we show that the static critical exponents of the model belong to the same universality class of the directed percolation.

A novel and efficient and low-cost methodology for purification of Macrotyloma axillare (Leguminosae) seed lectin.

The N-acetyl-galactosamine specific lectin from Macrotyloma axillare seeds (LMA) was purified by precipitation and ion exchange chromatography. The LMA 0.2 mol L(-1) fraction showed hemagglutinating activity on erythrocytes A1. The results for molecular mass determinations were about 28 kDa. The LMA pH-dependent assays showed best hemagglutinating activity at pH 6.0-8.0; being decreased at acidic/alkaline conditions and by EDTA treatment. LMA is a tetramer at pH 8.2 and a dimer at pH 4.0. Human erythrocytes from ABO system confirmed the A1 specificity for LMA. This new methodology is useful and easy, with low costs, for lectin purification in large amounts.

Alpha-synuclein, pesticides, and Parkinson disease: a case-control study.

BACKGROUND: Aggregation and fibrillization of the alpha-synuclein protein (encoded by the SNCA gene) may represent key events in the pathogenesis of Parkinson disease (PD). Variability in the length of a dinucleotide repeat sequence (REP1) within the SNCA promoter confers susceptibility to sporadic PD. Pesticide exposures may also confer susceptibility to PD. Our objective was to test possible joint effects of SNCA REP1 genotypes and pesticide exposures on the risk of PD. METHODS: This was a case-control study. Cases were recruited prospectively from the Department of Neurology of the Mayo Clinic, Rochester, MN, after June 1, 1996. The control subjects included unaffected siblings of cases and unrelated population control subjects. We assessed pesticide exposures by telephone interview and genotyped SNCA REP1. Odds ratios (ORs) and 95% CIs were determined using conditional logistic regression models. RESULTS: There were 833 case-control pairs. We observed an increased risk of PD with increasing SNCA REP1 bp length (OR, 1.18 for each score unit; 95% CI, 1.02-1.37; p = 0.03). Pesticide exposures were associated with PD in younger subjects only (lowest quartile of age at study,