Metabolomic and transcriptomic profiles after immune stimulation in the zebrafish testes.

Fish farms are prone to disease outbreaks and stress due to high-density rearing conditions in tanks and sea cages, adversely affecting growth, reproduction, and metabolism. To understand the molecular mechanisms affected in the gonads of breeder fish after an immune challenge, we investigated the metabolome and the transcriptome profiles in zebrafish testes after inducing an immune response. After 48 h of the immune challenge, ultra-high-performance liquid chromatography (LC-MS) and transcriptomic analysis by RNA-seq (Illumina) resulted in 20 different released metabolites and 80 differentially expressed genes. Among these, glutamine and succinic acid were the most abundant metabolites released and 27,5% of the genes belong to either the immune or reproduction systems. Pathway analysis based on metabolomic and transcriptomic crosstalk identified cad and iars genes that act simultaneously with succinate metabolite. This study deciphers interactions between reproduction and immune systems and provides a basis to improve protocols in generating more resistant broodstock.

Impact of Treatment with Unilateral Implant- Supported Fixed Partial Prosthesis on the Frequency of Masticatory Side Switches in Patients with Unilateral Posterior Missing Teeth.

This study aimed to determine the impact of treatment with an implant-supported fixed partial prosthesis (ISFPP) on the frequency of masticatory side switches in patients with unilateral posterior missing teeth. This was a prospective study of 30 patients with unilateral posterior missing teeth treated with one-, two-, or three-unit ISFPPs. Comparison was with 10 healthy individuals with complete natural dentitions. Each participant performed masticatory assays, which involved chewing pieces of silicon inside a latex bag, at baseline and at 3-months' follow-up. The frequency of masticatory side switches was reported as the masticatory side-switch index: the number of side switches divided by the maximum number of possible switches. Data were analyzed by Kruskal-Wallis, Mann-Whitney U test, or Wilcoxon test, as appropriate. At baseline, the masticatory side-switch index was lower only in patients with three missing teeth than in controls. At 3 months after treatment, a significant increase in the masticatory side-switch index was only observed in patients treated with three restorative units. Treatment with ISFPPs does not change the masticatory side-switch frequency in patients with unilateral posterior missing teeth, though it could increase it in patients with three missing teeth.

Immune genes, IL1ß and Casp9, show sexual dimorphic methylation patterns in zebrafish gonads.

There is crosstalk between the immune and reproductive systems in which sexual dimorphism is a common pattern in vertebrates. In recent years, epigenetics has emerged as a way to study the molecular mechanisms involved in gonadal development, those responsible for integrating environmental information that contribute to assigning a specific sexual phenotype (either an ovary or a testis). The knowledge of epigenetic mechanisms in certain molecular processes allows the development of epigenetic markers. In fish gonads, the existence of reproduction-immune system interactions is known, although the epigenetic mechanisms involved are far from clear. Here, we used the zebrafish (Danio rerio) as a model to study the DNA methylation patterns in gonads of two well-known innate immune genes: IL1ß and Casp9. DNA methylation levels were studied by a candidate gene approach at single nucleotide resolution and gene expression analyses were also carried out. Results showed that there was clear sexual dimorphism in the DNA methylation levels of the two immune genes studied, being significantly higher in the testes when compared to the ovaries. In summary, and although further research is needed, this paper presents sexual dimorphic methylation patterns of two immune-related genes, thus sex-biased differences in methylation profiles should considered when analyzing immune responses in fish. Data showed here can help to develop epimarkers with forthcoming applications in livestock and fish farming production, for example, in immune fish diseases or sexual control programs as epigenetic molecular tools to predict environmental pressure in the gonads.

Differential responses to environmental challenge by common carp Cyprinus carpio highlight the importance of coping style in integrative physiology.

Common carp Cyprinus carpio displaying proactive or reactive stress coping styles were acclimated to two environmental regimes (low oxygen and low temperature), and selected groups were tested for response to an inflammatory challenge (Escherichia coli lipopolysaccharide, LPS). Plasma glucose and lactate levels were measured, as were selected C. carpio-specific messenger (m)RNA transcript abundance, including cortisol receptor (CR), enolase (ENO), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and interleukin-1-beta (IL1ß) was measured in individual whole brain samples. Basal levels (in sham injected fish held in normoxic conditions at 25° C) of plasma lactate and glucose differed between coping styles, being significantly lower in proactive individuals. Both variables increased in response to LPS challenge, with the exception of plasma glucose in reactive fish held in hypoxia. Baseline levels of gene expression under control conditions were significantly different for GAPDH between behavioural phenotypes. The responses to experimental challenge were sometimes diametrically opposed between stress-coping styles in a transcript-specific manner. For CR and GAPDH, for example, the response to LPS injection in hypoxia were opposite between proactive and reactive animals. Proactive fish showed decreased CR and increased GAPDH, whereas reactive showed the opposite response. These results further highlight that screening for stress-coping styles prior to experiments in adaptive physiology can significantly affect the interpretation of data obtained. Further, this leads to a more finely tuned analytical output providing an improved understanding of variation in individual responses to both environmental and inflammatory challenge.

Epigenetic differences in the innate response after immune stimulation during zebrafish sex differentiation.

Infections are able to trigger epigenetic modifications; however, epigenetic-mediating infections in the immune system in fish is currently unavailable. Within this purpose, zebrafish were immune-stimulated with three lipopolysaccharides (LPS) during sex differentiation. Methylation patterns of three immune genes were studied by a candidate gene approach together with gene expression analysis, and in adulthood, sex ratios were determined. It was shown that the entrance of LPS was through the gills and accumulated in the pronephros. Significant hypomethylation levels of CASP9 and a significant CpG site for IL1ß after Pseudomonas aeruginosa LPS exposure were found. No methylation difference was observed for TNFα. Gene expression and correlation data differed among studied genes. Sex ratios showed a feminization in dose and LPS strain-dependent manner. Here, it is provided epigenetic regulatory mechanisms derived by innate response and the first evidence of possible epigenetic interactions between the immune and reproductive systems.

Characterization of the European Sea Bass (Dicentrarchus labrax) Gonadal Transcriptome During Sexual Development.

The European sea bass is one of the most important cultured fish in Europe and has a marked sexual growth dimorphism in favor of females. It is a gonochoristic species with polygenic sex determination, where a combination between still undifferentiated genetic factors and environmental temperature determines sex ratios. The molecular mechanisms responsible for gonadal sex differentiation are still unknown. Here, we sampled fish during the gonadal developmental period (110 to 350 days post fertilization, dpf), and performed a comprehensive transcriptomic study by using a species-specific microarray. This analysis uncovered sex-specific gonadal transcriptomic profiles at each stage of development, identifying larger number of differentially expressed genes in ovaries when compared to testis. The expression patterns of 54 reproduction-related genes were analyzed. We found that hsd17ß10 is a reliable marker of early ovarian differentiation. Further, three genes, pdgfb, snx1, and nfy, not previously related to fish sex differentiation, were tightly associated with testis development in the sea bass. Regarding signaling pathways, lysine degradation, bladder cancer, and NOD-like receptor signaling were enriched for ovarian development while eight pathways including basal transcription factors and steroid biosynthesis were enriched for testis development. Analysis of the transcription factor abundance showed an earlier increase in females than in males. Our results show that, although many players in the sex differentiation pathways are conserved among species, there are peculiarities in gene expression worth exploring. The genes identified in this study illustrate the diversity of players involved in fish sex differentiation and can become potential biomarkers for the management of sex ratios in the European sea bass and perhaps other cultured species.

Comprehensive transcriptomic analysis of the process of gonadal sex differentiation in the turbot (Scophthalmus maximus).

The turbot is a flatfish with a ZW/ZZ sex determination system but with a still unknown sex determining gene(s), and with a marked sexual growth dimorphism in favor of females. To better understand sexual development in turbot we sampled young turbot encompassing the whole process of gonadal differentiation and conducted a comprehensive transcriptomic study on its sex differentiation using a validated custom oligomicroarray. Also, the expression profiles of 18 canonical reproduction-related genes were studied along gonad development. The expression levels of gonadal aromatase cyp19a1a alone at three months of age allowed the accurate and early identification of sex before the first signs of histological differentiation. A total of 56 differentially expressed genes (DEG) that had not previously been related to sex differentiation in fish were identified within the first three months of age, of which 44 were associated with ovarian differentiation (e.g., cd98, gpd1 and cry2), and 12 with testicular differentiation (e.g., ace, capn8 and nxph1). To identify putative sex determining genes, ∼4.000 DEG in juvenile gonads were mapped and their positions compared with that of previously identified sex- and growth-related quantitative trait loci (QTL). Although no genes mapped to the previously identified sex-related QTLs, two genes (foxl2 and 17ßhsd) of the canonical reproduction-related genes mapped to growth-QTLs in linkage group (LG) 15 and LG6, respectively, suggesting that these genes are related to the growth dimorphism in this species.

Increase in prevalence of rhinoconjunctivitis but not asthma and atopic eczema in teenagers.

OBJECTIVE: We sought to assess changes in the prevalence of asthma and other allergic diseases in schoolchildren of Curitiba between 1995 and 2001. METHODS: Schoolchildren (aged 13-14) were evaluated by a self-reported written questionnaire of ISAAC protocol for symptoms of asthma and allergic diseases. The diagnoses of asthma, rhinoconjunctivitis and atopic eczema were considered according to the ISAAC protocol. Probable asthma was considered: more than 4 attacks of wheezing in the last 12 months; 1 to 3 attacks of wheezing in the last 12 months with dry cough at night, and/or with sleep disturbance due to wheezing, and/or with wheezing after exercise. The data obtained in 2001 were compared with the data obtained in 1995 for the same age group. RESULTS: We evaluated 2,946 schoolchildren in 1995 and 3.628 in 2001. The prevalence of symptoms of asthma, probable asthma, rhinoconjunctivitis and eczema in 1995 and 2001 was 18.4% and 18.7%, 11.6% and 12.4%, 14.2% and 17.2%, 3.8% and 3.7%, respectively. There was a 23.7% increase in the prevalence of rhinoconjunctivitis symptoms (p < 0.05). CONCLUSIONS: The prevalence of symptoms of asthma, probable asthma and eczema did not change over the past 6 years but a significant increase of the rhinoconjunctivitis symptoms was detected.

How could changes in diet explain changes in coronary heart disease mortality in Spain? The Spanish paradox.

We review and compare trends in coronary heart disease (CHD) and stroke mortality in Spain from 1966 to 1990 and changes in food consumption at national and regional levels. Since 1976, a decrease in cardiovascular disease (CVD) mortality in males and females has been observed, and standardized CHD mortality rates have fallen. Stroke mortality decreased during the same period. Trends in food consumption show increases in intakes of meat, dairy products, fish, and fruit, but decreases in consumption of olive oil, sugar, and all foods rich in carbohydrates. Although fat and saturated fat intakes increased, these changes were not accompanied by an increase in CHD mortality rates. This paradoxical situation can be explained by expanded access to clinical care, increased consumption of fruit and fish, improved control of hypertension, and a reduction in cigarette smoking. Diet appears to have an important role in this paradox, but it may not be as critical as other factors. Nevertheless, we suggest dietary guidelines for prevention of CHD in Spain.

Enamel corrosion produced by zinc phosphate dental cement.

The application of a phosphoric acid solution or of a zinc phosphate cement mix on enamel surface causes alteration of its structure. The lesions of the enamel are cavities of irregular shapes that might retain debris. In view of the high solubility of disintegration of the cement, those cavities can be considered potential sites of plaque installation and initiation of carious processes.

Enamel surface treated with zinc polyacrylate dental cement.

The surface of tooth enamel that had been left in touch with the liquid or the mix of a zinc polyacrylate dental cement was studied by scanning electron microscopy. Both treatments produced lesions on the enamel and were represented by unevenly distributed microcavities of varying extension. Topically applied SnF2 did not modify the aspect of the treated surface.

Comparative analysis of nutrition data from national, household, and individual levels: results from a WHO-CINDI collaborative project in Canada, Finland, Poland, and Spain.

STUDY OBJECTIVE: This project determined to what extent data on diet and nutrition, which were collected in a non-uniform manner, could be harmonised and pooled for international and national comparison. DESIGN: Direct comparisons of dietary data between studies were made using food balance sheets (FBS), household budget surveys (HBS), and individual dietary data (IDS); comparisons were also made within countries. Differences in study design and methodological approaches were taken into consideration. Data from research projects from the following four World Health Organisation (WHO) Countrywide Integrated Noncommunicable Disease Intervention (CINDI) countries were included-Canada, Finland, Poland, and Spain. MAIN RESULTS: FBS overestimated food consumption and nutrient intake compared to IDS. Results between HBS and IDS were quite similar, except for fish, meat, pulses and vegetables, which were underestimated by HBS, and sugar and honey and cereals, which were overestimated. Percentages of energy from fat, carbohydrates and proteins were higher when estimated from FBS, HBS, and IDS respectively. CONCLUSIONS: Results suggest that estimations from these three sources of dietary data are difficult to compare because they are measuring different levels of dietary information. The understanding of their relations may be important in formulating and evaluating a nutrition policy.

Obesity and other health determinants across Europe: the EURALIM project.

STUDY OBJECTIVE: EURALIM (EURope ALIMentation), a European collaborative study, aimed to determine and describe the extent to which European data on risk factor distributions from different populations could be pooled and harmonised in a common database for international comparisons. SETTING: Seven independent population-based surveys from six European countries (France, Italy, Northern Ireland/United Kingdom, Spain, Switzerland, the Netherlands). METHODS: Data for 18 381 women and 12 908 men aged 40-59 were pooled in a common database. Central statistical analyses on major cardiovascular risk factors were conducted with careful consideration of methodological issues, including differences in study designs, data assessment tools, and analytic techniques used. MAIN RESULTS: Because of the detected variability among methods used, direct comparisons of risk factor distributions and prevalences between studies were problematic. None the less, comparisons of within population contrasts by sex, age group, and other health determinants were considered to be meaningful and apt, as illustrated here for obesity. Results were targeted and disseminated to both the general public and public health professionals and framed in the context of a European information campaign. CONCLUSIONS: International and national comparisons between existing locally run studies are feasible and useful, but harmonisation methods need improvement. Development of an international risk factor surveillance programme based on decentralised data collection is warranted. In the meantime, risk factor contrasts across populations can be used as a basis for targeting needed public health intervention programmes.

Changing patterns of fat consumption in Spain.

Trends of fat and fat-containing food consumption in Spain are analysed; information was obtained from data collected at two different levels: household and individual. Three major household budget surveys conducted by the National Institute of Statistics in 1964, 1980-1981, and 1990-1991 show the trends of food and fat consumption in Spain; we can observe a decrease in cereals, potatoes and pulses and an increase in dairy products and meat, similar to that observed in other countries. According to these studies, the consumption of fat and saturated fat and the other vegetable oils have increased, while olive oil intake has decreased. Food and fat consumption in Spain presents a large variation between the Spanish regions. Six individual dietary studies conducted among adult free-living populations are reviewed; intakes of fat ranged from 90 to 110 g/person/day, and percentage of total energy from saturated fat from 12 to 15%. The rise in fat intake in Spain urges dietary interventions.

Sociodemographic and lifestyle determinants of food patterns in Spanish children and adolescents: the enKid study.

OBJECTIVE: To analyse prevailing food patterns among Spanish children and young people and their relationship to sociodemographic and lifestyle factors. DESIGN: Cross-sectional population survey. SETTING: Population study. Data were collected at participants' home addresses. SUBJECTS: Random sample of the Spanish population aged 2-24 y (n=3534; 1629 boys and 1905 girls). INTERVENTIONS: Food consumption was assessed by means of a 24-h recall and a food frequency questionnaire. Sociodemographic and lifestyle related data were collected by specially designed questionnaires, previously pretested and validated. All the information was collected during a personal interview by trained dietitians. DATA COLLECTION: May 1998-April 2000. RESULTS: Average consumption of fruit and vegetables was low. The youngest age group (2-5 y) showed the lowest proportions of inadequacy for the dairy group (P<0,001; chi(2)=39.11 boys; chi(2)=49.60 girls). Factor analysis identified five main components of dietary patterns. The 'Snacky' pattern was characterised by more frequent and higher consumption of bakery products (buns, cakes and biscuits), sweets, salted snacks and soft drinks. Higher intakes of fruit, vegetables and fish were associated to the 'Healthy' pattern. Children whose mother had a low level of education and those who spent more than 2 h daily watching TV were more likely to follow the 'Snacky' pattern. Girls were more likely to follow the 'Healthy' pattern, while children and young people whose mother had a lower level of education were less likely. CONCLUSION: Results from this study highlight the importance of enhancing school-based and community-based actions to promote healthy eating and physical activity addressed to children and young people.

Food preferences of Spanish children and young people: the enKid study.

OBJECTIVE: To describe the profile of food preferences, likes and dislikes of Spanish children and young people and analyse its connection with prevailing food patterns. DESIGN: Cross-sectional population survey. SETTING: Population study. Data were collected at participants' home address. SUBJECTS: Random sample of the Spanish population aged 2-24 y (n=3534; 1629 boys and 1905 girls). INTERVENTIONS: Food preferences, food consumption and practices as well as nutrition-related information were assessed by means of a multiple choice questionnaire. A food preference score was computed considering food items ranked as first, second or third choice within each food group. Data collection was carried out during May 1998-April 2000. RESULTS: Bananas and apples were the fruit items preferred by Spanish children and young people across all age and gender groups. Within the vegetable group tomato sauce and salads, particularly lettuce and tomato salad scored highest, followed by carrots in all age and gender groups. However, 47% (95% confidence interval 46-48%) of the sample reported dislike for vegetables and an additional 5.7% (95% confidence interval 4.9-6.5%) a dislike for fruit. The proportion of individuals with low consumption of vegetables or fruit was significantly higher among those reporting a dislike either for vegetables (chi(2)=127.69; P<0.001); fruit (chi(2)=24.62; P<0.001) or for both groups (chi(2)=81.53; P<0.001). CONCLUSION: There is a significant relationship between the likes/dislikes for fruits and vegetables and usual consumption of this food group among children and young people. Strategies addressed to improve acceptance for this food group should be considered when designing interventions aimed at promoting adequate consumption of fruits and vegetables among children and young people.

Olive oil and the Mediterranean diet: beyond the rhetoric.

OBJECTIVE: The purpose of this study was to analyze the association of food, nutrient and energy intakes with olive oil consumption in Spain. DESIGN: Cross-sectional study by face-to-face interview. SETTING: Population-based random sample derived from the Catalan Nutrition Survey. SUBJECTS: In total, 1600 individuals between 18 and 60 y of age. INTERVENTION: Two 24-h recalls were administered to measure food and nutrient intakes. Food consumption and nutrient intakes were analyzed comparing the highest and lowest quartiles of olive oil consumption. RESULTS: Those with the highest consumption of olive oil (greater than 13.5% of total calories, fourth quartile) consumed less cereal, baked goods, whole milk, sausages, candy, fruit juice and soft drinks, but more fish, eggs, vegetables and added fats, as compared to those with the lowest olive oil consumption (less than 6.8% of total calories, first quartile). The group with the greatest olive oil consumption also demonstrated high total fat intake, although saturated fats showed a lower percentage of total energy intake. Vitamin intake was more adequate in those with the highest consumption of olive oil. CONCLUSION: Olive oil is a key contributor to the healthy aspects attributed to the Mediterranean diet, and as such, nutritional objectives in Mediterranean countries should address reducing saturated fats, without modifying quantities of olive oil.

Nutrient adequacy and Mediterranean Diet in Spanish school children and adolescents.

OBJECTIVE: To evaluate dietary habits and nutritional status of Spanish school children and adolescents, and their relationship with the Mediterranean Diet. DESIGN: Cross-sectional study by face-to-face interview. SETTING: Free living children and adolescents of all Spanish regions. SUBJECTS: A random sample of 3166 people aged 6-24 y. METHODS: Home interviews conducted by a team of 43 dietitians included 24-h recalls (a second 24-h recall in 25% of the sample) and a short frequency questionnaire to determine the quality of the Mediterranean Diet (KIDMED). RESULTS: The participation rate was 68%. In general, the adequacy of the Mediterranean Diet rose with increased mean intakes of the majority of vitamins and minerals and decreased percentages of inadequate intakes (&<2/3 of the RNI) for calcium, magnesium, vitamin B(6) and C in both sexes, and iron and vitamin A only in females. CONCLUSIONS: This study demonstrates that the Mediterranean Diet contributes to nutritional quality, and also shows concomitant risks as the Mediterranean Diet deteriorates.

Population attitudes towards changing dietary habits and reliance on general practitioners in Spain.

OBJECTIVE: To investigate the relationship between population attitudes towards modifying food behaviour and reliance on General Practitioners (GPs) as nutrition educators. DESIGN: Personal interview in a random sample of the general population of the Canary Islands as part of the Canary Islands Nutrition Survey. SUBJECTS: 1747 individuals aged 6-74y. MAIN OUTCOME MEASURES: Attitudes towards changing food behaviour and reliance on GPs. RESULTS: Response rate was 67%. Physicians were the most reliable source of nutrition information with 79% of the population considering them as highly reliable. More than 60% of the population showed a favourable attitude towards increasing fruit and vegetables and towards decreasing alcohol, sugar and pastries. Reliance on GPs was associated with better attitudes towards increasing fruits, decreasing meat, pastries, sugar and losing weight. CONCLUSIONS: Population attitudes towards changing certain dietary behaviours were associated with having the greatest reliance on GPs.

Influence of sociodemographic factors in the prevalence of obesity in Spain. The SEEDO'97 Study.

OBJECTIVE: To analyse the influence of social and cultural factors in the prevalence of obesity in the Spanish adult population aged 25--60 y based on available population data. DESIGN: Pooled analysis of four cross-sectional nutrition surveys. SUBJECTS: A total of 5388 free-living subjects aged 25--60 y, respondents of the Nutritional Surveys carried out in four Spanish regions (Catalunya, Basque Country, Madrid and Valencia) from 1990 to 1994. The samples were pooled together and weighted to build a national random sample. MEASUREMENTS: Weight and height were measured on each individual by trained observers. Age, gender, educational level, occupation, habitat (rural/urban) and region were considered. Obesity was defined as body mass index > or = 30 kg/m(2). The protocol used in each survey was in accordance with the recommendations of the Spanish Society for the Study of Obesity (SEEDO). Logistic regression models were designed to analyse the influence of sociodemographic factors in the prevalence of obesity in men and women. RESULTS: The prevalence of obesity was higher in older age groups in men and women, odds ratio (OR) for every 10 y OR=1.40 (95% CI 1.39--1.41) for men and OR=1.86 (95% CI 1.85--1.87) for women. Logistic regression analysis adjusted for age showed higher obesity rates among low educated people, OR=1.80 (95% CI 1.78--1.81) in men and OR=2.36 (95% CI 2.29--2.42) in women (P<0.001). Among men the odds ratio for the prevalence of obesity in rural areas was OR=1.87 (95% CI 1.86--1.89), compared to cities. The geographical distribution showed higher obesity rates in the southeast. CONCLUSION: This study supports that obesity is a multifactorial problem. Older women with low educational level and low income seem to be the most susceptible group to weight gain. Therefore, Public Health Programs should consider this type of environmental factor when planning strategies aimed at preventing or reducing the problem of obesity in western societies.