Non-synonymous variation and protein structure of candidate genes associated with selection in farm and wild populations of turbot (Scophthalmus maximus).

Non-synonymous variation (NSV) of protein coding genes represents raw material for selection to improve adaptation to the diverse environmental scenarios in wild and livestock populations. Many aquatic species face variations in temperature, salinity and biological factors throughout their distribution range that is reflected by the presence of allelic clines or local adaptation. The turbot (Scophthalmus maximus) is a flatfish of great commercial value with a flourishing aquaculture which has promoted the development of genomic resources. In this study, we developed the first atlas of NSVs in the turbot genome by resequencing 10 individuals from Northeast Atlantic Ocean. More than 50,000 NSVs where detected in the ~ 21,500 coding genes of the turbot genome, and we selected 18 NSVs to be genotyped using a single Mass ARRAY multiplex on 13 wild populations and three turbot farms. We detected signals of divergent selection on several genes related to growth, circadian rhythms, osmoregulation and oxygen binding in the different scenarios evaluated. Furthermore, we explored the impact of NSVs identified on the 3D structure and functional relationship of the correspondent proteins. In summary, our study provides a strategy to identify NSVs in species with consistently annotated and assembled genomes to ascertain their role in adaptation.

Sunitinib-induced oxidative imbalance and retinotoxic effects in rats.

AIMS: Sunitinib (Su), a tyrosine kinase inhibitor, is one of the most commonly used anti-angiogenic drugs. Some studies have described retinal detachment and photoreceptor damage following systemic exposure to Su, despite beneficial effects achieved with local treatment of ocular pathologies. The aim of this study was to explore the role of NADPH oxidase system and oxidative stress in eyes from Su-treated animals. MAIN METHODS: Male Wistar rats were administered 25 mg Su/kg body weight/day incorporated in the chow for 3 weeks. Upon treatment completion, NADPH oxidase activity and ROS levels were measured in ocular tissue by chemiluminescence and dihydroethidium (DHE) staining, respectively. The expression of NADPH oxidase isoforms (NOX1, NOX2 and NOX4), antioxidant enzymes and endothelial/inducible nitric oxidase isoforms (eNOS/iNOS) in the eyecup and/or retina were measured via immunofluorescence, immunoblotting and RT-qPCR. KEY FINDINGS: NADPH oxidase activity/expression increased in eyecup and retinas from Su-treated rats. Immunohistofluorescence studies in retinal layer confirmed a higher signal of NADPH oxidase isoforms after Su treatment. Treated animals also presented with reductions in NO levels and eNOS expression, whereas iNOS was upregulated. Finally, a significant depletion of antioxidant enzyme glutathione peroxidase was measured in eyecups of rats following Su exposure, and the opposite pattern was seen for glutathione reductase and superoxide dismutase. SIGNIFICANCE: This study demonstrates that Su treatment is associated with NADPH oxidase-derived oxidative stress in the eye. Long-term treatment of Su should be properly monitored to avoid retinotoxic effects that might result in ocular pathologies and sight-threatening conditions.

Mechanism of Vascular Toxicity in Rats Subjected to Treatment with a Tyrosine Kinase Inhibitor.

Sunitinib (Su) is a tyrosine kinase inhibitor with antiangiogenic and antineoplastic effects that is recommended therapy for renal cell carcinoma, gastrointestinal stromal tumors, and pancreatic neuroendocrine tumors. Arterial hypertension is one of the adverse effects observed in the treatment with Su. The aim of this work was to deepen our understanding of the underlying mechanisms involved in the development of this side effect. Studies on endothelial function, vascular remodeling and nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) system were carried out in thoracic aortas from rats treated with Su for three weeks. Animals subjected to Su treatment presented with increased blood pressure and reduced endothelium-dependent vasodilation, the latter being reverted by NADPH oxidase blockade. Furthermore, vascular remodeling and stronger Masson trichrome staining, together with enhanced immunofluorescence signal for collagen 1 alpha 1 (Col1α1), were observed in aortas from treated animals. These results were accompanied by a significant elevation in superoxide anion production and the activity/protein/gene expression of NADPH oxidase isoforms (NOX1, NOX2, and NOX4), which was also prevented by NOX inhibition. Furthermore, a decrease in nitric oxide (NO) levels and endothelial nitric oxide synthase (eNOS) activation was observed in aortas from Su-treated animals. All these results indicate that endothelial dysfunction secondary to changes in vascular remodeling and oxidative stress might be responsible for the typical arterial hypertension that develops following treatment with Su.

Genomic Signatures After Five Generations of Intensive Selective Breeding: Runs of Homozygosity and Genetic Diversity in Representative Domestic and Wild Populations of Turbot (Scophthalmus maximus).

Massive genotyping of single nucleotide polymorphisms (SNP) has opened opportunities for analyzing the way in which selection shapes genomes. Artificial or natural selection usually leaves genomic signatures associated with selective sweeps around the responsible locus. Strong selective sweeps are most often identified either by lower genetic diversity than the genomic average and/or islands of runs of homozygosity (ROHi). Here, we conducted an analysis of selective sweeps in turbot (Scophthalmus maximus) using two SNP datasets from a Northeastern Atlantic population (36 individuals) and a domestic broodstock (46 individuals). Twenty-six families (∼ 40 offspring per family) from this broodstock and three SNP datasets applying differing filtering criteria were used to adjust ROH calling parameters. The best-fitted genomic inbreeding estimate (FROH) was obtained by the sum of ROH longer than 1 Mb, called using a 21,615 SNP panel, a sliding window of 37 SNPs and one heterozygous SNP per window allowed. These parameters were used to obtain the ROHi distribution in the domestic and wild populations (49 and 0 ROHi, respectively). Regions with higher and lower genetic diversity within each population were obtained using sliding windows of 37 SNPs. Furthermore, those regions were mapped in the turbot genome against previously reported genetic markers associated with QTL (Quantitative Trait Loci) and outlier loci for domestic or natural selection to identify putative selective sweeps. Out of the 319 and 278 windows surpassing the suggestive pooled heterozygosity thresholds (ZHp) in the wild and domestic population, respectively, 78 and 54 were retained under more restrictive ZHp criteria. A total of 116 suggestive windows (representing 19 genomic regions) were linked to either QTL for production traits, or outliers for divergent or balancing selection. Twenty-four of them (representing 3 genomic regions) were retained under stricter ZHp thresholds. Eleven QTL/outlier markers were exclusively found in suggestive regions of the domestic broodstock, 7 in the wild population and one in both populations; one (broodstock) and two (wild) of those were found in significant regions retained under more restrictive ZHp criteria in the broodstock and the wild population, respectively. Genome mining and functional enrichment within regions associated with selective sweeps disclosed relevant genes and pathways related to aquaculture target traits, including growth and immune-related pathways, metabolism and response to hypoxia, which showcases how this genome atlas of genetic diversity can be a valuable resource to look for candidate genes related to natural or artificial selection in turbot populations.

Basal functional status predicts one-year mortality after a heart failure hospitalization in elderly patients - The RICA prospective study.

BACKGROUND: Dependence for basic activities of the daily living (ADL) relates to adverse outcomes in elderly acute heart failure (AHF) patients. METHODS: We evaluated patients ≥75years admitted because of AHF, divided according to preadmission Barthel Index (BI) category: severe (BI 0-60), moderate (BI 61-90) and slight dependence or independence for basic ADL (BI 91-100). We compared their baseline characteristics and used logistic regression models to determine whether a BI≤60 confers higher one-year mortality risk. RESULTS: We included 2195 patients, mean age 83years; 57% women, Charlson Index 3, 65% with preserved left ventricular ejection fraction. Their median preadmission BI was 90 (65-100); 21.7% had BI≤60. Patients with BI≤60 were older, more often females, with higher comorbid and cognitive burden and more likely to be institutionalized. 560 patients (26%) died within the follow-up period. A preadmission BI≤60 was significantly associated with higher risk of 12-month mortality (HR 1.42, 95% CI 1.14-1.77) together with male sex (1.27, 1.04-1.54), valve disease (1.49, 1.20-1.83), worse preadmission NYHA class (1.44, 1.20-1.73), stage IV chronic kidney disease (1.70, 1.35-2.15), pulmonary edema (1.33, 1.01-1.76), no family support (1.47, 1.06-2.06), and higher Charlson Comorbidity Index (1.09, CI 1.05-1.13) and Pfeiffer cognitive screening questionnaire scores (1.10, 1.05-1.14). CONCLUSION: Among elderly AHF patients, the presence of severe (BI≤60) preadmission dependence for basic ADL confers a significant and independent risk of one-year post-discharge mortality.

Benefit of tolvaptan in the management of hyponatraemia in patients with diuretic-refractory congestive heart failure: the SEMI-SEC project.

AIMS: Hyponatraemia is an electrolyte disorder that occurs in advanced congestive heart failure (HF) and worsens prognosis. We explored the usefulness of tolvaptan, which has shown promising results in the treatment of this condition. METHODS AND RESULTS: This study is based on a retrospective national registry (2011-15) of patients hospitalized with refractory HF and hyponatraemia who agreed to receive tolvaptan when standard treatment was ineffective. The benefit of tolvaptan was analysed according to the following criteria: normalization ([Na+] ≥ 135 mmol/L) or increased sodium levels [Na+] ≥ 4 mEq/L on completion of treatment, and increase in urine output by 300 or 500 mL at 48 h. Factors associated with tolvaptan benefit were explored. A total of 241 patients were included, 53.9% of whom had ejection fraction <40%. All patients received concomitant loop diuretics. Initial tolvaptan dose was 17.2 ± 6.1 mg, and end dose was 26.4 ± 23.2 mg (duration 7.8 ± 8.6 days). Serum sodium concentrations increased significantly at 24-48 h, from 126.5 ± 6.2 mEq/L at baseline to 134.1 ± 6.1 mEq/L at the end of treatment (P < 0.0001). Weight fell by ~5 kg before discharge (P < 0.0001) and urine output increased 1.3-fold (P < 0.0001). Normal sodium levels and/or increases of 500 mL in urine output were achieved by 90.8% of patients (35.7% achieved both) and 94.8% increased to [Na+] ≥ 4 mEq/L and/or +300 mL in urine output (54.4% both). CONCLUSIONS: An increase in sodium levels and/or improvement in urine output was observed in patients admitted for HF and refractory hyponatraemia under tolvaptan treatment. Tolvaptan may be useful in this setting, in which no effective proven alternatives are available.

Inflammatory and fibrotic processes are involved in the cardiotoxic effect of sunitinib: Protective role of L-carnitine.

Sunitinib (Su) is currently approved for treatment of several malignances. However, along with the benefits of disease stabilization, cardiovascular toxicities have also been increasingly recognized. The aim of this study was to analyze which mechanisms are involved in the cardiotoxicity caused by Su, as well as to explore the potential cardioprotective effects of l-carnitine (LC). To this end, four groups of Wistar rats were used: (1) control; (2) rats treated with 400mg LC/kg/day; (3) rats treated with 25mg Su/kg/day; and (4) rats treated with LC+Su simultaneously. In addition, cultured rat cardiomyocytes were treated with an inhibitor of nuclear factor kappa B (NF-κB), in order to examine the role of this transcription factor in this process. An elevation in the myocardial expression of pro-inflammatory cytokines, together with an increase in the mRNA expression of NF-κB, was observed in Su-treated rats. These results were accompanied by an increase in the expression of pro-fibrotic factors, nitrotyrosine and NOX 2 subunit of NADPH oxidase; and by a decrease in that of collagen degradation factor. Higher blood pressure and heart rate levels were also found in Su-treated rats. All these alterations were inhibited by co-administration of LC. Furthermore, cardiotoxic effects of Su were blocked by NF-κB inhibition. Our results suggest that: (i) inflammatory and fibrotic processes are involved in the cardiac toxicity observed following treatment with Su; (ii) these processes might be mediated by the transcription factor NF-κB; (iii) LC exerts a protective effect against arterial hypertension, cardiac inflammation and fibrosis, which are all observed after Su treatment.

[Anemia in heart failure: its usefulness as prognosis or comorbidity marker: GESAIC-2 study]. / Anemia en la insuficiencia cardiaca: su utilidad como marcador de pronóstico o de comorbilidad: estudio GESAIC-2.

BACKGROUND AND OBJECTIVE: To assess the prognosis and etiology of anemia in heart failure (HF). PATIENTS AND METHODS: Prospective multicenter cohort of HF after one year of hospitalization. RESULTS: A total of 57 (27%) of the 211 patients died and 115 (67.8%) were readmitted. Mortality was higher in the group with anemia (31.8%) without statistical significance (P=.09), except for refractory HF mortality (P=.013). Predictors of HF mortality included Barthel index (hazard ratio [HR] 0.97, CI 95% 0.96 to 0.98) and serum creatinine at discharge (HR 2.28, 95% CI 1.51 to 3.45). For reentry, the Charlson index (OR 1.16, CI 95% 0.98 to 1.38), treatment with calcium channel blockers (OR 0.29, 95% CI 0.1 to 0.84) and not being treated with digoxin (OR 2.33, CI 95% 1.09 to 4.97), the latter with the greatest influence on readmission for HF (OR 3.07, CI 95% 1.39 to 6.79), along with not being a HF debut (OR 2.8, CI 95% 1.45 to 5.39). CONCLUSIONS: Anemia is an increased risk of mortality due to refractory HF, but not for readmission within the first year after an acute event.

Effect of melatonin administration on sleep, behavioral disorders and hypnotic drug discontinuation in the elderly: a randomized, double-blind, placebo-controlled study.

BACKGROUND AND AIMS: The aim of the study was to evaluate the effect of melatonin administration on sleep and behavioral disorders in the elderly and the facilitation of the discontinuation of regular hypnotic drugs. METHODS: This was a prospective, randomized, double-blind, placebo-controlled, crossover trial in a community-living population. Participants were 22 older adults (7 men, 15 women over 65) with a history of sleep disorder complaints. Fourteen of these subjects were receiving hypnotic drug therapy. Participants received 2 months of melatonin (5 mg/day) and 2 months of placebo. Sleep disorders were evaluated with the Northside Hospital Sleep Medicine Institute (NHSMI) test, discarding secondary insomnia and evaluating sleep quality. Behavioral disorders were evaluated with the Yesavage Geriatric Depression Scale (GDS) and Goldberg Anxiety Scale (GAS). Patients discontinuing hypnotic drugs were also recorded. RESULTS: Melatonin treatment for two months significantly improved sleep quality scores measured by the NHSMI test (1.78+/-0.40) when compared with both basal (3.72+/-0.45; p=0.001) and placebo (3.44+/-0.56; p=0.025) groups. Depression measured by GDS and anxiety measured by GAS also improved significantly after melatonin administration (p=0.043 and p=0.009, respectively). Nine out of 14 subjects receiving hypnotic drugs were able to discontinue this treatment during melatonin but not placebo administration; one discontinued hypnotic drugs during both melatonin and placebo administration, and four were unable to discontinue hypnotic therapy. CONCLUSIONS: The results of this study suggest that melatonin administration significantly improves sleep and behavioral disorders in the elderly and facilitates discontinuation of therapy with conventional hypnotic drugs.

The role of inflammatory markers in the cardioprotective effect of L-carnitine in L-NAME-induced hypertension.

BACKGROUND: The mechanism(s) underlying the effects of L-carnitine (beta-hydroxy-gamma-N-trimethylammonium-butyrate; LC) in cardiovascular diseases are not well clarified. Previous studies have demonstrated that oxidative stress and inflammation contribute to arterial hypertension, and antioxidant and/or anti-inflammatory therapies have been proposed. We hypothesized that LC might attenuate the hypertensive status through an inhibition of inflammation process. METHODS: Heart mRNA expression and plasma levels of inflammatory markers, interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha), were measured in rats that were made hypertensive with N(omega)-nitro-L-arginine methyl ester (L-NAME) and subjected to a simultaneous administration of LC. To clarify the role of the renin-angiotensin system (RAS) in this effect of LC, the activity and expression of angiotensin I-converting enzyme (ACE) as well as the expression of angiotensin II type I receptor (AT1R) in the heart were also determined. RESULTS: LC produced a significant, but not complete, reduction of blood pressure in L-NAME-treated rats. Plasma levels and heart expression of IL-1 beta, IL-6, and TNF-alpha showed an increase in the L-NAME group, which was reversed by LC treatment. The plasma ACE activity was not modified between normotensive and hypertensive rats although LC treatment produced a reduction of these values in the latter. Finally, protein and mRNA expression of ACE and AT1R was enhanced in the heart of L-NAME-treated animals, and LC reversed these values. CONCLUSIONS: The chronic administration of LC reduces blood pressure and attenuates the inflammatory process associated with arterial hypertension. LC might produce a partial inactivation in the RAS resulting in a reduction in the production and effects of angiotensin II.