Local drivers of heterogeneity in a tropical forest: epiphytic tank bromeliads affect the availability of soil resources and conditions and indirectly affect the structure of seedling communities.

Environmental heterogeneity is a key component in explaining the megadiversity of tropical forests. Despite its importance, knowledge about local drivers of environmental heterogeneity remains a challenge for ecologists. In Neotropical forests, epiphytic tank bromeliads store large amounts of water and nutrients in the tree canopy, and their tank overflow may create nutrient-rich patches in the soil. However, the effects of this nutrient flux on environmental heterogeneity and plant community structure in the understory remain unexplored. In a Brazilian coastal sandy forest, we investigated the effects of the presence of epiphytic tank bromeliads on throughfall chemistry, soil chemistry, soil litter biomass, light, and seedling community structure. In the presence of epiphytic tank bromeliads, the throughfall nitrogen concentration increased twofold, the throughfall phosphorus concentration increased threefold, and the soil patches had a 3.96% higher pH, a 50% higher calcium concentration, and 11.88% less light. By altering the availability of soil resources and conditions, the presence of bromeliads partially shifted the available niche spaces for plant species and indirectly affected the structure of the seedling communities, decreasing their diversity, density, and biomass. For the first time, we showed that the presence of tank bromeliads in the canopy can create characteristic soil patches in the understory, affecting the structure of seedling communities via fertilization. Our results reveal a novel local driver of environmental heterogeneity, reinforcing and expanding the key role of tank bromeliads both in nutrient cycling and plant community structuring of Neotropical coastal sandy forests.

Compositional response of Amazon forests to climate change.

Most of the planet's diversity is concentrated in the tropics, which includes many regions undergoing rapid climate change. Yet, while climate-induced biodiversity changes are widely documented elsewhere, few studies have addressed this issue for lowland tropical ecosystems. Here we investigate whether the floristic and functional composition of intact lowland Amazonian forests have been changing by evaluating records from 106 long-term inventory plots spanning 30 years. We analyse three traits that have been hypothesized to respond to different environmental drivers (increase in moisture stress and atmospheric CO2 concentrations): maximum tree size, biogeographic water-deficit affiliation and wood density. Tree communities have become increasingly dominated by large-statured taxa, but to date there has been no detectable change in mean wood density or water deficit affiliation at the community level, despite most forest plots having experienced an intensification of the dry season. However, among newly recruited trees, dry-affiliated genera have become more abundant, while the mortality of wet-affiliated genera has increased in those plots where the dry season has intensified most. Thus, a slow shift to a more dry-affiliated Amazonia is underway, with changes in compositional dynamics (recruits and mortality) consistent with climate-change drivers, but yet to significantly impact whole-community composition. The Amazon observational record suggests that the increase in atmospheric CO2 is driving a shift within tree communities to large-statured species and that climate changes to date will impact forest composition, but long generation times of tropical trees mean that biodiversity change is lagging behind climate change.

Differences in leaf thermoregulation and water use strategies between three co-occurring Atlantic forest tree species.

Given anticipated climate changes, it is crucial to understand controls on leaf temperatures including variation between species in diverse ecosystems. In the first study of leaf energy balance in tropical montane forests, we observed current leaf temperature patterns on 3 tree species in the Atlantic forest, Brazil, over a 10-day period and assessed whether and why patterns may vary among species. We found large leaf-to-air temperature differences (maximum 18.3 °C) and high leaf temperatures (over 35 °C) despite much lower air temperatures (maximum 22 °C). Leaf-to-air temperature differences were influenced strongly by radiation, whereas leaf temperatures were also influenced by air temperature. Leaf energy balance modelling informed by our measurements showed that observed differences in leaf temperature between 2 species were due to variation in leaf width and stomatal conductance. The results suggest a trade-off between water use and leaf thermoregulation; Miconia cabussu has more conservative water use compared with Alchornea triplinervia due to lower transpiration under high vapour pressure deficit, with the consequence of higher leaf temperatures under thermal stress conditions. We highlight the importance of leaf functional traits for leaf thermoregulation and also note that the high radiation levels that occur in montane forests may exacerbate the threat from increasing air temperatures.

Groundwater drawdown drives ecophysiological adjustments of woody vegetation in a semi-arid coastal ecosystem.

Predicted droughts and anthropogenic water use will increase groundwater lowering rates and intensify groundwater limitation, particularly for Mediterranean semi-arid ecosystems. These hydrological changes may be expected to elicit differential functional responses of vegetation either belowground or aboveground. Yet, our ability to predict the impacts of groundwater changes on these ecosystems is still poor. Thus, we sought to better understand the impact of falling water table on the physiology of woody vegetation. We specifically ask (a) how is woody vegetation ecophysiological performance affected by water table depth during the dry season? and (b) does the vegetation response to increasing depth to groundwater differ among water-use functional types? We examined a suite of physiological parameters and water-uptake depths of the dominant, functionally distinct woody vegetation along a water-table depth gradient in a Mediterranean semi-arid coastal ecosystem that is currently experiencing anthropogenic groundwater extraction pressure. We found that groundwater drawdown did negatively affect the ecophysiological performance of the woody vegetation. Across all studied environmental factors, depth to groundwater was the most important driver of ecophysiological adjustments. Plant functional types, independent of groundwater dependence, showed consistent declines in water content and generally reduced C and N acquisition with increasing depths to groundwater. Functional types showed distinct operating physiological ranges, but common physiological sensitivity to greater water table depth. Thus, although differences in water-source use exist, a physiological convergence appeared to happen among different functional types. These results strongly suggest that hydrological drought has an important impact on fundamental physiological processes, constraining the performance of woody vegetation under semi-arid conditions. By disentangling the functional responses and vulnerability of woody vegetation to groundwater limitation, our study establishes the basis for predicting the physiological responses of woody vegetation in semi-arid coastal ecosystems to groundwater drawdown.

Estimating and valuing the carbon release in scenarios of land-use and climate changes in a Brazilian coastal area.

Deforestation is a significant source of man-made carbon in the atmosphere, contributing to the greenhouse gas (GHG) effect. Although carbon releases are associated to the ecosystem functions of climate regulation and are essential ecological processes that sustain life, their incremental economic impact is difficult to estimate. Using the InVEST model, this work quantifies and assesses the value of the carbon balance generated by predictive land-use change (LUC) scenarios for the Northern Coast of São Paulo State, Brazil. In this case, carbon losses are explained by the suppression of natural vegetation and human intervention associated to global warming. We analyzed three scenarios: i) the more conservative "legal framework"; ii) the "status quo", which represents the current development level; and iii) the "new ventures", encompassing all new infrastructure projects in the region. The carbon losses in the "legal framework" and "status quo" scenarios are similar to those of past periods: around 3.7 million MgC in twenty years, pointing to net current values of US$ 47 million. However, carbon losses exceeded 7 million MgC when considering factors linked to the "new ventures" scenario, such as infrastructure logistics, oil-gas exploration and pressure on natural environments. In this case, monetary losses could amount to US$ 90 million in 20 years. Besides carbon release, results also highlight the large amount of carbon still stored in protected areas that is threatened by the regional economics dynamics and requires special attention from the public sector, management bodies and regulators.

Nitrogen dynamics in subtropical fringe and basin mangrove forests inferred from stable isotopes.

Mangroves exhibit low species richness compared to other tropical forests, but great structural and functional diversity. Aiming to contribute to a better understanding of the functioning of mangrove forests, we investigated nitrogen (N) dynamics in two physiographic types of mangroves (fringe and basin forests) in southeastern Brazil. Because fringe forests are under great influence of tidal flushing we hypothesized that these forests would exhibit higher N cycling rates in sediment and higher N losses to the atmosphere compared to basin forests. We quantified net N mineralization and nitrification rates in sediment and natural abundance of N stable isotopes (δ15N) in the sediment-plant-litter system. The fringe forest exhibited higher net N mineralization rates and δ15N in the sediment-plant-litter system, but net nitrification rates were similar to those of the basin forest. The results of the present study suggest that fringe forests exhibit higher N availability and N cycling in sediment compared to basin forests.

Clozapine-Loaded Polysorbate-Coated Polymeric Nanocapsules: Physico-Chemical Characterization and Toxicity Evaluation in Caenorhabditis elegans Model.

The aim of this work was to develop and characterize clozapine loaded polysorbate-coated polymeric nanocapsules and assess their toxicity in Caenorhabditis elegans, an invertebrate animal model. Formulations were prepared by nanoprecipitation method and characterized by particle size, zeta potential, pH, drug loading, entrapment efficiency and in vitro drug release. All nanocapsules prepared presented diameter around 140 nm, pH slightly acid and negative zeta potential. In vitro studies showed biphasic drug release from nanocapsules with decreasing of the release rate on nanoencapsulation. The t(1/2)beta of clozapine was 7.23 +/- 0.73 and 2.23 +/- 0.97 h for nanoencapsulated and free drug, respectively (p < 0.05), in pH 1.2 medium. Similar results were obtained in pH 6.8 buffer. Regarding toxicity evaluation, worms exposed to clozapine-loaded nanocapsules did not show the same mortality rate compared to others formulations, as the survival was significantly higher than the free drug treated-group. In addition, we observed that free clozapine decreased egg laying at the first reproductive day, whereas nanoencapsulated clozapine did not depict significant change of this parameter. Longevity assay showed no significant difference, demonstrating that the toxicological effects of clozapine observed in C. elegans are acute. In addition, we proved that free and nanoencapsulated clozapine were orally uptake by the worms, as determined by fluorescein-labeled nanocapsules. Then, the use of nanocapsules delayed the drug release and minimized the toxic effects of clozapine in worms, which can be used as a new animal model to evaluate the nanotoxicity of drug delivery systems.

Early recruitment responses to interactions between frequent fires, nutrients, and herbivory in the southern Amazon.

Understanding tropical forest diversity is a long-standing challenge in ecology. With global change, it has become increasingly important to understand how anthropogenic and natural factors interact to determine diversity. Anthropogenic increases in fire frequency are among the global change variables affecting forest diversity and functioning, and seasonally dry forest of the southern Amazon is among the ecosystems most affected by such pressures. Studying how fire will impact forests in this region is therefore important for understanding ecosystem functioning and for designing effective conservation action. We report the results of an experiment in which we manipulated fire, nutrient availability, and herbivory. We measured the effects of these interacting factors on the regenerative capacity of the ecotone between humid Amazon forest and Brazilian savanna. Regeneration density, diversity, and community composition were severely altered by fire. Additions of P and N + P reduced losses of density and richness in the first year post-fire. Herbivory was most important just after germination. Diversity was positively correlated with herbivory in unburned forest, likely because fire reduced the number of reproductive individuals. This contrasts with earlier results from the same study system in which herbivory was related to increased diversity after fire. We documented a significant effect of fire frequency; diversity in triennially burned forest was more similar to that in unburned than in annually burned forest, and the community composition of triennially burned forest was intermediate between unburned and annually burned areas. Preventing frequent fires will therefore help reduce losses in diversity in the southern Amazon's matrix of human-altered landscapes.

Clozapine linked to nanocapsules minimizes tissue and oxidative damage to biomolecules lipids, proteins and DNA in brain of rats Wistar.

Clozapine, atypical antipsychotic, can change oxidative stress parameters. It is known that reactive species, in excess, can have a crucial role in the etiology of diseases, as well as, can potentiating adverse effects induce by drugs. The nanocapsules have attracted attention as carriers of several drugs, with consequent reduction of adverse effects. This study aimed to evaluate histopathology and oxidative damage of biomolecules lipids, proteins and DNA in the brain of Wistar rats after treatment with nanocapsules containing clozapine. The study consisted of eight groups of male Wistar rats (n = 6): saline (SAL), free clozapine (CZP) (25 mg/Kg i.p.), blank uncoated nanocapsules (BNC), clozapine-loaded uncoated nanocapsules (CNC) (25 mg/Kg i.p.), blank chitosan-coated nanocapsules (BCSN), clozapine-loaded chitosan-coated nanocapsules (CCSN) (25 mg/Kg i.p.), blank polyethyleneglycol-coated nanocapsules (BPEGN), clozapine-loaded polyethyleneglycol-coated nanocapsules (CPEGN) (25 mg/Kg i.p.). The animals received the formulation once a day for seven consecutive days and euthanized in the eighth day. After euthanasia, the brain was collected and homogenate was processed for further analysis. The histopathology showed less brain tissue damage in nanocapsules-treated groups. The lipid peroxidation and carbonylation of proteins showed a significant increase (p < 0.05) induced by CZP. CNC and CPEGN groups obtained a reduction membrane of lipids damage and nanocapsules-treated groups showed significant improvement protein damage. CZP was able to induce genetic oxidative damage, while the nanocapsules causing less damage to DNA. The findings show that different coatings can act protecting target tissues decreasing oxidative damage, suggesting that the drug when linked to different nanocapsules is able to mitigate the harmful effects of clozapine.

Growth of children with end-stage renal disease undergoing daily hemodialysis.

BACKGROUND: The aim of this report is to describe the effect of daily hemodialysis on the growth of children with end-stage renal disease (ESRD). METHODS: We performed a prospective, observational study on 24 children with ESRD undergoing daily hemodialysis (DHD). The control group comprised 26 children on concurrent conventional hemodialysis (CHD), and the follow-up for both groups was 9.3 ± 3.0 months. No patient received growth hormone (GH) therapy. RESULTS: At the onset of the study, the height-for-age Z-score was -2.12 ± 1.54 in the CHD group and -2.84 ± 2.27 in the DHD group (p = 0.313). Assuming an increase of 0.5 standard deviation scores (SDS) of the height-for-age parameter as an improvement of growth, there were 33 % of patients in the DHD group and 8 % in the CHD group (p = 0.035). The cumulative probability of gain in height for age at 12 months was 40 % in the DHD group versus 15 % in the CHD group (p = 0.047). Also, 98 % of patients in the DHD group had an adequate total caloric intake, whereas 38 % in the CHD group reached this goal (p < 0.001). No patient left the study due to intensification of the dialysis modality. CONCLUSIONS: Our data show that the DHD favored a 0.5 SDS height gain in a third of patients without GH treatment. Dialysis intensification was not a cause for treatment dropouts, and DHD should be considered as a treatment for selected cases, especially small children.

The pace of life for forest trees.

Tree growth and longevity trade-offs fundamentally shape the terrestrial carbon balance. Yet, we lack a unified understanding of how such trade-offs vary across the world's forests. By mapping life history traits for a wide range of species across the Americas, we reveal considerable variation in life expectancies from 10 centimeters in diameter (ranging from 1.3 to 3195 years) and show that the pace of life for trees can be accurately classified into four demographic functional types. We found emergent patterns in the strength of trade-offs between growth and longevity across a temperature gradient. Furthermore, we show that the diversity of life history traits varies predictably across forest biomes, giving rise to a positive relationship between trait diversity and productivity. Our pan-latitudinal assessment provides new insights into the demographic mechanisms that govern the carbon turnover rate across forest biomes.

Interactions between repeated fire, nutrients, and insect herbivores affect the recovery of diversity in the southern Amazon.

Surface fires burn extensive areas of tropical forests each year, altering resource availability, biotic interactions, and, ultimately, plant diversity. In transitional forest between the Brazilian cerrado (savanna) and high stature Amazon forest, we took advantage of a long-term fire experiment to establish a factorial study of the interactions between fire, nutrient availability, and herbivory on early plant regeneration. Overall, five annual burns reduced the number and diversity of regenerating stems. Community composition changed substantially after repeated fires, and species common in the cerrado became more abundant. The number of recruits and their diversity were reduced in the burned area, but burned plots closed to herbivores with nitrogen additions had a 14 % increase in recruitment. Diversity of recruits also increased up to 50 % in burned plots when nitrogen was added. Phosphorus additions were related to an increase in species evenness in burned plots open to herbivores. Herbivory reduced seedling survival overall and increased diversity in burned plots when nutrients were added. This last result supports our hypothesis that positive relationships between herbivore presence and diversity would be strongest in treatments that favor herbivory--in this case herbivory was higher in burned plots which were initially lower in diversity. Regenerating seedlings in less diverse plots were likely more apparent to herbivores, enabling increased herbivory and a stronger signal of negative density dependence. In contrast, herbivores generally decreased diversity in more species rich unburned plots. Although this study documents complex interactions between repeated burns, nutrients, and herbivory, it is clear that fire initiates a shift in the factors that are most important in determining the diversity and number of recruits. This change may have long-lasting effects as the forest progresses through succession.

Long-term thermal sensitivity of Earth's tropical forests.

The sensitivity of tropical forest carbon to climate is a key uncertainty in predicting global climate change. Although short-term drying and warming are known to affect forests, it is unknown if such effects translate into long-term responses. Here, we analyze 590 permanent plots measured across the tropics to derive the equilibrium climate controls on forest carbon. Maximum temperature is the most important predictor of aboveground biomass (-9.1 megagrams of carbon per hectare per degree Celsius), primarily by reducing woody productivity, and has a greater impact per °C in the hottest forests (>32.2°C). Our results nevertheless reveal greater thermal resilience than observations of short-term variation imply. To realize the long-term climate adaptation potential of tropical forests requires both protecting them and stabilizing Earth's climate.

Nitrogen input by bamboos in neotropical forest: a new perspective.

BACKGROUND: Nitrogen (N) is an important macronutrient that controls the productivity of ecosystems and biological nitrogen fixation (BNF) is a major source of N in terrestrial systems, particularly tropical forests. Bamboo dominates theses forests, but our knowledge regarding the role of bamboo in ecosystem functioning remains in its infancy. We investigated the importance of a native bamboo species to the N cycle of a Neotropical forest. METHODS: We selected 100 sample units (100 m2 each) in a pristine montane Atlantic Forest, in Brazil. We counted all the clumps and live culms of Merostachys neesii bamboo and calculated the specific and total leaf area, as well as litter production and respective N content. Potential N input was estimated based on available data on BNF rates for the same bamboo species, whose N input was then contextualized using information on N cycling components in the study area. RESULTS: With 4,000 live culms ha-1, the native bamboo may contribute up to 11.7 kg N ha-1 during summer (January to March) and 19.6 kg N ha-1 in winter (July to September). When extrapolated for annual values, M. neesii could contribute more than 60 kg N ha-1y-1. DISCUSSION: The bamboo species' contribution to N input may be due to its abundance (habitat availability for microbial colonization) and the composition of the free-living N fixer community on its leaves (demonstrated in previous studies). Although some N is lost during decomposition, this input could mitigate the N deficit in the Atlantic Forest studied by at least 27%. Our findings suggest that M. neesii closely regulates N input and may better explain the high diversity and carbon stocks in the area. This is the first time that a study has investigated BNF using free-living N fixers on the phyllosphere of bamboo.

De Morsier syndrome associated with periventricular nodular heterotopia: case report.

INTRODUCTION: Septo-optic dysplasia (De Morsier syndrome) is defined as the association between optic nerve hypoplasia, midline central nervous system malformations and pituitary dysfunction. CASE REPORT: Third child born to nonconsanguineous parents, female, adequate pre-natal medical care, cesarean term delivery due to breech presentation, Apgar score 3 at the first minute and 8 at 5 minutes, symptomatic hypoglycemia at 18 hours. Neurological follow-up identified a delay in acquisition of motor and language developmental milestones. Epileptic generalized seizures began at 12 months and were controlled with phenobarbital. EEG was normal. MRI revealed agenesis of the pituitary stalk, hypoplasia of the optic chiasm and periventricular nodular heterotopia. Ophthalmologic evaluation showed bilateral optic disk hypoplasia. Endocrine function laboratory tests revealed primary hypothyroidism and hyperprolactinemia. CONCLUSION: The relevance of this case report relies on its uniqueness, since periventricular heterotopia had not been described in association with septo-optic dysplasia until 2006.

Clinical, laboratory and neuroimage findings in juvenile systemic lupus erythematosus presenting involvement of the nervous system.

OBJECTIVE: To characterize neurological involvement in juvenile systemic lupus erythe-matosus. METHOD: The charts of all patients with the diagnosis of systemic lupus erythematosus before the age of 16 years, followed at the Rheumatology Unit of Pequeno Príncipe Hospital, from January 1992 to January 2006, were retrospectively reviewed, highlighting neuropsychiatric aspects. RESULTS: Forty-seven patients were included. Neuropsychiatric syndromes were found 29 (61.7%): seizures (17 / 36.2%), intractable headache (7 / 14.9%), mood disorders (5 / 10.6%), cerebrovascular disease (4 / 8.5%), acute confusional state (3 / 6.4%), aseptic meningitis (3 / 6.4%), psychosis (3 / 6.4%), chorea (3 / 6.4%), Guillain-Barré syndrome (2 / 4.3%) and cranial neuropathy (1 / 2.1%). Morbidity indexes (SLEDAI and SLICC) were higher among patients with neuropsychiatric manifestations (p<0.05). CONCLUSION: Neuropsychiatric syndromes are frequent, and add significant morbidity to juvenile systemic lupus erythematosus.

Tropical forest light regimes in a human-modified landscape.

Light is the key energy input for all vegetated systems. Forest light regimes are complex, with the vertical pattern of light within canopies influenced by forest structure. Human disturbances in tropical forests impact forest structure and hence may influence the light environment and thus competitiveness of different trees. In this study, we measured vertical diffuse light profiles along a gradient of anthropogenic disturbance, sampling intact, logged, secondary, and fragmented sites in the biodiversity hot spot of the Atlantic forest, southeast Brazil, using photosynthetically active radiation sensors and a novel approach with estimations of vertical light profiles from hemispherical photographs. Our results show clear differences in vertical light profiles with disturbance: Fragmented forests are characterized by rapid light extinction within their low canopies, while the profiles in logged forests show high heterogeneity and high light in the mid-canopy despite decades of recovery. The secondary forest showed similar light profiles to intact forest, but with a lower canopy height. We also show that in some cases the upper canopy layer and heavy liana infestations can severely limit light penetration. Light extinction with height above the ground and depth below the canopy top was highest in fragmented forest and negatively correlated with canopy height. The novel, inexpensive, and rapid methods described here can be applied to other sites to quantify rarely measured vertical light profiles.

Lissencephaly, abnormal genitalia and refractory epilepsy: case report of XLAG syndrome.

INTRODUCTION: X-linked lissencephaly with ambiguous genitalia (XLAG) is a recently described genetic disorder caused by mutation in the aristaless-related homeobox (ARX) gene (Xp22.13). Patients present with lissencephaly, agenesis of the corpus callosum, refractory epilepsy of neonatal onset, acquired microcephaly and male genotype with ambiguous genitalia. CASE REPORT: Second child born to healthy nonconsanguineous parents, presented with seizures within the first hour of life that remained refractory to phenobarbital, phenytoin and midazolam. Examination identified microcephaly, axial hypotonia, pyramidal signs and ambiguous genitalia. EEG showed disorganized background activity and seizures starting at the right midtemporal, central and occipital regions. MRI showed diffuse pachygyria, moderate thickening of the cortex, enlarged ventricles, agenesis of the corpus callosum and septum pellucidum. Karyotype showed a 46,XY genotype. Additional findings were hypercalciuria, vesicoureteral reflux, patent ductus arteriosus and chronic diarrhea.

Perinatal and Hemodynamic Evaluation of Sildenafil Citrate for Preeclampsia Treatment: A Randomized Controlled Trial.

OBJECTIVE: To evaluate whether therapy with sildenafil citrate prolongs gestation in women with preeclampsia. METHODS: In a randomized double-blind, placebo-controlled trial, 100 singleton pregnancies with preeclampsia between 24 and 33 weeks of gestation were randomized to 50 mg oral sildenafil citrate every 8 hours or placebo. The primary outcome was prolongation of pregnancy from randomization to delivery. Secondary outcomes were changes in resistance indices of uterine, umbilical, and middle cerebral arteries by Doppler, fetal and maternal complications, and adverse neonatal outcomes. Power analysis estimated that to detect a difference of 5 days in pregnancy duration, 43 patients would have to be randomized to each group. RESULTS: From June 2013 to October 2015, 50 patients were randomized to each group. Pregnancy duration was on average 4 days longer (14.4 days, 95% confidence interval [CI] 12.5-16.6 days compared with 10.4 days, 95% CI 8.4-12.3 days, P=.008) and percent reduction in pulsatility indices of uterine and umbilical arteries higher (22.5% and 18.5%, compared with placebo 2.1% and 2.5%, P<.001) for patients treated with sildenafil compared with placebo. Maternal blood pressure before and 24 hours after randomization was lower with sildenafil (sildenafil: 100.3±5.6 mm Hg compared with 116.4±5.1 mm Hg, P<.05; placebo: 110.6±6.2 mm Hg compared with 114.7±6.5 mm Hg, P=.21). There was no difference in perinatal morbidity, mortality, or adverse effects between groups. CONCLUSION: Therapy with sildenafil citrate was associated with pregnancy prolongation of approximately 4 days compared with placebo in women with preeclampsia. CLINICAL TRIAL REGISTRATION: Brazilian Registry of Clinical Trials, www.ensaiosclinicos.gov.br, RBR-8qj4p5.