Evaluation of graphene oxide toxicity by fet test in the presence of humic acid.

Sustainable development of nanotechnology requires the deep evaluation of the safety of its products. Despite nanotoxicological studies have been intensified in recent years, gaps remain in the methods used to assess the nanotechnology risks. These gaps are due to the complex nanomaterials behavior in the environment, especially in the presence of organic matter and depending on the nanomaterial characteristics. Studies indicate that humic acid present in the aquatic environment can increase the stability of nanomaterial dispersions and may change its toxicity to aquatic organisms

[¹⁸F]fluorination of biorelevant arylboronic acid pinacol ester scaffolds synthesized by convergence techniques

Aim: The development of small molecules through convergent multicomponent reactions (MCR) has been boosted during the last decade due to the ability to synthesize, virtually without any side-products, numerous small drug-like molecules with several degrees of structural diversity.(1) The association of positron emission tomography (PET) labeling techniques in line with the “one-pot” development of biologically active compounds has the potential to become relevant not only for the evaluation and characterization of those MCR products through molecular imaging, but also to increase the library of radiotracers available. Therefore, since the [18F]fluorination of arylboronic acid pinacol ester derivatives tolerates electron-poor and electro-rich arenes and various functional groups,(2) the main goal of this research work was to achieve the 18F-radiolabeling of several different molecules synthesized through MCR. Materials and Methods: [18F]Fluorination of boronic acid pinacol esters was first extensively optimized using a benzaldehyde derivative in relation to the ideal amount of Cu(II) catalyst and precursor to be used, as well as the reaction solvent. Radiochemical conversion (RCC) yields were assessed by TLC-SG. The optimized radiolabeling conditions were subsequently applied to several structurally different MCR scaffolds comprising biologically relevant pharmacophores (e.g. β-lactam, morpholine, tetrazole, oxazole) that were synthesized to specifically contain a boronic acid pinacol ester group. Results: Radiolabeling with fluorine-18 was achieved with volumes (800 μl) and activities (≤ 2 GBq) compatible with most radiochemistry techniques and modules. In summary, an increase in the quantities of precursor or Cu(II) catalyst lead to higher conversion yields. An optimal amount of precursor (0.06 mmol) and Cu(OTf)2(py)4 (0.04 mmol) was defined for further reactions, with DMA being a preferential solvent over DMF. RCC yields from 15% to 76%, depending on the scaffold, were reproducibly achieved. Interestingly, it was noticed that the structure of the scaffolds, beyond the arylboronic acid, exerts some influence in the final RCC, with electron-withdrawing groups in the para position apparently enhancing the radiolabeling yield. Conclusion: The developed method with high RCC and reproducibility has the potential to be applied in line with MCR and also has a possibility to be incorporated in a later stage of this convergent “one-pot” synthesis strategy. Further studies are currently ongoing to apply this radiolabeling concept to fluorine-containing approved drugs whose boronic acid pinacol ester precursors can be synthesized through MCR (e.g. atorvastatin)

Advanced Automata Minimization

We present an efficient algorithm to reduce the size of nondeterministic Buchi word automata, while retaining their language. Additionally, we describe methods to solve PSPACE-complete automata problems like universality, equivalence and inclusion for much larger instances (1-3 orders of magnitude) than before. This can be used to scale up applications of automata in formal verification tools and decision procedures for logical theories. The algorithm is based on new transition pruning techniques. These use criteria based on combinations of backward and forward trace inclusions. Since these relations are themselves PSPACE-complete, we describe methods to compute good approximations of them in polynomial time. Extensive experiments show that the average-case complexity of our algorithm scales quadratically. The size reduction of the automata depends very much on the class of instances, but our algorithm consistently outperforms all previous techniques by a wide margin. We tested our algorithm on Buchi automata derived from LTL-formulae, many classes of random automata and automata derived from mutual exclusion protocols, and compared its performance to the well-known automata tool GOAL.Comment: 15 page

Proton and Helium Spectra from the CREAM-III Flight

Primary cosmic-ray elemental spectra have been measured with the balloon-borne Cosmic Ray Energetics And Mass (CREAM) experiment since 2004. The third CREAM payload (CREAM-III) flew for 29 days during the 2007-2008 Antarctic season. Energies of incident particles above 1 TeV are measured with a calorimeter. Individual elements are clearly separated with a charge resolution of ~0.12 e (in charge units) and ~0.14 e for protons and helium nuclei, respectively, using two layers of silicon charge detectors. The measured proton and helium energy spectra at the top of the atmosphere are harder than other existing measurements at a few tens of GeV. The relative abundance of protons to helium nuclei is 9.53+-0.03 for the range of 1 TeV/n to 63 TeV/n. The ratio is considerably smaller than other measurements at a few tens of GeV/n. The spectra become softer above ~20 TeV. However, our statistical uncertainties are large at these energies and more data are needed

Zebrafish exposure to graphene oxide is related to behavior changes.

Graphene oxide (GO) has been explored to application in areas such as pharmaceutical and environmental sciences. However, GO safety is still unclear. Beyond that, environmental factors, as the presence of humic substances in aquatic environment, can affect nanomaterials behavior and its toxicological effects. The motor development of zebrafish embryo has been strongly characterized and established to neurotoxicity studies. A previous study of our group revealed that the exposure of zebrafish embryos to GO is related to a reduction in total body length and in acetylcholinesterase activity. Morphological and biochemical changes can be related to behavior abnormalities and affect organism?s survival, leading to serious environmental consequences. Thus, the aim of this study was to investigate the implications of GO exposure in zebrafish behavior under environmental conditions. Zebrafish embryos (n=48/group) were exposed to GO (100 mg/L) in the presence or absence of humic acid (HA, 20 mg/L) during 5 days. Larvae were not feed during the experiment and were exposed under a 14/10 h light/dark cycle, 26 ± 1oC. Twelve larvae per group were individually allocated in a 96 well-plate containing clean water and acclimated during 30 min. The following behavior?s biomarkers were monitored during 30 min through DanioVision video-tracking system: total time in movement, distance moved and swimming velocity. At the end of the experiment, larvae were fixed with PFA 4% and its total length and yolk sac were measured through a stereomicroscope. The mortality in all groups was below 10% and no malformation was observed. The statistical analysis (Two-way ANOVA followed by Bonferroni test) showed that, independently of HA presence, larvae exposed to GO dispended more time in movement, swam a greater distance, and showed higher swimming velocity than controls. The larvae exposed to GO, with or without HA, were about 3.8% shorter than control. There was no difference among groups related to yolk sac. The effect can be related to GO adsorption to larvae body that can act as tactile stimuli. Our previous study supports the hypothesis that GO can cause impairment in neuromuscular development. Further experiments are being performed to clarify these observations

Characterisation of LTR-Retrotransposons of Stevia rebaudiana and Their Use for the Analysis of Genetic Variability

Stevia rebaudiana is one of the most important crops belonging to the Asteraceae family. Stevia is cultivated all over the world as it represents a valid natural alternative to artificial sweeteners thanks to its leaves, which produce steviol glycosides that have high sweetening power and reduced caloric value. In this work, the stevia genome sequence was used to isolate and characterise full-length long-terminal repeat retrotransposons (LTR-REs), which account for more than half of the genome. The Gypsy retrotransposons were twice as abundant as the Copia ones. A disproportionate abundance of elements belonging to the Chromovirus/Tekay lineage was observed among the Gypsy elements. Only the SIRE and Angela lineages represented significant portions of the genome among the Copia elements. The dynamics with which LTR-REs colonised the stevia genome were also estimated; all isolated full-length elements turned out to be relatively young, with a proliferation peak around 1–2 million years ago. However, a different analysis conducted by comparing sequences encoding retrotranscriptase showed the occurrence of an older period in which there was a lot of LTR-RE proliferation. Finally, a group of isolated full-length elements belonging to the lineage Angela was used to analyse the genetic variability in 25 accessions of S. rebaudiana using the Inter-Retrotransposon Amplified Polymorphism (IRAP) protocol. The obtained fingerprints highlighted a high degree of genetic variability and were used to study the genomic structures of the different accessions. It was hypothesised that there are four ancestral subpopulations at the root of the analysed accessions, which all turned out to be admixed. Overall, these data may be useful for genome sequence annotations and for evaluating genetic variability in this species, which may be useful in stevia breeding

Variations of training load, monotony, and strain and dose-response relationships with maximal aerobic speed, maximal oxygen uptake, and isokinetic strength in professional soccer players

This study aimed to identify variations in weekly training load, training monotony, and training strain across a 10-week period (during both, pre- and in-season phases); and to analyze the dose-response relationships between training markers and maximal aerobic speed (MAS), maximal oxygen uptake, and isokinetic strength. Twenty-seven professional soccer players (24.9±3.5 years old) were monitored across the 10-week period using global positioning system units. Players were also tested for maximal aerobic speed, maximal oxygen uptake, and isokinetic strength before and after 10 weeks of training. Large positive correlations were found between sum of training load and extension peak torque in the right lower limb (r = 0.57, 90%CI[0.15;0.82]) and the ratio agonist/antagonist in the right lower limb (r = 0.51, [0.06;0.78]). It was observed that loading measures fluctuated across the period of the study and that the load was meaningfully associated with changes in the fitness status of players. However, those magnitudes of correlations were small-to-large, suggesting that variations in fitness level cannot be exclusively explained by the accumulated load and loading profile

Perspectivas do uso do teste com embriões de zebrafish no âmbito da nanotoxicologia.

O teste com embriões de peixes (Fish Embryo Toxicity Test - FET) pode ser utilizado em substituição ao teste agudo com peixes de acordo com recomendação da União Europeia. Assim, a experimentação científica com embriões de zebrafish tem crescido vertiginosamente, beneficiada por diversas características deste modelo experimental, como a transparência dos ovos e o rápido desenvolvimento embrionário. Da mesma forma, a nanotoxicologia tem explorado intensamente este modelo, devido ao reduzido volume de nanomateriais (NMs) consumidos e descartados na realização dos testes. Por outro lado, a singularidade do comportamento dos NMs nos distintos sistemas biológicos demanda adaptações nos protocolos toxicológicos já existentes. As características do meio de exposição, como pH e força iônica combinadas às características dos NMs, como área e carga de superfície, afetam consideravelmente o comportamento dos mesmos em suspensão, podendo ser motivo de variabilidade nos resultados. Como ainda não há um consenso e conhecimento suficiente que sustentem propostas regulatórias para o uso de NMs, é necessário o desenvolvimento e validação de protocolos confiáveis, robustos e reprodutíveis para avaliar os potenciais riscos da produção, manipulação e uso de NMs

Exploring the mechanisms of graphene oxide toxicity to zebrafish embryo.

Many applications of graphene oxide (GO) are still in the research phase, but its promising uses have raised concerns about their risks to human health and the environment. Environmental factors, as the presence of humic substances in aquatic environment, can affect nanomaterials behavior and its toxicological effects. A previous study of our group revealed that the exposure of zebrafish embryos to GO is related to a reduction in total body length and in acetylcholinesterase activity. Thus, the aim of this study was to investigate the implications of GO exposure in muscular development and acetylcholinesterase expression. Zebrafish embryos (n=48/group) were exposed to GO (100 mg/L) in the presence or absence of humic acid (HA, 20 mg/L) during 5 days. Larvae were not feed during the experiment and were exposed under a 14/10 h light/dark cycle, 26 ± 1oC. The mortality in all groups was below 10% and no malformation was observed. At the end of the experiment, alive larvae were fixed with PFA 4% to evaluate its muscular morphology (confocal microscopy, SHG technique) or frozen in liquid nitrogen to perform RT-PCR analysis (AchE expression). Measurements of sarcomere and somite length were performed. No change in muscular tissue was observed, neither differences in AchE expression among groups were detected. So, further experiments have to be performed to clarify the mechanisms of graphene toxicity. FAPESP grants 2014/01995-9, 2014/12891-0, 2014/15640-8