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

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

Downregulation Of 14q32 Micrornas In Primary Human Desmoplastic Medulloblastoma

Medulloblastoma (MB) is one of the most common pediatric cancers, likely originating from abnormal development of cerebellar progenitor neurons. MicroRNA (miRNA) has been shown to play an important role in the development of the central nervous system. Microarray analysis was used to investigate miRNA expression in desmoplastic MB from patients diagnosed at a young age (1 or 2 years old). Normal fetal or newborn cerebellum was used as control. A total of 84 differentially expressed miRNAs (64 downregulated and 20 upregulated) were found. Most downregulated miRNAs (32/64) were found to belong to the cluster of miRNAs at the 14q32 locus, suggesting that this miRNA locus is regulated as a module in MB. Possible mechanisms of 14q32 miRNAs downregulation were investigated by the analysis of publicly available gene expression data sets. First, expression of estrogen-related receptor-γ (ESRRG), a reported positive transcriptional regulator of some 14q32 miRNAs, was found downregulated in desmoplastic MB. Second, expression of the parentally imprinted gene MEG3 was lower in MB in comparison to normal cerebellum, suggesting a possible epigenetic silencing of the 14q32 locus. miR-129-5p (11p11.2/7q32.1), miR-206 (6p12.2), and miR-323-3p (14q32.2), were chosen for functional studies in DAOY cells. Overexpression of miR-129-5p using mimics decreased DAOY proliferation. 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The role of glial cells and apoptosis of enteric neurones in the neuropathology of intractable slow transit constipation

BACKGROUND: Idiopathic slow transit constipation is one of the most severe and often intractable forms of constipation. As motor abnormalities are thought to play an important pathogenetic role, studies have been performed on the colonic neuroenteric system, which rules the motor aspects of the viscus. AIMS: We hypothesised that important neuropathological abnormalities of the large bowel are present, that these are not confined to the interstitial cells of Cajal and ganglion cells, and that the previously described reduction of enteric neurones, if confirmed, might be related to an increase in programmed cell death (apoptosis). PATIENTS AND METHODS: Surgical specimens from 26 severely constipated patients were assessed by conventional and immunohistochemical methods. Specific staining for enteric neurones, glial cells, interstitial cells of Cajal, and fibroblast‐like cells associated with the latter were used. In addition, gangliar cell apoptosis was evaluated by means of indirect and direct techniques. Data from patients were compared with those obtained in 10 controls. RESULTS: Severely constipated patients displayed a significant decrease in enteric gangliar cells, glial cells, and interstitial cells of Cajal. Fibroblast‐like cells associated with the latter did not differ significantly between patients and controls. Patients had significantly more apoptotic enteric neurones than controls. CONCLUSION: Severely constipated patients have important neuroenteric abnormalities, not confined to gangliar cells and interstitial cells of Cajal. The reduction of enteric neurones may in part be due to increased apoptotic phenomena

Exploring the mechanisms of graphene oxide behavioral and morphological changes in zebrafish.

Abstract: The presence of natural organic matter such as humic acid (HA) can influence the behavior of graphene oxide (GO) in the aquatic environment. In this study, zebrafish embryos were analyzed after 5 and 7 days of exposure to GO (100 mg L-1) and HA (20 mg L?1) alone or together. The results indicated that, regardless of the presence of HA, larvae exposed to GO for 5 days showed an increase in locomotor activity, reduction in the yolk sac size, and total length and inhibition of AChE activity, but there was no difference in enzyme expression. The statistical analysis indicated that the reductions in total larval length, yolk sac size, and AChE activity in larvae exposed to GO persisted in relation to the control group, but there was a recovery of these parameters in groups also exposed to HA. Larvae exposed to GO for 7 days did not show significant differences in locomotor activity, but the RT-PCR gene expression analysis evidenced an increase in the AChE expression. Since the embryos exposed to GO showed a reduction in overall length, they were submitted to confocal microscopy and their muscle tissue configuration investigated. No changes were observed in the muscle tissue. The results indicated that HA is associated with the toxicity risk modulation by GO and that some compensatory homeostasis mechanisms may be involved in the developmental effects observed in zebrafish.Made available in DSpace on 2019-11-29T00:38:10Z (GMT). No. of bitstreams: 1 CastroExplorinGraphene2019.pdf: 1588063 bytes, checksum: 442617336ba12bcfbf2332ea5e36872c (MD5) Previous issue date: 2019bitstream/item/205788/1/Castro-Explorin-Graphene-2019.pd

Normal ATXN3 allele but not CHIP polymorphisms modulates age at onset in Machado-Joseph Disease

Background: Age at onset (AO) in Machado-Joseph disease (MJD) is closely associated with the length of the CAG repeat at the mutant ATXN3 allele, but there are other intervening factors. Experimental evidence indicates that the normal ATXN3 allele and the C-terminal heat shock protein 70 (Hsp70)-interacting protein (CHIP) may be genetic modifiers of AO in MJD. Methods: To investigate this hypothesis, we determined the length of normal and expanded CAG repeats at the ATXN3 gene in 210 unrelated patients with MJD. In addition, we genotyped five single nucleotide polymorphisms (SNPs) within the CHIP gene. We first compared the frequencies of the different genotypes in two subgroups of patients who were highly discordant for AO after correction for the length of the expanded CAG allele. The possible modifier effect of each gene was then evaluated in a stepwise multiple linear regression model. Results: AO was associated with the length of the expanded CAG allele (r2 = 0.596, p<0.001). Frequencies of the normal CAG repeats at the ATXN3 gene and of CHIP polymorphisms did not differ significantly between groups with highly discordant ages at onset. However, addition of the normal allele improved the model fit for prediction of AO (r2 = 0.604, p=0.014). Indeed, we found that the normal CAG allele at ATXN3 had a positive independent effect on AO. Conclusion: The normal CAG repeat at the ATXN3 gene has a small but significant influence on AO of MJD