Alterações comportamentais no modelo animal de autismo induzido por exposição pré-natal ao ácido valpróico

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Inibição da aromatase por ácido valpróico no autismo

O autismo pertence ao Transtorno do Espectro do Autismo (TEA), e é caracterizado pelo comprometimento da comunicação, interação social, comportamento estereotipado e restritos interesses do indivíduo. A etiologia do autismo ainda é desconhecida, mas diversos estudos apontam para uma origem multifatorial. No autismo, alguns componentes endógenos encontram-se alterados. Dentre esses, a testosterona destaca-se por apresentar níveis séricos elevados. Diversas isoenzimas do citocromo P450 apresentam atividade de detoxificação de fármacos. A aromatase (CYP19A1) é uma isoenzima do citocromo P450, responsável pela biossíntese de estrogênios a partir de precursores androgênicos. A reação catalítica ocorre em um processo de três etapas que resultam na aromatização de um dos anéis que formam o esqueleto da estrutura básica dos esteroides. A aromatase encontra-se principalmente na placenta e nos folículos dos ovários e em menores concentrações no fígado, músculo e cérebro. O ácido valpróico (VPA) é um fármaco da classe de anticonvulsivantes e é também utilizado como estabilizador de humor. Entretanto, essa classe apresenta-se como um grupo de substâncias com potencial teratogênico quando utilizados por gestantes está relacionado ao aumento dos riscos de surgimento de autismo. O VPA é um dos principais fármacos dessa classe atuando como inibidor de aromatase. O uso do VPA em mulheres está associado a efeitos adversos como ovários policísticos, anovulação e hiperandrogenismo. O objetivo deste trabalho foi avaliar o mecanismo de inibição da aromatase pelo VPA, relacionado ao Autismo. Para isso, aplicação de ferramentas de Bioinformática para a modelagem da estrutura molecular do VPA, otimização da estrutura, docking no sítio ativo da aromatase e análise dos principais resíduos envolvidos na inibição da enzima foram utilizados. Como resultado, a análise sobre os principais resíduos envolvidos na inibição da enzima pelo VPA, sugere que a Arg115 é o resíduo fundamental para que ocorra a inibição enzimática. A energia de interação com o VPA é significativa frente a interação que ocorre com precursores androgênicos. O impedimento estérico gerado pelo ancoramento do VPA a um resíduo não envolvido na aromatização do precursor androgênico, revela o mecanismo de inibição da enzima pelo VPA. Os resultados apresentados nesse estudo sugerem que os níveis séricos elevados de testosterona podem ter como fundamento a inibição da CYP19A1 através do uso do VPA. Além disso, os resultados obtidos auxiliam na elucidação estrutural dos fatores principais no desenvolvimento de novos e promissores inibidores seletivos desta enzima. O mecanismo de ação explicado através de metodologia de bioinformática contribui de maneira significativa nos avanços dos estudos sobre a eitologia do autismo

Estudo de biotransformação de fármacos através de Cunninghamella elegans ATCC 9245

Os fungos filamentosos apresentam sistema enzimático semelhante à via de metabolismo enzimático formado pelo citocromo P450 de mamíferos. Através desse sistema, reações de fase I (oxidativa) e fase II (conjugação) biotransformam diferentes classes de fármacos com o objetivo de facilitar a excreção desses pelo organismo. Os estudos de biotransformação de fármacos surgem como uma alternativa aos estudos farmacológicos e de desenvolvimento de novos candidatos a fármacos com investimento reduzido. Além disso, permitem uma possibilidade de produção de padrões analíticos. O objetivo deste estudo foi avaliar a capacidade de biotransformação dos fármacos linagliptina, vildagliptina, ticagrelor, rivaroxabana e metronidazol utilizando o fungo filamentoso Cunninghamella elegans ATCC 9245. Para o monitoramento do processo de biotransformação deste estudo, amostras de biotransformação e de controle de degradação do fármaco foram coletadas e tratadas com diferentes solventes orgânicos a fim de otimizar o processo de extração líquido-líquido dos fármacos e metabólitos formados. O acompanhamento da formação dos metabólitos foi realizado por cromatografia líquida de alta eficiência (CLAE) acoplada à espectrometria de massas (EM) ou ultravioleta (UV). A biotransformação dos fármacos metronidazol e ticagrelor resultou num consumo do fármaco em cultivo e formação de novos picos cromatográficos de estruturas químicas. Os resultados relacionados à linagliptina e rivaroxabana demonstram que ocorre um consumo dos fármacos pela C. elegans, representado pela redução das concentrações dos fármacos. Entretanto, houve aumento das concentrações após determinado período de incubação. Devido às características químicas da vildagliptina, a extração líquido-líquido não se apresentou adequada para a execução desta etapa dificultando as análises. Este trabalho ainda apresenta o desenvolvimento e validação da metodologia bioanalítica por CLAE aplicada ao metronidazol por ser o único fármaco a apresentar a formação de metabólito detectável por CLAE-UV.Filamentous fungi model can be used for simulating the mammalian metabolism of many molecules of pharmacological importance. The common way of metabolizing drugs involves the alteration of functional groups, as well documented via the cytochrome P450 enzymes. Reactions of phase I (oxidative) and phase II (conjugation) can biotransform different classes of drugs with the objective of facilitating the excretion of the organism. Drug biotransformation studies appear as an alternative to pharmacological studies of metabolites, development of new drug candidates with reduced investment as well as the most efficient production of chemical structures involves and drug quality control studies. The objective of this study was to evaluate the capacity of drugs biotransformation to linagliptin, vildagliptin, ticagrelor, rivaroxaban and metronidazole through the filamentous fungus Cunninghamella elegans ATCC 9245. For the monitoring of the biotransformation process, samples of biotransformation assay and drug degradation control were collected and treated with different organic solvents to optimize the liquid-liquid extraction process of the formed drugs and metabolites. The monitoring of metabolite formation was performed by high-performance liquid chromatography (HPLC) coupled to mass or ultraviolet (UV) spectrophotometry. The results of the biotransformation of metronidazole and ticagrelor drugs showed drug consumption in culture and formation of new chromatographic peaks of chemical structures not elucidated. Results related to linagliptin and rivaroxaban demonstrate that they occur in a consumption of drugs by C. elegans ATCC 9245, represented by the reduction of drug concentrations in the culture medium, with the subsequent return as initial concentrations of the experiments. Due to the chemical characteristics of vildagliptin, a liquid-liquid extraction did not present adequate for an execution of this stage and difficult the analyses. This work also presents the development and the validation of the bioanalytical methodology applied to HPLC for metronidazole because it is the only drug to present a metabolite formation detectable by HPLC-UV

Resistance of endodontically treated roots restored with different fibre post systems with or without post space preparation: in vitro analysis and SEM investigation.

Aim: To compare the mechanical resistance to fracture of two conical post systems placed with no preparation of the root canal with that of double taper fibre posts seated in endodontically treated single roots after standard post space preparation using dedicated drills. Methodology: Thirty fibre posts with double (G1, n = 10, DT Light Post) and single taper (G2, n = 10, SurgiPost Multiconical; G3, n = 10, Tech ES Endoshape) were luted with self-adhesive cement in endodontically treated single roots using different post space preparation techniques. The bonded posts were experimentally loaded until failure and the maximum load to fracture was registered. Fracture patterns were qualitatively evaluated and SEM analysis was performed to assess the quality of endodontic treatments and cementation. Data were statistically analysed by means of one-way ANOVA. Results: The mean maximum load to fracture was 165.05 23.46 N in G1, 151.52 16.23 N in G2 and 129.09 15.25 N in G3. Statistically significant differences were pointed out between G1 and G3 (p < 0.01) and G2 and G3 (p < 0.05). No root fractures were evidenced. SEM analyses showed slightly thicker cement ayers at the apical and middle thirds of single taper posts (G2 and G3). Conclusions: DT Light Post and SurgiPost Multiconical fibre posts showed similar properties in terms of mechanical resistance to fracture and higher than those of Tech ES Endoshape. Unrestorable root fractures did not occur with any of the tested posts

Rifampicin: biotransformation study using the fungus Cunninghamella elegans and monitoring through UHPLC-MS

Drug biotransformation studies appear as an alternative to pharmacological investigations of metabolites, development of new drug candidates with reduced investment and most efficient production. The objective of this study was to evaluate the capacity of biotransformation of Rifampicin (RIF) by the filamentous fungus Cunninghamella elegans as a microbial model of mammalian metabolism. In 120 h, C. elegans transformed the drug into the following two metabolites: rifampicin quinone and novel metabolite. The products of rifampicin formed in vitro were monitored by HPLC-PDA, being identified through UHPLC–QTOF/MS. Metabolites were characterized according to their chromatographic profile, mass fragments and UV spectral data. The major metabolic pathways of rifampicin transformed by the fungus were oxidation, demethylation and mono-oxidation. The microbial transformation of RIF showed the potential of Cunninghamella species to produce RIF metabolites. This process can be used for a cost effective method for both known and unknown metabolite production

pH effect on stability and kinetics degradation of nitazoxanide in solution

Stability studies correspond to a set of tests designed to assess changes in the quality of a given drug over time and under the influence of a number of factors. Among these factors, pH plays an important role, due to the catalytic effect that hydronium and hydroxide ions can play in several reactions. In the present study, the degradation kinetics of nitazoxanide was evaluated over a wide pH range, and the main degradation product generated was identified by LC-MS/MS. Nitazoxanide showed first-order degradation kinetics in the pH range of 0.01 to 10.0 showing greater stability between pH 1.0 and 4.0. The degradation rate constant calculated for these pH was 0.0885 x 10-2 min-1 and 0.0689 x 10-2 min-1, respectively. The highest degradation rate constant value was observed at pH 10.0 (0.7418 x 10-2 min-1) followed by pH 0.01 (0.5882 x 10-2 min-1). A major degradation product (DP-1) was observed in all conditions tested. Through LC-MS/MS analysis, DP-1 was identified as a product of nitazoxanide deacetylation. The effect of pH on the stability of nitazoxanide and the kinetic data obtained contribute to a better understanding of the intrinsic stability characteristics of nitazoxanide

HLA-DP Allele-Specific T Cell Responses to Beryllium Account for DP-Associated Susceptibility to Chronic Beryllium Disease

Abstract Occupational exposure to small molecules, such as metals, is frequently associated with hypersensitivity reactions. Chronic beryllium (Be) disease (CBD) is a multisystem granulomatous disease that primarily affects the lung, and occurs in ∼3% of individuals exposed to this element. Immunogenetic studies have demonstrated a strong association between CBD and possession of alleles of HLA-DP containing glutamic acid (Glu) at position 69 in the HLA-DPβ-chain. T cell clones were raised from three patients with CBD in whom exposure occurred 10 and 30 years previously. Of 25 Be-specific clones that were obtained, all were restricted by HLA-DP alleles with Glu at DPβ69. Furthermore, the proliferative responses of the clones were absolutely dependent upon DPβ Glu69 in that a single amino acid substitution at this position abolished the response. As befits a disease whose pathogenesis involves a delayed type hypersensitivity response, the large majority of Be-specific clones secreted IFN-γ (Th1) and little or no IL-4 (Th2) cytokines. This study provides insights into the molecular basis of DP2-associated susceptibility to CBD

BIOTRANSFORMATION OF METRONIDAZOLE BY CUNNINGHAMELLA ELEGANS ATCC 9245

Drug biotransformation studies appear as an alternative to pharmacological studies of metabolites, development of new drug candidates with reduced investment as well as the most efficient production of chemical structures involves and drug quality control studies. A wide range of reactions in biotransformations process is catalyzed by microorganisms. Fungi can be considered as a promising source of new biotransformation reactions. The aim of this study was to evaluate the capacity of metronidazole biotransformation through the filamentous fungus Cunninghamella elegans ATCC 9245. The monitoring of metabolite formation was performed by high-performance liquid chromatography (HPLC) coupled to ultraviolet (UV) spectrophotometry. The results of the biotransformation of metronidazole showed drug consumption in culture and the formation of four new chromatographic peaks of chemical structures not elucidated. The method showed it became linear over 10-70 μg/mL (r = 0.999953). Accuracy, precision and stability studies agree with international guidelines.  Results are consistent in accordance with the principles of green chemistry as the experimental conditions had a low environmental impact, and few solvents use. 

TSPO acts as an immune resistance gene involved in the T cell mediated immune control of glioblastoma

Glioblastoma (GB) IDH-wildtype is the most malignant primary brain tumor. It is particularly resistant to current immunotherapies. Translocator protein 18 kDa (TSPO) is upregulated in GB and correlates with malignancy and poor prognosis, but also with increased immune infiltration. Here, we studied the role of TSPO in the regulation of immune resistance of human GB cells. The role of TSPO in tumor immune resistance was experimentally determined in primary brain tumor initiating cells (BTICs) and cell lines through genetic manipulation of TSPO expression and subsequent cocultures with antigen specific cytotoxic T cells and autologous tumor-infiltrating T cells. Death inducing intrinsic and extrinsic apoptotic pathways affected by TSPO were investigated. TSPO-regulated genes mediating apoptosis resistance in BTICs were identified through gene expression analysis and subsequent functional analyses. TSPO transcription in primary GB cells correlated with CD8+ T cell infiltration, cytotoxic activity of T cell infiltrate, expression of TNFR and IFNGR and with the activity of their downstream signalling pathways, as well as with the expression of TRAIL receptors. Coculture of BTICs with tumor reactive cytotoxic T cells or with T cell-derived factors induced TSPO up-regulation through T cell derived TNFα and IFNγ. Silencing of TSPO sensitized BTICs against T cell-mediated cytotoxicity. TSPO selectively protected BTICs against TRAIL-induced apoptosis by regulating apoptosis pathways. TSPO also regulated the expression of multiple genes associated with resistance against apoptosis. We conclude that TSPO expression in GB is induced through T cell-derived cytokines TNFα and IFNγ and that TSPO expression protects GB cells against cytotoxic T cell attack through TRAIL. Our data thereby provide an indication that therapeutic targeting of TSPO may be a suitable approach to sensitize GB to immune cell-mediated cytotoxicity by circumventing tumor intrinsic TRAIL resistance

Adjunctive rifampicin for Staphylococcus aureus bacteraemia (ARREST): a multicentre, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Staphylococcus aureus bacteraemia is a common cause of severe community-acquired and hospital-acquired infection worldwide. We tested the hypothesis that adjunctive rifampicin would reduce bacteriologically confirmed treatment failure or disease recurrence, or death, by enhancing early S aureus killing, sterilising infected foci and blood faster, and reducing risks of dissemination and metastatic infection. METHODS: In this multicentre, randomised, double-blind, placebo-controlled trial, adults (≥18 years) with S aureus bacteraemia who had received ≤96 h of active antibiotic therapy were recruited from 29 UK hospitals. Patients were randomly assigned (1:1) via a computer-generated sequential randomisation list to receive 2 weeks of adjunctive rifampicin (600 mg or 900 mg per day according to weight, oral or intravenous) versus identical placebo, together with standard antibiotic therapy. Randomisation was stratified by centre. Patients, investigators, and those caring for the patients were masked to group allocation. The primary outcome was time to bacteriologically confirmed treatment failure or disease recurrence, or death (all-cause), from randomisation to 12 weeks, adjudicated by an independent review committee masked to the treatment. Analysis was intention to treat. This trial was registered, number ISRCTN37666216, and is closed to new participants. FINDINGS: Between Dec 10, 2012, and Oct 25, 2016, 758 eligible participants were randomly assigned: 370 to rifampicin and 388 to placebo. 485 (64%) participants had community-acquired S aureus infections, and 132 (17%) had nosocomial S aureus infections. 47 (6%) had meticillin-resistant infections. 301 (40%) participants had an initial deep infection focus. Standard antibiotics were given for 29 (IQR 18-45) days; 619 (82%) participants received flucloxacillin. By week 12, 62 (17%) of participants who received rifampicin versus 71 (18%) who received placebo experienced treatment failure or disease recurrence, or died (absolute risk difference -1·4%, 95% CI -7·0 to 4·3; hazard ratio 0·96, 0·68-1·35, p=0·81). From randomisation to 12 weeks, no evidence of differences in serious (p=0·17) or grade 3-4 (p=0·36) adverse events were observed; however, 63 (17%) participants in the rifampicin group versus 39 (10%) in the placebo group had antibiotic or trial drug-modifying adverse events (p=0·004), and 24 (6%) versus six (2%) had drug interactions (p=0·0005). INTERPRETATION: Adjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S aureus bacteraemia. FUNDING: UK National Institute for Health Research Health Technology Assessment