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The endocannabinoid system (ECS) refers to a complex cell-signaling system highly conserved among species formed by numerous receptors, lipid mediators (endocannabinoids) and synthetic and degradative enzymes. It is widely distributed throughout the body including the CNS, where it participates in synaptic signaling, plasticity and neurodevelopment. Besides, the olfactory ensheathing glia (OEG) present in the olfactory system is also known to play an important role in the promotion of axonal growth and/or myelination. Therefore, both OEG and the ECS promote neurogenesis and oligodendrogenesis in the CNS. Here, we investigated if the ECS is expressed in cultured OEG, by assessing the main markers of the ECS through immunofluorescence, western blotting and qRT-PCR and quantifying the content of endocannabinoids in the conditioned medium of these cells. After that, we investigated whether the production and release of endocannabinoids regulate the differentiation of oligodendrocytes co-cultured with hippocampal neurons, through Sholl analysis in oligodendrocytes expressing O4 and MBP markers. Additionally, we evaluated through western blotting the modulation of downstream pathways such as PI3K/Akt/mTOR and ERK/MAPK, being known to be involved in the proliferation and differentiation of oligodendrocytes and activated by CB1, which is the major endocannabinoid responsive receptor in the brain. Our data show that OEG expresses key genes of the ECS, including the CB1 receptor, FAAH and MAGL. Besides, we were able to identify AEA, 2-AG and AEA related mediators palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), in the conditioned medium of OEG cultures. These cultures were also treated with URB597 10-9 M, a FAAH selective inhibitor, or JZL184 10-9 M, a MAGL selective inhibitor, which led to the increase in the concentrations of OEA and 2-AG in the conditioned medium. Moreover, we found that the addition of OEG conditioned medium (OEGCM) enhanced the complexity of oligodendrocyte process branching in hippocampal mixed cell cultures and that this effect was inhibited by AM251 10-6 M, a CB1 receptor antagonist. However, treatment with the conditioned medium enriched with OEA or 2-AG did not alter the process branching complexity of premyelinating oligodendrocytes, while decreased the branching complexity in mature oligodendrocytes. We also observed no change in the phosphorylation of Akt and ERK 44/42 in any of the conditions used. In conclusion, our data show that the ECS modulates the number and maturation of oligodendrocytes in hippocampal mixed cell cultures.
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Discoidin domain receptor 1 (DDR1) is a tyrosine kinase receptor expressed in epithelial cells from different tissues in which collagen binding activates pleiotropic functions. In the brain, DDR1 is mainly expressed in oligodendrocytes (OLs), the function of which is unclear. Whether collagen can activate DDR1 in OLs has not been studied. Here, we assessed the expression of DDR1 during in vitro OL differentiation, including collagen IV incubation, and the capability of collagen IV to induce DDR1 phosphorylation. Experiments were performed using two in vitro models of OL differentiation: OLs derived from adult rat neural stem cells (NSCs) and the HOG16 human oligodendroglial cell line. Immunocytofluorescence, western blotting, and ELISA were performed to analyze these questions. The differentiation of OLs from NSCs was addressed using oligodendrocyte transcription factor 2 (Olig2) and myelin basic protein (MBP). In HOG16 OLs, collagen IV induced DDR1 phosphorylation through slow and sustained kinetics. In NSC-derived OLs, DDR1 was found in a high proportion of differentiating cells (MBP+/Olig2+), but its protein expression was decreased in later stages. The addition of collagen IV did not change the number of DDR1+/MBP+ cells but did accelerate OL branching. Here, we provide the first demonstration that collagen IV mediates the phosphorylation of DDR1 in HOG16 cells and that the in vitro co-expression of DDR1 and MBP is associated with accelerated branching during the differentiation of primary OLs.
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Receptor com Domínio Discoidina 1 , Receptores Proteína Tirosina Quinases , Ratos , Humanos , Animais , Receptor com Domínio Discoidina 1/metabolismo , Ligantes , Colágeno Tipo IV/metabolismo , Oligodendroglia/metabolismoRESUMO
U-Omp19 is a bacterial protease inhibitor from Brucella abortus that inhibits gastrointestinal and lysosomal proteases, enhancing the half-life and immunogenicity of co-delivered antigens. U-Omp19 is a novel adjuvant that is in preclinical development with various vaccine candidates. However, the molecular mechanisms by which it exerts these functions and the structural elements responsible for these activities remain unknown. In this work, a structural, biochemical, and functional characterization of U-Omp19 is presented. Dynamic features of U-Omp19 in solution by NMR and the crystal structure of its C-terminal domain are described. The protein consists of a compact C-terminal beta-barrel domain and a flexible N-terminal domain. The latter domain behaves as an intrinsically disordered protein and retains the full protease inhibitor activity against pancreatic elastase, papain and pepsin. This domain also retains the capacity to induce CD8+ T cells in vivo of U-Omp19. This information may lead to future rationale vaccine designs using U-Omp19 as an adjuvant to deliver other proteins or peptides in oral formulations against infectious diseases, as well as to design strategies to incorporate modifications in its structure that may improve its adjuvanticity.
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Current pharmacological therapies against demyelinating diseases are not quite satisfactory to promote remyelination. Epidermal growth factor (EGF) can expand the population of oligodendrocyte precursor cells (OPCs) that may help with the remyelination process, but its delivery into the injured tissue is still a biomedical challenge. Gold nanoparticles (GNPs) may be a useful tool for drug delivery into the brain. To evaluate remyelination in the septal nucleus, we administered intracerebral GNPs coupled with EGF (EGF-GNPs). C57BL6/J mice were demyelinated with 0.4% cuprizone (CPZ) and divided into several groups: Sham, Ctrl, GNPs, EGF, and EGF-GNPs. We evaluated the remyelination process at two time-points: 2 weeks and 3 weeks post-injection (WPI) of each treatment. We used the rotarod for evaluating motor coordination. Then, we did a Western blot analysis myelin-associated proteins: CNPase, MAG, MOG, and MBP. EGF-GNPs increase the expression of CNPase, MAG, and MOG at 2 WPI. At 3 WPI, we found that the EGF-GNPs treatment improves motor coordination and increases MAG, MOG, and MBP. EGF-GNPs enhance the expression of myelin-associated proteins and improve the motor coordination in mice. Thus, EGF-associated GNPs may be a promising pharmacological vehicle for delivering long-lasting drugs into the brain.
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A prospective cohort study was conducted to measure the concentration levels of three primary phthalate metabolites (MBP, MEHP, MEP) during pregnancy in a group of women from the State of Mexico. The urinary concentration levels of the three phthalate primary metabolites were measured by gas chromatography mass spectrometry during the first, second and third trimesters of pregnancy. The geometric mean and 95 % CI for MBP was 20.38 µg/mL (15.35-27.09); for MEHP 13.43 µg/mL (8.93-20.20), and MEP 52.47 µg/mL (39.88-69.04) adjusted to one g of creatinine. No significant trends were observed among the studied metabolites during the pregnancy period. MBP was higher in less educated women, while women who resided in industrialized zones showed higher levels of MEHP and MEP than women from non-industrialized zones. Consumption of plastic bottled beverages was associated with MBP and MEHP phthalate exposure. Women who used non-registered brands of plastic food containers for storage or for microwave oven use showed the highest levels of MBP and MEP phthalates. The pregnant women in our study were exposed to the three studied primary phthalate metabolites, and this could present a risk to their newborns. To better integrate public health policies, major exploration of potential exposure sources and effects at the regional level is required.
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This study was performed to evaluate the effect of monobutyl phthalate (MBP) on GPR30-activated pathways in Sertoli cells. Additionally, we tested if GIM-1 (Panax ginseng metabolite) modulates MBP action. Human Sertoli cells (HSeC lineage) were exposed to MBP and/or GIM-1 for 30 min, 1, 12, and 48 h. Four experimental treatments were performed: control (DEMEM/F12 medium), MBP, GIM-1, and MBP + GIM-1. The results indicate that MBP activates GPR30, PKA, Src, EGFR, and the ERK1/2 proteins, while GIM-1 inhibits PKA, Src, ERK1/2, and the AKT pathway. MBP also enhances Cofilin expression, decreasing F-actin intensity on the cell surface in a short time. The combined exposure demonstrated a functional antagonism between compounds. Collectively, these data show that MBP activates GPR30 in Sertoli cells, and GIM-1 modulates this response, playing a protective role in Sertoli cells exposed to MBP.
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Citoproteção/efeitos dos fármacos , Disruptores Endócrinos/toxicidade , Panax , Ácidos Ftálicos/toxicidade , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células de Sertoli/efeitos dos fármacos , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Células de Sertoli/metabolismo , Quinases da Família src/metabolismoRESUMO
The use of grasses as cover crops in the off-season of cash crops under no-till has been largely adopted. However, soil phosphorus (P) uptake was previously shown to be reduced when ruzigrass is introduced in the rotation, affecting the viability and sustainability of this cropping system. The objective of this study was to assess the effect of ruzigrass on soil P availability and desorption kinetics under different P fertilizer application rates. A long-term field experiment where soybean (Glycine max) has been grown in rotation with ruzigrass (Urochloa ruziziensis) or fallow for 10â¯years, with the application of 0, 13, and 26â¯kgâ¯ha-1 of P, was evaluated for two consecutive years. Soil P desorption kinetics was assessed using diffusive equilibrium (DET) and gradient in thin films (DGT) techniques, as well as the DGT-induced fluxes in soils model (DIFS). Microbial biomass P (MBP) was assessed to verify if soil solution P (PDET) was reduced due to immobilization by microorganisms. Ruzigrass reduced MBP and PDET especially when P fertilizer was applied. The concentration of labile P (PDGT) was also lower after ruzigrass than in fallow. The soil ability to resupply P to soil solution was lower after ruzigrass regardless of P rates due to a slower desorption in response to the perturbation imposed by DGT. Growing ruzigrass as cover crop in the soybean off-season decreases soil P availability regardless of P fertilizer application rates by fundamentally reducing P mobility and P resupply from soil solid phase into soil solution.
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Constructs containing partial coding sequences of myosin A, myosin B, and glideosome-associated protein (50 kDa) of Plasmodium falciparum were used to challenge several strategies designed in order to improve the production and solubility of recombinant proteins in Escherichia coli. Assays were carried out inducing expression in a late log phase culture, optimizing the inductor concentration, reducing the growth temperature for induced cultures, and supplementing additives in the lysis buffer. In addition, recombinant proteins were expressed as fusion proteins with three different tags (6His, GST, and MBP) in four different E. coli strains. We found that the only condition that consistently produced soluble proteins was the use of MBP as a fusion tag, which became a valuable tool for detecting the proteins used in this study and did not caused any interference in protein-protein interaction assays (Far Western Blot). Besides, we found that BL21-pG-KJE8 strain did not improve the solubility of any of the recombinant protein produced, while the BL21-CodonPlus(DE3)-RIL strain improved the expression of some of them independent of the rare codon content. Proteins with rare codons occurring at high frequencies (¼ 10%) were expressed efficiently in strains that do not supplement tRNAs for these triplets.
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Escherichia coli/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Simulação por Computador , Eletroforese em Gel de Poliacrilamida , Histidina/genética , Oligopeptídeos/genética , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , SolubilidadeRESUMO
Hypoxic-ischemic (HI) injury is an important cause of death and disabilities. Despite all improvements in neonatal care, the number of children who suffer some kind of injury during birth has remained stable in the last decade. A great number of studies have shown alterations in neural cells and many animal models have been proposed in the last 5 decades. Robinson et al. (2005) proposed an HI model in which the uterine arteries are temporarily clamped on the 18th gestation day. The findings were quite similar to the ones observed in postmortem studies. The white matter is clearly damaged, and a great amount of astrogliosis takes place both in the gray and white matters. Motor changes were also found but no data regarding the cerebellum, an important structure related to motor performance, was presented. Using this model, we have shown an increased level of iNOS at P0 and microgliosis and astrogliosis at P9, and astrogliosis at P23 (up to 4 weeks from the insult). NO is important in migration, maturation, and synaptic plasticity, but in exacerbated levels it may also contribute to cellular and tissue damage. We have also evaluated oligodendroglia development in the cerebellum. At P9 in HI animals, we found a decrease in the number of PDGFRα+ cells and an apparent delay in myelination, suggesting a failure in oligodendroglial progenitors migration/maturation and/or in the myelination process. These results point to an injury in cerebellar development that might help to explain the motor problems in HI.
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Cerebelo/patologia , Gliose/patologia , Hipóxia-Isquemia Encefálica/patologia , Neurônios Motores/patologia , Oligodendroglia/patologia , Efeitos Tardios da Exposição Pré-Natal/patologia , Animais , Animais Recém-Nascidos , Cerebelo/metabolismo , Feminino , Expressão Gênica , Gliose/genética , Gliose/metabolismo , Humanos , Hipóxia-Isquemia Encefálica/genética , Hipóxia-Isquemia Encefálica/metabolismo , Camundongos , Neurônios Motores/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Oligodendroglia/metabolismo , Gravidez , Efeitos Tardios da Exposição Pré-Natal/genética , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Ratos , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Substância Branca/metabolismo , Substância Branca/patologiaRESUMO
OBJECTIVES: This study aimed to investigate peripheral blood gene expression in ultra-high-risk subjects (UHR) compared to first-episode psychosis individuals (FEP) and healthy controls (HC). METHODS: We enrolled 22 UHR, 66 FEP and 67 HC and investigated the expression of 12 genes using Taqman assays. We used the Univariate General Linear Model, as well as Bonferroni correction for multiple comparisons. RESULTS: We found that UFD1L (ubiquitin fusion degradation 1 like (yeast)) gene was upregulated in UHR group compared to HC and FEP (P = 3.44 × 10-6 ; P = 9.41 × 10-6). MBP (myelin basic protein) was downregulated in UHR compared to FEP (P = 6.07 × 10-6). DISC1 (disrupted in schizophrenia 1) was also upregulated in UHR compared to FEP but lost statistical significance when corrected for age. CONCLUSIONS: These genes are directly related to neurodevelopmental processes and have been associated to schizophrenia. Recent findings described that DISC1 overexpression can disrupt MBP expression, thus, we think that these alterations in UHR individuals could be associated with a common process. UFD1L showed a different pattern of expression only for UHR group, suggesting that they can be under an acute endoplasmatic reticulum stress, demanding elevated levels of Ufd1. Further studies can improve knowledge on disease progression and putative targets to preventive strategies.
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Exposure to endocrine disrupting chemicals such as bisphenol A (BPA) and phthalates is prevalent among children and adolescents, but little is known regarding important sources of exposure at these sensitive life stages. In this study, we measured urinary concentrations of BPA and nine phthalate metabolites in 108 Mexican children aged 8-13 years. Associations of age, time of day, and questionnaire items on external environment, water use, and food container use with specific gravity-corrected urinary concentrations were assessed, as were questionnaire items concerning the use of 17 personal care products in the past 48-h. As a secondary aim, third trimester urinary concentrations were measured in 99 mothers of these children, and the relationship between specific gravity-corrected urinary concentrations at these two time points was explored. After adjusting for potential confounding by other personal care product use in the past 48-h, there were statistically significant (p<0.05) positive associations in boys for cologne/perfume use and monoethyl phthalate (MEP), mono(3-carboxypropyl) phthalate (MCPP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), and mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), and in girls for colored cosmetics use and mono-n-butyl phthalate (MBP), mono(2-ethylhexyl) phthalate (MEHP), MEHHP, MEOHP, and mono(2-ethyl-5-carboxypentyl) phthalate (MECPP), conditioner use and MEP, deodorant use and MEP, and other hair products use and MBP. There was a statistically significant positive trend for the number of personal care products used in the past 48-h and log-MEP in girls. However, there were no statistically significant associations between the analytes and the other questionnaire items and there were no strong correlations between the analytes measured during the third trimester and at 8-13 years of age. We demonstrated that personal care product use is associated with exposure to multiple phthalates in children. Due to rapid development, children may be susceptible to impacts from exposure to endocrine disrupting chemicals; thus, reduced or delayed use of certain personal care products among children may be warranted.
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Compostos Benzidrílicos/urina , Exposição Ambiental/estatística & dados numéricos , Poluentes Ambientais/urina , Fenóis/urina , Ácidos Ftálicos/urina , Adolescente , Biomarcadores/urina , Criança , Cosméticos/metabolismo , Exposição Ambiental/análise , Feminino , Humanos , Masculino , MéxicoRESUMO
OBJECTIVE: To examine associations of urinary phthalate levels with blood pressure (BP) and serum triglyceride and lipoprotein levels in children. STUDY DESIGN: We performed a cross-sectional analysis of a subsample of US children aged 6-19 years who participated in the National Health and Nutrition Examination Survey between 2003 and 2008. We quantified exposure to 3 families of phthalates--low molecular weight, high molecular weight and di-2-ethylhexylphthalate (DEHP)--based on molar concentration of urinary metabolites. We assessed descriptive, bivariate, and multivariate associations with BP and lipid levels. RESULTS: Controlling for an array of sociodemographic and behavioral factors, as well as diet and body mass index, levels of metabolites of DEHP, a phthalate commonly found in processed foods, were associated with higher age-, sex-, and height-standardized BP. For each log unit (roughly 3-fold) increase in DEHP metabolites, a 0.041 SD unit increase in systolic BP z-score was identified (P = .047). Metabolites of low molecular weight phthalates commonly found in cosmetics and personal care products were not associated with BP. Phthalate metabolites were not associated with triglyceride levels, high-density lipoprotein level, or prehypertension. CONCLUSIONS: Dietary phthalate exposure is associated with higher systolic BP in children and adolescents. Further work is needed to confirm these associations, as well as to evaluate opportunities for intervention.
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Exposição Ambiental/efeitos adversos , Poluentes Ambientais/efeitos adversos , Hipertensão/induzido quimicamente , Ácidos Ftálicos/efeitos adversos , Pré-Hipertensão/induzido quimicamente , Adolescente , Biomarcadores/sangue , Biomarcadores/urina , Criança , Estudos Transversais , Dislipidemias/induzido quimicamente , Exposição Ambiental/análise , Monitoramento Ambiental , Poluentes Ambientais/urina , Feminino , Humanos , Hipertensão/sangue , Hipertensão/urina , Modelos Lineares , Lipoproteínas HDL/sangue , Modelos Logísticos , Masculino , Análise Multivariada , Inquéritos Nutricionais , Ácidos Ftálicos/urina , Pré-Hipertensão/sangue , Pré-Hipertensão/urina , Triglicerídeos/sangue , Estados Unidos , Adulto JovemRESUMO
We have previously demonstrated that aTf (apotransferrin) accelerates maturation of OLs (oligodendrocytes) in vitro as well as in vivo. The purpose of this study is to determine whether aTf plays a functional role in a model of H/I (hypoxia/ischaemia) in the neonatal brain. Twenty-four hours after H/I insult, neonatal rats were intracranially injected with aTf and the effects of this treatment were evaluated in the CC (corpus callosum) as well as the SVZ (subventricular zone) at different time points. Similar to previous studies, the H/I event produced severe demyelination in the CC. Demyelination was accompanied by microglial activation, astrogliosis and iron deposition. Ferritin levels increased together with lipid peroxidation and apoptotic cell death. Histological examination after the H/I event in brain tissue of aTf-treated animals (H/I aTF) revealed a great number of mature OLs repopulating the CC compared with saline-treated animals (H/I S). ApoTf treatment induced a gradual increase in MBP (myelin basic protein) and myelin lipid staining in the CC reaching normal levels after 15 days. Furthermore, significant increase in the number of OPCs (oligodendroglial progenitor cells) was found in the SVZ of aTf-treated brains compared with H/I S. Specifically, there was a rise in cells positive for OPC markers, i.e. PDGFRα and SHH(+) cells, with a decrease in cleaved-caspase-3(+) cells compared with H/I S. Additionally, neurospheres from aTf-treated rats were bigger in size and produced more O4/MBP(+) cells. Our findings indicate a role for aTf as a potential inducer of OLs in neonatal rat brain in acute demyelination caused by H/I and a contribution to the differentiation/maturation of OLs and survival/migration of SVZ progenitors after demyelination in vivo.