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BACKGROUND AND OBJECTIVE: In the European Union, the record of cocaine-related seizures indicates an expanding supply. The purity has also been increasing. The health impact of these trends remains poorly documented, in particular, the changes and clinical manifestations of intoxication in young children. We attempted to evaluate the trend in French pediatric admissions for cocaine intoxication/exposure over an 11-year period (2010-2020). METHODS: A retrospective, national, multicenter, study of a pediatric cohort. All children less than 15 years of age admitted to a tertiary-level pediatric emergency unit for proven cocaine intoxication (compatible symptoms and positive toxicological screening) during the reference period were included. RESULTS: Seventy-four children were included. Forty-six percent were less than 6 years old. Annual admissions increased by a factor of 8 over 11 years (+700%) and 57% of all cases were admitted in the last two years. The main clinical signs were neurologic (59%) followed by cardiovascular symptoms (34%). Twelve patients were transferred to the pediatric intensive care unit. Factors significantly associated with the risk of being transferred to the pediatric intensive care unit were initial admission to the pediatric resuscitation area (P < 0.001), respiratory impairment (P < 0.01), mydriasis (P < 0.01), cardiovascular symptoms (P = 0.014), age of less than 2 years (P = 0.014). Blood and/or urine toxicological screening isolated eighteen other substances besides cocaine in 46 children (66%). CONCLUSION: Children are collateral victims of the changing trends in cocaine availability, use and purity. Admissions of intoxicated children to pediatric emergency departments are more frequent and there is an increase in severe presentations. Therefore, this is a growing public health concern.
Assuntos
Cocaína , Criança , Humanos , Pré-Escolar , Estudos Retrospectivos , Convulsões , Hospitalização , Serviço Hospitalar de EmergênciaRESUMO
SUMOylation is a dynamic posttranslational modification, that provides fine-tuning of protein function involved in the cellular response to stress, differentiation, and tissue development. In the adrenal cortex, an emblematic endocrine organ that mediates adaptation to physiological demands, the SUMOylation gradient is inversely correlated with the gradient of cellular differentiation raising important questions about its role in functional zonation and the response to stress. Considering that SUMO-specific protease 2 (SENP2), a deSUMOylating enzyme, is upregulated by Adrenocorticotropic Hormone (ACTH)/cAMP-dependent Protein Kinase (PKA) signalling within the zona fasciculata, we generated mice with adrenal-specific Senp2 loss to address these questions. Disruption of SENP2 activity in steroidogenic cells leads to specific hypoplasia of the zona fasciculata, a blunted reponse to ACTH and isolated glucocorticoid deficiency. Mechanistically, overSUMOylation resulting from SENP2 loss shifts the balance between ACTH/PKA and WNT/ß-catenin signalling leading to repression of PKA activity and ectopic activation of ß-catenin. At the cellular level, this blocks transdifferentiation of ß-catenin-positive zona glomerulosa cells into fasciculata cells and sensitises them to premature apoptosis. Our findings indicate that the SUMO pathway is critical for adrenal homeostasis and stress responsiveness.
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Transdiferenciação Celular , Cisteína Endopeptidases , Glucocorticoides , Animais , Camundongos , Córtex Suprarrenal/metabolismo , Corticosteroides/metabolismo , Hormônio Adrenocorticotrópico/metabolismo , beta Catenina/metabolismo , Transdiferenciação Celular/genética , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Glucocorticoides/metabolismo , Via de Sinalização WntRESUMO
Unlike most cancers, adrenocortical carcinomas (ACCs) are more frequent in women than in men, but the underlying mechanisms of this sexual dimorphism remain elusive. Here, we show that inactivation of Znrf3 in the mouse adrenal cortex, recapitulating the most frequent alteration in ACC patients, is associated with sexually dimorphic tumor progression. Although female knockouts develop metastatic carcinomas at 18 months, adrenal hyperplasia regresses in male knockouts. This male-specific phenotype is associated with androgen-dependent induction of senescence, recruitment, and differentiation of highly phagocytic macrophages that clear out senescent cells. In contrast, in females, macrophage recruitment is delayed and dampened, which allows for aggressive tumor progression. Consistently, analysis of TCGA-ACC data shows that phagocytic macrophages are more prominent in men and are associated with better prognosis. Together, these data show that phagocytic macrophages are key players in the sexual dimorphism of ACC that could be previously unidentified allies in the fight against this devastating cancer.
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Neoplasias do Córtex Suprarrenal , Carcinoma Adrenocortical , Neoplasias do Córtex Suprarrenal/genética , Neoplasias do Córtex Suprarrenal/patologia , Carcinoma Adrenocortical/genética , Carcinoma Adrenocortical/patologia , Androgênios , Animais , Feminino , Masculino , Camundongos , PrognósticoRESUMO
Carney complex is a rare familial multineoplastic syndrome predisposing to endocrine and nonendocrine tumors due to inactivating mutations of PRKAR1A, leading to perturbations of the cAMPâprotein kinase A signaling pathway. Skin lesions are the most common manifestation of Carney complex, including lentigines, blue nevi, and cutaneous myxomas in unusual locations such as oral and genital mucosa. Unlike endocrine disorders, the pathogenesis of skin lesions remains unexplained. In this study, we show that embryonic invalidation of the Prkar1a gene in steroidogenic factor-1âexpressing cells leads to the development of familial skin pigmentation alterations, reminiscent of those in patients with Carney complex. Immunohistological and molecular analyses, coupled with genetic monitoring of recombinant cell lineages in mouse skin, suggest that familial lentiginosis and myxomas occur in skin areas specifically enriched in dermal melanocytes. In lentigines- and blue neviâprone areas from mutant mice and patients, Prkar1a/PRKAR1A invalidation occurs in a subset of dermal fibroblasts capable of inducing, under the influence of protein kinase A signaling, the production of promelanogenic EDN3 and hepatocyte GF signals. Our model strongly suggests that the origin of the typical Carney complex cutaneous lesions is the result of noncell-autonomous promelanogenic activity of a dermal fibroblast population sharing a community of origin with steroidogenic factor-1 lineage.
Assuntos
Complexo de Carney , Lentigo , Mixoma , Nevo Azul , Dermatopatias , Animais , Camundongos , Complexo de Carney/genética , Complexo de Carney/patologia , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Mixoma/genética , Mixoma/patologia , Síndrome , Lentigo/patologiaRESUMO
Appetite traits have multifactorial origins. In association with environmental and genetic factors, they could become problematic and lead to Feeding or Eating Disorders (FED). As the DSM-5 classification is not suitable for pediatric FED, another way to describe eating behavior is to distinguish the clinical profiles of "small eater" and "big eater". The aim of this study was to identify socio-demographic and medical factors associated with these profiles, and to compare problematic and non-problematic profiles. From the Pedianut study, we analyzed socio-demographic, medical and family history data among 401 children according to 4 age groups (<1 year n = 101, 1-6 years n = 99, 6-12 years n = 100, 12-18 years n = 101). The information collected on eating behavior made it possible to define small eater profile (SEP) and big eater profile (BEP) using predefined grids. BEP was more frequent in adolescents (35.6%), and SEP was more frequent in children aged 1-6 years (34.3%). BEP was associated with having separated parents, being male and the oldest sibling (p < 0.05). Problematic BEP was associated with eating while watching television, being a girl, and having sensory disorders (p < 0.05). SEP was associated, whatever age, with non-breastfeeding, chronic illness, psychological history, sensory disorders, language delays (in the 1-6 year age group), and family history of FED (in the adolescent group) (p < 0.05). This analysis of factors associated with eater profile opens new perspectives for research on risk factors associated with eating traits, which warrants further study in larger populations to delineate transition from healthy to problematic eating.
Assuntos
Comportamento Alimentar , Transtornos da Alimentação e da Ingestão de Alimentos , Adolescente , Criança , Estudos de Coortes , Transtornos da Alimentação e da Ingestão de Alimentos/epidemiologia , Feminino , Humanos , Lactente , Masculino , Inquéritos e QuestionáriosRESUMO
Large-cell calcifying Sertoli cell tumors (LCCSCTs) are among the most frequent lesions occurring in male Carney complex (CNC) patients. Although they constitute a key diagnostic criterion for this rare multiple neoplasia syndrome resulting from inactivating mutations of the tumor suppressor PRKAR1A, leading to unrepressed PKA activity, LCCSCT pathogenesis and origin remain elusive. Mouse models targeting Prkar1a inactivation in all somatic populations or separately in each cell type were generated to decipher the molecular and paracrine networks involved in the induction of CNC testis lesions. We demonstrate that the Prkar1a mutation was required in both stromal and Sertoli cells for the occurrence of LCCSCTs. Integrative analyses comparing transcriptomic, immunohistological data and phenotype of mutant mouse combinations led to the understanding of human LCCSCT pathogenesis and demonstrated PKA-induced paracrine molecular circuits in which the aberrant WNT4 signal production is a limiting step in shaping intratubular lesions and tumor expansion both in a mouse model and in human CNC testes.
Assuntos
Complexo de Carney/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células de Sertoli/citologia , Neoplasias Testiculares/metabolismo , Proteína Wnt4/metabolismo , Animais , Apoptose , Complexo de Carney/genética , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Genes Supressores de Tumor , Humanos , Masculino , Camundongos , Camundongos Knockout , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Comunicação Parácrina , Fenótipo , Pigmentação , Túbulos Seminíferos/metabolismo , Testículo/metabolismo , TranscriptomaRESUMO
Feeding and Eating Disorders (FED) are mostly described in infants and adolescents but are less well-known in children. Information on the prevalence of FED in the general pediatric population is still limited. The aim of this study was to estimate the prevalence and the care pathway of FED in a population aged 0-18 years old, using the Diagnostic and Statistical Manual of Mental Disorders (DSM)-5 classification. Two physicians interviewed 401 families using a questionnaire including demographics, BMI, dietary behavior data, and age-appropriate screening tools. Qualitative and quantitative variables were compared using the Chi2 test and Student's t-test, respectively. After a headcount adjustment based on the French population by age group, the estimated prevalence rate was 3% [95%CI (1.7-5.1)] for Avoidant and Restrictive Food Intake Disorder (ARFID), and 9.7% [95%CI (7.2-13.0)] for Unspecified FED (UFED), which included other restrictive and compulsive FED. The median age for ARFID was 4.8 years (0.8-9 years), and 7.5 years (0.6-17 years) for UFED. The interviews did not identify cases of anorexia, bulimia, binge eating disorder, other specified FED, pica or rumination. Only 15.2% of children with an FED were receiving medical care. The development of validated pediatric screening tools, as well as the training of health professionals in children FED is necessary.
Assuntos
Transtornos da Alimentação e da Ingestão de Alimentos/epidemiologia , Adolescente , Transtorno Alimentar Restritivo Evitativo , Transtorno da Compulsão Alimentar , Bulimia Nervosa , Criança , Pré-Escolar , Estudos Transversais , Manual Diagnóstico e Estatístico de Transtornos Mentais , França/epidemiologia , Humanos , Lactente , Recém-Nascido , Prevalência , Inquéritos e QuestionáriosRESUMO
SUMOylation is a highly conserved and dynamic post-translational mechanism primarily affecting nuclear programs for adapting organisms to stressful challenges. Alteration of SUMOylation cycles leads to severe developmental and homeostatic defects and malignancy, but signals coordinating SUMOylation are still unidentified. The adrenal cortex is a zonated endocrine gland that controls body homeostasis and stress response. Here, we show that in human and in mouse adrenals, SUMOylation follows a decreasing centripetal gradient that mirrors cortical differentiation flow and delimits highly and weakly SUMOylated steroidogenic compartments, overlapping glomerulosa, and fasciculata zones. Activation of PKA signaling by acute hormonal treatment, mouse genetic engineering, or in Carney complex results in repression of small ubiquitin-like modifier (SUMO) conjugation in the inner cortex by coordinating expression of SUMO pathway inducers and repressors. Conversely, genetic activation of canonical wingless-related integration site signaling maintains high SUMOylation potential in the outer neoplastic cortex. Thus, SUMOylation is tightly regulated by signaling pathways that orchestrate adrenal zonation and diseases.-Dumontet, T., Sahut-Barnola, I., Dufour, D., Lefrançois-Martinez, A.-M., Berthon, A., Montanier, N., Ragazzon, B., Djari, C., Pointud, J.-C., Roucher-Boulez, F., Batisse-Lignier, M., Tauveron, I., Bertherat, J., Val, P., Martinez, A. Hormonal and spatial control of SUMOylation in the human and mouse adrenal cortex.
Assuntos
Córtex Suprarrenal/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Processamento de Proteína Pós-Traducional/fisiologia , Sumoilação/fisiologia , Córtex Suprarrenal/efeitos dos fármacos , Córtex Suprarrenal/ultraestrutura , Neoplasias do Córtex Suprarrenal/patologia , Hormônio Adrenocorticotrópico/administração & dosagem , Animais , Complexo de Carney/metabolismo , Linhagem Celular Tumoral , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Preparações de Ação Retardada , Dexametasona/análogos & derivados , Dexametasona/farmacologia , Feminino , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Neoplasias/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sumoilação/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/fisiologia , Zona Fasciculada/efeitos dos fármacos , Zona Fasciculada/metabolismo , Zona Glomerulosa/efeitos dos fármacos , Zona Glomerulosa/metabolismo , beta Catenina/deficiência , beta Catenina/genéticaRESUMO
Nucleotides play a role in inflammation processes: cAMP and cGMP in the endothelial barrier function, ADP in platelet aggregation, ATP and UTP in vasodilatation and/or vasoconstriction of blood vessels, UDP in macrophages activation. The aim of this study is to develop and validate a LC/MS-MS method able to quantify simultaneously nine nucleotides (AMP, cAMP, ADP, ATP, GMP, cGMP, UMP, UDP and UTP) in biological matrixes (cells and plasma). The method we developed, has lower LOQ's than others and has the main advantage to quantify all nucleotides within one single injection in less than 10â¯min. The measured nucleotides concentrations obtained with this method are similar to those obtained with assay kits commercially available. Analysis of plasma and red blood cells from healthy donors permits to estimate the physiological concentration of those nucleotides in human plasma and red blood cells, such information being poorly available in the literature. Furthermore, the protocol presented in this paper allowed us to observe that AMP, ADP, ATP concentrations are modified in human red blood cells and plasma after a venous stasis of 4â¯min compared to physiological blood circulation. Therefore, this specific method enables future studies on nucleotides implications in chronic inflammatory diseases but also in other pathologies where nucleotides are implicated in.
Assuntos
Cromatografia Líquida/métodos , Nucleotídeos/sangue , Espectrometria de Massas em Tandem/métodos , Linhagem Celular , Células Endoteliais/química , Eritrócitos/química , Humanos , Sensibilidade e EspecificidadeRESUMO
Dietary restriction (DR) is thought to exert its beneficial effects on healthspan at least partially by a senolytic and senostatic action, i.e. by reducing frequencies of cells with markers of DNA damage and senescence in multiple tissues. Due to its importance in metabolic and inflammation regulation, fat is a prime tissue for health span determination as well as a prime target for DR. We aimed to determine here whether the beneficial effects of DR would be retained over a subsequent period of ad libitum (AL) feeding. Male mice were kept under either 40% DR or AL feeding regimes from 3 to 12â¯months of age and then either switched back to the opposite feeding regimen or kept in the same state for another 3â¯months. Visceral adipose tissue from 4 to 5 mice per group for all conditions was analysed for markers of senescence (adipocyte size, γH2A.X, p16, p21) and inflammation (e.g. IL-6, TNFα, IL-1ß) using immuno-staining or qPCR. Macrophages were detected by immunohistochemistry. We found that both 9 and 12â¯months DR (long term) as well as 3â¯month (short term, mid-life onset) DR reduced the number of cells harbouring DNA damage and adipocyte size (area and perimeter) in visceral adipocytes with similar efficiency. Importantly, beneficial health markers induced by DR such as small adipocyte size and low DNA damage were maintained for at least 3â¯month after termination of DR, demonstrating that the previously identified 'metabolic memory' of the DR state in male mice extends to senescence markers in visceral fat.
Assuntos
Adipócitos/citologia , Restrição Calórica , Senescência Celular , Dano ao DNA , Gordura Intra-Abdominal/metabolismo , Animais , Biomarcadores , Inflamação/metabolismo , Macrófagos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos AnimaisRESUMO
Photoactive ruthenium-based complexes are actively studied for their biological applications as potential theragnostic agents against cancer. One major issue of these inorganic complexes is to penetrate inside cells in order to fulfil their function, either sensing the internal cell environment or exert a photocytotoxic activity. The use of lipophilic ligands allows the corresponding ruthenium complexes to passively diffuse inside cells but limits their structural and photophysical properties. Moreover, this strategy does not provide any cell selectivity. This limitation is also faced by complexes anchored on cell-penetrating peptides. In order to provide a selective cell targeting, we developed a multivalent system composed of a photoreactive ruthenium(II) complex tethered to a calix[4]arene platform bearing multiple RGD-containing cyclopentapeptides. Extensive photophysical and photochemical characterizations of this Ru(II)-calixarene conjugate as well as the study of its photoreactivity in the presence of guanosine monophosphate have been achieved. The results show that the ruthenium complex should be able to perform efficiently its photoinduced cytotoxic activity, once incorporated into targeted cancer cells thanks to the multivalent platform.
RESUMO
This article present data related to the publication entitled "Native and myeloperoxidase-oxidized low-density lipoproteins act in synergy to induce release of resolvin-D1 from endothelial cells" (Dufour et al., 2018). The supporting materials include results obtained by Mox-LDLs stimulated macrophages and investigation performed on scavenger receptors. Linear regressions (RvD1 vs age of mice and RvD1 vs CL-Tyr/Tyr) and Data related to validation were also presented. The interpretation of these data and further extensive insights can be found in Dufour et al. (2018) [1].
RESUMO
BACKGROUND AND AIMS: Oxidation of native low-density lipoproteins (LDLs-nat) plays an important role in the development of atherosclerosis. A major player in LDL-nat oxidation is myeloperoxidase (MPO), a heme enzyme present in azurophil granules of neutrophils and monocytes. MPO produces oxidized LDLs called Mox-LDLs, which cause a pro-inflammatory response in human microvascular endothelial cells (HMEC), monocyte/macrophage activation and formation of foam cells. Resolvin D1 (RvD1) is a compound derived from the metabolism of the polyunsaturated fatty acid DHA, which promotes resolution of inflammation at the ng/ml level. METHODS: In the present study, we used liquid chromatography-mass spectrometry (LC-MS/MS) to investigate the synthesis of RvD1 and its precursors - 17(S)-hydroxy docosahexaenoic acid (17S-HDHA) and docosahexaenoic acid (DHA) - by HMEC, in the presence of several concentrations of Mox-LDLs, copper-oxidized-LDLs (Ox-LDLs), and native LDLs or in mouse plasma. The LC-MS/MS method has been validated and applied to cell supernatants and plasma to measure production of RvD1 and its precursors in several conditions. RESULTS: Mox-LDLs played a significant role in the synthesis of RvD1 and 17S-HDHA from DHA compared to Ox-LDLs. Moreover, Mox-LDLs and LDLs-nat acted in synergy to produce RvD1. In addition, different correlations were found between RvD1 and M1 macrophages, age of mice or Cl-Tyr/Tyr ratio. CONCLUSIONS: These results suggest that although Mox-LDLs are known to be pro-inflammatory and deleterious in the context of atherosclerosis, they are also able to induce a pro-resolution effect by induction of RvD1 from HMEC. Finally, our data also suggest that HMEC can produce RvD1 on their own.
Assuntos
Ácidos Docosa-Hexaenoicos/biossíntese , Células Endoteliais/citologia , Lipoproteínas LDL/sangue , Peroxidase/metabolismo , Animais , Aterosclerose/metabolismo , Calibragem , Linhagem Celular , Cromatografia Líquida , Cobre , Humanos , Inflamação , Limite de Detecção , Lipídeos/sangue , Macrófagos , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Oxigênio , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Protein carbamylation by cyanate is a post-translational modification associated with several (patho)physiological conditions, including cardiovascular disorders. However, the biochemical pathways leading to protein carbamylation are incompletely characterized. This work demonstrates that the heme protein myeloperoxidase (MPO), which is secreted at high concentrations at inflammatory sites from stimulated neutrophils and monocytes, is able to catalyze the two-electron oxidation of cyanide to cyanate and promote the carbamylation of taurine, lysine, and low-density lipoproteins. We probed the role of cyanide as both electron donor and low-spin ligand by pre-steady-state and steady-state kinetic analyses and analyzed reaction products by MS. Moreover, we present two further pathways of carbamylation that involve reaction products of MPO, namely oxidation of cyanide by hypochlorous acid and reaction of thiocyanate with chloramines. Finally, using an in vivo approach with mice on a high-fat diet and carrying the human MPO gene, we found that during chronic exposure to cyanide, mimicking exposure to pollution and smoking, MPO promotes protein-bound accumulation of carbamyllysine (homocitrulline) in atheroma plaque, demonstrating a link between cyanide exposure and atheroma. In summary, our findings indicate that cyanide is a substrate for MPO and suggest an additional pathway for in vivo cyanate formation and protein carbamylation that involves MPO either directly or via its reaction products hypochlorous acid or chloramines. They also suggest that chronic cyanide exposure could promote the accumulation of carbamylated proteins in atherosclerotic plaques.
Assuntos
Cianatos , Cianetos , Peroxidase , Placa Aterosclerótica/enzimologia , Carbamilação de Proteínas , Animais , Citrulina/análogos & derivados , Citrulina/química , Citrulina/genética , Citrulina/metabolismo , Cianatos/química , Cianatos/metabolismo , Cianetos/química , Cianetos/metabolismo , Humanos , Camundongos , Camundongos Knockout , Oxirredução , Peroxidase/química , Peroxidase/genética , Peroxidase/metabolismo , Placa Aterosclerótica/genética , Placa Aterosclerótica/patologiaRESUMO
BACKGROUND AND OBJECTIVES: In France, cannabis consumption is illegal. The health impact of its increasing use and higher tetrahydrocannabinol (THC) concentrations is still poorly documented, particularly that of unintentional pediatric intoxications. We sought to evaluate the French national trend of admissions for unintentional cannabis intoxication in children over an 11-year period (2004-2014). METHODS: A retrospective, national, multicenter, observational study of a pediatric cohort. All children aged <6 years admitted to a tertiary-level pediatric emergency department (PED) for proven cannabis intoxication (compatible symptoms and positive toxicological screening results) during the reference period were included. RESULTS: Twenty-four PEDs participated in our study; 235 children were included, and 71% of the patients were 18 months old or younger. Annual admissions increased by a factor of 13. Hashish resin was the main form ingested (72%). During the study period, the evolution was characterized by a national increase in intoxications, younger intoxicated children (1.28 ± 0.4 vs 1.7 ± 0.7 years, P = .005), and more comas (n = 38) (P = .05, odds ratio 3.5 [1.02-11.8]). Compared with other intoxications, other PED admissions, and the same age population, cannabis-related admissions were greater. There was a potential link between the increased incidence of comas and increased THC concentration in resin seized in France over the period. CONCLUSIONS: Children are collateral victims of changing trends in cannabis use and a prevailing THC concentration. Intoxicated children are more frequent, are younger, and have intoxications that are more severe. This raises a real issue of public health.
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Cannabis/efeitos adversos , Hospitalização/estatística & dados numéricos , Abuso de Maconha/epidemiologia , Transtornos Relacionados ao Uso de Substâncias/epidemiologia , Criança , Pré-Escolar , Serviço Hospitalar de Emergência/estatística & dados numéricos , Feminino , França/epidemiologia , Humanos , Incidência , Lactente , Masculino , Estudos RetrospectivosRESUMO
Myeloperoxidase (MPO) is able to promote several kinds of damage and is involved in mechanisms leading to various diseases such as atherosclerosis or cancers. An example of these damages is the chlorination of nucleic acids, which is considered as a specific marker of the MPO activity. Since 5-chlorocytidine has been recently shown in healthy donor plasmas, this study aimed at discovering if these circulating modified nucleosides could be incorporated into RNA and DNA and if their presence impacts the ability of enzymes involved in the incorporation, transcription, and translation processes. Experimentations, which were carried out in vitro with endothelial and prostatic cells, showed a large penetration of all chloronucleosides but an exclusive incorporation of 5-chlorocytidine into RNA. However, no incorporation into DNA was observed. This specific incorporation is accompanied by an important reduction of translation yield. Although, in vitro, DNA polymerase processed in the presence of chloronucleosides but more slowly than in control conditions, ribonucleotide reductase could not reduce chloronucleotides prior to the replication. This reduction seems to be a limiting step, protecting DNA from chloronucleoside incorporation. This study shows the capacity of transcription enzyme to specifically incorporate 5-chlorocytidine into RNA and the loss of capacity-complete or partial-of different enzymes, involved in replication, transcription or translation, in the presence of chloronucleosides. Questions remain about the long-term impact of such specific incorporation in the RNA and such decrease of protein production on the cell viability and function.
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Células Endoteliais/citologia , Líquido Extracelular/química , Nucleosídeos/química , Próstata/citologia , RNA/análise , Células Cultivadas , Cloro/química , Citidina/química , Halogenação , Humanos , Masculino , Nucleosídeos/sangue , Peroxidase/metabolismo , Biossíntese de Proteínas , RNA/química , Transcrição GênicaRESUMO
Human myeloperoxidase (MPO) plays an important role in innate immunity but also aggravates tissue damage by oxidation of biomolecules at sites of inflammation. As a result from a recent high-throughput virtual screening approach for MPO inhibitors, bis-2,2'-[(dihydro-1,3(2H,4H) pyrimidinediyl)bis(methylene)]phenol was detected as a promising lead compound for inhibition of the MPO-typical two-electron oxidation of chloride to hypochlorous acid (IC50 = 0.5 µM). In the present pharmacomodulation study, 37 derivatives of this lead compound were designed and synthesized driven by comprehensive docking studies and the impact on the chlorination activity of MPO. We describe the structural requirements for optimum (i) binding to the heme periphery and (ii) inhibition capacity. Finally, the best three inhibitors (bis-arylalkylamine derivatives) were probed for interaction with the MPO redox intermediates Compound I and Compound II. Determined apparent bimolecular rate constants together with determination of reduction potential and nucleophilicity of the selected compounds allowed us to propose a mechanism of inhibition. The best inhibitor was found to promote the accumulation of inactive form of MPO-Compound II and has IC50 = 54 nM, demonstrating the successful approach of the drug design. Due to the similarity of ligand interactions between MPO and serotonine transporter, the selectivity of this inhibitor was also tested on the serotonin transporter providing a selectivity index of 14 (KiSERT/IC50MPO).
Assuntos
Aminas/síntese química , Aminas/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Peroxidase/antagonistas & inibidores , Aminas/química , Aminas/metabolismo , Técnicas de Química Sintética , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Halogenação , Humanos , Cinética , Simulação de Acoplamento Molecular , Oxirredução , Peroxidase/química , Peroxidase/metabolismo , Conformação Proteica , Inibidores Seletivos de Recaptação de Serotonina/síntese química , Inibidores Seletivos de Recaptação de Serotonina/química , Inibidores Seletivos de Recaptação de Serotonina/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Relação Estrutura-AtividadeRESUMO
Myeloperoxidase promotes several kinds of damage and is involved in the development of various diseases (as atherosclerosis and cancers). An example of these damage is the chlorination of nucleic acids, which is considered as a specific marker of the MPO activity on those acids. This study aimed to develop and validate a method to analyze oxidized and MPO-specific chlorinated nucleosides in biological matrixes (cells, tissues and plasma). Although a lot of methods to quantify oxidized or chlorinated nucleosides have already been established, none of them took into account all these derivatives together. The new method used a Triple Quadrupole mass spectrometer fitted with a Jet Stream electrospray ionization source. This approach has two advantages compared with existing LC/MSMS analyses: it includes MPO-induced modifications in a unique analysis and obtains a better sensitivity. Our optimized method reached LOQs of 1.50pg and 1.42pg respectively for oxoG and oxo(d)G, being 4 times more sensitive than previous methods, and LOQs of 1.39pg, 1.30pg and 63.4 fg respectively for 5-chlorocytidine, 5-chloro-2'-deoxycytidine and 8-chloroguanosine. Developed method is also 25 times more sensitive for chloroguanosine than the best existing method. Nevertheless, this method is not specific enough for 8-chloro-(2'-deoxy)adenosine analysis. Examples of applications demonstrate the interest of this validated method. Indeed analysis of plasma from healthy donors highlighted exclusively the presence of 5-chlorocytidine (1.0±0.2nM) whereas analysis of treated endothelial cells by HOCl showed chlorination of guanosine and cytidine in cytoplasmic pools and chlorination of (deoxy)cytidine in DNA and RNA. In conclusion, this study shows that 5-chloro-2'-deoxycytidine, 5-chlorocytidine and 8-chloroguanosine are good markers allowing us to detect the MPO activity in biological fluids. The robust, specific and sensitive developed method enables future studies on MPO implications in human diseases.
Assuntos
Espectrometria de Massas em Tandem , Cromatografia Líquida , Desoxicitidina/análogos & derivados , Guanosina/análogos & derivados , PeroxidaseRESUMO
In the last decades, proteomics has largely progressed. Mass spectrometry and liquid chromatography (LC) are generally used in proteomics. These techniques enable proper separation of peptides and good identification and/or quantification of them. Later, nano-scaled liquid chromatography, improvements of mass spectrometry resolution and sensitivity brought huge advancements. Enhancements in chemistry of chromatographic columns also brought interesting results. In the present work, the potency of identification of proteins by different nano-chip columns was studied and compared with classical LC column. The present study was applied to cardiovascular field where proteomics has shown to be highly helpful in research of new biomarkers. Protein extracts from atheroma plaques were used and proteomics data were compared. Results show that fewer spectra were acquired by the mass spectrometer when nano-chip columns were used instead of the classical ones. However, approximately 40% more unique peptides were identified by the recently optimized chip named Polaris-HR-chip-3C18 column, and 20% more proteins were identified. This fact leads to the identification of more low-abundance proteins. Many of them are involved in atheroma plaque development such as apolipoproteins, ceruloplasmin, etc. In conclusion, present data shows that recent developments of nanoLC column chemistry and dimensions enabled the improved detection and identification of low-abundance proteins in atheroma plaques. Several of them are of major interest in the field of cardiovascular disease.