RESUMO
We set out to determine whether the C-terminus (amino acids 481-798) of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α, UniProt Q9UBK2), a regulatory metabolic protein involved in mitochondrial biogenesis, and respiration, is an arginine methyltransferase substrate. Arginine methylation by protein arginine methyltransferases (PRMTs) alters protein function and thus contributes to various cellular processes. In addition to confirming methylation of the C-terminus by PRMT1 as described in the literature, we have identified methylation by another member of the PRMT family, PRMT7. We performed in vitro methylation reactions using recombinant mammalian PRMT7 and PRMT1 at 37, 30, 21, 18, and 4 °C. Various fragments of PGC-1α corresponding to the C-terminus were used as substrates, and the methylation reactions were analyzed by fluorography and mass spectrometry to determine the extent of methylation throughout the substrates, the location of the methylated PGC-1α arginine residues, and finally, whether temperature affects the deposition of methyl groups. We also employed two prediction programs, PRmePRed and MePred-RF, to search for putative methyltransferase sites. Methylation reactions show that arginine residues R548 and R753 in PGC-1α are methylated at or below 30 °C by PRMT7, while methylation by PRMT1 was detected at these same residues at 30 °C. Computational approaches yielded additional putative methylarginine sites, indicating that since PGC-1α is an intrinsically disordered protein, additional methylated arginine residues have yet to be experimentally verified. We conclude that temperature affects the extent of arginine methylation, with more methylation by PRMT7 occurring below physiological temperature, uncovering an additional control point for PGC-1α.
Assuntos
Arginina , Fatores de Transcrição , Animais , Metilação , Arginina/metabolismo , Temperatura , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Mamíferos/metabolismoRESUMO
In late 2003, a major breakthrough in our understanding of the mechanisms that govern reproduction occurred with the identification of the reproductive roles of kisspeptins, encoded by the Kiss1 gene, and their receptor, Gpr54 (aka, Kiss1R). The discovery of this unsuspected reproductive facet attracted an extraordinary interest and boosted an intense research activity, in human and model species, that, in a relatively short period, established a series of basic concepts on the physiological roles of kisspeptins. Such fundamental knowledge, gathered in these early years of kisspeptin research, set the scene for the more recent in-depth dissection of the intimacies of the neuronal networks involving Kiss1 neurons, their precise mechanisms of regulation and the molecular underpinnings of the function of kisspeptins as pivotal regulators of all key aspects of reproductive function, from puberty onset to pulsatile gonadotropin secretion and the metabolic control of fertility. While no clear temporal boundaries between these two periods can be defined, in this review we will summarize the most prominent advances in kisspeptin research occurred in the last ten years, as a means to provide an up-dated view of the state of the art and potential paths of future progress in this dynamic, and ever growing domain of Neuroendocrinology.
Assuntos
Kisspeptinas , Reprodução , Hormônio Liberador de Gonadotropina/metabolismo , Humanos , Kisspeptinas/metabolismo , Sistemas Neurossecretores/metabolismo , Puberdade/metabolismo , Receptores Acoplados a Proteínas G , Receptores de Kisspeptina-1/metabolismo , Reprodução/fisiologiaRESUMO
Macroalgae are an extensive resource for the obtention of bioactive compounds, mainly phenolic compounds, phlorotannins, and pigments. Fucoxanthin (Fx) is the most abundant pigment present in brown algae and has shown several useful bioactivities that can be used to fortify products in the food and cosmetic industries. Nevertheless, to date, there is still insufficient literature reporting on the extraction yield of Fx from U. pinnatifida species from green technologies. In this regard, the present study aims to optimize the extraction conditions to obtain the highest Fx yield from U. pinnatifida through emerging techniques, namely microwave-assisted extraction (MAE) and ultrasound-assisted extraction (UAE). These methods will be compared with the conventional methodologies of heat-assisted extraction (HAE) and Soxhlet-assisted extraction (SAE). According to our results, even though the extraction yield could be slightly higher when using MAE than UAE, the Fx concentration obtained from the alga was double when using UAE. Thus, the Fx ratio in the final extract reached values of 124.39 mg Fx/g E. However, the optimal conditions should also be considered since UAE needed 30 min to perform the extraction, whereas MAE was able to obtain 58.83 mg Fx/g E in only 3 min and 2 bar, meaning less energy expenditure and minimum cost function. To our knowledge, this study obtains the highest concentrations of Fx ever reported (58.83 mg Fx/g E for MAE and 124.39 mg Fx/g E for UAE), with low energy consumption and short times (3.00 min for MAE and 35.16 min for UAE). Any of these results could be selected for further experiments and proposed for industrial scaling-up.
Assuntos
Phaeophyceae , Undaria , Micro-Ondas , XantofilasRESUMO
STUDY QUESTION: Does direct kisspeptin signaling in the oocyte have a role in the control of follicular dynamics and ovulation? SUMMARY ANSWER: Kisspeptin signaling in the oocyte plays a relevant physiological role in the direct control of ovulation; oocyte-specific ablation of kisspeptin receptor, Gpr54, induces a state of premature ovulatory failure in mice that recapitulates some features of premature ovarian insufficiency (POI). WHAT IS KNOWN ALREADY: Kisspeptins, encoded by the Kiss1 gene, are essential for the control of ovulation and fertility, acting primarily on hypothalamic GnRH neurons to stimulate gonadotropin secretion. However, kisspeptins and their receptor, Gpr54, are also expressed in the ovary of different mammalian species, including humans, where their physiological roles remain contentious and poorly characterized. STUDY DESIGN, SIZE, DURATION: A novel mouse line with conditional ablation of Gpr54 in oocytes, named OoGpr54-/-, was generated and studied in terms of follicular and ovulatory dynamics at different age-points of postnatal maturation. A total of 59 OoGpr54-/- mice and 47 corresponding controls were analyzed. In addition, direct RNA sequencing was applied to ovarian samples from 8 OoGpr54-/- and 7 control mice at 6 months of age, and gonadotropin priming for ovulatory induction was conducted in mice (N = 7) from both genotypes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Oocyte-selective ablation of Gpr54 in the oocyte was achieved in vivo by crossing a Gdf9-driven Cre-expressing transgenic mouse line with a Gpr54 LoxP mouse line. The resulting OoGpr54-/- mouse line was subjected to phenotypic, histological, hormonal and molecular analyses at different age-points of postnatal maturation (Day 45, and 2, 4, 6 and 10-11 months of age), in order to characterize the timing of puberty, ovarian follicular dynamics and ovulation, with particular attention to identification of features reminiscent of POI. The molecular signature of ovaries from OoGpr54-/- mice was defined by direct RNA sequencing. Ovulatory responses to gonadotropin priming were also assessed in OoGpr54-/- mice. MAIN RESULTS AND THE ROLE OF CHANCE: Oocyte-specific ablation of Gpr54 caused premature ovulatory failure, with some POI-like features. OoGpr54-/- mice had preserved puberty onset, without signs of hypogonadism. However, already at 2 months of age, 40% of OoGpr54-/- females showed histological features reminiscent of ovarian failure and anovulation. Penetrance of the phenotype progressed with age, with >80% and 100% of OoGpr54-/- females displaying complete ovulatory failure by 6- and 10 months, respectively. This occurred despite unaltered hypothalamic Gpr54 expression and gonadotropin levels. Yet, OoGpr54-/- mice had decreased sex steroid levels. While the RNA signature of OoGpr54-/- ovaries was dominated by the anovulatory state, oocyte-specific ablation of Gpr54 significantly up- or downregulated of a set of 21 genes, including those encoding pituitary adenylate cyclase-activating polypeptide, Wnt-10B, matrix-metalloprotease-12, vitamin A-related factors and calcium-activated chloride channel-2, which might contribute to the POI-like state. Notably, the anovulatory state of young OoGpr54-/- mice could be rescued by gonadotropin priming. LARGE SCALE DATA: N/A. . LIMITATIONS, REASONS FOR CAUTION: Conditional ablation of Gpr54 in oocytes unambiguously caused premature ovulatory failure in mice; yet, the ultimate molecular mechanisms for such state of POI can be only inferred on the basis of RNAseq data and need further elucidation, since some of the molecular changes observed in OoGpr54-/- ovaries were secondary to the anovulatory state. Direct translation of mouse findings to human disease should be made with caution since, despite the conserved expression of Kiss1/kisspeptin and Gpr54 in rodents and humans, our mouse model does not recapitulate all features of common forms of POI. WIDER IMPLICATIONS OF THE FINDINGS: Deregulation of kisspeptin signaling in the oocyte might be an underlying, and previously unnoticed, cause for some forms of POI in women. STUDY FUNDING/COMPETING INTEREST(S): This work was primarily supported by a grant to M.P. and M.T.-S. from the FiDiPro (Finnish Distinguished Professor) Program of the Academy of Finland. Additional financial support came from grant BFU2017-83934-P (M.T.-S.; Ministerio de Economía y Competitividad, Spain; co-funded with EU funds/FEDER Program), research funds from the IVIRMA International Award in Reproductive Medicine (M.T.-S.), and EFSD Albert Renold Fellowship Programme (S.T.R.). The authors have no conflicts of interest to declare in relation to the contents of this work. TRIAL REGISTRATION NUMBER: N/A.
Assuntos
Anovulação , Kisspeptinas , Animais , Feminino , Humanos , Kisspeptinas/genética , Mamíferos/metabolismo , Camundongos , Oócitos/metabolismo , OvulaçãoRESUMO
Tropical montane forests are threatened by uncontrolled fire events because of agricultural expansion. Consequently, deforested areas frequently are dominated by the bracken fern, Pteridium spp., for long periods, and forest regeneration is limited. Despite considerable research on bracken-dominated ecosystems, little is known about the relationship between bracken mycorrhizal fungi and tree seedlings. Arbuscular mycorrhizal fungi (AMF) form symbiotic relationships with terrestrial plants, providing nutrients and protection against pathogens and promoting seedling growth and establishment. Therefore, AMF inoculum have high potential for forest restoration programs. Here, we compare the species diversity of AMF spores, root colonization, and seedling growth of Clusia trochiformis 1 year after the addition of different liquefied root inocula: forest conspecific, forest heterospecific, and from Pteridium rhizomes. Thirteen morphospecies of arbuscular mycorrhizal fungi were identified on the roots of C. trochiformis, and Glomus spp. were the most abundant in all treatments. No differences were observed in spore species richness and diversity among treatments, but spore density was the highest subsequent to the Pteridium inoculum. There was no significant difference in mycorrhizal root colonization and seedling growth of C. trochiformis among inoculated treatments. We found a positive relation between root colonization and total biomass. This study shows that the AMF communities in bracken areas and forests present similar characteristics and that the bracken fern does not limit AMF inoculum potential, favouring seedling growth of Clusia.
Assuntos
Glomeromycota , Micorrizas , Pteridium , Ecossistema , Florestas , Raízes de Plantas , Microbiologia do SoloRESUMO
The effects of phosphorylation of histone H3 at serine 10 have been studied in the context of other posttranslational modifications such as lysine methylation. We set out to investigate the impact of phosphoserine-10 on arginine-8 methylation. We performed methylation reactions using peptides based on histone H3 that contain a phosphorylated serine and compared the extent of arginine methylation with unmodified peptides. Results obtained via fluorography indicate that peptides containing a phosphorylated serine-10 inhibit deposition of methyl groups to arginine-8 residues. To further explore the effects of phosphoserine on neighboring arginine residues, we physically characterized the non-covalent interactions between histone H3 phosphoserine-10 and arginine-8 using 31P NMR spectroscopy. A salt bridge was detected between the negatively charged phosphoserine-10 and the positively charged unmodified arginine-8 residue. This salt bridge was not detected when arginine-8 was symmetrically dimethylated. Finally, molecular simulations not only confirm the presence of a salt bridge but also identify a subset of electrostatic interactions present when arginine is replaced with alanine. Taken together, our work suggests that the negatively charged phosphoserine maximizes its interactions. By limiting its exposure and creating new contacts with neighboring residues, it will inhibit deposition of neighboring methyl groups, not through steric hindrance, but by forming intrapeptide interactions that may mask substrate recognition. Our work provides a mechanistic framework for understanding the role of phosphoserine on nearby amino acid residues and arginine methylation.
Assuntos
Arginina/metabolismo , Histonas/metabolismo , Fosfosserina/metabolismo , Processamento de Proteína Pós-Traducional , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Arginina/química , Histonas/química , Humanos , Metilação , Simulação de Dinâmica Molecular , Fosfosserina/química , Eletricidade Estática , Xenopus laevisRESUMO
Dopamine is likely the most studied modulatory neurotransmitter, in great part due to characteristic motor deficits in Parkinson's disease that arise after the degeneration of the dopaminergic neurons in the substantia nigra pars compacta (SNc). The SNc, together with the ventral tegmental area (VTA), play a key role modulating motor responses through the basal ganglia. In contrast to the large amount of existing literature addressing the mammalian dopaminergic system, comparatively little is known in other vertebrate groups. However, in the last several years, numerous studies have been carried out in basal vertebrates, allowing a better understanding of the evolution of the dopaminergic system, especially the SNc/VTA. We provide an overview of existing research in basal vertebrates, mainly focusing on lampreys, belonging to the oldest group of extant vertebrates. The lamprey dopaminergic system and its role in modulating motor responses have been characterized in significant detail, both anatomically and functionally, providing the basis for understanding the evolution of the SNc/VTA in vertebrates. When considered alongside results from other early vertebrates, data in lampreys show that the key role of the SNc/VTA dopaminergic neurons modulating motor responses through the basal ganglia was already well developed early in vertebrate evolution.
Assuntos
Dopamina/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Atividade Motora/efeitos dos fármacos , Vias Neurais/efeitos dos fármacos , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , Animais , Cardiotônicos/farmacologiaRESUMO
Tannins are polyphenolic compounds naturally found in vegetables. Their presence in nature has prompted their historical use in many different ways. The revision of their traditional utilization has allowed their further modification aiming for an industrial application. Sometimes these modifications have implied the addition of harmful substances such as formaldehyde, classified as a carcinogen of category B1. In other cases, these natural tannins have been replaced by synthetic compounds that threaten human and animal health and damage the environment. Therefore, currently, both academy and industry are searching for the substitution of these unsafe complexes by the increasing inclusion of tannins, natural molecules that can be obtained from several and diverse renewable resources, modified using harmless additives. To achieve promising results, cost-efficient and eco-friendly extraction methods have been designed. Once these green alternatives have been isolated, they have been successfully applied to many fields with very assorted aims of utilization such as coagulants, adhesives, floatation agents, tannings, dyes, additives, or biomolecules. Therefore, this review offers a global vision of the full process that involves the tannin's technological application including an overview of the most relevant tannin sources, effective extraction methods, and their utilization in very diverse fields.
Assuntos
Extratos Vegetais/farmacologia , Taninos/farmacologia , Adesivos/farmacologia , Animais , Anticoagulantes/farmacologia , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Antivirais/farmacologia , HumanosRESUMO
BACKGROUND: Ectopic pregnancy is a life-threatening condition for which novel screening tools that would enable early accurate diagnosis would improve clinical outcomes. Kisspeptins, encoded by KISS1, play an essential role in human reproduction, at least partially by regulating placental function and possibly embryo implantation. Kisspeptin levels are elevated massively in normal pregnancy and reportedly altered in various gestational pathologic diseases. Yet, the pathophysiologic role of KISS1/kisspeptin in ectopic pregnancy has not been investigated previously. OBJECTIVE: The purpose of this study was to evaluate changes of KISS1/kisspeptin levels in ectopic pregnancy and their underlaying molecular mechanisms and to ascertain the diagnostic implications of these changes. STUDY DESIGN: A total of 122 women with normal pregnancy who underwent voluntary termination of pregnancy and 84 patients who experienced tubal ectopic pregnancy were recruited. Measurements of plasma kisspeptins and KISS1 expression analyses in human embryonic/placental tissue were conducted in ectopic pregnancy and voluntary termination of pregnancy control subjects during the early gestational window (<12 weeks). Putative microRNA regulators of KISS1 were predicted in silico, followed by expression analyses of selected microRNAs and validation of repressive interactions in vitro. Circulating levels of these microRNAs were also assayed in ectopic pregnancy vs voluntary termination of pregnancy. RESULTS: Circulating kisspeptins gradually increased during the first trimester of normal pregnancy but were reduced markedly in ectopic pregnancy. This profile correlated with the expression levels of KISS1 in human embryonic/placental tissue, which increased in voluntary termination of pregnancy but remained suppressed in ectopic pregnancy. Bioinformatic predictions and expression analyses identified miR-27b-3p and miR-324-3p as putative repressors of KISS1 in human embryonic/placental tissue at <12 weeks gestation, when expression of microRNAs was low in voluntary termination of pregnancy control subjects but significantly increased in ectopic pregnancy. Yet, a significant repressive interaction was documented only for miR-324-3p, occurring at the predicted 3'-UTR of KISS1. Interestingly, circulating levels of miR-324-3p, but not of miR-27b-3p, were suppressed distinctly in ectopic pregnancy, despite elevated tissue expression of the pre-microRNA. A decision-tree model that used kisspeptin and miR-324-3p levels was successful in discriminating ectopic pregnancy vs voluntary termination of pregnancy, with a receiver-operating characteristic area under the curve of 0.95±0.02 (95% confidence interval). CONCLUSION: Our results document a significant down-regulation of KISS1/kisspeptins in early stages of ectopic pregnancy via, at least partially, a repressive interaction with miR-324-3p. Our data identify circulating kisspeptins and miR-324-3p as putative biomarkers for accurate screening of ectopic pregnancy at early gestational ages.
Assuntos
Embrião de Mamíferos/metabolismo , Kisspeptinas/metabolismo , MicroRNAs/metabolismo , Placenta/metabolismo , Gravidez Ectópica/diagnóstico , Biomarcadores/metabolismo , Estudos de Casos e Controles , Árvores de Decisões , Regulação para Baixo , Diagnóstico Precoce , Feminino , Idade Gestacional , Humanos , Kisspeptinas/genética , Gravidez , Gravidez Ectópica/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
In this work we describe not previously explored binding studies on the reversible interaction of benzoxaborole with ligands of medical and pharmaceutical interest such as nucleosidic drugs gemcitabine and capecitabine, as well as the hydrophobic chemotherapeutic doxorubicin. We include functional derivatives of benzoxaborole such as a near infrared fluorescent boronolectine, Cy-Bx, The dynamic covalent interaction in physiological conditions was assessed by spectroscopic techniques yielding moderate to high binding affinities. The cytotoxic activity of the drugs upon conjugation to the boronolectins was evaluated revealing significant influence of the bioconjugation status on the cellular viability. The availability of the conjugate for cellular uptake and localization in the model cancer cell line HeLa was assessed by fluorescence imaging. Benzoxaborole and the fluorescent boronolectin Cy-Bx, proved to be versatile conjugation tools for 1,2 and 1,3-diol containing pharmacophores as well as bioisosteric forms such as 1,2-hydroxyamino, envisioning these small boronolectins as components in systems for drug release with tracking capability.
Assuntos
Antineoplásicos/farmacologia , Benzamidas/química , Compostos de Boro/farmacologia , Doxorrubicina/farmacologia , Nucleosídeos/química , Nucleosídeos/metabolismo , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos/química , Compostos de Boro/química , Proliferação de Células , Doxorrubicina/química , Liberação Controlada de Fármacos , Corantes Fluorescentes/química , Células HeLa , Humanos , Monossacarídeos/químicaRESUMO
Persea americana (avocado) is a fruit consumed worldwide; however, since avocado leaves are apparently a natural ingredient that can be used as a traditional medicine, they can be a potential source of bioactive compounds. This study aimed to analyze the antioxidant activity of seven Mexican avocado leaf extracts by DPPHâ¢, ABTSâ¢+, and lipid peroxidation (LPO), and to identify the compound profile by liquid chromatography coupled to mass spectrometry/electron spray ionization. The highest free radical-scavenging activity was observed for Platano Delgado and Criollo 6 avocado cultivars havin IC50 values of 271.86 ± 13.69 and 269.56 ± 6.53 for DPPH⢠and ABTSâ¢+ radicals, respectively, while the best result for lipid oxidation inhibition was registered in Criollo 6 cultivar extract. In this study forty-one compounds were detected in avocado leaves of the the seven cultivars analyzed, and of these compounds, eighteen phenolics were identified for first time in such plant material. The present study demonstrated that Mexican cultivars of Persea americana possess diverse polyphenolic compounds with strong antioxidant activity, which might be useful in the food and pharmaceutical industries.
Assuntos
Antioxidantes/química , Persea/química , Extratos Vegetais/química , Polifenóis/química , Benzotiazóis/química , Compostos de Bifenilo/química , Sequestradores de Radicais Livres/química , Peroxidação de Lipídeos/efeitos dos fármacos , Picratos/química , Folhas de Planta/química , Ácidos Sulfônicos/químicaRESUMO
A novel fluorescent molecular probe is reported, which is able to detect glycoproteins, especially mucins, with high sensitivity and with a turn-on response along with a large Stokes shift (>130â nm), within the biologically active window. The probe contains an aminotricarbocyanine as the fluorescent reporter with a linked benzoboroxole as the recognition unit, which operates through a dynamic covalent reaction between the boronic hemiester residue of the receptor and cis-diols of the analyte. The superior selectivity of the probe is displayed by the labeling of mucins present in Calu-3 cells. The new benzoboroxole fluorescent derivative gathers together key properties to make it a highly rated molecular probe: specificity, excellent solubility in water, and off-on near infrared emission. This probe is expected to be an excellent tool for imaging intracellular mucin to evaluate mucus-related diseases as well as a sensing strategy towards glycosylated structures with a high potential for theranostics approaches in biological samples.
Assuntos
Corantes Fluorescentes/química , Glicoproteínas/análise , Mucinas/análise , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Compostos de Boro/química , Células Epiteliais/efeitos dos fármacos , Humanos , Estrutura MolecularRESUMO
The total phenolic compounds content, free radical-scavenging capacity and capsaicinoid content in populations of wild Piquin chili (C. annuum) were studied. Aqueous and hydroalcoholic extracts from nine ecotypes were evaluated. High contents of phenolic compounds and free radical-scavenging capacities were observed for both extracts; however, the values that were found for the hydroalcoholic phase were substantially higher. LC-MS analysis allowed for the detection of 32 compounds, where apigenin-8-C-glucoside followed by vanillic acid 1-O-ß-o-glucopyranosylester (Isomer I or II) and 7-ethoxy-4-methylcoumarin were the most widely distributed; they were found in more than 89% of the ecotypes. The diversity of identified phenolic compounds was different among ecotypes, allowing them to be distinguished by chemical diversity, free radical-scavenging capacities and heat Scoville units. The total capsaicinoid content was higher in Population I (23.5 mg/g DW) than in Populations II and III, which had contents of 15.3 and 10.7 mg/g DW, respectively. This variability could lead to phytochemical exploitation and the conservation of the natural populations of wild chili.
Assuntos
Capsicum/química , Sequestradores de Radicais Livres/isolamento & purificação , Fenóis/isolamento & purificação , Capsaicina , Cromatografia Líquida , Sequestradores de Radicais Livres/química , Sequestradores de Radicais Livres/farmacologia , Espectrometria de Massas , Estrutura Molecular , Fenóis/química , Fenóis/farmacologia , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologiaRESUMO
A cornerstone of modern biomedical research is the use of mouse models to explore basic pathophysiological mechanisms, evaluate new therapeutic approaches, and make go or no-go decisions to carry new drug candidates forward into clinical trials. Systematic studies evaluating how well murine models mimic human inflammatory diseases are nonexistent. Here, we show that, although acute inflammatory stresses from different etiologies result in highly similar genomic responses in humans, the responses in corresponding mouse models correlate poorly with the human conditions and also, one another. Among genes changed significantly in humans, the murine orthologs are close to random in matching their human counterparts (e.g., R(2) between 0.0 and 0.1). In addition to improvements in the current animal model systems, our study supports higher priority for translational medical research to focus on the more complex human conditions rather than relying on mouse models to study human inflammatory diseases.
Assuntos
Genômica , Inflamação/genética , Doença Aguda , Adolescente , Adulto , Animais , Queimaduras/genética , Queimaduras/patologia , Modelos Animais de Doenças , Endotoxemia/genética , Endotoxemia/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/genética , Fatores de Tempo , Ferimentos e Lesões/genética , Ferimentos e Lesões/patologia , Adulto JovemRESUMO
INTRODUCTION: We wished to characterize the relationship of advanced age to clinical outcomes and to transcriptomic responses after severe blunt traumatic injury with hemorrhagic shock. METHODS: We performed epidemiological, cytokine, and transcriptomic analyses on a prospective, multi-center cohort of 1,928 severely injured patients. RESULTS: We found that there was no difference in injury severity between the aged (age ≥55, n = 533) and young (age <55, n = 1395) cohorts. However, aged patients had more comorbidities. Advanced age was associated with more severe organ failure, infectious complications, ventilator days, and intensive care unit length of stay, as well as, an increased likelihood of being discharged to skilled nursing or long-term care facilities. Additionally, advanced age was an independent predictor of a complicated recovery and 28-day mortality. Acutely after trauma, blood neutrophil genome-wide expression analysis revealed an attenuated transcriptomic response as compared to the young; this attenuated response was supported by the patients' plasma cytokine and chemokine concentrations. Later, these patients demonstrated gene expression changes consistent with simultaneous, persistent pro-inflammatory and immunosuppressive states. CONCLUSIONS: We concluded that advanced age is one of the strongest non-injury related risk factors for poor outcomes after severe trauma with hemorrhagic shock and is associated with an altered and unique peripheral leukocyte genomic response. As the general population's age increases, it will be important to individualize prediction models and therapeutic targets to this high risk cohort.
Assuntos
Perfilação da Expressão Gênica , Avaliação de Resultados da Assistência ao Paciente , Choque Hemorrágico/epidemiologia , Ferimentos não Penetrantes/epidemiologia , Adulto , Fatores Etários , Idoso , Estudos de Casos e Controles , Quimiocinas/sangue , Estudos de Coortes , Comorbidade , Citocinas/sangue , Florida/epidemiologia , Genoma Humano , Humanos , Infecções/epidemiologia , Unidades de Terapia Intensiva , Tempo de Internação/estatística & dados numéricos , Assistência de Longa Duração , Pessoa de Meia-Idade , Insuficiência de Múltiplos Órgãos/epidemiologia , Análise Multivariada , Neutrófilos/metabolismo , Alta do Paciente , Respiração Artificial/estatística & dados numéricos , Instituições de Cuidados Especializados de EnfermagemRESUMO
The mammalian protein arginine methyltransferase 7 (PRMT7) has been implicated in roles of transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation, and metastasis. However, the type of reaction that it catalyzes and its substrate specificity remain controversial. In this study, we purified a recombinant mouse PRMT7 expressed in insect cells that demonstrates a robust methyltransferase activity. Using a variety of substrates, we demonstrate that the enzyme only catalyzes the formation of ω-monomethylarginine residues, and we confirm its activity as the prototype type III protein arginine methyltransferase. This enzyme is active on all recombinant human core histones, but histone H2B is a highly preferred substrate. Analysis of the specific methylation sites within intact histone H2B and within H2B and H4 peptides revealed novel post-translational modification sites and a unique specificity of PRMT7 for methylating arginine residues in lysine- and arginine-rich regions. We demonstrate that a prominent substrate recognition motif consists of a pair of arginine residues separated by one residue (RXR motif). These findings will significantly accelerate substrate profile analysis, biological function study, and inhibitor discovery for PRMT7.
Assuntos
Histonas/química , Processamento de Proteína Pós-Traducional/fisiologia , Proteína-Arginina N-Metiltransferases/química , Motivos de Aminoácidos , Animais , Arginina/química , Arginina/genética , Arginina/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Lisina/química , Lisina/genética , Lisina/metabolismo , Metilação , Camundongos , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Especificidade por SubstratoRESUMO
OBJECTIVES: Determining the prevalence of children in day-care centres (DCCs) carrying faecal extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae and molecularly characterizing those belonging to the Escherichia coli species. METHODS: Stools were collected from children's diapers (January-April 2012) in randomly chosen DCCs and plated onto ChromID ESBL. Colonies growing on this medium were identified by the Vitek 2 system and tested for antibiotic susceptibility and for ESBL production by the double-disc synergy test. ESBL genotypes were determined as well as phylogenetic groups, ERIC-2 (enterobacterial repetitive intergenic consensus) PCR profiles and sequence types (STs) for the E. coli isolates. Serotypes, virotypes, fimH alleles, ESBL-carrying plasmids and PFGE patterns were determined for the ST131 E. coli isolates. RESULTS: Among 419 children from 25 participating DCCs, 1 was colonized by CTX-M-15-producing Klebsiella pneumoniae and 27 (6.4%) by E. coli, which all produced CTX-M enzymes [CTX-M-15 (37%), CTX-M-1 (26%), CTX-M-14 (22%), CTX-M-27 (11%) and CTX-M-22 (4%)]. The 27 E. coli isolates, 55.5% belonging to group B2, displayed 20 ERIC-2 PCR profiles and 16 STs. The ST131 E. coli isolates were dominant (44%), displayed serotypes O25b:H4 and O16:H5, fimH alleles 30 and 41 and virotypes A and C. According to the PFGE patterns, one strain of E. coli ST131 producing a CTX-M-15 enzyme carried by an IncF F2:A1:B- plasmid had spread within one DCC. CONCLUSIONS: This study shows a notable prevalence (6.4%) of DCC children with faecal CTX-M-producing E. coli isolates comprising a high proportion of E. coli ST131 isolates, suggesting that these children might be a reservoir of this clone.
Assuntos
Creches , Infecções por Escherichia coli/microbiologia , Escherichia coli/classificação , Escherichia coli/isolamento & purificação , Fezes/microbiologia , beta-Lactamases/metabolismo , Pré-Escolar , Análise por Conglomerados , Estudos Transversais , Escherichia coli/enzimologia , Feminino , França/epidemiologia , Genótipo , Humanos , Lactente , Masculino , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Tipagem Molecular , Filogenia , Prevalência , Sorotipagem , beta-Lactamases/genéticaRESUMO
The present study was carried out to evaluate the prevalence of the clonal subgroup O16:H5-ST131 and the H30 and H30-Rx subclones among E. coli isolates causing extraintestinal infections and to know their virulence potential. The ST131 clonal group accounted for 490 (16%) of the 2995 isolates obtained from clinical samples in five Spanish hospitals during the study period (2005-2012). Among those 490 ST131 isolates, 456 belonged to serotype O25b:H4, 27 to O16:H5 and seven were O-non-typeable:H4 (ONT:H4). All 27 O16:H5 isolates showed fimH41, whereas fimH30 and fimH22 alleles were the most frequently detected among O25b:H4 isolates. The majority (381/490; 78%) of ST131 isolates belonged to H30 subclone, and 302 of 381 (79%) H30 isolates belonged to the H30-Rx subclone. Of the 27 O16:H5 isolates, 48% produced CTX-M-14; however, none produced CTX-M-15. In contrast, 46% of O25b:H4 isolates produced CTX-M-15 while only 2% produced CTX-M-14. More than a half of the O16:H5 isolates (56%) showed the ExPEC status which was significantly more prevalent within O25b:H4 isolates (81%) (P<0.01), especially among H30-Rx (97%) isolates. In the present study, a modified virotype scheme was applied within which approximately half (52%) of the O16:H5 isolates showed the C1 specific virotype. Despite their low virulence-gene score (mean of virulence genes 6.4 versus 8.5 in O25b:H4 isolates), six out of the 10 O16:H5 isolates assayed showed high virulence in the mouse model of sepsis (killed 90-100% of mice challenged). Furthermore, four O16:H5 isolates of virotypes A and C1, carrying K2 variant of group II capsule, showed lethality at 24h. Thus, certain O16:H5 fimH41 isolates show a similar in vivo virulence to that reported with the highly virulent O25b:H4 H30-Rx isolates (Mora et al., PLOS ONE 2014, e87025), supporting their potential virulence for humans.
Assuntos
Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Escherichia coli/classificação , Escherichia coli/genética , Tipagem Molecular , Sorogrupo , Fatores de Virulência/genética , Animais , Modelos Animais de Doenças , Escherichia coli/isolamento & purificação , Feminino , Genótipo , Humanos , Camundongos , Epidemiologia Molecular , Sepse/microbiologia , Espanha/epidemiologia , Análise de Sobrevida , VirulênciaRESUMO
Targeting drugs to the central nervous system (CNS) is challenging due to the presence of the blood-brain barrier (BBB). The cutting edge in nanotechnology generates optimism to overcome the growing challenges in biomedical sciences through the effective engineering of nanogels. The primary objective of the present report was to develop and characterize a biocompatible natural chitosan (CS)-based NG that can be tracked thanks to the tricarbocyanine (CNN) fluorescent probe addition on the biopolymer backbone. FTIR shed light on the chemical groups involved in the CS and CNN interactions and between CNN-CS and tripolyphosphate, the cross-linking agent. Both in vitro and in vivo experiments were carried out to determine if CS-NGs can be utilized as therapeutic delivery vehicles directed towards the brain. An ionic gelation method was chosen to generate cationic CNN-CS-NG. DLS and TEM confirmed that these entities' sizes fell into the nanoscale. CNN-CS-NG was found to be non-cytotoxic, as determined in the SH-SY5Y neuroblastoma cell line through biocompatibility assays. After cellular internalization, the occurrence of an endo-lysosomal escape (a crucial event for an efficient drug delivery) of CNN-CS-NG was detected. Furthermore, CNN-CS-NG administered intraperitoneally to female CF-1 mice were detected in different brain regions after 2 h of administration, using fluorescence microscopy. To conclude, the obtained findings in the present report can be useful in the field of neuro-nanomedicine when designing drug vehicles with the purpose of delivering drugs to the CNS.
RESUMO
The pupillary light reflex (PLR) adapts the amount of light reaching the retina, protecting it and improving image formation. Two PLR mechanisms have been described in vertebrates. First, the pretectum receives retinal inputs and projects to the Edinger-Westphal nucleus (EWN), which targets the ciliary ganglion through the oculomotor nerve (nIII). Postganglionic fibers enter the eye-globe, traveling to the iris sphincter muscle. Additionally, some vertebrates exhibit an iris-intrinsic PLR mechanism mediated by sphincter muscle cells that express melanopsin inducing muscle contraction. Given the high degree of conservation of the lamprey visual system, we investigated the mechanisms underlying the PLR to shed light onto their evolutionary origins. Recently, a PLR mediated by melanopsin was demonstrated in lampreys, suggested to be brain mediated. Remarkably, we found that PLR is instead mediated by direct retino-iridal cholinergic projections. This retina-mediated PLR acts synergistically with an iris-intrinsic mechanism that, as in other vertebrates, is mediated by melanopsin and has contribution of gap junctions between muscle fibers. In contrast, we show that lampreys lack the brain-mediated PLR. Our results suggest that two eye-intrinsic PLR mechanisms were present in early vertebrate evolution, whereas the brain-mediated PLR has a more recent origin.