RESUMEN
G protein-coupled receptors (GPCRs) relay extracellular stimuli into specific cellular functions. Cells express many different GPCRs, but all these GPCRs signal to only a few second messengers such as cAMP. It is largely unknown how cells distinguish between signals triggered by different GPCRs to orchestrate their complex functions. Here, we demonstrate that individual GPCRs signal via receptor-associated independent cAMP nanodomains (RAINs) that constitute self-sufficient, independent cell signaling units. Low concentrations of glucagon-like peptide 1 (GLP-1) and isoproterenol exclusively generate highly localized cAMP pools around GLP-1- and ß2-adrenergic receptors, respectively, which are protected from cAMP originating from other receptors and cell compartments. Mapping local cAMP concentrations with engineered GPCR nanorulers reveals gradients over only tens of nanometers that define the size of individual RAINs. The coexistence of many such RAINs allows a single cell to operate thousands of independent cellular signals simultaneously, rather than function as a simple "on/off" switch.
Asunto(s)
Receptores Acoplados a Proteínas G , Transducción de Señal , Fenómenos Fisiológicos Celulares , AMP Cíclico , Péptido 1 Similar al Glucagón , Receptores Adrenérgicos beta 2 , Receptores Acoplados a Proteínas G/química , Sistemas de Mensajero SecundarioRESUMEN
Cells relay a plethora of extracellular signals to specific cellular responses by using only a few second messengers, such as cAMP. To explain signaling specificity, cAMP-degrading phosphodiesterases (PDEs) have been suggested to confine cAMP to distinct cellular compartments. However, measured rates of fast cAMP diffusion and slow PDE activity render cAMP compartmentalization essentially impossible. Using fluorescence spectroscopy, we show that, contrary to earlier data, cAMP at physiological concentrations is predominantly bound to cAMP binding sites and, thus, immobile. Binding and unbinding results in largely reduced cAMP dynamics, which we term "buffered diffusion." With a large fraction of cAMP being buffered, PDEs can create nanometer-size domains of low cAMP concentrations. Using FRET-cAMP nanorulers, we directly map cAMP gradients at the nanoscale around PDE molecules and the areas of resulting downstream activation of cAMP-dependent protein kinase (PKA). Our study reveals that spatiotemporal cAMP signaling is under precise control of nanometer-size domains shaped by PDEs that gate activation of downstream effectors.
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Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Transducción de Señal , Análisis de la Célula Individual/métodos , Simulación por Computador , AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/química , Citoplasma/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Modelos Moleculares , Hidrolasas Diéster Fosfóricas/química , Unión Proteica , Dominios Proteicos , Proteínas Recombinantes , Análisis Espacio-Temporal , Espectrometría de FluorescenciaRESUMEN
Louis Pasteur once famously said 'in the fields of observation chance favors only the prepared mind'. Much of chance is being in the right place at the right time. This is particularly true in the crowded molecular environment of the cell where being in the right place is often more important than timing. Although Brownian motion argues that enzymes will eventually bump into substrates, this probability is greatly enhanced if both molecules reside in the same subcellular compartment. However, activation of cell signaling enzymes often requires the transmission of chemical signals from extracellular stimuli to intracellular sites of action. This review highlights new developments in our understanding of cAMP generation and the 3D utilization of this second messenger inside cells.
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AMP Cíclico , Transducción de Señal , Transducción de Señal/fisiologíaRESUMEN
G protein-coupled receptors are the largest and pharmacologically most important receptor family and are involved in the regulation of most cell functions. Most of them reside exclusively at the cell surface, from where they signal via heterotrimeric G proteins to control the production of second messengers such as cAMP and IP3 as well as the activity of several ion channels. However, they may also internalize upon agonist stimulation or constitutively reside in various intracellular locations. Recent evidence indicates that their function differs depending on their precise cellular localization. This is because the signals they produce, notably cAMP and Ca2+, are mostly bound to cell proteins that significantly reduce their mobility, allowing the generation of steep concentration gradients. As a result, signals generated by the receptors remain confined to nanometer-sized domains. We propose that such nanometer-sized domains represent the basic signaling units in a cell and a new type of target for drug development.
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Desarrollo de Medicamentos , Transducción de Señal , Humanos , Membrana CelularRESUMEN
BACKGROUND: Numerous children present with early wheeze symptoms, yet solely a subgroup develops childhood asthma. Early identification of children at risk is key for clinical monitoring, timely patient-tailored treatment, and preventing chronic, severe sequelae. For early prediction of childhood asthma, we aimed to define an integrated risk score combining established risk factors with genome-wide molecular markers at birth, complemented by subsequent clinical symptoms/diagnoses (wheezing, atopic dermatitis, food allergy). METHODS: Three longitudinal birth cohorts (PAULINA/PAULCHEN, n = 190 + 93 = 283, PASTURE, n = 1133) were used to predict childhood asthma (age 5-11) including epidemiological characteristics and molecular markers: genotype, DNA methylation and mRNA expression (RNASeq/NanoString). Apparent (ap) and optimism-corrected (oc) performance (AUC/R2) was assessed leveraging evidence from independent studies (Naïve-Bayes approach) combined with high-dimensional logistic regression models (LASSO). RESULTS: Asthma prediction with epidemiological characteristics at birth (maternal asthma, sex, farm environment) yielded an ocAUC = 0.65. Inclusion of molecular markers as predictors resulted in an improvement in apparent prediction performance, however, for optimism-corrected performance only a moderate increase was observed (upto ocAUC = 0.68). The greatest discriminate power was reached by adding the first symptoms/diagnosis (up to ocAUC = 0.76; increase of 0.08, p = .002). Longitudinal analysis of selected mRNA expression in PASTURE (cord blood, 1, 4.5, 6 years) showed that expression at age six had the strongest association with asthma and correlation of genes getting larger over time (r = .59, p < .001, 4.5-6 years). CONCLUSION: Applying epidemiological predictors alone showed moderate predictive abilities. Molecular markers from birth modestly improved prediction. Allergic symptoms/diagnoses enhanced the power of prediction, which is important for clinical practice and for the design of future studies with molecular markers.
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Asma , Humanos , Asma/epidemiología , Asma/genética , Asma/diagnóstico , Femenino , Masculino , Niño , Preescolar , Factores de Riesgo , Estudios Longitudinales , Metilación de ADN , Biomarcadores , Cohorte de NacimientoRESUMEN
G-protein-coupled receptors (GPCRs) are key signaling proteins that mostly function as monomers, but for several receptors constitutive dimer formation has been described and in some cases is essential for function. Using single-molecule microscopy combined with super-resolution techniques on intact cells, we describe here a dynamic monomer-dimer equilibrium of µ-opioid receptors (µORs), where dimer formation is driven by specific agonists. The agonist DAMGO, but not morphine, induces dimer formation in a process that correlates both temporally and in its agonist- and phosphorylation-dependence with ß-arrestin2 binding to the receptors. This dimerization is independent from, but may precede, µOR internalization. These data suggest a new level of GPCR regulation that links dimer formation to specific agonists and their downstream signals.
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Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Imagen Individual de Molécula/métodos , Animales , Células CHO , Cricetulus , Encefalina Ala(2)-MeFe(4)-Gli(5)/química , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Transferencia Resonante de Energía de Fluorescencia , Morfina/química , Morfina/farmacología , Mutación , Naloxona/química , Naloxona/farmacología , Naltrexona/análogos & derivados , Naltrexona/química , Naltrexona/farmacología , Antagonistas de Narcóticos/química , Antagonistas de Narcóticos/farmacología , Fosforilación , Multimerización de Proteína , Receptores Opioides mu/antagonistas & inhibidores , Receptores Opioides mu/genética , beta-Arrestinas/metabolismoRESUMEN
BACKGROUND: Childhood wheeze represents a first symptom of asthma. Early identification of children at risk for wheeze related to 17q12-21 variants and their underlying immunological mechanisms remain unknown. We aimed to assess the influence of 17q12-21 variants and mRNA expression at birth on the development of wheeze. METHODS: Children were classified as multitrigger/viral/no wheeze until six years of age. The PAULINA/PAULCHEN birth cohorts were genotyped (n = 216; GSA-chip). mRNA expression of 17q21 and innate/adaptive genes was measured (qRT-PCR) in cord blood mononuclear cells. Expression quantitative trait loci (eQTL) and mediation analyses were performed. Genetic variation of 17q12-21 asthma-single nucleotide polymorphisms (SNPs) was summarized as the first principal component (PC1) and used to classify single SNP effects on gene expression as (locus)-dependent/independent eQTL SNPs. RESULTS: Core region risk variants (IKZF3, ZPBP2, GSDMB, ORMDL3) were associated with multitrigger wheeze (OR: 3.05-5.43) and were locus-dependent eQTL SNPs with higher GSDMA, TLR2, TLR5, and lower TGFB1 expression. Increased risk of multitrigger wheeze with rs9303277 was in part mediated by TLR2 expression. Risk variants distal to the core region were mainly locus-independent eQTL SNPs with decreased CD209, CD86, TRAF6, RORA, and IL-9 expression. Distinct immune signatures in cord blood were associated either with multitrigger wheeze (increased innate genes, e.g., TLR2, IPS1, LY75) or viral wheeze (decreased NF-κB genes, e.g., TNFAIP3 and TNIP2). CONCLUSION: Locus-dependent eQTL SNPs (core region) associated with increased inflammatory genes (primarily TLR2) at birth and subsequent multitrigger wheeze indicate that early priming and imbalance may be crucial for asthma pathophysiology. Locus-independent eQTL SNPs (mainly distal region, rs1007654) may be involved in the initiation of dendritic cell activation/maturation (TRAF6) and interaction with T cells (CD209, CD86). Identifying potential mechanistic pathways at birth may point to critical key points during early immune development predisposing to asthma.
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Asma , Sangre Fetal , Proteínas Adaptadoras Transductoras de Señales/genética , Asma/epidemiología , Asma/genética , Niño , Cromosomas Humanos Par 17 , Proteínas del Huevo , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Recién Nacido , Proteínas de la Membrana/genética , Proteínas de Neoplasias/genética , Polimorfismo de Nucleótido Simple , Proteínas Citotóxicas Formadoras de Poros , Ruidos Respiratorios/genéticaRESUMEN
BACKGROUND: While childhood asthma prevalence is rising in Westernized countries, farm children are protected. The mitogen-activated protein kinase (MAPK) pathway with its negative regulator dual-specificity phosphatase-1 (DUSP1) is presumably associated with asthma development. OBJECTIVES: We aimed to investigate the role of MAPK signaling in childhood asthma and its environment-mediated protection, including a representative selection of 232 out of 1062 children from two cross-sectional cohorts and one birth cohort study. METHODS: Peripheral blood mononuclear cells (PBMC) from asthmatic and healthy children were cultured upon stimulation with farm-dust extracts or lipopolysaccharide. In subgroups, gene expression was analyzed by qPCR (PBMCs, cord blood) and NanoString technology (dendritic cells). Protein expression of phosphorylated MAPKs was measured by mass cytometry. Histone acetylation was investigated by chromatin immunoprecipitation. RESULTS: Asthmatic children expressed significantly less DUSP1 (p = .006) with reduced acetylation at histone H4 (p = .012) compared with healthy controls. Farm-dust stimulation upregulated DUSP1 expression reaching healthy levels and downregulated inflammatory MAPKs on gene and protein levels (PBMCs; p ≤ .01). Single-cell protein analysis revealed downregulated pMAPKs upon farm-dust stimulation in B cells, NK cells, monocytes, and T-cell subpopulations. CONCLUSION: Lower DUSP1 baseline levels in asthmatic children and anti-inflammatory regulation of MAPK in several immune cell types by farm-dust stimulation indicate a regulatory function for DUSP1 for future therapy contributing to anti-inflammatory characteristics of farming environments.
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Asma , Leucocitos Mononucleares , Asma/epidemiología , Asma/genética , Niño , Estudios de Cohortes , Estudios Transversales , Humanos , Proteínas Quinasas Activadas por MitógenosRESUMEN
Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.
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Anticuerpos Monoclonales/química , Productos Biológicos , Biofarmacia/métodos , Anticuerpos Monoclonales/metabolismo , Glicómica/métodos , Glicopéptidos/metabolismo , Glicosilación , Humanos , Laboratorios , Polisacáridos/metabolismo , Procesamiento Proteico-Postraduccional , Proteómica/métodosRESUMEN
G protein-coupled receptors (GPCRs) are key biological switches that transmit both internal and external stimuli into the cell interior. Among the GPCRs, the "light receptor" rhodopsin has been shown to activate with a rearrangement of the transmembrane (TM) helix bundle within â¼1 ms, while all other receptors are thought to become activated within â¼50 ms to seconds at saturating concentrations. Here, we investigate synchronous stimulation of a dimeric GPCR, the metabotropic glutamate receptor type 1 (mGluR1), by two entirely different methods: (i) UV light-triggered uncaging of glutamate in intact cells or (ii) piezo-driven solution exchange in outside-out patches. Submillisecond FRET recordings between labels at intracellular receptor sites were used to record conformational changes in the mGluR1. At millimolar ligand concentrations, the initial rearrangement between the mGluR1 subunits occurs at a speed of τ1 â¼ 1-2 ms and requires the occupancy of both binding sites in the mGluR1 dimer. These rapid changes were followed by significantly slower conformational changes in the TM domain (τ2 â¼ 20 ms). Receptor deactivation occurred with time constants of â¼40 and â¼900 ms for the inter- and intrasubunit conformational changes, respectively. Together, these data show that, at high glutamate concentrations, the initial intersubunit activation of mGluR1 proceeds with millisecond speed, that there is loose coupling between this initial step and activation of the TM domain, and that activation and deactivation follow a cyclic pathway, including-in addition to the inactive and active states-at least two metastable intermediate states.
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Receptores Acoplados a Proteínas G/metabolismo , Dimerización , Células HEK293 , Humanos , Cinética , Receptores Acoplados a Proteínas G/efectos de la radiaciónRESUMEN
Several features common to a Western lifestyle, including obesity and low levels of physical activity, are known risk factors for gastrointestinal cancers. There is substantial evidence suggesting that diet markedly affects the composition of the intestinal microbiota. Moreover, there is now unequivocal evidence linking dysbiosis to cancer development. However, the mechanisms by which high-fat diet (HFD)-mediated changes in the microbial community affect the severity of tumorigenesis in the gut remain to be determined. Here we demonstrate that an HFD promotes tumour progression in the small intestine of genetically susceptible, K-ras(G12Dint), mice independently of obesity. HFD consumption, in conjunction with K-ras mutation, mediated a shift in the composition of the gut microbiota, and this shift was associated with a decrease in Paneth-cell-mediated antimicrobial host defence that compromised dendritic cell recruitment and MHC class II molecule presentation in the gut-associated lymphoid tissues. When butyrate was administered to HFD-fed K-ras(G12Dint) mice, dendritic cell recruitment in the gut-associated lymphoid tissues was normalized, and tumour progression was attenuated. Importantly, deficiency in MYD88, a signalling adaptor for pattern recognition receptors and Toll-like receptors, blocked tumour progression. The transfer of faecal samples from HFD-fed mice with intestinal tumours to healthy adult K-ras(G12Dint) mice was sufficient to transmit disease in the absence of an HFD. Furthermore, treatment with antibiotics completely blocked HFD-induced tumour progression, suggesting that distinct shifts in the microbiota have a pivotal role in aggravating disease. Collectively, these data underscore the importance of the reciprocal interaction between host and environmental factors in selecting a microbiota that favours carcinogenesis, and they suggest that tumorigenesis is transmissible among genetically predisposed individuals.
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Carcinogénesis/efectos de los fármacos , Dieta Alta en Grasa/efectos adversos , Grasas de la Dieta/efectos adversos , Disbiosis/inducido químicamente , Disbiosis/microbiología , Neoplasias Intestinales/microbiología , Obesidad , Animales , Antibacterianos/farmacología , Butiratos/farmacología , Progresión de la Enfermedad , Mucosa Intestinal/inmunología , Neoplasias Intestinales/inducido químicamente , Intestinos/efectos de los fármacos , Intestinos/microbiología , Ratones , Obesidad/inducido químicamente , Obesidad/microbiología , PrebióticosRESUMEN
BACKGROUND: Childhood asthma prevalence is significantly greater in urban areas compared with rural/farm environments. Murine studies have shown that TNF-α-induced protein 3 (TNFAIP3; A20), an anti-inflammatory regulator of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, mediates environmentally induced asthma protection. OBJECTIVE: We aimed to determine the role of TNFAIP3 for asthma development in childhood and the immunomodulatory effects of environmental factors. METHODS: In a representative selection of 250 of 2168 children from 2 prospective birth cohorts and 2 cross-sectional studies, we analyzed blood cells of healthy and asthmatic children from urban and rural/farm environments from Europe and China. PBMCs were stimulated ex vivo with dust from "asthma-protective" farms or LPS. NF-κB signaling-related gene and protein expression was assessed in PBMCs and multiplex gene expression assays (NanoString Technologies) in isolated dendritic cells of schoolchildren and in cord blood mononuclear cells from newborns. RESULTS: Anti-inflammatory TNFAIP3 gene and protein expression was consistently decreased, whereas proinflammatory Toll-like receptor 4 expression was increased in urban asthmatic patients (P < .05), reflecting their increased inflammatory status. Ex vivo farm dust or LPS stimulation restored TNFAIP3 expression to healthy levels in asthmatic patients and shifted NF-κB signaling-associated gene expression toward an anti-inflammatory state (P < .001). Farm/rural children had lower expression, indicating tolerance induction by continuous environmental exposure. Newborns with asthma at school age had reduced TNFAIP3 expression at birth, suggesting TNFAIP3 as a possible biomarker predicting subsequent asthma. CONCLUSION: Our data indicate TNFAIP3 as a key regulator during childhood asthma development and its environmentally mediated protection. Because environmental dust exposure conferred the anti-inflammatory effects, it might represent a promising future agent for asthma prevention and treatment.
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Asma/sangre , Exposición a Riesgos Ambientales/efectos adversos , Regulación de la Expresión Génica , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/sangre , Asma/inmunología , Asma/patología , Asma/prevención & control , Biomarcadores/sangre , Niño , Preescolar , Estudios Transversales , Femenino , Humanos , Lactante , Recién Nacido , Estudios Longitudinales , Masculino , Estudios Prospectivos , Receptor Toll-Like 4/sangre , Receptor Toll-Like 4/inmunología , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
BACKGROUND: Associations between childhood asthma phenotypes and genetic, immunological, and environmental factors have been previously established. Yet, strategies to integrate high-dimensional risk factors from multiple distinct data sets, and thereby increase the statistical power of analyses, have been hampered by a preponderance of missing data and lack of methods to accommodate them. METHODS: We assembled questionnaire, diagnostic, genotype, microarray, RT-qPCR, flow cytometry, and cytokine data (referred to as data modalities) to use as input factors for a classifier that could distinguish healthy children, mild-to-moderate allergic asthmatics, and nonallergic asthmatics. Based on data from 260 German children aged 4-14 from our university outpatient clinic, we built a novel multilevel prediction approach for asthma outcome which could deal with a present complex missing data structure. RESULTS: The optimal learning method was boosting based on all data sets, achieving an area underneath the receiver operating characteristic curve (AUC) for three classes of phenotypes of 0.81 (95%-confidence interval (CI): 0.65-0.94) using leave-one-out cross-validation. Besides improving the AUC, our integrative multilevel learning approach led to tighter CIs than using smaller complete predictor data sets (AUC = 0.82 [0.66-0.94] for boosting). The most important variables for classifying childhood asthma phenotypes comprised novel identified genes, namely PKN2 (protein kinase N2), PTK2 (protein tyrosine kinase 2), and ALPP (alkaline phosphatase, placental). CONCLUSION: Our combination of several data modalities using a novel strategy improved classification of childhood asthma phenotypes but requires validation in external populations. The generic approach is applicable to other multilevel data-based risk prediction settings, which typically suffer from incomplete data.
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Asma/epidemiología , Asma/etiología , Susceptibilidad a Enfermedades , Exposición a Riesgos Ambientales , Adolescente , Área Bajo la Curva , Biomarcadores , Niño , Preescolar , Citocinas/metabolismo , Susceptibilidad a Enfermedades/inmunología , Predisposición Genética a la Enfermedad , Humanos , Inmunofenotipificación , Curva ROCRESUMEN
BACKGROUND: In Middle Europe ultrasonography is the standard method used to screen for developmental dysplasia of the hip in infants. Our aim was to determine whether manual fixation of the child is equivalent to Graf's technique regarding image quality. METHODS: This randomized trial was conducted at a free-standing general pediatric outpatient clinic in Vienna, Austria. Healthy infants in the 1st and between the 6th and 8th week of life with no hip malalignment were included. After randomization, Group 1 was examined using Graf's fixation device and participants in Group 2 were fixated on the examination couch by their parents. In a second step, all images underwent a blinded evaluation. RESULTS: A total of 117 babies (Group 1: n = 62, Group 2: n = 54, excluded: n = 1) were examined and 230 images (Group 1: n = 122, Group 2: n = 108) were evaluated, of which 225 were sonographically normal. Two images, showing a type IIa right hip and a type IIa + left hip respectively, were excluded. One participant had to be excluded as the respective images showed two pathologic hip joints. Two images in Group 1 and three in Group 2 were not evaluable. No statistical association between image quality (11 quality criteria and overall evaluability) and fixation technique (0.12 ≤ p ≤ 1.0 or constant) was found. CONCLUSIONS: Considering sonographically normal hip joints, we found no evidence that manual fixation differed from Graf's technique regarding image quality. In future studies, hip pathologies should be included and discomfort of infants and parents during the examination should be addressed. TRIAL REGISTRATION: German Clinical Trials Register, ID: DRKS00015694 ), registered retrospectively on October 7th, 2018.
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Luxación Congénita de la Cadera/diagnóstico por imagen , Luxación Congénita de la Cadera/terapia , Diseño de Equipo , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Ultrasonografía/instrumentación , Ultrasonografía/métodosRESUMEN
BACKGROUND: Hypernatraemia is common in inpatients and is associated with substantial morbidity. Its differential diagnosis is challenging, and delayed treatment may have devastating consequences. The most important hormone for the regulation of water homeostasis is arginine vasopressin, and copeptin, the C-terminal portion of the precursor peptide of arginine vasopressin, might be a reliable new parameter with which to assess the underlying cause of hypernatraemia. METHODS: In this prospective, multicentre, observational study conducted in two tertiary referral centres in Switzerland, 92 patients with severe hyperosmolar hypernatraemia (Na+ > 155 mmol/L) were included. After a standardised diagnostic evaluation, the underlying cause of hypernatraemia was identified and copeptin levels were measured. RESULTS: The most common aetiology of hypernatraemia was dehydration (DH) (n = 65 [71%]), followed by salt overload (SO) (n = 20 [22%]), central diabetes insipidus (CDI) (n = 5 [5%]) and nephrogenic diabetes insipidus (NDI) (n = 2 [2%]). Low urine osmolality was indicative for patients with CDI and NDI (P < 0.01). Patients with CDI had lower copeptin levels than patients with DH or SO (both P < 0.01) or those with NDI. Copeptin identified CDI with an AUC of 0.99 (95% CI 0.97-1.00), and a cut-off value ≤ 4.4pmol/L showed a sensitivity of 100% and a specificity of 99% to predict CDI. Similarly, urea values were lower in CDI than in DH or SO (P < 0.05 and P < 0.01, respectively) or NDI. The AUC for diagnosing CDI was 0.98 (95% CI 0.96-1.00), and a cut-off value < 5.05 mmol/L showed high specificity and sensitivity for the diagnosis of CDI (98% and 100%, respectively). Copeptin and urea could not differentiate hypernatraemia induced by DH from that induced by SO (P = 0.66 and P = 0.30, respectively). CONCLUSIONS: Copeptin and urea reliably identify patients with CDI and are therefore helpful tools for therapeutic management in patients with severe hypernatraemia. TRIALS REGISTRATION: ClinicalTrials.gov, NCT01456533 . Registered on 20 October 2011.
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Glicopéptidos/análisis , Anciano , Anciano de 80 o más Años , Área Bajo la Curva , Diagnóstico Diferencial , Femenino , Escala de Coma de Glasgow , Glicopéptidos/sangre , Glicopéptidos/uso terapéutico , Hospitalización/estadística & datos numéricos , Humanos , Hipernatremia/mortalidad , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Puntuación Fisiológica Simplificada Aguda , Estadísticas no Paramétricas , SuizaRESUMEN
Antihypertensive therapy in chronic kidney disease: what targets and how to reach them Abstract. The primary treatment target for lowering cardiovascular risk in chronic renal failure (as well as in the general hypertensive population) is to lower sitting office blood pressure. Since the physiological nocturnal blood pressure drop ("dipping") is frequently absent or even reversed in chronic renal failure (occasionally causing nocturnal blood pressures to be higher than at daytime), 24 hour blood pressure monitoring needs to be liberally used. Systolic office blood pressures should be 130 to 139 mm Hg, diastolic 80 to 89 mm Hg, and 24 hour blood pressures 10 mm Hg lower. In kidney diseases with relevant proteinuria (> 0.5 - 1.0 g / 24 h) and in diabetics with microalbuminuria (> 30 mg / 24 h), proteinuria reduction is an important second treatment goal, because this slows progressive loss of renal function. If there still is relevant proteinuria or (in diabetics, microalbuminuria) after reaching "cardiovascular" blood pressure goals, further lowering of blood pressure below 130 mm Hg systolic should be attempted, if tolerated. Orthostasis, syncope and diastolic blood pressures < 70 mm Hg must by all means be avoided. In addition, there is no demonstrable benefit of lowering systolic blood pressure < 130 mm Hg if eGFR is < 20 ml / min / 1.73 m2. Since intake and excretion of sodium chloride play a central role in the pathogenesis of hypertension in renal failure, habitual salt overconsumers (taking more than approximately 9 g NaCl / 24 h) need to reduce their intake. For the same reason, diuretics are mandatory components of antihypertensive treatment in chronic renal failure, even if there are no signs of volume expansion at all. At eGFRs < 30 ml / min / 1.73 m2, thiazide type diuretics should replaced or supplemented by loop diuretics (e. g. torasemide). SGLT2 inhibitors are adjuvant diuretics in diabetics with an eGFR > 30 ml / min / 1.73 m2. ACE inhibitors or angiotensin 2 receptor antagonists are only mandatory if proteinuria exceeds 0.5 to 1.0 g / 24 h (or in diabetics with microalbuminuria), but not generally in chronic renal failure. At least two antihypertensive drugs are almost always required; at least one should be taken in the evening.
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Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Antihipertensivos/uso terapéutico , Presión Sanguínea/efectos de los fármacos , Hipertensión , Insuficiencia Renal Crónica , Humanos , Hipertensión/tratamiento farmacológicoRESUMEN
Protean agonists are of great pharmacological interest as their behavior may change in magnitude and direction depending on the constitutive activity of a receptor. Yet, this intriguing phenomenon has been poorly described and understood, due to the lack of stable experimental systems and design strategies. In this study, we overcome both limitations: First, we demonstrate that modulation of the ionic strength in a defined experimental set-up allows for analysis of G protein-coupled receptor activation in the absence and presence of a specific amount of spontaneous receptor activity using the muscarinic M2 acetylcholine receptor as a model. Second, we employ this assay system to show that a dualsteric design principle, that is, molecular probes, carrying two pharmacophores to simultaneously adopt orthosteric and allosteric topography within a G protein-coupled receptor, may represent a novel approach to achieve protean agonism. We pinpoint three molecular requirements within dualsteric compounds that elicit protean agonism at the muscarinic M2 acetylcholine receptor. Using radioligand-binding and functional assays, we posit that dynamic ligand binding may be the mechanism underlying protean agonism of dualsteric ligands. Our findings provide both new mechanistic insights into the still enigmatic phenomenon of protean agonism and a rationale for the design of such compounds for a G protein-coupled receptor.
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Ingeniería de Proteínas , Receptores Acoplados a Proteínas G/agonistas , Regulación Alostérica , Animales , Células CHO , Cricetinae , Cricetulus , Humanos , Ligandos , Unión Proteica , Receptor Muscarínico M2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , TrometaminaRESUMEN
G protein-coupled receptors constitute the largest family of membrane receptors and modulate almost every physiological process in humans. Binding of agonists to G protein-coupled receptors induces a shift from inactive to active receptor conformations. Biophysical studies of the dynamic equilibrium of receptors suggest that a portion of receptors can remain in inactive states even in the presence of saturating concentrations of agonist and G protein mimetic. However, the molecular details of agonist-bound inactive receptors are poorly understood. Here we use the model of bitopic orthosteric/allosteric (i.e. dualsteric) agonists for muscarinic M2 receptors to demonstrate the existence and function of such inactive agonist·receptor complexes on a molecular level. Using all-atom molecular dynamics simulations, dynophores (i.e. a combination of static three-dimensional pharmacophores and molecular dynamics-based conformational sampling), ligand design, and receptor mutagenesis, we show that inactive agonist·receptor complexes can result from agonist binding to the allosteric vestibule alone, whereas the dualsteric binding mode produces active receptors. Each agonist forms a distinct ligand binding ensemble, and different agonist efficacies depend on the fraction of purely allosteric (i.e. inactive) versus dualsteric (i.e. active) binding modes. We propose that this concept may explain why agonist·receptor complexes can be inactive and that adopting multiple binding modes may be generalized also to small agonists where binding modes will be only subtly different and confined to only one binding site.
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Simulación de Dinámica Molecular , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/química , Regulación Alostérica , Animales , Células CHO , Cricetinae , Cricetulus , Humanos , Ligandos , Receptor Muscarínico M2/metabolismoRESUMEN
OBJECTIVE: Hyponatraemia is common and its differential diagnosis is challenging. Commonly used diagnostic algorithms have limited diagnostic accuracy. Copeptin, the c-terminal portion of the precursor peptide of arginine vasopressin might help in the differential diagnosis of hyponatraemia. DESIGN: Prospective multicentre observational study. PATIENTS/METHODS: A total of 298 patients admitted with profound hypoosmolar hyponatraemia (Na < 125 mmol/l) were evaluated. Three experts uninvolved in the patients' care determined the aetiology of hyponatraemia after standardized diagnostic evaluation. RESULTS: Hyponatraemia differential diagnoses were as follows: syndrome of inappropriate antidiuresis (SIAD), 106 patients (35·6%); 'diuretic-induced', 72 (24·2%); 'hypovolaemic', 59 (19·8%); 'hypervolaemic', 33 (11·1%); primary polydipsia (PP), 24 (8·1%); and cortisol deficiency, 4 (1·3%). Copeptin levels <3·9 pmol/l identified patients with PP with high specificity (91%). Further, copeptin levels >84 pmol/l were highly predictive for hypovolaemic hyponatraemia (specificity: 90%). Urinary sodium levels and copeptin/urinary sodium ratio in patients with SIAD were higher and lower as compared to other hyponatraemia aetiologies (P < 0·0001). However, the specificity to identify SIAD was moderate for both parameters (31% and 61%). Fractional uric acid excretion (FEUA ) and fractional urea excretion (FEurea ) were higher in patients with SIAD compared to other hyponatraemia aetiologies (both P < 0·0001). FEurea values >55% and FEUA values >12% had a specificity of 96% and 77% to detect patients with SIAD. These results remained similar after excluding patients taking diuretics. CONCLUSIONS: Overall, there is only limited diagnostic utility of copeptin in the differential diagnosis of profound hyponatraemia. Very low copeptin levels are seen in patients with PP and highest copeptin levels in hypovolaemic hyponatraemia. To discriminate between SIAD and other hyponatraemia aetiologies, FEurea and FEUA levels are valuable irrespective of diuretics use.