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The MYC oncoprotein binds to promoter-proximal regions of virtually all transcribed genes and enhances RNA polymerase II (Pol II) function, but its precise mode of action is poorly understood. Using mass spectrometry of both MYC and Pol II complexes, we show here that MYC controls the assembly of Pol II with a small set of transcription elongation factors that includes SPT5, a subunit of the elongation factor DSIF. MYC directly binds SPT5, recruits SPT5 to promoters, and enables the CDK7-dependent transfer of SPT5 onto Pol II. Consistent with known functions of SPT5, MYC is required for fast and processive transcription elongation. Intriguingly, the high levels of MYC that are expressed in tumors sequester SPT5 into non-functional complexes, thereby decreasing the expression of growth-suppressive genes. Altogether, these results argue that MYC controls the productive assembly of processive Pol II elongation complexes and provide insight into how oncogenic levels of MYC permit uncontrolled cellular growth.
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Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-myc/genética , RNA Polimerase II/genética , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Quinases Ciclina-Dependentes/genética , Chaperonas de Histonas/genética , Humanos , Neoplasias/genética , Regiões Promotoras Genéticas , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Sleep is controlled by homeostatic mechanisms, which drive sleep after wakefulness, and a circadian clock, which confers the 24-h rhythm of sleep. These processes interact with each other to control the timing of sleep in a daily cycle as well as following sleep deprivation. However, the mechanisms by which they interact are poorly understood. We show here that hugin+ neurons, previously identified as neurons that function downstream of the clock to regulate rhythms of locomotor activity, are also targets of the sleep homeostat. Sleep deprivation decreases activity of hugin+ neurons, likely to suppress circadian-driven activity during recovery sleep, and ablation of hugin+ neurons promotes sleep increases generated by activation of the homeostatic sleep locus, the dorsal fan-shaped body (dFB). Also, mutations in peptides produced by the hugin+ locus increase recovery sleep following deprivation. Transsynaptic mapping reveals that hugin+ neurons feed back onto central clock neurons, which also show decreased activity upon sleep loss, in a Hugin peptide-dependent fashion. We propose that hugin+ neurons integrate circadian and sleep signals to modulate circadian circuitry and regulate the timing of sleep.
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Relógios Circadianos/fisiologia , Proteínas de Drosophila/metabolismo , Neurônios/fisiologia , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Sono/fisiologia , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Feminino , Homeostase , Locomoção , Mutação , Privação do Sono , Vigília/fisiologiaRESUMO
The mitotic kinase AURORA-A is essential for cell cycle progression and is considered a priority cancer target. Although the catalytic activity of AURORA-A is essential for its mitotic function, recent reports indicate an additional non-catalytic function, which is difficult to target by conventional small molecules. We therefore developed a series of chemical degraders (PROTACs) by connecting a clinical kinase inhibitor of AURORA-A to E3 ligase-binding molecules (for example, thalidomide). One degrader induced rapid, durable and highly specific degradation of AURORA-A. In addition, we found that the degrader complex was stabilized by cooperative binding between AURORA-A and CEREBLON. Degrader-mediated AURORA-A depletion caused an S-phase defect, which is not the cell cycle effect observed upon kinase inhibition, supporting an important non-catalytic function of AURORA-A during DNA replication. AURORA-A degradation induced rampant apoptosis in cancer cell lines and thus represents a versatile starting point for developing new therapeutics to counter AURORA-A function in cancer.
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Antineoplásicos/química , Aurora Quinase A/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Proteólise/efeitos dos fármacos , Talidomida/química , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose/efeitos dos fármacos , Aurora Quinase A/genética , Benzazepinas/química , Domínio Catalítico , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Replicação do DNA/efeitos dos fármacos , Desenho de Fármacos , Feminino , Humanos , Masculino , Terapia de Alvo Molecular , Polietilenoglicóis/química , Ligação Proteica , Conformação ProteicaRESUMO
BACKGROUND: Existing research shows significant effects of pedometer interventions on students' physical activity (PA). The current study investigates motivational aspects of quantitative increases in PA in the course of a school-based pedometer intervention for adolescents using qualitative data analysis. In addition, critical issues regarding pedometer use in adolescence were examined. METHOD: Data were obtained in the "läuft." evaluation trial. Students of the intervention group received pedometers and took part in class competitions over a time period of 12 weeks. Qualitative data were collected through focus groups (n = 34) with students of the intervention group as well as through in-depth interviews with teachers (n = 33) who implemented the intervention. A grounded theory approach was used to analyze qualitative data. RESULTS: Five major motivational aspects for subsequent increases in PA were examined among students participating in the "läuft." PA program issues: (1) affordance of the pedometer, (2) awareness of the own PA, (3) self-determination with regard to time and type of PA, (4) individual goal setting, and (5) social comparison and competition. CONCLUSIONS: Findings provide a deeper understanding of which aspects of pedometer interventions might be especially important in adolescence and help design effective future interventions for the promotion of PA.
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Actigrafia , Exercício Físico , Motivação , Adolescente , Feminino , Grupos Focais , Promoção da Saúde/métodos , Humanos , Masculino , Pesquisa Qualitativa , EstudantesRESUMO
ß-Casofensin is a bioactive milk peptide that modulates the intestinal barrier, particularly through its action on goblet cells. ß-Casofensin corresponds to fragment (f) 94-123 of the bovine ß-casein (ß-CN) A2 variant. Fifteen genetic variants of bovine ß-CN (A1-3, B-G, H1-2, I-L) are known, of which the A2, A1, and B forms are the most common. These variants differ from each other by the substitution of one or more amino acids, some of which are localized in f94 to 123. The aim of our study was to compare the intestinal effects of ß-casofensin A2 and its 3 main variants: A1, A3, and B. For this purpose, a solution (0.1 µM; 10 µL/g of body weight, postnatal d 10-20) containing ß-casofensin A2, one of its variants (A1, A3, or B), or drinking water (control; CT) was administered to rat pups orally. After euthanasia (postnatal d 20), intestinal segments were collected for biochemical and histochemical analysis and also used to determine paracellular permeability to fluorescein isothiocyanate-labeled 4-kDa dextran in an Ussing chamber. We also studied the direct effects of ß-casofensin A2 and its A1 variant on the paracellular permeability of jejunum segments of adult rats. ß-Casofensin A2 and its B variant significantly increased the population of goblet cells compared with the CT, A1, and A3 groups. The mucin 2 mRNA level was significantly higher in the ß-casofensin A2 group than in the CT, A3, and B groups. Our results also revealed that the protein expression of zonula occludens-1 and occludin was reduced in the jejunum of rats in the A1, A3, and B groups compared with the CT group. However, the A1 variant was the only peptide to decrease jejunal permeability compared with the CT group. This variant, tested directly in the apical compartment of an Ussing chamber at a concentration of 0.1 nM, also reduced jejunal permeability. In conclusion, the substitution of a single amino acid alters the effect of ß-CN sequence f94 to 123 on goblet cells and on intestinal permeability. A genetic polymorphism of ß-CN can affect the biological activity of peptides derived from this protein. These data should be taken into account in the production of bioactive foods.
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Caseínas/química , Leite/química , Animais , Bovinos , Variação Genética , Mucosa Intestinal/metabolismo , Peptídeos , RatosRESUMO
High-protein diets are known to reduce adiposity in the context of high carbohydrate and Western diets. However, few studies have investigated the specific high-protein effect on lipogenesis induced by a high-sucrose (HS) diet or fat deposition induced by high-fat feeding. We aimed to determine the effects of high protein intake on the development of fat deposition and partitioning in response to high-fat and/or HS feeding. A total of thirty adult male Wistar rats were assigned to one of the six dietary regimens with low and high protein, sucrose and fat contents for 5 weeks. Body weight (BW) and food intake were measured weekly. Oral glucose tolerance tests and meal tolerance tests were performed after 4th and 5th weeks of the regimen, respectively. At the end of the study, the rats were killed 2 h after ingestion of a calibrated meal. Blood, tissues and organs were collected for analysis of circulating metabolites and hormones, body composition and mRNA expression in the liver and adipose tissues. No changes were observed in cumulative energy intake and BW gain after 5 weeks of dietary treatment. However, high-protein diets reduced by 20 % the adiposity gain induced by HS and high-sucrose high-fat (HS-HF) diets. Gene expression and transcriptomic analysis suggested that high protein intake reduced liver capacity for lipogenesis by reducing mRNA expressions of fatty acid synthase (fasn), acetyl-CoA carboxylase a and b (Acaca and Acacb) and sterol regulatory element binding transcription factor 1c (Srebf-1c). Moreover, ketogenesis, as indicated by plasma ß-hydroxybutyrate levels, was higher in HS-HF-fed mice that were also fed high protein levels. Taken together, these results suggest that high-protein diets may reduce adiposity by inhibiting lipogenesis and stimulating ketogenesis in the liver.
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Tecido Adiposo/metabolismo , Dieta Hiperlipídica/efeitos adversos , Proteínas Alimentares/administração & dosagem , Sacarose Alimentar/efeitos adversos , Lipogênese , Ácido 3-Hidroxibutírico/sangue , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Adiposidade , Animais , Glicemia/metabolismo , Composição Corporal , Peso Corporal , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Gorduras na Dieta/administração & dosagem , Sacarose Alimentar/administração & dosagem , Ingestão de Energia , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Grelina/sangue , Teste de Tolerância a Glucose , Hipotálamo/metabolismo , Leptina/sangue , Fígado/metabolismo , Masculino , Músculo Esquelético/metabolismo , Ratos , Ratos Wistar , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Triglicerídeos/sangueRESUMO
We aim to externally validate the Ottawa subarachnoid hemorrhage (OSAH) clinical decision rule. This rule identifies patients with acute nontraumatic headache who require further investigation. We conducted a medical record review of all patients presenting to the emergency department (ED) with headache from January 2011 to November 2013. Per the OSAH rule, patients with any of the following predictors require further investigation: age 40 years or older, neck pain, stiffness or limited flexion, loss of consciousness, onset during exertion, or thunderclap. The rule was applied following the OSAH rule criteria. Patients were followed up for repeat visits within 7 days of initial presentation. Data were electronically harvested from the electronic medical record and manually abstracted from individual patient charts using a standardized data abstraction form. Calibration between trained reviewers was performed periodically. A total of 5034 ED visits with acute headache were reviewed for eligibility. There were 1521 visits that met exclusion criteria, and 3059 had headache of gradual onset or time to maximal intensity greater than or equal to 1 hour. The rule was applied to 454 patients (9.0%). There were 9 cases of subarachnoid hemorrhage (SAH), yielding an incidence of 2.0% (95% confidence interval [CI], 1.0%-3.9%) in the eligible cohort. The sensitivity for SAH was 100% (95% CI, 62.9%-100%); specificity, 7.6% (95% CI, 5.4%-10.6%); positive predictive value, 2.1% (95% CI 1.0%-4.2%); and negative predictive value, 100% (95% CI, 87.4%-100%). The OSAH rule was 100% sensitive for SAH in the eligible cohort. However, its low specificity and applicability to only a minority of ED patients with headache (9%) reduce its potential impact on practice.
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Técnicas de Apoio para a Decisão , Cefaleia/etiologia , Hemorragia Subaracnóidea/diagnóstico , Doença Aguda , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Serviço Hospitalar de Emergência , Feminino , Cefaleia/diagnóstico , Humanos , Masculino , Pessoa de Meia-Idade , Cervicalgia/etiologia , Reprodutibilidade dos Testes , Estudos Retrospectivos , Sensibilidade e Especificidade , Hemorragia Subaracnóidea/complicações , Inconsciência/etiologia , Adulto JovemRESUMO
The objective of this study was to evaluate the effect of increasing protein intake, at the expense of carbohydrates, on intrahepatic lipids (IHLs), circulating triglycerides (TGs), and body composition in healthy humans consuming a high-fat, hypercaloric diet. A crossover randomized trial with a parallel control group was performed. After a 2-wk run-in period, participants were assigned to either the control diet [n = 10; 27.8 energy percent (en%) fat, 16.9 en% protein, 55.3 en% carbohydrates] for 4 wk or a high-fat, hypercaloric diet (n = 17; >2 MJ/d) crossover trial with 2 periods of 2 wk, with either high-protein (HP) (37.7 en% fat, 25.7 en% protein, 36.6 en% carbohydrates) or normal-protein (NP) (39.4 en% fat, 15.4 en% protein, 45.2 en% carbohydrates) content. Measurements were performed after 2 wk of run-in (baseline), 2 wk of intervention (period 1), and 4 wk of intervention (period 2). A trend toward lower IHL and plasma TG concentrations during the HP condition compared with the NP condition was observed (IHL: 0.35 ± 0.04% vs. 0.51 ± 0.08%, P = 0.08; TG: 0.65 ± 0.03 vs. 0.77 ± 0.05 mmol/L, P = 0.07, for HP and NP, respectively). Fat mass was significantly lower (10.6 ± 1.72 vs. 10.9 ± 1.73 kg; P = 0.02) with the HP diet than with the NP diet, whereas fat-free mass was higher (55.7 ± 2.79 vs. 55.2 ± 2.80 kg; P = 0.003). This study indicated that an HP, high-fat, hypercaloric diet affects lipid metabolism. It tends to lower the IHL and circulating TG concentrations and significantly lowers fat mass and increases fat-free mass compared with an NP, high-fat, hypercaloric diet. This trail was registered at www.clinicaltrials.gov as NCT01354626.
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Dieta Hiperlipídica , Proteínas Alimentares/administração & dosagem , Ingestão de Energia , Metabolismo dos Lipídeos , Tecido Adiposo , Composição Corporal , Peso Corporal , Estudos Cross-Over , Carboidratos da Dieta/administração & dosagem , Gorduras na Dieta/administração & dosagem , Metabolismo Energético , Feminino , Humanos , Resistência à Insulina , Masculino , Triglicerídeos/sangue , Adulto JovemRESUMO
Circadian cycles of sleep:wake and gene expression change with age in all organisms examined. Metabolism is also under robust circadian regulation, but little is known about how metabolic cycles change with age and whether these contribute to the regulation of behavioral cycles. To address this gap, we compared cycling of metabolites in young and old Drosophila and found major age-related variations. A significant model separated the young metabolic profiles by circadian timepoint, but could not be defined for the old metabolic profiles due to the greater variation in this dataset. Of the 159 metabolites measured in fly heads, we found 17 that cycle by JTK analysis in young flies and 17 in aged. Only four metabolites overlapped in the two groups, suggesting that cycling metabolites are distinct in young and old animals. Among our top cyclers exclusive to young flies were components of the pentose phosphate pathway (PPP). As the PPP is important for buffering reactive oxygen species, and overexpression of glucose-6-phosphate dehydrogenase (G6PD), a key component of the PPP, was previously shown to extend lifespan in Drosophila, we asked if this manipulation also affects sleep:wake cycles. We found that overexpression in circadian clock neurons decreases sleep in association with an increase in cellular calcium and mitochondrial oxidation, suggesting that altering PPP activity affects neuronal activity. Our findings elucidate the importance of metabolic regulation in maintaining patterns of neural activity, and thereby sleep:wake cycles.
Assuntos
Relógios Circadianos , Drosophila , Animais , Drosophila/metabolismo , Sono , Espécies Reativas de Oxigênio/metabolismo , Via de Pentose Fosfato , Ritmo CircadianoRESUMO
The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting the brain from pathogens. The inherent barrier properties of the BBB pose a challenge for therapeutic drug delivery into the CNS to treat neurological diseases. Impaired BBB function has been related to neurological disease. Cerebral amyloid angiopathy (CAA), the deposition of amyloid in the cerebral vasculature leading to a compromised BBB, is a co-morbidity in most cases of Alzheimer's disease (AD), suggesting that BBB dysfunction or breakdown may be involved in neurodegeneration. Due to limited access to human BBB tissue, the mechanisms that contribute to proper BBB function and BBB degeneration remain unknown. To address these limitations, we have developed a human pluripotent stem cell-derived BBB (iBBB) by incorporating endothelial cells, pericytes, and astrocytes in a 3D matrix. The iBBB self-assembles to recapitulate the anatomy and cellular interactions present in the BBB. Seeding iBBBs with amyloid captures key aspects of CAA. Additionally, the iBBB offers a flexible platform to modulate genetic and environmental factors implicated in cerebrovascular disease and neurodegeneration, to investigate how genetics and lifestyle affect disease risk. Finally, the iBBB can be used for drug screening and medicinal chemistry studies to optimize therapeutic delivery to the CNS. In this protocol, we describe the differentiation of the three types of cells (endothelial cells, pericytes, and astrocytes) arising from human pluripotent stem cells, how to assemble the differentiated cells into the iBBB, and how to model CAA in vitro using exogenous amyloid. This model overcomes the challenge of studying live human brain tissue with a system that has both biological fidelity and experimental flexibility, and enables the interrogation of the human BBB and its role in neurodegeneration.
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Doença de Alzheimer , Angiopatia Amiloide Cerebral , Humanos , Barreira Hematoencefálica/metabolismo , Células Endoteliais/metabolismo , Encéfalo/metabolismo , Sistema Nervoso Central/metabolismo , Doença de Alzheimer/metabolismoRESUMO
Aging is associated with a number of physiologic changes including perturbed circadian rhythms; however, mechanisms by which rhythms are altered remain unknown. To test the idea that circulating factors mediate age-dependent changes in peripheral rhythms, we compared the ability of human serum from young and old individuals to synchronize circadian rhythms in culture. We collected blood from apparently healthy young (age 25-30) and old (age 70-76) individuals and used the serum to synchronize cultured fibroblasts. We found that young and old sera are equally competent at driving robust ~24h oscillations of a luciferase reporter driven by clock gene promoter. However, cyclic gene expression is affected, such that young and old sera drive cycling of different genes. While genes involved in the cell cycle and transcription/translation remain rhythmic in both conditions, genes identified by STRING and IPA analyses as associated with oxidative phosphorylation and Alzheimer's Disease lose rhythmicity in the aged condition. Also, the expression of cycling genes associated with cholesterol biosynthesis increases in the cells entrained with old serum. We did not observe a global difference in the distribution of phase between groups, but find that peak expression of several clock controlled genes (PER3, NR1D1, NR1D2, CRY1, CRY2, and TEF) lags in the cells synchronized with old serum. Taken together, these findings demonstrate that age-dependent blood-borne factors affect peripheral circadian rhythms in cells and have the potential to impact health and disease via maintaining or disrupting rhythms respectively.
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(1) Background: A tryptic hydrolysate of bovine αs1-casein (CH) exerts anxiolytic-like properties in many species, including humans. This is mainly related to the presence of α-casozepine (α-CZP), which yields these properties in rodents. This study evaluates, in a rat model, the roles of the vagus nerve and the benzodiazepine binding site of GABAA receptors in the mode of action of CH. (2) Methods: The conditioned defensive burying test was used to evaluate anxiety. (3) Results: Participation of the vagus nerve in the mode of action of CH was excluded, as the global anxiety score in vagotomised rats was not significantly different from that of non-vagotomised animals. The blocking of the binding sites of benzodiazepines with flumazenil antagonised CH anxiolytic-like properties. (4) Conclusions: The vagus nerve does not play a role in the anxiolytic-like properties of CH. On the other hand, this anxiolytic-like activity relies on the benzodiazepine binding site of the GABAA receptors. This result is consistent with previous in vitro studies and, more specifically with the discovery of α-CZP, the peptide responsible for the anxiolytic-like properties of CH.
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Ansiolíticos , Animais , Ansiolíticos/farmacologia , Ansiedade/tratamento farmacológico , Benzodiazepinas/farmacologia , Sítios de Ligação , Caseínas/metabolismo , Bovinos , Fragmentos de Peptídeos , Ratos , Receptores de GABA-A/metabolismo , Nervo Vago/metabolismo , Ácido gama-AminobutíricoRESUMO
Cellular growth is a fundamental process of life and must be precisely controlled in multicellular organisms. Growth is crucially controlled by the number of functional ribosomes available in cells. The production of new ribosomes depends critically on the activity of RNA polymerase (RNAP) II in addition to the activity of RNAP I and III, which produce ribosomal RNAs. Indeed, the expression of both, ribosomal proteins and proteins required for ribosome assembly (ribosomal biogenesis factors), is considered rate-limiting for ribosome synthesis. Here, we used genetic screening to identify novel transcriptional regulators of cell growth genes by fusing promoters from a ribosomal protein gene (Rpl18) and from a ribosomal biogenesis factor (Fbl) with fluorescent protein genes (RFP, GFP) as reporters. Subsequently, both reporters were stably integrated into immortalized mouse fibroblasts, which were then transduced with a genome-wide sgRNA-CRISPR knockout library. Subsequently, cells with altered reporter activity were isolated by FACS and the causative sgRNAs were identified. Interestingly, we identified two novel regulators of growth genes. Firstly, the exon junction complex protein RBM8A controls transcript levels of the intronless reporters used here. By acute depletion of RBM8A protein using the auxin degron system combined with the genome-wide analysis of nascent transcription, we showed that RBM8A is an important global regulator of ribosomal protein transcripts. Secondly, we unexpectedly observed that the glycolytic enzyme aldolase A (ALDOA) regulates the expression of ribosomal biogenesis factors. Consistent with published observations that a fraction of this protein is located in the nucleus, this may be a mechanism linking transcription of growth genes to metabolic processes and possibly to metabolite availability.
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In this report, we describe a case of bilateral lactational mastitis in a primigravid, Spanish-speaking woman who exclusively pumped breast milk for a hospitalized, critically ill infant in the NICU within a free-standing children's hospital. The case follows her clinical presentation, assessments, diagnostics, and therapeutic interventions during the 45-day postpartum period. This case report highlights the situational and environmental context of the woman's experiences and emphasizes potential disconnections of care. Regarding her mastitis, the maternity care providers relied on the woman to provide all relevant information without knowledge of her hospitalized infant's health status. Traditionally during the postpartum period, infants hospitalized in the NICU and their mothers are cared for by separate provider teams. Clinicians must acknowledge that when women transition from recent patients to parent visitors after birth, they will likely have ongoing medical, obstetric, and psychosocial care needs.
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Extração de Leite , Mastite , Serviços de Saúde Materna , Aleitamento Materno , Criança , Feminino , Humanos , Lactente , Recém-Nascido , Unidades de Terapia Intensiva Neonatal , Mastite/diagnóstico , Mastite/terapia , Leite Humano , Mães/psicologia , GravidezRESUMO
The blood-brain barrier (BBB) is critical for neural function. We report here circadian regulation of the BBB in mammals. Efflux of xenobiotics by the BBB oscillates in mice, with highest levels during the active phase and lowest during the resting phase. This oscillation is abrogated in circadian clock mutants. To elucidate mechanisms of circadian regulation, we profiled the transcriptome of brain endothelial cells; interestingly, we detected limited circadian regulation of transcription, with no evident oscillations in efflux transporters. We recapitulated the cycling of xenobiotic efflux using a human microvascular endothelial cell line to find that the molecular clock drives cycling of intracellular magnesium through transcriptional regulation of TRPM7, which appears to contribute to the rhythm in efflux. Our findings suggest that considering circadian regulation may be important when therapeutically targeting efflux transporter substrates to the CNS.
Assuntos
Barreira Hematoencefálica/metabolismo , Relógios Circadianos , Xenobióticos/metabolismo , Fatores de Transcrição ARNTL/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Humanos , Magnésio/metabolismo , Camundongos Endogâmicos C57BL , Modelos Biológicos , Permeabilidade , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Especificidade por Substrato , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismoRESUMO
The western dietary pattern is known for its frequent meals rich in saturated fat and protein, resulting in a postprandial state for a large part of the day. Therefore, our aim was to investigate the postprandial glucose and lipid metabolism in response to high (HP) or normal (NP) protein, high-fat hypercaloric diet and to identify early biomarkers of protein intake and hepatic lipid accumulation. In a crossover design, 17 healthy subjects were randomly assigned to consume a HP or NP hypercaloric diet for two weeks. In parallel, a control group (CD; n = 10) consumed a weight-maintaining control diet. Biomarkers of postprandial lipid and glucose metabolism were measured in 24 h urine and in plasma before and following a meal challenge. The metabolic profile of urine but not plasma, showed increased excretion of 13C, carnitine and short chain acyl-carnitines after adaptation to the HP diet. Urinary excretion of decatrienoylcarnitine and octenoylcarnitine increased after adaptation to the NP diet. Our results suggest that the higher excretion of short-chain urinary acyl-carnitines could facilitate the elimination of excess fat of the HP diet and thereby reduce hepatic fat accumulation previously reported, whereas the higher excretion medium-chains acyl-carnitine could be early biomarkers of hepatic lipid accumulation.
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Carnitina/análogos & derivados , Dieta Hiperlipídica/efeitos adversos , Dieta Rica em Proteínas/efeitos adversos , Dieta Ocidental/efeitos adversos , Síndrome Metabólica/diagnóstico , Adulto , Biomarcadores/urina , Carnitina/metabolismo , Carnitina/urina , Estudos Cross-Over , Gorduras na Dieta/efeitos adversos , Gorduras na Dieta/metabolismo , Proteínas Alimentares/metabolismo , Ingestão de Energia/fisiologia , Feminino , Glucose/metabolismo , Voluntários Saudáveis , Humanos , Metabolismo dos Lipídeos/fisiologia , Fígado/metabolismo , Masculino , Síndrome Metabólica/etiologia , Síndrome Metabólica/metabolismo , Síndrome Metabólica/urina , Período Pós-Prandial/fisiologia , Eliminação Renal/fisiologia , Adulto JovemRESUMO
Lung cancer is the most common cancer worldwide and the leading cause of cancer-related deaths in both men and women. Despite the development of novel therapeutic interventions, the 5-year survival rate for non-small cell lung cancer (NSCLC) patients remains low, demonstrating the necessity for novel treatments. One strategy to improve translational research is the development of surrogate models reflecting somatic mutations identified in lung cancer patients as these impact treatment responses. With the advent of CRISPR-mediated genome editing, gene deletion as well as site-directed integration of point mutations enabled us to model human malignancies in more detail than ever before. Here, we report that by using CRISPR/Cas9-mediated targeting of Trp53 and KRas, we recapitulated the classic murine NSCLC model Trp53 fl/fl :lsl-KRas G12D/wt . Developing tumors were indistinguishable from Trp53 fl/fl :lsl-KRas G12D/ wt -derived tumors with regard to morphology, marker expression, and transcriptional profiles. We demonstrate the applicability of CRISPR for tumor modeling in vivo and ameliorating the need to use conventional genetically engineered mouse models. Furthermore, tumor onset was not only achieved in constitutive Cas9 expression but also in wild-type animals via infection of lung epithelial cells with two discrete AAVs encoding different parts of the CRISPR machinery. While conventional mouse models require extensive husbandry to integrate new genetic features allowing for gene targeting, basic molecular methods suffice to inflict the desired genetic alterations in vivo. Utilizing the CRISPR toolbox, in vivo cancer research and modeling is rapidly evolving and enables researchers to swiftly develop new, clinically relevant surrogate models for translational research.
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Introduction: MamaCare is an adaptation of the CenteringPregnancy group prenatal care model designed to support women when the pregnancy has been complicated by the presence of known congenital anomalies. The lactation-related outcomes of participants were unknown. Materials and Methods: This is a retrospective descriptive cohort study describing the lactation-related outcomes of participants of MamaCare over 43 months. Data collection included abstraction of maternal demographic data, maternal group prenatal care session participation data, maternal pregnancy and delivery data, as well as documented lactation and breastfeeding data. Results: The total cohort consisted of 92 dyads. Of the 81 women who received an individualized prenatal nutrition consultation, 65 (80.2%) voiced a prenatal feeding goal of human milk and breastfeeding only, while the remaining 16 (19.8%) expressed goals of combination feeding (human milk and infant formula). For the surviving 85 infants, 91.8% of the mothers initiated lactation on the delivery day and the remaining 7 women initiated lactation on postpartum day 1. Also at the time of discharge from the initial intensive care unit stay, 87.1% of infants were receiving maternal human milk. Discussion: For families who participate in MamaCare, not only do they form a sense of community and learn about human milk and breastfeeding before delivery, the MamaCare facilitators and presenters normalize their experience to help MamaCare participants best meet their personal breastfeeding goals as well.
Assuntos
Aleitamento Materno , Anormalidades Congênitas , Lactação , Mães/psicologia , Cuidado Pré-Natal/métodos , Adolescente , Adulto , Estudos de Coortes , Feminino , Humanos , Lactente , Pessoa de Meia-Idade , Gravidez , Diagnóstico Pré-Natal , Estudos RetrospectivosRESUMO
α-Casozepine (α-CZP) is an anxiolytic-like bioactive decapeptide derived from bovine αs1casein. The N-terminal peptide YLGYL was previously identified after proteolysis of the original peptide in an in vitro digestion model. Its putative anxiolytic-like properties were evaluated in a Swiss mice model using a light/dark box (LDB) after an intraperitoneal injection (0.5 mg/kg). The effect of YLGYL on c-Fos expression in brain regions linked to anxiety regulation was afterwards evaluated via immunofluorescence and compared to those of α-CZP and diazepam, a reference anxiolytic benzodiazepine. YLGYL elicited some anxiolytic-like properties in the LDB, similar to αCZP and diazepam. The two peptides displayed some strong differences compared with diazepam in terms of c-Fos expression modulation in the prefontal cortex, the amygdala, the nucleus of the tractus solitarius, the periaqueductal grey, and the raphe magnus nucleus, implying a potentially different mode of action. Additionally, YLGYL modulated c-Fos expression in the amygdala and in one of the raphe nuclei, displaying a somewhat similar pattern of activation as α-CZP. Nevertheless, some differences were also spotted between the two peptides, making it possible to formulate the hypothesis that these peptides could act differently on anxiety regulation. Taken together, these results showed that YLGYL could contribute to the in vivo overall action of αCZP.
Assuntos
Ansiolíticos/farmacologia , Ansiedade/tratamento farmacológico , Encéfalo/metabolismo , Caseínas/farmacologia , Fragmentos de Peptídeos/farmacologia , Tonsila do Cerebelo , Animais , Benzodiazepinas/farmacologia , Bovinos , Diazepam/metabolismo , Diazepam/farmacologia , Masculino , Camundongos , Proteínas Proto-Oncogênicas c-fos/metabolismoRESUMO
The model of group prenatal care was initially developed to include peer support and to improve education and health-promoting behaviors during pregnancy. This model has since been adapted for populations with unique educational needs. Mama Care is an adaptation of the CenteringPregnancy Model of prenatal care. Mama Care is situated within a national and international referral center for families with prenatally diagnosed fetal anomalies. In December 2013, the Center for Fetal Diagnosis and Treatment at Children's Hospital of Philadelphia began offering a model of group prenatal care to women whose pregnancies are affected by a prenatal diagnosis of a fetal anomaly. The model incorporates significant adaptations of CenteringPregnancy in order to accommodate these women, who typically transition their care from community-based settings to the Center for Fetal Diagnosis and Treatment in the late second or early third trimester. Unique challenges associated with caring for families within a referral center include a condensed visit schedule, complex social needs such as housing and psychosocial support, as well as an increased need for antenatal surveillance and frequent preterm birth. Outcomes of the program are favorable and suggest group prenatal care models can be developed to support the needs of patients with prenatally diagnosed fetal anomalies.