RESUMO
BACKGROUND & AIMS: The health benefit of human milk (HM) for preterm infant development is known but the role of human milk oligosaccharides (HMOs) contained in HM remains underexplored. We explored the relationship between exposure to HMOs contained in mother's milk and growth and neurodevelopment at 2-years corrected age in preterm infants. METHODS: Exclusively breastfed preterm infants born between 27 and 34 weeks of gestation were enrolled in a monocentric prospective observational study, LACTACOL. Samples of breast milk were collected once a week for 7 weeks after birth. HMOs and sialic acid were measured by liquid chromatography. Age and Stages questionnaire (ASQ) version 2 was used to assess 2-year neurodevelopmental outcome. We analyzed the relationship between HMO content and (i) infant neurodevelopment at 2-years, and (ii) growth outcome at discharge and at 2 years. A secondary analysis was performed among Secretor(+) Lewis(+) mothers. Only associations with a false discovery rate of 10% or less according to the Benjamini-Hochberg procedure were considered significant. RESULTS: 137 preterm infants (mean gestational age of 31.3 ± 1.7 weeks, mean birth weight of 1494 g ± 336 g) born to 117 mothers (mean age of 30.8 ± 5.0 years) were enrolled. Total HMOs and most individual HMOs and sialic acid concentrations decreased with advancing postnatal age, except for lacto-N-fucopentaose-III and 3-fucosyllactose, which increased. Total HMOs were positively correlated with neonatal length growth (adjusted p = 0.012). Neither total HMOs nor any individual HMO correlated with ASQ score in the overall cohort. However, lacto-N-fucopentaose-III (LNFP-III) was significantly associated with total ASQ score (adjusted p ≤ 0.015) among the 104 infants born to Secretor(+) Lewis(+) mothers. CONCLUSIONS: In this exploratory study in very preterm infants, total HMOs and most individual HMOs, except LNFP-III, decreased with advancing postnatal age. Neither the concentration of total HMOs nor that of any individual HMO were associated with ASQ score at 2 years, except for LNFP-III in Secretor(+) Lewis(+) mothers.
Assuntos
Recém-Nascido Prematuro , Leite Humano , Adulto , Aleitamento Materno , Criança , Feminino , Humanos , Lactente , Recém-Nascido , Leite Humano/química , Ácido N-Acetilneuramínico/análise , OligossacarídeosRESUMO
The roles of nitric oxide (NO) and endothelial NO synthase (eNOS) in the regulation of angiogenesis are well documented. However, the involvement of eNOS in the sprouting of endothelial tip-cells at the vascular front during sprouting angiogenesis remains poorly defined. In this study, we show that downregulation of eNOS markedly inhibits VEGF-stimulated migration of endothelial cells but increases their polarization, as evidenced by the reorientation of the Golgi in migrating monolayers and by the fewer filopodia on tip cells at ends of sprouts in endothelial cell spheroids. The effect of eNOS inhibition on EC polarization was prevented in Par3-depleted cells. Importantly, downregulation of eNOS increased the expression of polarity genes, such as PARD3B, PARD6A, PARD6B, PKCΖ, TJP3, and CRB1 in endothelial cells. In retinas of eNOS knockout mice, vascular development is retarded with decreased vessel density and vascular branching. Furthermore, tip cells at the extremities of the vascular front have a marked reduction in the number of filopodia per cell and are more oriented. In a model of oxygen-induced retinopathy (OIR), eNOS deficient mice are protected during the initial vaso-obliterative phase, have reduced pathological neovascularization, and retinal endothelial tip cells have fewer filopodia. Single-cell RNA sequencing of endothelial cells from OIR retinas revealed enrichment of genes related to cell polarity in the endothelial tip-cell subtype of eNOS deficient mice. These results indicate that inhibition of eNOS alters the polarity program of endothelial cells, which increases cell polarization, regulates sprouting angiogenesis and normalizes pathological neovascularization during retinopathy.
Assuntos
Neovascularização Patológica , Neovascularização Fisiológica , Óxido Nítrico Sintase Tipo III/fisiologia , Retina/metabolismo , Neovascularização Retiniana , Vasos Retinianos , Animais , Bovinos , Linhagem Celular , Movimento Celular , Polaridade Celular , Células Endoteliais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Retina/citologia , Retina/patologia , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Vasos Retinianos/citologia , Vasos Retinianos/patologiaRESUMO
BACKGROUND: Feeding supplemented mother milk during hospital stay improves neurodevelopment in preterm infants. Yet the composition of mother milk varies widely between subjects. The relationship between this variation and outcome is unknown. OBJECTIVE: To determine whether the protein content in native breast milk (BM) correlates with 2-year infant outcome. DESIGN: In a monocentric prospective observational study, LACTACOL, preterm infants born between 28 and 34 weeks of gestation, whose mothers decided to exclusively breastfeed, were enrolled during the first week of life. Samples of expressed breast milk obtained at several times of the day were pooled over a 24-h period, and such pool was used for macronutrient analysis, using mid-infrared analyzer. Age and Stages questionnaire (ASQ) was used to assess 2-year neurodevelopmental outcome. We analyzed the relationship between protein content in BM, and (i) infant neurodevelopment at 2-year (primary outcome), and (ii) growth until 2-year (secondary outcome). RESULTS: 138 infants were enrolled. The main analysis concerned 130 infants (including 40 twin infants) and 110 mothers with BM samples collected at week 3 after birth. Native BM samples were ranked in three tertiles of protein content (g/100 ml): 0.91 ± 0.09 (lower), 1.14 ± 0.05 (middle) and 1.40 ± 0.15 (upper); 48, 47 and 35 infants were ranked, respectively, in these three tertiles. Infants in the upper tertile were more often singleton (P = 0.012) and were born with lower birth weight and head circumference Z-scores (P = 0.005 and 0.002, respectively). Differences in weight and head circumference were no longer observed at 2-year. ASQ score at age 2 did not differ between the three tertiles (P = 0.780). Sensitivity analyses with imputations, including all 138 infants, confirmed the main analysis as well as analyses based on fortified BM as exposure. CONCLUSIONS: Protein content of BM (native or fortified) is not associated with preterm infant neurodevelopment at 2-year. Higher protein content was associated with a lower birth weight.
Assuntos
Proteínas do Leite , Leite Humano , Pré-Escolar , Feminino , Alimentos Fortificados , Humanos , Lactente , Fórmulas Infantis , Fenômenos Fisiológicos da Nutrição do Lactente , Recém-Nascido , Recém-Nascido Prematuro , Aumento de PesoRESUMO
OBJECTIVE: Previous studies have assessed breastfeeding-support programmes. Among these, osteopathic manipulative treatment (OMT) is a frequently used approach, although without strong evidence of efficacy. METHODS: A double-blind randomised controlled trial was conducted between July 2013 and March 2016. Breastfed term infants were eligible if one of the following criteria was met: suboptimal breastfeeding behaviour, maternal cracked nipples or maternal pain. The infants were randomly assigned to the intervention or the control group. The intervention consisted of two sessions of early OMT, while in the control group, the manipulations were performed on a doll behind a screen. The primary outcome was the exclusive breastfeeding rate at 1 month, which was assessed in an intention-to-treat analysis. Randomisation was computer generated and only accessible to the osteopath practitioner. The parents, research assistants and paediatricians were masked to group assignment. RESULTS: One hundred twenty-eight mother-infant dyads were randomised, with 64 assigned to each group. In each group, five infants were lost to follow-up. In the intervention group, 31 of 59 (53%) of infants were still exclusively breast fed at 1 month vs 39 of 59 (66%) in the control group, (OR 0.55, 95% CI 0.26 to 1.17; p=0.12). After adjustment for suboptimal breastfeeding behaviour, caesarean section, use of supplements and breast shields, the adjusted OR was 0.44 (95% CI 0.17 to 1.11; p=0.08). No adverse effects were reported in either group. CONCLUSION: OMT did not improve exclusive breast feeding at 1 month. TRIAL REGISTRATION NUMBER: NCT01890668.
Assuntos
Aleitamento Materno , Cuidado do Lactente , Osteopatia/métodos , Relações Mãe-Filho , Adulto , Aleitamento Materno/métodos , Aleitamento Materno/psicologia , Método Duplo-Cego , Feminino , Humanos , Cuidado do Lactente/métodos , Cuidado do Lactente/psicologia , Recém-Nascido , Análise de Intenção de Tratamento , Masculino , Comportamento Materno , Avaliação de Processos e Resultados em Cuidados de Saúde , Resultado do TratamentoRESUMO
Context: The incidence of early-onset neonatal infection has greatly decreased, but a new diagnostic approach is needed to avoid overdiagnosis and overtreatment. The aim of this study was to assess the potential impact of an algorithm incorporating umbilical-cord-blood procalcitonin (PCT) level on neonatal antibiotics prescription rate as compared with current practice. Material and methods: We conducted a prospective study in three maternity wards in France. All term and preterm neonates with the usual risk factors for neonatal group B Streptococcus infection were eligible for umbilical-cord-blood PCT testing. We compared the proportion of neonates who were exposed early to antibiotics (before 6 days of life) to that of neonates for whom antibiotics prescription would be indicated according to the PCT-based algorithm. Results: Among the 3,080 neonates included, 1 neonate presented with certain infection and 38 neonates with probable infection. The global antibiotics prescription rate was 4.6% [95% confidence interval (CI), 4.1-5]. With the PCT-based algorithm, the potential decrease in prescription rate would be 1.8% (95% CI, 1.3-2.3), corresponding to a 39% (95% CI, 37.3-40.7) relative reduction in antibiotics exposure (p < 0.05). Conclusion: These results suggest that the umbilical-cord-blood PCT-based algorithm could significantly help the clinicians in their antibiotic prescription decision to decrease neonatal antibiotics exposure as compared with current practice. If validated in a larger interventional randomized study, this approach could help clinicians stratify the risk of early-onset neonatal infection and initiate early antibiotics treatment in newborns at high risk of infection while limiting the deleterious effects of useless prescriptions in non-infected newborns.
RESUMO
Angiopoietin-1 (Ang-1) is an important proangiogenic factor also involved in the maintenance of endothelial-barrier integrity. The small GTPase Rap1 is involved in the regulation of adherens junctions through VE-cadherin-mediated adhesion, and in endothelial permeability. While many studies established that Rap1 activation is critical for endothelial cell-cell adhesions, its roles in the antipermeability effects of Ang-1 are ill-defined. Thus, we determined the contribution of Rap1 to Ang-1-stimulated angiogenic effects on endothelial cells (ECs). We found that Rap1 is activated following Ang-1 stimulation and is required for the antipermeability effects of Ang-1 on EC monolayers. Our results also revealed that Rap1 is necessary for EC sprouting stimulated by Ang-1 but had no significant effect on Ang-1-induced EC migration and adhesion. In contrast, downregulation of VE-cadherin markedly increased the adhesiveness of ECs to the substratum, which resulted in inhibition of Ang-1-stimulated migration. These results revealed that Rap1 is central to the effects of Ang-1 at intercellular junctions of ECs, whereas VE-cadherin is also involved in the adhesion of ECs to the extracellular matrix.
Assuntos
Angiopoietina-1/farmacologia , Aorta/fisiologia , Adesão Celular , Comunicação Celular , Endotélio Vascular/fisiologia , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Bovinos , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Transdução de Sinais , Proteínas rap1 de Ligação ao GTP/genéticaRESUMO
BACKGROUND: Cell reprogramming is a promising avenue for cell-based therapies as it allows for the generation of multipotent, unipotent, or mature somatic cells without going through a pluripotent state. While the use of autologous cells is considered ideal, key challenges for their clinical translation include the ability to reproducibly generate sufficient quantities of cells within a therapeutically relevant time window. METHODS: We performed transfection of three distinct human somatic starting populations of cells with a non-integrating synthetic plasmid expressing Musashi 1 (MSI1), Neurogenin 2 (NGN2), and Methyl-CpG-Binding Domain 2 (MBD2). The resulting directly reprogrammed neural precursor cells (drNPCs) were examined in vitro using RT-qPCR, karyotype analysis, immunohistochemistry, and FACS at early and late time post-transfection. Electrophysiology (patch clamp) was performed on drNPC-derived neurons to determine their capacity to generate action potentials. In vivo characterization was performed following transplantation of drNPCs into two animal models (Shiverer and SCID/Beige mice), and the numbers, location, and differentiation profile of the transplanted cells were examined using immunohistochemistry. RESULTS: Human somatic cells can be directly reprogrammed within two weeks to neural precursor cells (drNPCs) by transient exposure to Msi1, Ngn2, and MBD2 using non-viral constructs. The drNPCs generate all three neural cell types (astrocytes, oligodendrocytes, and neurons) and can be passaged in vitro to generate large numbers of cells within four weeks. drNPCs can respond to in vivo differentiation and migration cues as demonstrated by their migration to the olfactory bulb and contribution to neurogenesis in vivo. Differentiation profiles of transplanted cells onto the corpus callosum of myelin-deficient mice reveal the production of oligodendrocytes and astrocytes. CONCLUSIONS: Human drNPCs can be efficiently and rapidly produced from donor somatic cells and possess all the important characteristics of native neural multipotent cells including differentiation into neurons, astrocytes, and oligodendrocytes, and in vivo neurogenesis and myelination.
Assuntos
Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Eletrofisiologia , Citometria de Fluxo , Humanos , Cariótipo , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Neurogênese/genética , Neurogênese/fisiologia , Neurônios/citologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Remielinização/genética , Remielinização/fisiologiaRESUMO
Binding of angiopoietin-1 (Ang-1) to its receptor Tie2 on endothelial cells (ECs) promotes vessel barrier integrity and angiogenesis. Here, we identify PAK2 and paxillin as critical targets of Ang-1 responsible for EC migration, polarization, and sprouting. We found that Ang-1 increases PAK2-dependent paxillin phosphorylation and remodeling of focal adhesions and that PAK2 and paxillin are required for EC polarization, migration, and angiogenic sprouting in response to Ang-1. Our findings show that Ang-1 triggers Cdc42 activation at the leading edges of migrating ECs, which is dependent on PAK2 and paxillin expression. We also established that the polarity protein Par3 interacts with Cdc42 in response to Ang-1 in a PAK2- and paxillin-dependent manner. Par3 is recruited at the leading edges of migrating cells and in focal adhesion, where it forms a signaling complex with PAK2 and paxillin in response to Ang-1. These results show that Ang-1 triggers EC polarization and angiogenic sprouting through PAK2-dependent paxillin activation and remodeling of focal adhesions, which are necessary for local activation of Cdc42 and the associated polarity complex. We have shown that PAK2 controls a signaling pathway important for angiogenic sprouting that links focal adhesions to polarity signaling in ECs.
Assuntos
Angiopoietina-1/metabolismo , Paxilina/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Quinases Ativadas por p21/metabolismo , Animais , Aorta/metabolismo , Bovinos , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Adesões Focais/metabolismo , Neovascularização Fisiológica , Fosforilação , Receptor TIE-2 , Transdução de Sinais , Quinases Ativadas por p21/fisiologiaRESUMO
Ankyloglossia is a congenital oral anomaly characterized by the presence of a hypertrophic and short lingual frenulum. Mutations in the gene encoding the transcription factor TBX22 have been involved in isolated ankyloglossia and X-linked cleft palate. The knockout of Lgr5 in mice results in ankyloglossia. Here, we report a five-generation family including patients with severe ankyloglossia and missing lower central incisors. Two members of this family also exhibited congenital anorectal malformations. In this report, male-to-male transmission was in favor of an autosomal dominant inheritance, which allowed us to exclude the X-linked TBX22 gene. Linkage analysis using short tandem repeat markers located in the vicinity of LGR5 excluded this gene as a potential candidate. These results indicate genetic heterogeneity for ankyloglossia. Further investigations with additional families are required in order to identify novel candidate genes.
Assuntos
Anquiloglossia/patologia , Genes Dominantes , Anormalidades Dentárias/patologia , Anquiloglossia/genética , Feminino , Genes Ligados ao Cromossomo X , Humanos , Recém-Nascido , Masculino , Mutação , Linhagem , Fenótipo , Anormalidades Dentárias/genéticaRESUMO
Human milk is recommended for feeding preterm infants. The current pilot study aims to determine whether breast-milk lipidome had any impact on the early growth-pattern of preterm infants fed their own mother's milk. A prospective-monocentric-observational birth-cohort was established, enrolling 138 preterm infants, who received their own mother's breast-milk throughout hospital stay. All infants were ranked according to the change in weight Z-score between birth and hospital discharge. Then, we selected infants who experienced "slower" (n = 15, -1.54 ± 0.42 Z-score) or "faster" (n = 11, -0.48 ± 0.19 Z-score) growth; as expected, although groups did not differ regarding gestational age, birth weight Z-score was lower in the "faster-growth" group (0.56 ± 0.72 vs. -1.59 ± 0.96). Liquid chromatography-mass spectrometry lipidomic signatures combined with multivariate analyses made it possible to identify breast-milk lipid species that allowed clear-cut discrimination between groups. Validation of the selected biomarkers was performed using multidimensional statistical, false-discovery-rate and ROC (Receiver Operating Characteristic) tools. Breast-milk associated with faster growth contained more medium-chain saturated fatty acid and sphingomyelin, dihomo-γ-linolenic acid (DGLA)-containing phosphethanolamine, and less oleic acid-containing triglyceride and DGLA-oxylipin. The ability of such biomarkers to predict early-growth was validated in presence of confounding clinical factors but remains to be ascertained in larger cohort studies.
Assuntos
Desenvolvimento Infantil , Cabeça/crescimento & desenvolvimento , Recém-Nascido Prematuro/crescimento & desenvolvimento , Lipídeos/análise , Leite Humano/química , Aumento de Peso , Fatores Etários , Peso ao Nascer , Estatura , Índice de Massa Corporal , Cefalometria , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , França , Idade Gestacional , Humanos , Recém-Nascido , Projetos Piloto , Estudos Prospectivos , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Fatores de TempoRESUMO
BACKGROUND AND OBJECTIVES: In developed countries, where omphalitis has become rare and related mortality nil, benefits of antiseptic use in umbilical cord care have not been demonstrated. We aimed to assess the noninferiority of dry care compared with antiseptics in France where antiseptic use is widespread. METHODS: We conducted a noninferiority, cluster-randomized, 2-period crossover trial, in 6 French university maternity units including all infants born after 36 weeks' gestation. Maternity units were randomly assigned to provide either their usual antiseptic care or a dry care umbilical cord method for a 4-month period, and then units switched to the alternate cord cleansing method for a 4-month period. The primary outcome was neonatal omphalitis, adjudicated by an independent blinded committee based on all available photographs, clinical, and bacteriological data. We used a noninferiority margin of 0.4%. Analysis was performed per protocol and by intention to treat. RESULTS: Among 8698 participants, omphalitis occurred in 3 of 4293 (0.07%) newborns in the dry care group and in none of the 4404 newborns in the antiseptic care group (crude difference: 0.07; 95% confidence interval: -0.03 to 0.21). Late neonatal infection, parental appreciation of difficulty in care, and time to separation of the cord were not significantly different between the 2 groups. CONCLUSIONS: Dry cord was noninferior to the use of antiseptics in preventing omphalitis in full-term newborns in a developed country. Antiseptic use in umbilical cord care is therefore unnecessary, constraining, and expensive in high-income countries and may be replaced by dry care.
Assuntos
Anti-Infecciosos Locais/administração & dosagem , Cuidado do Lactente/métodos , Infecção da Ferida Cirúrgica/prevenção & controle , Cordão Umbilical/efeitos dos fármacos , Cordão Umbilical/cirurgia , Análise por Conglomerados , Estudos Cross-Over , Feminino , França , Humanos , Recém-Nascido , MasculinoAssuntos
Mutação/genética , Miotonia Congênita/genética , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Criança , Pré-Escolar , Família , Humanos , Lactente , Masculino , Músculo Esquelético/diagnóstico por imagem , Músculo Esquelético/patologia , Miotonia Congênita/diagnóstico por imagem , Miotonia Congênita/patologia , LinhagemRESUMO
BACKGROUND: Human milk composition analysis seems essential to adapt human milk fortification for preterm neonates. The Miris human milk analyzer (HMA), based on mid-infrared methodology, is convenient for a unique determination of macronutrients. However, HMA measurements are not totally comparable with reference methods (RMs). OBJECTIVE: The primary aim of this study was to compare HMA results with results from biochemical RMs for a large range of protein, fat, and carbohydrate contents and to establish a calibration adjustment. METHODS: Human milk was fractionated in protein, fat, and skim milk by covering large ranges of protein (0-3 g/100 mL), fat (0-8 g/100 mL), and carbohydrate (5-8 g/100 mL). For each macronutrient, a calibration curve was plotted by linear regression using measurements obtained using HMA and RMs. RESULTS: For fat, 53 measurements were performed, and the linear regression equation was HMA = 0.79RM + 0.28 (R(2) = 0.92). For true protein (29 measurements), the linear regression equation was HMA = 0.9RM + 0.23 (R(2) = 0.98). For carbohydrate (15 measurements), the linear regression equation was HMA = 0.59RM + 1.86 (R(2) = 0.95). A homogenization step with a disruptor coupled to a sonication step was necessary to obtain better accuracy of the measurements. Good repeatability (coefficient of variation < 7%) and reproducibility (coefficient of variation < 17%) were obtained after calibration adjustment. CONCLUSION: New calibration curves were developed for the Miris HMA, allowing accurate measurements in large ranges of macronutrient content. This is necessary for reliable use of this device in individualizing nutrition for preterm newborns.
Assuntos
Carboidratos da Dieta/análise , Gorduras na Dieta/análise , Proteínas do Leite/análise , Leite Humano/química , Espectrofotometria Infravermelho/normas , Calibragem , Humanos , Modelos Lineares , Padrões de Referência , Reprodutibilidade dos Testes , Espectrofotometria Infravermelho/instrumentaçãoRESUMO
Diagnostic of early neonatal infection remains a serious challenge. Since clinical symptoms and biological markers are neither sensitive nor specific, a lot of newborns suspected of infection, undergo biological analysis and empirical antibiotic treatment awaiting bacteriological results. However the prevalence of the disease has dramatically decreased since the last twenty years, subsequently to the generalization of the per partum antibioprophylaxis strategy. Because the possible deleterious effects of neonatal antibiotic treatment are well described now, it appears very urgent to restrict their use to the infected newborns only. Recent studies underline the benefit of using procalcitonin (PCT) to diferentiate virai infections of bacterial infections. PCT in blood cord could become a new and efficient marker to help neonatologists taking care of infection-suspected newborns. An evidence based approach is necessary, combining anamnestic, clinical and biological data as PCT to identify the very low risk newborns population and to limit the neonatal antibiotic prescriptions.
Assuntos
Antibacterianos/uso terapêutico , Infecções Bacterianas/diagnóstico , Infecções Bacterianas/tratamento farmacológico , Calcitonina/sangue , Sangue Fetal , Algoritmos , Biomarcadores/sangue , Diagnóstico Precoce , Humanos , Prescrição Inadequada/prevenção & controle , Recém-NascidoRESUMO
BACKGROUND AND AIMS: Human breast milk is an extremely dynamic fluid containing many biologically-active components which change throughout the feeding period and throughout the day. We designed a miRNA assay on minimized amounts of raw milk obtained from mothers of preterm infants. We investigated changes in miRNA expression within month 2 of lactation and then over the course of 24 hours. MATERIALS AND METHODS: Analyses were performed on pooled breast milk, made by combining samples collected at different clock times from the same mother donor, along with time series collected over 24 hours from four unsynchronized mothers. Whole milk, lipids or skim milk fractions were processed and analyzed by qPCR. We measured hsa-miR-16-5p, hsa-miR-21-5p, hsa-miR-146-5p, and hsa-let-7a, d and g (all -5p). Stability of miRNA endogenous controls was evaluated using RefFinder, a web tool integrating geNorm, Normfinder, BestKeeper and the comparative ΔΔCt method. RESULTS: MiR-21 and miR-16 were stably expressed in whole milk collected within month 2 of lactation from four mothers. Analysis of lipids and skim milk revealed that miR-146b and let-7d were better references in both fractions. Time series (5H-23H) allowed the identification of a set of three endogenous reference genes (hsa-let-7d, hsa-let-7g and miR-146b) to normalize raw quantification cycle (Cq) data. We identified a daily oscillation of miR-16-5p. PERSPECTIVES: Our assay allows exploring miRNA levels of breast milk from mother with preterm baby collected in time series over 48-72 hours.
Assuntos
MicroRNAs/análise , Leite Humano/química , Reação em Cadeia da Polimerase em Tempo Real/métodos , Ritmo Circadiano , Feminino , Humanos , Recém-Nascido Prematuro , Reação em Cadeia da Polimerase em Tempo Real/normasRESUMO
The first weeks of life are a period of great immaturity and vulnerability. While pathologies and their semiology are very specific in this period, doctors must quickly identify therapeutic emergencies but also reassure parents when symptoms are physiological for age. Jaundice, baby crying, fever or breastfeeding constitute various medical challenges for the primary care physician, especially after birth; in an area of early back home.
Assuntos
Estado Terminal , Emergências , Recém-Nascido , Aleitamento Materno , Estado Terminal/epidemiologia , Estado Terminal/terapia , Choro , Febre/congênito , Febre/terapia , Humanos , Entrevistas como Assunto , PaisRESUMO
Spatial organization of the plasma membrane is an essential feature of the cellular response to external stimuli. Receptor organization at the cell surface mediates transmission of extracellular stimuli to intracellular signalling molecules and effectors that impact various cellular processes including cell differentiation, metabolism, growth, migration and apoptosis. Membrane domains include morphologically distinct plasma membrane invaginations such as clathrin-coated pits and caveolae, but also less well-defined domains such as lipid rafts and the galectin lattice. In the present chapter, we will discuss interaction between caveolae, lipid rafts and the galectin lattice in the control of cancer cell signalling.
Assuntos
Caveolina 1/metabolismo , Receptores ErbB/metabolismo , Galectina 3/metabolismo , Regulação Neoplásica da Expressão Gênica , Microdomínios da Membrana/metabolismo , Transdução de Sinais , Proteínas Sanguíneas , Caveolina 1/genética , Colesterol/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Vesículas Revestidas por Clatrina/ultraestrutura , Receptores ErbB/genética , Adesões Focais/metabolismo , Adesões Focais/ultraestrutura , Galectina 3/genética , Galectinas , Humanos , Microdomínios da Membrana/ultraestrutura , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Células Tumorais CultivadasRESUMO
Bisphenol A (BPA) is an industrial chemical widely used in the production of polycarbonate and epoxy resins. Identified as an endocrine-disrupting chemical (EDC), BPA is a matter of existing or ongoing restrictive regulations and then is increasingly being replaced by other analogues used as BPA's substitutes. Human biomonitoring studies focusing on both BPA and emerging related analogues consequently appear as a requirement either for documenting the efficiency of regulatory actions toward BPA and for fuelling incoming risk assessment studies toward BPA's substitutes. In particular, the increasing concern about the late effects consecutive to early exposures naturally identify human breast milk as a target biological matrix of interest for priority exposure assessment focused on critical sub-populations such as pregnant women, fetuses, and/or newborns. In this context, an accurate and sensitive analytical method based on gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) was developed for the quantification of 18 "BPA-like" compounds in breast milk samples at trace levels (<0.05 µg kg(-1)). The method includes a preliminary protein precipitation step followed by two successive solid-phase extraction (SPE) stages. Quantification of the targeted compounds was achieved according to the isotopic dilution method using (13)C12-BPA as internal standard. The method was validated according to current EU guidelines and criteria. Linearity (R (2)) was better than 0.99 for each molecule within the concentration range 0-5 µg kg(-1). The detection and quantification limits ranged from 0.001 to 0.030 µg kg(-1) and from 0.002 to 0.050 µg kg(-1), respectively. The analytical method was successfully applied to the first set of human breast milk samples (n = 30) originating from French women in the Region Pays-de-la-Loire. The measured levels of BPA were found in the Assuntos
Compostos Benzidrílicos/química
, Cromatografia Gasosa-Espectrometria de Massas/métodos
, Leite Humano/química
, Fenóis/química
, Espectrometria de Massas em Tandem/métodos
, Feminino
, Humanos
, Estrutura Molecular
, Gravidez