RESUMEN
OBJECTIVE: To examine the associations between exposure to gestational diabetes mellitus (GDM) and maternal glycemic markers during pregnancy and offspring behaviors at 3 and 5 years. We hypothesized that exposure to maternal hyperglycemia would be associated with more behavioral problems in offspring. METHODS: We included 548 mother-child pairs from the prospective pre-birth Gen3G cohort (Canada). Glycemic markers were measured during a 75 g oral glucose tolerance test (OGTT) in the second trimester of pregnancy. Based on OGTT, we classified 59 women (10.8%) as having GDM according to international diagnostic criteria. Mothers reported offspring behavior using the Strengths and Difficulties Questionnaire (SDQ) at 3 and 5 years, and the Child Behavior Checklist (CBCL) at 5 years. We used linear mixed models and multivariate regression to assess the associations between GDM or glycemic markers and children's behavior, adjusted for child sex and age, and maternal demographic factors, body mass index and family history of diabetes. RESULTS: Exposure to GDM was associated with higher SDQ externalizing scores at 3 and 5 years [B = 1.12, 95% CI (0.14, 2.10)] in fully adjusted linear mixed models. These results were supported by the CBCL at 5 years. Higher levels of maternal glucose at 1 h and 2 h during OGTT were associated with greater SDQ externalizing scores. Fasting glucose levels were not associated with child behavior scores. We did not observe associations between glycemic markers and internalizing behaviors. CONCLUSIONS: Exposure to higher levels of maternal glycemia during pregnancy was associated with more externalizing behaviors in children at 3 and 5 years.
What is already known on this subject? Prenatal exposure to gestational diabetes mellitus (GDM) has been linked to a higher risk of long-term consequences in offspring including metabolic problems and cognitive difficulties. However, prior studies examining associations between GDM and behavior in children reported mixed results. What this study adds? We reported associations between exposure to maternal GDM and post-OGTT hyperglycemia during pregnancy and greater levels of externalizing behaviors in children at 3 and 5 years of age. Our results underscore the importance of early detection of behavioral problems in children.
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Diabetes Gestacional , Hiperglucemia , Embarazo , Humanos , Femenino , Diabetes Gestacional/epidemiología , Estudios Prospectivos , Prueba de Tolerancia a la Glucosa , Glucosa , Hiperglucemia/epidemiologíaRESUMEN
BACKGROUND: Fetal exposure to maternal excess adiposity and hyperglycemia is risk factors for childhood adverse metabolic outcomes. Using data from a prospective pre-birth cohort, we aimed to further understand the prenatal determinants of fetal metabolic programming based on analyses of maternal adiposity and glycemic traits across pregnancy with childhood metabolomic profiles. METHODS: This study included 330 mother-child pairs from the Gen3G cohort with information on maternal adiposity and glycemic markers at 5-16 (visit 1) and 24-30 (visit 2) weeks of pregnancy. At mid-childhood (4.8-7.2 years old), we collected fasting plasma and measured 1116 metabolites using an untargeted approach. We constructed networks of interconnected metabolites using a weighted-correlation network analysis algorithm. We estimated Spearman's partial correlation coefficients of maternal adiposity and glycemic traits across pregnancy with metabolite networks and individual metabolites, adjusting for maternal age, gravidity, race/ethnicity, history of smoking, and child's sex and age at blood collection for metabolite measurement. RESULTS: We identified a network of 16 metabolites, primarily glycero-3-phosphoethanolamines (GPE) at mid-childhood that showed consistent negative correlations with maternal body mass index, waist circumference, and body-fat percentage at visits 1 and 2 (ρadjusted = -0.14 to -0.21) and post-challenge glucose levels at visit 2 (ρadjusted = -0.10 to -0.13), while positive correlations with Matsuda index (ρadjusted = 0.13). Within this identified network, 1-palmitoyl-2-decosahexaenoyl-GPE and 1-stearoyl-2-decosahexaenoyl-GPE appeared to be driving the associations. In addition, a network of 89 metabolites, primarily phosphatidylcholines, plasmalogens, sphingomyelins, and ceramides showed consistent negative correlations with insulin at visit 1 and post-challenge glucose at visit 2, while positive correlation with adiponectin at visit 2. CONCLUSIONS: Prenatal exposure to maternal higher adiposity and hyperglycemic traits and lower insulin sensitivity markers were associated with a unique metabolomic pattern characterized by low serum phospho- and sphingolipids in mid-childhood.
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Glucemia , Metaboloma , Obesidad Materna , Efectos Tardíos de la Exposición Prenatal/metabolismo , Adiposidad , Índice de Masa Corporal , Canadá , Niño , Preescolar , Estudios de Cohortes , Femenino , Humanos , Hiperglucemia , Resistencia a la Insulina , Fosfolípidos/sangre , Embarazo , Estudios Prospectivos , Factores de Riesgo , Esfingolípidos/sangre , Circunferencia de la CinturaRESUMEN
BACKGROUND: Maternal insulin resistance is associated with greater maternal inflammation during pregnancy, but its relation to inflammation in offspring remains unclear. The goal of this study was to assess the relationship of gestational insulin resistance and other glycemic markers with offspring inflammation at birth and at 5 years of age. METHODS: We included 653 mother-child pairs from the prospective pre-birth Gen3G cohort. We examined maternal insulin and glucose levels measured during the second trimester of pregnancy, from which we derived the homeostatic model of assessment of insulin resistance (HOMA-IR) and the Matsuda index. We assessed offspring inflammation at birth and at 5 years of age by measuring plasma tumor necrosis factor-α (TNFα) concentrations. We conducted multivariable regression models to evaluate associations of each insulin and glucose marker with offspring inflammation adjusting for confounding variables. RESULTS: Higher levels of fasting insulin were associated with lower TNFα levels at birth (-0.78, 95% CI [-1.45, -0.11]), in the fully adjusted model. We observed similar associations with the HOMA-IR and opposite direction with the Matsuda index. We did not find persistence of the association between maternal fasting insulin and offspring TNFα at 5 years of age. CONCLUSIONS: Greater maternal insulin resistance during pregnancy was associated with lower cord blood TNFα levels in newborns. The mechanisms by which maternal insulin resistance may promote lower inflammatory levels in newborns are not fully understood and more research is needed to deepen our understanding of these mechanisms.
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Inflamación/patología , Resistencia a la Insulina , Biomarcadores/metabolismo , Glucemia/metabolismo , Preescolar , Femenino , Humanos , Recién Nacido , Inflamación/sangre , Insulina/metabolismo , Masculino , Análisis Multivariante , Embarazo , Estudios Prospectivos , Análisis de Regresión , Factor de Necrosis Tumoral alfa/sangreRESUMEN
De novo germline mutations arise preferentially in male owing to fundamental differences between spermatogenesis and oogenesis. Post-meiotic chromatin remodeling in spermatids results in the elimination of most of the nucleosomal supercoiling and is characterized by transient DNA fragmentation. Using three alternative methods, DNA from sorted populations of mouse spermatids was used to confirm that double-strand breaks (DSB) are created in elongating spermatids and repaired at later steps. Specific capture of DSB was used for whole-genome mapping of DSB hotspots (breakome) for each population of differentiating spermatids. Hotspots are observed preferentially within introns and repeated sequences hence are more prevalent in the Y chromosome. When hotspots arise within genes, those involved in neurodevelopmental pathways become preferentially targeted reaching a high level of significance. Given the non-templated DNA repair in haploid spermatids, transient DSBs formation may, therefore, represent an important component of the male mutation bias and the etiology of neurological disorders, adding to the genetic variation provided by meiosis.
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Ensamble y Desensamble de Cromatina/genética , Roturas del ADN de Doble Cadena , Fragmentación del ADN , Espermátides/metabolismo , Animales , Ensayo Cometa , ADN/genética , ADN/metabolismo , Reparación del ADN , Masculino , Meiosis/genética , Ratones Endogámicos C57BL , Nucleosomas/genéticaRESUMEN
Transient DNA breaks and evidence of DNA damage response have recently been reported during the chromatin remodeling process in haploid spermatids, creating a potential window of enhanced genetic instability. We used flow cytometry to achieve separation of differentiating spermatids into four highly purified populations using transgenic mice harboring 160 CAG repeats within exon 1 of the human Huntington disease gene (HTT). Trinucleotic repeat expansion was found to occur immediately following the chromatin remodeling steps, confirming the genetic instability of the process and pointing to the origin of paternal anticipation observed in some trinucleotidic repeats diseases.
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Ensamble y Desensamble de Cromatina , Inestabilidad Genómica , Espermátides/metabolismo , Repeticiones de Trinucleótidos , Animales , Citometría de Flujo , Masculino , Ratones , Espermatogénesis/genética , Expansión de Repetición de TrinucleótidoRESUMEN
OBJECTIVE: This study identified metabolite modules associated with adiposity and body fat distribution in childhood using gold-standard measurements. METHODS: We used cross-sectional data from 329 children at mid-childhood (age 5.3 ± 0.3 years; BMI 15.7 ± 1.5 kg/m2) from the Genetics of Glucose regulation in Gestation and Growth (Gen3G), a prospective pre-birth cohort. We quantified 1038 plasma metabolites and measured body composition using the gold-standard dual-energy x-ray absorptiometry (DXA), in addition to skinfold, waist circumference, and BMI. We applied weighted-correlation network analysis to identify a network of highly correlated metabolite modules. Spearman's partial correlations were applied to determine the associations of adiposity with metabolite modules and individual metabolites with false discovery rate (FDR) correction. RESULTS: We identified a 'green' module of 120 metabolites, primarily comprised of lipids (mostly sphingomyelins and phosphatidylcholine), that showed positive correlations (all FDR p < 0.05) with DXA estimates of total and truncal fat (ρadjusted = 0.11-0.19), skinfold measures (ρadjusted = 0.09-0.26), and BMI and waist circumference (ρadjusted = 0.15 and 0.18, respectively). These correlations were similar when stratified by sex. Within this module, sphingomyelin (d18:2/14:0, d18:1/14:1)*, a sphingomyelin sub-specie that is an important component of cell membranes, showed the strongest associations. CONCLUSIONS: A module of metabolites was associated with adiposity measures in childhood.
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Absorciometría de Fotón , Adiposidad , Composición Corporal , Humanos , Femenino , Masculino , Adiposidad/fisiología , Estudios Transversales , Preescolar , Niño , Estudios Prospectivos , Metabolómica , Índice de Masa Corporal , Obesidad Infantil/sangre , Obesidad Infantil/genética , Metaboloma , Circunferencia de la CinturaRESUMEN
BACKGROUND: Colorectal cancer (CRC) is a major cause of cancer mortality in the world. One of the most widely used screening tests for CRC is the immunochemical fecal occult blood test (iFOBT), which detects human hemoglobin from patient's stool sample. Although it is highly efficient in detecting blood from patients with gastro-intestinal lesions, such as polyps and cancers, the iFOBT has a high rate of false positive discovery. Recent studies suggested gut bacteria as a promising noninvasive biomarker for improving the diagnosis of CRC. In this study, we examined the composition of gut bacteria using iFOBT leftover from patients undergoing screening test along with a colonoscopy. METHODS: After collecting data from more than 800 patients, we considered 4 groups for this study. The first and second groups were respectively "healthy" in which the patients had either no blood in their stool or had blood but no lesions. The third and fourth groups of patients had both blood in their stools with precancerous and cancerous lesions and considered either as low-grade and high-grade lesion groups, respectively. An amplification of 16S rRNA (V4 region) gene was performed, followed by sequencing along with various statistical and bioinformatic analysis. RESULTS: We analyzed the composition of the gut bacteriome at phylum, class, genus, and species levels. Although members of the Firmicute phylum increased in the 3 groups compared to healthy patients, the phylum Actinobacteriota was found to decrease. Moreover, Blautia obeum and Anaerostipes hadrus from the phylum Firmicutes were increased and Collinsella aerofaciens from phylum Actinobacteriota was found decreased when healthy group is compared to the patients with high-grade lesions. Finally, among the 5 machine learning algorithms used to perform our analysis, both elastic net (AUC > 0.7) and random forest (AUC > 0.8) performs well in differentiating healthy patients from 3 other patient groups having blood in their stool. CONCLUSION: Our study integrates the iFOBT screening tool with gut bacterial composition to improve the prediction of CRC lesions.
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Neoplasias Colorrectales , Humanos , Neoplasias Colorrectales/patología , Sangre Oculta , ARN Ribosómico 16S/genética , Detección Precoz del Cáncer , Tamizaje MasivoRESUMEN
At the sequence level, genetic diversity is provided by de novo transmittable mutations that may act as a substrate for natural selection. The gametogenesis process itself is considered more likely to induce endogenous mutations and a clear male bias has been demonstrated from recent next-generation sequencing analyses. As new experimental evidence accumulates, the post-meiotic events of the male gametogenesis (spermiogenesis) appear as an ideal context to induce de novo genetic polymorphism transmittable to the next generation. It may prove to be a major component of the observed male mutation bias. As spermatids undergo chromatin remodeling, transient endogenous DNA double-stranded breaks are produced and trigger a DNA damage response. In these haploid cells, one would expect that the non-templated, DNA end-joining repair processes may generate a repertoire of sequence alterations in every sperm cell potentially transmittable to the next generation. This may therefore represent a novel physiological mechanism contributing to genetic diversity and evolution.
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Daño del ADN/genética , Reparación del ADN/genética , Células Germinativas/citología , Espermatogénesis/genética , Espermatozoides/citología , Variación Genética , Haploidia , Humanos , Masculino , Mutación , Polimorfismo Genético , Espermatozoides/metabolismoRESUMEN
BACKGROUND: Childhood obesity has been associated with prenatal exposure to maternal hyperglycaemia, but we lack understanding about maternal insulin physiologic components that contribute to this association. OBJECTIVES: Evaluate the association between maternal insulin sensitivity during pregnancy and adiposity measures in childhood. METHODS: In 422 mother-child pairs, we tested associations between maternal insulin sensitivity measures at ~26 weeks of pregnancy and child adiposity measures, including dual-energy X-ray absorptiometry body composition and anthropometry (body mass index and waist circumference) at ~5 years. We used linear regression analyses to adjust for maternal age, ethnicity, gravidity, first-trimester body mass index, and child sex and age at mid-childhood. RESULTS: In early pregnancy, maternal mean age was 28.6 ± 4.3 years and median body mass index was 24.1 kg/m2 . Lower maternal insulin sensitivity indices were correlated with greater child adiposity based on anthropometry measures and on dual-energy X-ray absorptiometry total and trunk % fat in univariate associations (r = -0.122 to -0.159). Lower maternal insulin sensitivity was specifically associated with higher dual-energy X-ray absorptiometry trunk % fat (n = 359 for Matsuda; ß = -0.034 ± 0.013; p = 0.01) after adjustment for covariates, including maternal body mass index. CONCLUSIONS: Maternal insulin sensitivity during pregnancy may contribute to increased risk for higher offspring central adiposity in middle childhood.
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Resistencia a la Insulina , Obesidad Infantil , Niño , Embarazo , Femenino , Humanos , Adulto Joven , Adulto , Obesidad Infantil/epidemiología , Obesidad Infantil/genética , Glucosa , Adiposidad , Estudios Prospectivos , Índice de Masa Corporal , Obesidad Abdominal/epidemiología , Absorciometría de FotónRESUMEN
Exposure to maternal hyperglycemia in utero has been associated with adverse metabolic outcomes in offspring. However, few studies have investigated the relationship between maternal hyperglycemia and offspring cortisol levels. We assessed associations of gestational diabetes mellitus (GDM) with cortisol biomarkers in two longitudinal prebirth cohorts: Project Viva included 928 mother-child pairs and Gen3G included 313 mother-child pairs. In Project Viva, GDM was diagnosed in N = 48 (5.2%) women using a two-step procedure (50 g glucose challenge test, if abnormal followed by 100 g oral glucose tolerance test [OGTT]), and in N = 29 (9.3%) women participating in Gen3G using one-step 75 g OGTT. In Project Viva, we measured cord blood glucocorticoids and child hair cortisol levels during mid-childhood (mean (SD) age: 7.8 (0.8) years) and early adolescence (mean (SD) age: 13.2 (0.9) years). In Gen3G, we measured hair cortisol at 5.4 (0.3) years. We used multivariable linear regression to examine associations of GDM with offspring cortisol, adjusting for child age and sex, maternal prepregnancy body mass index, education, and socioeconomic status. We additionally adjusted for child race/ethnicity in the cord blood analyses. In both Project Viva and Gen3G, we observed null associations of GDM and maternal glucose markers in pregnancy with cortisol biomarkers in cord blood at birth (ß = 16.6 nmol/L, 95% CI -60.7, 94.0 in Project Viva) and in hair samples during childhood (ß = -0.56 pg/mg, 95% CI -1.16, 0.04 in Project Viva; ß = 0.09 pg/mg, 95% CI -0.38, 0.57 in Gen3G). Our findings do not support the hypothesis that maternal hyperglycemia is related to hypothalamic-pituitary-adrenal axis activity.
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Diabetes Gestacional , Hiperglucemia , Efectos Tardíos de la Exposición Prenatal , Embarazo , Recién Nacido , Adolescente , Humanos , Femenino , Niño , Masculino , Glucocorticoides/efectos adversos , Hidrocortisona , Glucosa , Sangre Fetal/metabolismo , Sistema Hipotálamo-Hipofisario/metabolismo , Efectos Tardíos de la Exposición Prenatal/metabolismo , Sistema Hipófiso-Suprarrenal , Diabetes Gestacional/diagnóstico , Cabello/metabolismo , Biomarcadores , Hiperglucemia/diagnóstico , Hiperglucemia/etiología , Glucemia/metabolismoRESUMEN
Lower cord blood leptin levels have been associated with lower and higher adiposity in childhood and associations seem to differ according to the child's age, methods of adiposity assessment and sex. Our aim was to investigate sex-specific associations of cord blood leptinemia with childhood adiposity at birth, 3 and 5 years of age. We measured cord blood leptin using Luminex immunoassays in 520 offspring from the Gen3G cohort. We tested associations between cord blood leptin and body mass index (BMI) z-score, skinfolds thicknesses (SFT), and body composition using dual-energy X-ray absorptiometry, adjusted for confounders. At birth, girls had almost twice as much leptin in cord blood as boys (15.5 [8.9; 25.6] vs. 8.6 [4.9; 15.0] ng/mL; p < 0.0001) as well as significantly greater adiposity. Lower levels of cord blood leptin were associated with higher sum of SFT (ß = −0.05 ± 0.02; p = 0.03) and higher BMI z-score (ß= −0.22 ± 0.08; p = 0.01) in 3-year-old boys only. We did not observe these associations at age 5, or in girls. Our results suggest a sexual dimorphism in the programming of leptin sensitivity and childhood adiposity, but further observational and functional studies are needed to better understand the role of leptin in early life.
RESUMEN
BACKGROUND: Exposure to maternal hyperglycaemia in pregnancy has been associated with childhood obesity. Leptin regulation might be involved in this 'adiposity programming' and may depend on timing of exposure. OBJECTIVES: To investigate associations of maternal glycaemia at different periods in pregnancy with childhood adiposity and leptin levels at 5 years of age. METHODS: In a prospective pre-birth cohort, we measured maternal glucose levels after a 50 g oral glucose challenge test at first trimester (9.8 ± 2.3 weeks) and during a 75 g oral glucose tolerance test at second trimester (26.5 ± 0.9 weeks). We followed up children at 5 years; we measured anthropometry and body composition using dual-energy X-ray absorptiometry (DXA). We measured fasting leptin levels using immunoassays (Luminex) in 328 children. We conducted linear regression analyses, adjusting for potential confounders. RESULTS: Maternal glycaemia at first trimester was associated with childhood leptin levels at 5 years, independently of maternal pre-pregnancy BMI and other confounders (ß = .09 ± .04; P = .03). Higher post-load glucose levels at second trimester were associated with greater total body fat percentage measured by DXA (1 hour-glucose: ß = .010 ± .004; P = .03 and 2 hours-glucose: ß = .016 ± .005; P = .002), but not with leptin levels. CONCLUSIONS: Our results suggest that programming of leptin regulation may be sensitive to maternal hyperglycaemia specifically in early pregnancy.
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Adiposidad , Glucemia , Leptina/sangre , Obesidad Infantil , Cohorte de Nacimiento , Índice de Masa Corporal , Preescolar , Femenino , Humanos , Madres , Obesidad Infantil/epidemiología , Embarazo , Efectos Tardíos de la Exposición Prenatal , Estudios ProspectivosRESUMEN
For most organisms, iron is an essential nutrient that is both difficult to acquire from the environment and toxic at high concentration. Therefore, to avoid deprivation or over-abundance of iron, bacteria and eukaryotes have developed a tight regulatory system to keep the metal within a narrow concentration range. Recent work in the bacteria Escherichia coli and in Pseudomonas aeruginosa has demonstrated that small regulatory RNAs function post-transcriptionally to repress iron-using proteins, thereby ensuring that limited iron resources are allocated to crucial cellular functions during iron starvation. Following this discovery, a parallel mechanism that uses a protein and not a small RNA was described in the budding yeast Saccharomyces cerevisiae under iron restriction. The common characteristics of these three different organisms suggest a novel mechanism of iron homeostasis.
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Homeostasis , Hierro/metabolismo , Animales , Regulación hacia Abajo , Regulación de la Expresión Génica , Humanos , ARN/genética , Proteínas Represoras/metabolismoRESUMEN
Herein, we investigated the survival roles of Fak, Src, MEK/Erk, and PI3-K/Akt-1 in intestinal epithelial cancer cells (HCT116, HT29, and T84), in comparison to undifferentiated and differentiated intestinal epithelial cells (IECs). We report that: (1) cancer cells display striking anoikis resistance, as opposed to undifferentiated/differentiated IECs; (2) under anoikis conditions and consequent Fak down-activation, cancer cells nevertheless exhibit sustained Fak-Src interactions and Src/MEK/Erk activation, unlike undifferentiated/differentiated IECs; however, HCT116 and HT29 cells exhibit a PI3-K/Akt-1 down-activation, as undifferentiated/differentiated IECs, whereas T84 cells do not; (3) cancer cells require MEK/Erk for survival, as differentiated (but not undifferentiated) IECs; however, T84 cells do not require Fak and HCT116 cells do not require PI3-K/Akt-1, in contrast to the other cells studied; (4) Src acts as a cornerstone in Fak-mediated signaling to MEK/Erk and PI3-K/Akt-1 in T84 cells, as in undifferentiated IECs, whereas PI3-K/Akt-1 is Src-independent in HCT116, HT29 cells, as in differentiated IECs; and (5) EGFR activity inhibition abrogates anoikis resistance in cancer cells through a loss of Fak-Src interactions and down-activation of Src/MEK/Erk (T84, HCT116, HT29 cells) and PI3-K/Akt-1 (T84 cells). Hence, despite distinctions in signaling behavior not necessarily related to undifferentiated or differentiated IECs, intestinal epithelial cancer cells commonly display an EGFR-mediated sustained activation of Src under anoikis conditions. Furthermore, such sustained Src activation confers anoikis resistance at least in part through a consequent sustenance of Fak-Src interactions and MEK/Erk activation, thus not only overriding Fak-mediated signaling to MEK/Erk and/or PI3-K/Akt-1, but also the requirement of Fak and/or PI3-K/Akt-1 for survival.
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Anoicis , Receptores ErbB , Quinasa 1 de Adhesión Focal/metabolismo , Neoplasias Intestinales/patología , Transducción de Señal , Familia-src Quinasas/metabolismo , Diferenciación Celular , Línea Celular , Línea Celular Tumoral , Células Epiteliales/citología , Células Epiteliales/patología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Mucosa Intestinal/citología , Neoplasias Intestinales/enzimología , Neoplasias Intestinales/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Iron is one of the most important metals in the metabolism of many organisms, including bacteria, in which it serves as a cofactor in multiple enzymatic reactions. Most of the earlier research on iron regulation in bacteria has focused on the transcriptional regulator Fur and its effect on the many genes involved in iron uptake. More recent work demonstrates the essential role of a small regulatory RNA, RyhB, in iron metabolism. RyhB downregulates a large number of transcripts encoding iron-using proteins, resulting in redistribution of the intracellular iron. Recent advances have been made in the understanding of the small RNAs that modulate the intracellular iron usage in different organisms such as, Escherichia coli, Pseudomonas aeruginosa, Vibrio cholerae, Shigella flexneri and cyanobacteria.
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Bacterias/metabolismo , Hierro/metabolismo , ARN Bacteriano/metabolismo , ARN no Traducido/metabolismo , Bacterias/genética , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Proteínas Represoras/genéticaRESUMEN
The molecular determinants which dictate survival and apoptosis/anoikis in human intestinal crypt cells remain to be fully understood. To this effect, the roles of beta1 integrin/Fak/Src signaling to the PI3-K/Akt-1, MEK/Erk, and p38 pathways, were investigated. The regulation of six Bcl-2 homologs (Bcl-2, Mcl-1, Bcl-X(L), Bax, Bak, Bad) was likewise analyzed. We report that: (1) Anoikis causes a down-activation of Fak, Src, Akt-1 and Erk1/2, a loss of Fak-Src association, and a sustained/enhanced activation of p38beta, which is required as apoptosis/anoikis driver; (2) PI3-K/Akt-1 up-regulates the expression of Bcl-X(L) and Mcl-1, down-regulates Bax and Bak, drives Bad phosphorylation (both serine112/136 residues) and antagonizes p38beta activation; (3) MEK/Erk up-regulates Bcl-2, drives Bad phosphorylation (serine112 residue), but does not antagonize p38bactivation; (4) PI3-K/Akt-1 is required for survival, whereas MEK/Erk is not; (5) Src acts as a cornerstone in the engagement of both pathways by beta1 integrins/Fak, and is crucial for survival; and (6) beta1 integrins/Fak and/or Src regulate Bcl-2 homologs as both PI3-K/Atk-1 and MEK/Erk combined. Hence, beta1 integrin/Fak/Src signaling translates into integrated mediating functions of p38beta activation and regulation of Bcl-2 homologs by PI3-K/Akt-1 and MEK/Erk, consequently determining their requirement (or not) for survival.
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Anoicis/fisiología , Supervivencia Celular/fisiología , Quinasa 1 de Adhesión Focal/fisiología , Integrina beta1/fisiología , Mucosa Intestinal/fisiología , Proteína Oncogénica pp60(v-src)/fisiología , Transducción de Señal/fisiología , Células Cultivadas , Regulación hacia Abajo , Humanos , Mucosa Intestinal/citología , Quinasas Quinasa Quinasa PAM/fisiología , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteína Quinasa 11 Activada por Mitógenos/fisiología , Proteína Quinasa 3 Activada por Mitógenos/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Regulación hacia ArribaRESUMEN
DNA double-strand breaks (DSBs) represent a major threat to the genetic integrity of the cell. Knowing both their genome-wide distribution and number is important for a better assessment of genotoxicity at a molecular level. Available methods may have underestimated the extent of DSBs as they are based on markers specific to those undergoing active repair or may not be adapted for the large diversity of naturally occurring DNA ends. We have established conditions for an efficient first step of DNA nick and gap repair (NGR) allowing specific determination of DSBs by end labeling with terminal transferase. We used DNA extracted from HeLa cells harboring an I-SceI cassette to induce a targeted nick or DSB and demonstrated by immunocapture of 3'-OH that a prior step of NGR allows specific determination of loci-specific or genome wide DSBs. This method can be applied to the global determination of DSBs using radioactive end labeling and can find several applications aimed at understanding the distribution and kinetics of DSBs formation and repair.
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Mapeo Cromosómico/métodos , ADN Nucleotidilexotransferasa/genética , Reparación del ADN , ADN/genética , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Genoma Humano , Proteínas de Saccharomyces cerevisiae/genética , ADN/metabolismo , Roturas del ADN de Doble Cadena , ADN Nucleotidilexotransferasa/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Sitios Genéticos , Células HeLa , Humanos , Cinética , Radioisótopos de Fósforo/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , TransfecciónRESUMEN
The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to the next generation. So far, molecular studies of this morphological transition have been hampered by the lack of a method allowing adequate separation of these important steps of spermatid differentiation for subsequent analyses. Earlier attempts at proper gating of these cells using flow cytometry may have been difficult because of a peculiar increase in DNA fluorescence in spermatids undergoing chromatin remodeling. Based on this observation, we provide details of a simple flow cytometry scheme, allowing reproducible purification of four populations of mouse spermatids fixed with ethanol, each representing a different state in the nuclear remodeling process. Population enrichment is confirmed using step-specific markers and morphological criterions. The purified spermatids can be used for genomic and proteomic analyses.
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Citometría de Flujo/métodos , Espermátides/clasificación , Animales , Diferenciación Celular/fisiología , ADN/análisis , Masculino , Ratones , Proteómica/métodos , Espermátides/química , Espermátides/citología , EspermatogénesisRESUMEN
During the haploid phase of spermatogenesis, spermatids undergo a complex remodeling of the paternal genome involving the finely orchestrated replacement of histones by the highly-basic protamines. The associated striking change in DNA topology is characterized by a transient surge of both single- and double-stranded DNA breaks in the whole population of spermatids which are repaired before spermiation. These transient DNA breaks are now considered part of the normal differentiation program of these cells. Despite an increasing interest in the study of spermiogenesis in the last decade and the potential threat to the haploid genome, the origin of these DNA breaks still remains elusive. This review briefly outlines the current hypotheses regarding possible mechanisms that may lead to such transient DNA fragmentation including torsional stress, enzyme-induced breaks, apoptosis-like processes or oxidative stress. A better understanding of the origin of these DNA breaks will lead to further investigations on the genetic instability and mutagenic potential induced by the chromatin remodeling.
Lors de la phase haploïde de la spermatogenèse, les spermatides subissent un remodelage complexe du génome paternel impliquant un remplacement finement orchestré des histones par des protamines hautement basiques. Le changement topologique de l'ADN associé est caractérisé par une augmentation transitoire de cassures simple et double brins de l'ADN dans l'entière population des spermatides qui sont réparées avant la spermiation. Ces cassures transitoires de l'ADN sont maintenant considérées comme faisant partie du processus normal de différenciation de ces cellules. Malgré un intérêt croissant dans l'étude de la spermiogenèse ces 10 dernières années et la menace potentielle pour le génome haploïde, l'origine de ces cassures d'ADN reste encore incertaine. Cette revue décrit brièvement les hypothèses actuelles concernant les mécanismes possibles qui pourraient mener à cette fragmentation transitoire de l'ADN incluant le stress torsionnel, les cassures enzymatiques, des processus semblables à l'apoptose et le stress oxidatif. Une meilleure compréhension de l'origine de ces cassures d'ADN mènerait à des études approfondies concernant l'instabilité génétique et le potentiel mutagène induit par le remodelage de la chromatine.
RESUMEN
Determination of cellular DNA damage has so far been limited to global assessment of genome integrity whereas nucleotide-level mapping has been restricted to specific loci by the use of specific primers. Therefore, only limited DNA sequences can be studied and novel regions of genomic instability can hardly be discovered. Using a well-characterized yeast model, we describe a straightforward strategy to map genome-wide DNA strand breaks without compromising nucleotide-level resolution. This technique, termed "damaged DNA immunoprecipitation" (dDIP), uses immunoprecipitation and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL) to capture DNA at break sites. When used in combination with microarray or next-generation sequencing technologies, dDIP will allow researchers to map genome-wide DNA strand breaks as well as other types of DNA damage and to establish a clear profiling of altered genes and/or intergenic sequences in various experimental conditions. This mapping technique could find several applications for instance in the study of aging, genotoxic drug screening, cancer, meiosis, radiation and oxidative DNA damage.