Sex-specific programming effects of parental obesity in pre-implantation embryonic development.

Background: Obesity is a global rising problem with epidemiological dimension. Obese parents can have programming effects on their offspring leading to obesity and associated diseases in later life. This constitutes a vicious circle.

Late-pregnancy dysglycemia in obese pregnancies after negative testing for gestational diabetes and risk of future childhood overweight: An interim analysis from a longitudinal mother-child cohort study.

Background: Maternal pre-conception obesity is a strong risk factor for childhood overweight. However, prenatal mechanisms and their effects in susceptible gestational periods that contribute to this risk are not well understood. We aimed to assess the impact of late-pregnancy dysglycemia in obese pregnancies with negative testing for gestational diabetes mellitus (GDM) on long-term mother-child outcomes.

Modification of the fatty acid composition of an obesogenic diet improves the maternal and placental metabolic environment in obese pregnant mice.

Peri-conceptional exposure to maternal obesogenic nutrition is associated with in utero programming of later-life overweight and metabolic disease in the offspring. We aimed to investigate whether dietary intervention with a modified fatty acid quality in an obesogenic high-calorie (HC) diet during the preconception and gestational phases can improve unfavourable effects of an adipogenic maternal environment. In NMRI mice, peri-conceptional and gestational obesity was induced by feeding a HC diet (controls), and they were compared with dams on a fat-modified (Fat-mod) HC diet of the same energy content but enriched with medium-chain fatty acids (MCFAs) and adjusted to a decreased ratio of n-6 to n-3 long-chain polyunsaturated fatty acids (LC-PUFAs).

The leukemogenic fusion gene MLL-AF9 alters microRNA expression pattern and inhibits monoblastic differentiation via miR-511 repression.

Background: In this study we explored the role of microRNAs (miRNAs) as mediators of leukemogenic effects of the fusion gene MLL-AF9, which results from a frequent chromosomal translocation in infant and monoblastic acute myeloid leukemia (AML).

Methods: We performed a specific and efficient knockdown of endogenous MLL-AF9 in the human monoblastic AML cell line THP1.

Results: The knockdown associated miRNA expression profile revealed 21 MLL-AF9 dependently expressed miRNAs.

Obese Nondiabetic Pregnancies and High Maternal Glycated Hemoglobin at Delivery as an Indicator of Offspring and Maternal Postpartum Risks: The Prospective PEACHES Mother-Child Cohort.

Background: We investigated whether obese pregnant women negative for gestational diabetes (GDM) still experience dysglycemia, as indicated by high glycated hemoglobin (Hb A1c) at delivery, and whether this impacts offspring and long-term maternal outcomes.

Methods: Data of 462 mother-child pairs of our prospective Programming of Enhanced Adiposity Risk in Childhood - Early Screening (PEACHES) cohort study were analyzed. Of 885 obese and normal-weight pregnancies prospectively enrolled after GDM testing according to the International Association of Diabetes and Pregnancy Study Groups criteria, 462 GDM-negative mothers and their offspring were investigated.

RNAi-mediated silencing of MLL-AF9 reveals leukemia-associated downstream targets and processes.

Background: The translocation t(9;11)(p22;q23) leading to the leukemogenic fusion gene MLL-AF9 is a frequent translocation in infant acute myeloid leukemia (AML). This study aimed to identify genes and molecular processes downstream of MLL-AF9 (alias MLL-MLLT3) which could assist to develop new targeted therapies for such leukemia with unfavorable prognosis.

Methods: In the AML cell line THP1 which harbors this t(9;11) translocation, endogenous MLL-AF9 was silenced via siRNA while ensuring specificity of the knockdown and its efficiency on functional protein level.

Digital image analysis approach for lipid droplet size quantitation of Oil Red O-stained cultured cells.

A simple approach was developed for the quantification of lipid droplet size and frequency distribution in images acquired by standard light microscopy. Oil Red O-stained cell images were thresholded for the lipid droplet signal using the freely available imaging software ImageJ. Watershed algorithms allowed analyzing the area of each individual lipid droplet.

Cellular signaling of amino acids towards mTORC1 activation in impaired human leucine catabolism.

The regulation of cell growth and protein biosynthesis is triggered by the mammalian target of rapamycin complex 1 (mTORC1) responding to amino acids, especially leucine. The molecular mechanisms linking leucine to mTORC1 activation are not well understood. We analyzed whether the free intracellular leucine availability, a metabolite of leucine catabolism or the process of leucine oxidation activates mTORC1 signaling.

Changing metabolic signatures of amino acids and lipids during the prediabetic period in a pig model with impaired incretin function and reduced β-cell mass.

Diabetes is generally diagnosed too late. Therefore, biomarkers indicating early stages of β-cell dysfunction and mass reduction would facilitate timely counteraction. Transgenic pigs expressing a dominant-negative glucose-dependent insulinotropic polypeptide receptor (GIPR(dn)) reveal progressive deterioration of glucose control and reduction of β-cell mass, providing a unique opportunity to study metabolic changes during the prediabetic period.

In situ assay of fatty acid β-oxidation by metabolite profiling following permeabilization of cell membranes.

Quantitative analysis of mitochondrial FA β-oxidation (FAO) has drawn increasing interest for defining lipid-induced metabolic dysfunctions, such as in obesity-induced insulin resistance, and evaluating pharmacologic strategies to improve β-oxidation function. The aim was to develop a new assay to quantify β-oxidation function in intact mitochondria and with a low amount of cell material. Cell membranes of primary human fibroblasts were permeabilized with digitonin prior to a load with FFA substrate.

Pahenu1 is a mouse model for tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency and promotes analysis of the pharmacological chaperone mechanism in vivo.

The recent approval of sapropterin dihydrochloride, the synthetic form of 6[R]-l-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)), for the treatment of phenylketonuria (PKU) as the first pharmacological chaperone drug initiated a paradigm change in the treatment of monogenetic diseases. Symptomatic treatment is now replaced by a causal pharmacological therapy correcting misfolding of the defective phenylalanine hydroxylase (PAH) in numerous patients. Here, we disclose BH(4) responsiveness in Pah(enu1), a mouse model for PAH deficiency.

Stability of acylcarnitines and free carnitine in dried blood samples: implications for retrospective diagnosis of inborn errors of metabolism and neonatal screening for carnitine transporter deficiency.

Objective: Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) is increasingly used in newborn screening programs. Acylcarnitine profiles from dried blood spots (DBS) are used to detect fatty acid oxidation disorders, carnitine cycle disorders, and organic acidurias. Stored dried blood is also a valuable source for postmortem investigations to unravel the cause of unexplained death in early childhood.

X-linked adrenoleukodystrophy phenotype is independent of ABCD2 genotype.

Strikingly variable clinical phenotypes can be found in X-linked adrenoleukodystrophy (X-ALD) even with the same ABCD1 mutation. ABCD2 is the closest homolog to ABCD1. Since ABCD2 overexpression complements the loss of ABCD1 in vivo and in vitro, we have investigated the possible role of the ABCD2 gene locus as determinant of X-ALD phenotypes.

Loss of function in phenylketonuria is caused by impaired molecular motions and conformational instability.

A significant share of patients with phenylalanine hydroxylase (PAH) deficiency benefits from pharmacological doses of tetrahydrobiopterin (BH(4)), the natural PAH cofactor. Phenylketonuria (PKU) is hypothesized to be a conformational disease, with loss of function due to protein destabilization, and the restoration of enzyme function that is observed in BH(4) treatment might be transmitted by correction of protein misfolding. To elucidate the molecular basis of functional impairment in PAH deficiency, we investigated the impact of ten PAH gene mutations identified in patients with BH(4)-responsiveness on enzyme kinetics, stability, and conformation of the protein (F55L, I65S, H170Q, P275L, A300S, S310Y, P314S, R408W, Y414C, Y417H).

Cleavage of CXCR1 on neutrophils disables bacterial killing in cystic fibrosis lung disease.

Interleukin-8 (IL-8) activates neutrophils via the chemokine receptors CXCR1 and CXCR2. However, the airways of individuals with cystic fibrosis are frequently colonized by bacterial pathogens, despite the presence of large numbers of neutrophils and IL-8. Here we show that IL-8 promotes bacterial killing by neutrophils through CXCR1 but not CXCR2.

Newborn screening for 3-methylcrotonyl-CoA carboxylase deficiency: population heterogeneity of MCCA and MCCB mutations and impact on risk assessment.

New technology enables expansion of newborn screening (NBS) of inborn errors aimed to prevent adverse outcome. In conditions with a large share of asymptomatic phenotypes, the potential harm created by NBS must carefully be weighed against benefit. Policies vary throughout the United States, Australia, and Europe due to limited data on outcome and treatability of candidate screening conditions.

Disease manifestations and X inactivation in heterozygous females with Fabry disease.

Aim: Fabry disease is an X-linked lysosomal storage disorder characterized by an accumulation of neutral glycosphingolipids in multiple organ systems caused by alpha-galactosidase A deficiency due to mutations in the GLA gene. The majority of heterozygous females show the characteristic signs and symptoms of the disease, and some of them are severely affected. The current hypothesis for the occurrence of disease manifestations in females is skewed X inactivation favouring the mutant GLA allele.

Mitochondrial targeting signals and mature peptides of 3-methylcrotonyl-CoA carboxylase.

Inherited deficiency of 3-methylcrotonyl-CoA carboxylase (MCC), an enzyme of leucine degradation, is an organic acidemia detectable by expanded newborn screening with a variable phenotype that ranges from asymptomatic to death in infancy. Here, we show that the two subunits of the enzyme (MCCalpha; MCCbeta) are imported into the mitochondrial matrix by the classical pathway involving cleavable amino-terminal targeting presequences. We identified the cleavage sites (Tyr41/Thr42 and Ala22/Tyr23 for MCCalpha and MCCbeta, respectively) of the targeting signals and the amino-termini of the mature polypeptides of MCC and propionyl-CoA carboxylase, a mitochondrial paralog.

Effect of amino acid infusion on human postoperative colon protein synthesis in situ.

Background: Amino acids are an integral part of parenteral nutrition because of their anabolic action helping to conserve body protein after surgical stress. At the gastrointestinal tract, an adequate supply of amino acids may be particularly important because of the gut's high rate of protein turnover, cell division, and proliferation. However, no information is available about the effects of amino acids on human intestinal protein metabolism after surgery.

Reduced incidence of severe metabolic crisis or death in children with medium chain acyl-CoA dehydrogenase deficiency homozygous for c.985A>G identified by neonatal screening.

The incidence of severe metabolic crises in medium chain acyl-CoA dehydrogenase deficiency (MCADD) patients homozygous for the common c.985A>G mutation, who had been identified by neonatal screening, was assessed prospectively and compared to retrospective cohort data in unscreened patients with identical genotypes. Logrank test showed a significant reduction of severe metabolic crises in the screened cohort (p<0.

Population spectrum of ACADM genotypes correlated to biochemical phenotypes in newborn screening for medium-chain acyl-CoA dehydrogenase deficiency.

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most frequent inherited defect of fatty acid oxidation, with a significant morbidity and mortality in undiagnosed patients. Adverse outcomes can effectively be prevented by avoiding metabolic stress and following simple dietary measures. Therefore, prospective newborn screening (NBS) is being proposed for this condition.

Rapid second-tier molecular genetic analysis for congenital adrenal hyperplasia attributable to steroid 21-hydroxylase deficiency.

Background: Neonatal screening for steroid 21-hydroxylase (CYP21) deficiency is performed to identify congenital adrenal hyperplasia (CAH). The immunologic assay for 17alpha-hydroxyprogesterone (17-OHP) has a high rate of false positives. We assessed the potential for increasing the specificity for CAH by use of a second step involving analysis of the CYP21 gene.

Simultaneous quantitative and allele-specific expression analysis with real competitive PCR.

Background: For a diploid organism such as human, the two alleles of a particular gene can be expressed at different levels due to X chromosome inactivation, gene imprinting, different local promoter activity, or mRNA stability. Recently, imbalanced allelic expression was found to be common in human and can follow Mendelian inheritance. Here we present a method that employs real competitive PCR for allele-specific expression analysis.