200 research outputs found
Prenatal screening of sialic acid storage disease and confirmation in cultured fibroblasts by LC-MS/MS
Sialic acid storage disease (SASD) is an inborn error resulting from defects in the lysosomal membrane protein sialin. The SASD phenotypical spectrum ranges from a severe presentation, infantile sialic acid storage disease (ISSD) which may present as hydrops fetalis, to a relatively mild form, Salla disease. Screening for SASD is performed by determination of free sialic acid (FSA) in urine or amniotic fluid supernatant (AFS). Subsequent diagnosis of SASD is performed by quantification of FSA in cultured fibroblasts and by mutation analysis of the sialin gene, SLC17A5. We describe simple quantitative procedures to determine FSA as well as conjugated sialic acid in AFS, and FSA in cultured fibroblasts, using isotope dilution (13C3-sialic acid) and multiple reaction monitoring LC-ESI-MS/MS. The whole procedure can be performed in 2–4 h. Reference values in AFS were 0–8.2 μmol/L for 15–25 weeks of gestation and 3.2-12.0 μmol/L for 26–38 weeks of gestation. In AFS samples from five fetuses affected with ISSD FSA was 23.9-58.9 μmol/L demonstrating that this method is able to discriminate ISSD pregnancies from normal ones. The method was also validated for determination of FSA in fibroblast homogenates. FSA in SASD fibroblasts (ISSD; 20–154 nmol/mg protein, intermediate SASD; 12.9-15.1 nmol/mg, Salla disease; 5.9-7.4 nmol/mg) was clearly elevated compared to normal controls (0.3-2.2 nmol/mg). In conclusion, we report simple quantitative procedures to determine FSA in AFS and cultured fibroblasts improving both prenatal diagnostic efficacy for ISSD as well as confirmatory testing in cultured fibroblasts following initial screening in urine or AFS
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Analysis of thermally-degrading, confined HMX
The response of a thermally-degrading, confined HMX pellet is analyzed using a Reactive Elastic-Plastic (REP) constitutive model which is founded on the collapse and growth of internal inclusions resulting from physical and chemical processes such as forced displacement, thermal expansion, and/or decomposition. Axial stress predictions compare adequately to data. Deficiencies in the model and future directions are discussed
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Examination of phase transformations and decomposition chemistry in thermally aged thin-film explosives
To develop predictive models for the response of weapon systems to abnormal thermal environments, such as cookoff, an improved understanding of temperature-dependent thermophysical phenomena (such as phase transformations) and decomposition chemistry in totally confined explosive samples is needed. It is particularly important to examine the effects of maintaining-intimate contact between the decomposition products and the remaining condensed-phase explosive during slow reaction at elevated temperatures since confinement of the decomposition products may significantly affect thermophysical phenomena and decomposition reaction rates. The purpose of this work has been to examine experimentally the effects on condensed-phase chemistry which result when decomposition products remain in intimate contact with the reacting explosive during isothermal aging experiments at temperatures below those of the DTA exotherm for the explosive. To provide confinement, minimize vapor space, and permit condensed-phase chemical analysis, experiments were done using thin-film samples of the explosive, which were pressed and sealed between two infrared-transmitting windows, so that condensed-phase chemistry could be monitored using infrared spectroscopy. Experiments were done with NC, HMX, HMX-NC composite, and RDX samples. Results from the experiments with NC showed that for some decomposition mechanisms, the reaction rates for confined samples compared favorably with published reaction rates from unconfined samples. However, the results also demonstrated that for other mechanisms, the reaction rates were significantly affected by confinement of the decomposition products. The experiments with HMX and RDX indicated that some decomposition occurred at temperatures well below the temperatures of the respective DTA exotherms, and the experiments with HMX-NC composite samples showed some interaction between NC and HMX at temperatures as low as 150{degrees}C
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Mechanical Response and Decomposition of Thermally Degraded Energetic Materials: Experiments and Model Simulations
We report progress of a continuing effort to characterize and simulate the response of energetic materials (EMs), primarily HMX-based, under conditions leading to cookoff. Our experiments include mechanical-effects testing of HMX and FIMX with binder at temperatures nearing decomposition thresholds. Additional experiments have focused on decomposition of these EMs under confinement, measuring evolution of gas products and observing the effect of pressurization on the solid. Real-time measurements on HMX show abrupt changes that maybe due to sudden void collapse under increasing load. Postmortem examination shows significant internal damage to the pellets, including voids and cracks. These experiments have been used to help develop a constitutive model for pure HMX. Unconfined uniaxial compression tests were performed on HMX and LX-14 to examine the effect of binders on the deviatoric strength of EM pellets, and to assess the need of including deviatoric terms in the model. A scale-up experiment will be described that is being developed to validate the model and provide additional diagnostics
Mammalian Target of Rapamycin Is a Therapeutic Target for Murine Ovarian Endometrioid Adenocarcinomas with Dysregulated Wnt/β-Catenin and PTEN
Despite the fact that epithelial ovarian cancers are the leading cause of death from gynecological cancer, very little is known about the pathophysiology of the disease. Mutations in the WNT and PI3K pathways are frequently observed in the human ovarian endometrioid adenocarcinomas (OEAs). However, the role of WNT/β-catenin and PTEN/AKT signaling in the etiology and/or progression of this disease is currently unclear. In this report we show that mice with a gain-of-function mutation in β-catenin that leads to dysregulated nuclear accumulation of β-catenin expression in the ovarian surface epithelium (OSE) cells develop indolent, undifferentiated tumors with both mesenchymal and epithelial characteristics. Combining dysregulated β-catenin with homozygous deletion of PTEN in the OSE resulted in development of significantly more aggressive tumors, which was correlated with inhibition of p53 expression and cellular senescence. Induced expression of both mTOR kinase, a master regulator of proliferation, and phosphorylation of its downstream target, S6Kinase was also observed in both the indolent and aggressive mouse tumors, as well as in human OEA with nuclear β-catenin accumulation. Ectopic allotransplants of the mouse ovarian tumor cells with a gain-of-function mutation in β-catenin and PTEN deletion developed into tumors with OEA histology, the growth of which were significantly inhibited by oral rapamycin treatment. These studies demonstrate that rapamycin might be an effective therapeutic for human ovarian endometrioid patients with dysregulated Wnt/β-catenin and Pten/PI3K signaling
A Mitochondrial Kinase Complex Is Essential to Mediate an ERK1/2-Dependent Phosphorylation of a Key Regulatory Protein in Steroid Biosynthesis
ERK1/2 is known to be involved in hormone-stimulated steroid synthesis, but its exact roles and the underlying mechanisms remain elusive. Both ERK1/2 phosphorylation and steroidogenesis may be triggered by cAMP/cAMP-dependent protein kinase (PKA)-dependent and-independent mechanisms; however, ERK1/2 activation by cAMP results in a maximal steroidogenic rate, whereas canonical activation by epidermal growth factor (EGF) does not. We demonstrate herein by Western blot analysis and confocal studies that temporal mitochondrial ERK1/2 activation is obligatory for PKA-mediated steroidogenesis in the Leydig-transformed MA-10 cell line. PKA activity leads to the phosphorylation of a constitutive mitochondrial MEK1/2 pool with a lower effect in cytosolic MEKs, while EGF allows predominant cytosolic MEK activation and nuclear pERK1/2 localization. These results would explain why PKA favors a more durable ERK1/2 activation in mitochondria than does EGF. By means of ex vivo experiments, we showed that mitochondrial maximal steroidogenesis occurred as a result of the mutual action of steroidogenic acute regulatory (StAR) protein –a key regulatory component in steroid biosynthesis-, active ERK1/2 and PKA. Our results indicate that there is an interaction between mitochondrial StAR and ERK1/2, involving a D domain with sequential basic-hydrophobic motifs similar to ERK substrates. As a result of this binding and only in the presence of cholesterol, ERK1/2 phosphorylates StAR at Ser232. Directed mutagenesis of Ser232 to a non-phosphorylable amino acid such as Ala (StAR S232A) inhibited in vitro StAR phosphorylation by active ERK1/2. Transient transfection of MA-10 cells with StAR S232A markedly reduced the yield of progesterone production. In summary, here we show that StAR is a novel substrate of ERK1/2, and that mitochondrial ERK1/2 is part of a multimeric protein kinase complex that regulates cholesterol transport. The role of MAPKs in mitochondrial function is underlined
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Reactive wave growth in shock-compressed thermally degraded high explosives
The authors have performed experiments to study the effect of thermal degradation on shock sensitivity and growth to detonation of several high-density plastic bonded explosives, confined in stainless steel cells. Assemblies were heated in situ in the target chamber of a light-gas gun. Confinement was varied to allow, in some cases, for thermal expansion of the explosive, and in other cases to vent the decomposition gases. Particle velocity profiles were measured using VISAR at a LiF window interface. Results for the IHE PBX-9502 showed that its sensitivity to shock initiation could be dramatically increased or decreased depending on the confinement conditions during heating. Effects were much less pronounced for PBX-9404 and PBX-9501
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