Effects of Thyroxine Exposure on Osteogenesis in Mouse Calvarial Pre-Osteoblasts

The incidence of craniosynostosis is one in every 1,800-2500 births. The gene-environment model proposes that if a genetic predisposition is coupled with environmental exposures, the effects can be multiplicative resulting in severely abnormal phenotypes. At present, very little is known about the role of gene-environment interactions in modulating craniosynostosis phenotypes, but prior evidence suggests a role for endocrine factors. Here we provide a report of the effects of thyroid hormone exposure on murine calvaria cells. Murine derived calvaria cells were exposed to critical doses of pharmaceutical thyroxine and analyzed after 3 and 7 days of treatment. Endpoint assays were designed to determine the effects of the hormone exposure on markers of osteogenesis and included, proliferation assay, quantitative ALP activity assay, targeted qPCR for mRNA expression of Runx2, Alp, Ocn, and Twist1, genechip array for 28,853 targets, and targeted osteogenic microarray with qPCR confirmations. Exposure to thyroxine stimulated the cells to express ALP in a dose dependent manner. There were no patterns of difference observed for proliferation. Targeted RNA expression data confirmed expression increases for Alp and Ocn at 7 days in culture. The genechip array suggests substantive expression differences for 46 gene targets and the targeted osteogenesis microarray indicated 23 targets with substantive differences. 11 gene targets were chosen for qPCR confirmation because of their known association with bone or craniosynostosis (Col2a1, Dmp1, Fgf1, 2, Igf1, Mmp9, Phex, Tnf, Htra1, Por, and Dcn). We confirmed substantive increases in mRNA for Phex, FGF1, 2, Tnf, Dmp1, Htra1, Por, Igf1 and Mmp9, and substantive decreases for Dcn. It appears thyroid hormone may exert its effects through increasing osteogenesis. Targets isolated suggest a possible interaction for those gene products associated with calvarial suture growth and homeostasis as well as craniosynostosis. © 2013 Cray et al

Numerical simulation of high pressure release and dispersion of hydrogen into air with real gas model

Hydrogen is a renewable and clean source of energy, and it is a good replacement for the current fossil fuels. Nevertheless, hydrogen should be stored in high-pressure reservoirs to have sufficient energy. An in-house code is developed to numerically simulate the release of hydrogen from a high-pressure tank into ambient air with more accuracy. Real gas models are used to simulate the flow since high-pressure hydrogen deviates from ideal gas law. Beattie–Bridgeman and Abel Noble equations are applied as real gas equation of state. A transport equation is added to the code to calculate the concentration of the hydrogen–air mixture after release. The uniqueness of the code is to simulate hydrogen in air release with the real gas model. Initial tank pressures of up to 70 MPa are simulated

A Non-Derivatized Assay for the Simultaneous Detection of Amino Acids, Acylcarnitines, Succinylacetone, Creatine, and Guanidinoacetic Acid in Dried Blood Spots via Tandem Mass Spectrometry

Guanidinoacetate methyltransferase (GAMT) deficiency is an autosomal recessive genetic disorder which results in global developmental delay and intellectual disability. There is evidence that early treatment prevents intellectual disability and seizures. GAMT deficiency is now being discussed as a potential addition to the U.S. Recommended Uniform Screening Panel (RUSP); the availability of suitable screening methods must be considered. A neonatal screening derivatized method to quantify creatine (CRE) and guanidinoacetic acid (GAA) in dried blood spots by tandem mass spectrometry (MS/MS) has been described. Its key feature is the ability to detect CRE and GAA in the same extract generated from neonatal dried blood spots (DBS’s) during amino acids (AA) and acylcarnitines (AC) analysis. More laboratories are adopting non-derivatized MS/MS screening methods. We describe an improved, non-derivatized DBS extraction and MS/MS analytical method (AAAC-GAMT) that incorporates quantitation of CRE and GAA into routine analysis of amino acids, acylcarnitines, and succinylacetone. The non-derivatized AAAC-GAMT method performs comparably to the stand-alone GAMT and non-derivatized AAAC screening methods, supporting its potential suitability for high-throughput GAMT neonatal screening

Targeted Osteogenic MicroArray, Genes Dysregulated after Thyroxine Treatment.

<p>Targeted Osteogenic MicroArray, Genes Dysregulated after Thyroxine Treatment.</p

Quantitative Polymerase Chain Reaction Primer Data.

<p>Quantitative Polymerase Chain Reaction Primer Data.</p

Gene-Chip MicroArray, Genes Dysregulated after 7 days of Thyroxine Treatment.

<p>Gene-Chip MicroArray, Genes Dysregulated after 7 days of Thyroxine Treatment.</p

<i>Proliferation</i>: Cell proliferation assay after thyroxine treatment.

<p>Reference line indicates control (untreated) response and experimental group plotted as percent response compared to reference (error bars = standard error of the mean). Note the increases in proliferation after 3 and 7 days of treatment with the exception of the highest dose after 7 days.</p

<i>Gene Expression</i>: Fold change mRNA for markers of osteoblastogenesis after thyroxine treatment for 3 or 7 days (error bars = standard error of the mean fold change).

<p>Note greater than two-fold up-regulations (reference lines) for alkaline phosphatase and osteocalcin.</p