Sensitive detection of BRAF V600E mutation by Amplification Refractory Mutation System (ARMS)-PCR

BACKGROUND: BRAF mutations occur in approximately 8% of all human cancers and approach 50% in melanoma and papillary carcinoma of thyroid. These mutations provide potentially valuable diagnostic, prognostic and treatment response prediction markers. A sensitive, specific, low-cost assay to detect these mutations is needed. RESULTS: To detect BRAF V600E mutation in formalin-fixed, paraffin-embedded (FFPE) tissue, we developed a method using Amplification Refractory Mutation System (ARMS)-PCR. This method was designed to amplify three products in a single reaction tube: a 200 bp common product serving as an amplification control, a 144 bp BRAF V600E specific product, and a 97 bp wild-type (wt) specific product. The sensitivity of this method was determined to be as low as 0.5% for the BRAF V600E allele in a wild-type background. This method was successfully validated in 72 thyroid tumors. It detected V600E mutation in 22 out of 33 (67%) of the conventional papillary thyroid carcinoma (PTC), 8 out of 12 (75%) of the tall-cell variant of PTC, whereas none of the 10 follicular variant of PTC showed BRAF V600E mutation. In addition, none of the 14 follicular adenomas and 3 follicular carcinomas had BRAF V600E mutation. As a comparison method, direct dideoxy sequencing found only 27 out of 30 (90%) mutations detected by ARMS-PCR method, suggesting that this ARMS-PCR method has higher sensitivity. CONCLUSIONS: Our ARMS-PCR method provides a new tool for rapid detection of BRAF V600E mutation. Our results indicate that ARMS-PCR is more sensitive than automated dideoxy sequencing in detecting low BRAF V600E allele burdens in FFPE tumor specimen. The strategy of this ARMS-PCR design may be adapted for early detection of point mutations of a variety of biomarker genes

SIRT1 Promotes N-Myc Oncogenesis through a Positive Feedback Loop Involving the Effects of MKP3 and ERK on N-Myc Protein Stability

The N-Myc oncoprotein is a critical factor in neuroblastoma tumorigenesis which requires additional mechanisms converting a low-level to a high-level N-Myc expression. N-Myc protein is stabilized when phosphorylated at Serine 62 by phosphorylated ERK protein. Here we describe a novel positive feedback loop whereby N-Myc directly induced the transcription of the class III histone deacetylase SIRT1, which in turn increased N-Myc protein stability. SIRT1 binds to Myc Box I domain of N-Myc protein to form a novel transcriptional repressor complex at gene promoter of mitogen-activated protein kinase phosphatase 3 (MKP3), leading to transcriptional repression of MKP3, ERK protein phosphorylation, N-Myc protein phosphorylation at Serine 62, and N-Myc protein stabilization. Importantly, SIRT1 was up-regulated, MKP3 down-regulated, in pre-cancerous cells, and preventative treatment with the SIRT1 inhibitor Cambinol reduced tumorigenesis in TH-MYCN transgenic mice. Our data demonstrate the important roles of SIRT1 in N-Myc oncogenesis and SIRT1 inhibitors in the prevention and therapy of N-Myc–induced neuroblastoma

Comparative study of topological Hall effect and skyrmions in NiMnIn and NiMnGa

A nonequilibrium rapid-quenching method has been used to fabricate NiMnIn and NiMnGa alloys that are chemically and morphologically similar but crystallographically and physically very different. NiMnGa crystallizes in a Ni2In-type hexagonal structure, whereas NiMnIn is a cubic Heusler alloy. Both alloys yield a topological Hall effect contribution corresponding to bubble-type skyrmion spin structures, but it occurs in much lower magnetic fields in NiMnIn as compared to NiMnGa. The effect is unrelated to net Dzyaloshinskii-Moriya interactions, which are absent in both alloys due to their inversion-symmetric crystal structures. Based on magnetic-force microscopy, we explain the difference between the two alloys by magnetocrystalline anisotropy and uniaxial and cubic anisotropies yielding full-fledged and reduced topological Hall effects, respectively. Since NiMnIn involves small magnetic fields (0.02–0.3 kOe) at and above room temperature, it is of potential interest in spin electronics

Band offsets of atomic-layer-deposited Al2O3 on GaAs and the effects of surface treatment

The metal gate/high-k dielectric/III-V semiconductor band alignment is one of the most technologically important parameters. We report the band offsets of the Al/Al2O3/GaAs structure and the effect of GaAs surface treatment. The energy barrier at the Al2O3 and sulfur-passivated GaAs interface is found to be 3.0 +/- 0.1 eV whereas for the unpassivated or NH4OH-treated GaAs is 3.6 eV. At the Al/Al2O3 interface, all samples yield the same barrier height of 2.9 +/- 0.2 eV. With a band gap of 6.4 +/- 0.05 eV for Al2O3, the band alignments at both Al2O3 interfaces are established. (C) 2008 American Institute of Physics