Novel ACADVL variants resulting in mitochondrial defects in long-chain acyl-CoA dehydrogenase deficiency / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The pathogenesis of very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is highly heterogeneous and still unclear. Additional novel variants have been recently detected in the population. The molecular and cellular effects of these previously unreported variants are still poorly understood and require further characterization. To address this problem, we have evaluated the various functions and biochemical consequences of six novel missense variants that lead to mild VLCAD deficiency. Marked deficiencies in fatty acid oxidation (FAO) and other mitochondrial defects were observed in cells carrying one of these six variants (c.541C>T, c.863T>G, c.895A>G, c.1238T>C, c.1276G>A, and c.1505T>A), including reductions in mitochondrial respiratory-chain function and adenosine triphosphate (ATP) production, and increased levels of mitochondrial reactive oxygen species (ROS). Intriguingly, higher apoptosis levels were found in cells carrying the mutant VLCAD under glucose-limited stress. Moreover, the stability of the mutant homodimer was disturbed, and major conformational changes in each mutant VLCAD structure were predicted by molecular dynamics (MD) simulation. The data presented here may provide valuable information for improving management of diagnosis and treatment of VLCAD deficiency and for a better understanding of the general molecular bases of disease variability.

In vitro culture and characterization of alveolar bone osteoblasts isolated from type 2 diabetics

In order to understand the mechanisms of poor osseointegration following dental implants in type 2 diabetics, it is important to study the biological properties of alveolar bone osteoblasts isolated from these patients. We collected alveolar bone chips under aseptic conditions and cultured them in vitro using the tissue explants adherent method. The biological properties of these cells were characterized using the following methods: alkaline phosphatase (ALP) chemical staining for cell viability, Alizarin red staining for osteogenic characteristics, MTT test for cell proliferation, enzyme dynamics for ALP contents, radio-immunoassay for bone gla protein (BGP) concentration, and ELISA for the concentration of type I collagen (COL-I) in the supernatant. Furthermore, we detected the adhesion ability of two types of cells from titanium slices using non-specific immunofluorescence staining and cell count. The two cell forms showed no significant difference in morphology under the same culture conditions. However, the alveolar bone osteoblasts received from type 2 diabetic patients had slower growth, lower cell activity and calcium nodule formation than the normal ones. The concentration of ALP, BGP and COL-I was lower in the supernatant of alveolar bone osteoblasts received from type 2 diabetic patients than in that received from normal subjects (P < 0.05). The alveolar bone osteoblasts obtained from type 2 diabetic patients can be successfully cultured in vitro with the same morphology and biological characteristics as those from normal patients, but with slower growth and lower concentration of specific secretion and lower combining ability with titanium than normal ones.

Detection of aberrant p16 methylation in the serum of colorectal cancer patients / 中华预防医学杂志

<p><b>OBJECTIVES</b>To detect aberrant p16 promoter methylation in serum of patients with colorectal cancer (CRC), and to explore the possibility of using this assay in early detection or as a prognostic marker.</p><p><b>METHODS</b>Methylation-specific PCR was used to detect p16 methylation in DNA extracted from 52 CRCs and corresponding serum samples. Serum samples from 34 patients with adenomatous polyps and 10 healthy individuals were used as controls. The association of p16 hypermethylation in serum DNA of CRC patients with clinicopathological characteristics was analyzed.</p><p><b>RESULTS</b>p16 methylation was found in 38% (20 of 52) of CRC tissues. Among the 20 patients with aberrant methylation in the tumor tissues, similar changes were also detected in the serum of 14 (70%) patients. No methylated p16 sequences were detected in the peripheral serum of the 32 CRC patients without these changes in the tumor, in 34 paitents with adenomatous polyps, or in 10 healthy controls. Clinicopathological analysis revealed that p16 methylation in serum was significantly associated with later Dukes' stage (chi(2) = 5.7, P = 0.03).</p><p><b>CONCLUSION</b>This assay offers a potential means for the serum-based detection and/or monitoring of CRC patients.</p>