Photocatalytic Nanofabrication and Intracellular Raman Imaging of Living Cells with Functionalized AFM Probes.

Atomic force microscopy (AFM) is an effective platform for in vitro manipulation and analysis of living cells in medical and biological sciences. To introduce additional new features and functionalities into a conventional AFM system, we investigated the photocatalytic nanofabrication and intracellular Raman imaging of living cells by employing functionalized AFM probes. Herein, we investigated the effect of indentation speed on the cell membrane perforation of living HeLa cells based on highly localized photochemical oxidation with a catalytic titanium dioxide (TiO2)-functionalized AFM probe. On the basis of force-distance curves obtained during the indentation process, the probability of cell membrane perforation, penetration force, and cell viability was determined quantitatively. Moreover, we explored the possibility of intracellular tip-enhanced Raman spectroscopy (TERS) imaging of molecular dynamics in living cells via an AFM probe functionalized with silver nanoparticles in a homemade Raman system integrated with an inverted microscope. We successfully demonstrated that the intracellular TERS imaging has the potential to visualize distinctly different features in Raman spectra between the nucleus and the cytoplasm of a single living cell and to analyze the dynamic behavior of biomolecules inside a living cell.

TYK2 Promoter Variant and Diabetes Mellitus in the Japanese.

BACKGROUND: Recently, natural mutation of Tyrosine kinase 2 (Tyk2) gene has been shown to determine susceptibility to murine virus-induced diabetes. In addition, a previous human genome-wide study suggested the type 1 diabetes (T1D) susceptibility region to be 19p13, where the human TYK2 gene is located (19p13.2). METHODS: Polymorphisms of TYK2 gene at the promoter region and exons were studied among 331 healthy controls, and 302 patients with T1D and 314 with type 2 diabetes (T2D) in the Japanese. FINDINGS: A TYK2 promoter haplotype with multiple genetic polymorphisms, which are in complete linkage disequilibrium, named TYK2 promoter variant, presenting decreased promoter activity, is associated with an increased risk of not only T1D (odds ratio (OR), 2.4; 95% confidence interval (CI), 1.2 to 4.6; P = 0.01), but also T2D (OR, 2.1; 95% CI, 1.1 to 4.1; P = 0.03). The risk is high in patients with T1D associated with flu-like syndrome at diabetes onset and also those without anti-glutamic acid decarboxylase autoantibody. INTERPRETATION: The TYK2 promoter variant is associated with an overall risk for diabetes, serving a good candidate as a virus-induced diabetes susceptibility gene in humans. FUNDING: Ministry of Education, Culture, Sports, Science and Technology and of Health, Labor and Welfare of Japan.

Reduced Tyk2 gene expression in ß-cells due to natural mutation determines susceptibility to virus-induced diabetes.

Accumulating evidence suggests that viruses play an important role in the development of diabetes. Although the diabetogenic encephalomyocarditis strain D virus induces diabetes in restricted lines of inbred mice, the susceptibility genes to virus-induced diabetes have not been identified. We report here that novel Tyrosine kinase 2 (Tyk2) gene mutations are present in virus-induced diabetes-sensitive SJL and SWR mice. Mice carrying the mutant Tyk2 gene on the virus-resistant C57BL/6 background are highly sensitive to virus-induced diabetes. Tyk2 gene expression is strongly reduced in Tyk2-mutant mice, associated with low Tyk2 promoter activity, and leads to decreased expression of interferon-inducible genes, resulting in significantly compromised antiviral response. Tyk2-mutant pancreatic ß-cells are unresponsive even to high dose of Type I interferon. Reversal of virus-induced diabetes could be achieved by ß-cell-specific Tyk2 gene expression. Thus, reduced Tyk2 gene expression in pancreatic ß-cells due to natural mutation is responsible for susceptibility to virus-induced diabetes.