Development of a one-dimensional Wolter mirror for an advanced Kirkpatrick-Baez mirror

To realize achromatic full-field hard X-ray microscopy with a resolution better than 100 nm, we studied an imaging system consisting of an elliptical mirror and a hyperbolic mirror. The figure accuracies of the elliptical and hyperbolic mirrors required to obtain diffraction-limited resolution were investigated using a wave-optical simulator, and then elliptical and hyperbolic mirrors were precisely fabricated, following the criterion of the figure accuracies. Experiments to form a demagnified image of a one-dimensional slit installed 45 m upstream were conducted using the imaging system at an X-ray energy of 11.5 keV at BL29XUL of SPring-8. The system could form a demagnified image with the best resolution of 78 nm. In addition, the field of view to obtain a resolution better than 200 nm was 4.2 micron. © 2010 SPIE.S. Matsuyama, T. Wakioka, H. Mimura, T. Kimura, N. Kidani, Y. Sano, Y. Nishino, K. Tamasaku, M. Yabashi, T. Ishikawa, and K. Yamauchi "Development of a one-dimensional Wolter mirror for an advanced Kirkpatrick-Baez mirror", Proc. SPIE 7802, Advances in X-Ray/EUV Optics and Components V, 780202 (27 August 2010); https://doi.org/10.1117/12.860405.SPIE Optical Engineering + Applications, 2010, San Diego, California, United State

A Quantitative Image Cytometry Technique for Time Series or Population Analyses of Signaling Networks

Background: Modeling of cellular functions on the basis of experimental observation is increasingly common in the field of cellular signaling. However, such modeling requires a large amount of quantitative data of signaling events with high spatio-temporal resolution. A novel technique which allows us to obtain such data is needed for systems biology of cellular signaling. Methodology/Principal Findings: We developed a fully automatable assay technique, termed quantitative image cytometry (QIC), which integrates a quantitative immunostaining technique and a high precision image-processing algorithm for cell identification. With the aid of an automated sample preparation system, this device can quantify protein expression, phosphorylation and localization with subcellular resolution at one-minute intervals. The signaling activities quantified by the assay system showed good correlation with, as well as comparable reproducibility to, western blot analysis. Taking advantage of the high spatio-temporal resolution, we investigated the signaling dynamics of the ERK pathway in PC12 cells. Conclusions/Significance: The QIC technique appears as a highly quantitative and versatile technique, which can be a convenient replacement for the most conventional techniques including western blot, flow cytometry and live cell imaging

Sprouty2 and Spred1-2 Proteins Inhibit the Activation of the ERK Pathway Elicited by Cyclopentenone Prostanoids

Sprouty and Spred proteins have been widely implicated in the negative regulation of the fibroblast growth factor receptor-extracellular regulated kinase (ERK) pathway. In considering the functional role of these proteins, we explored their effects on ERK activation induced by cyclopentenone prostanoids, which bind to and activate Ras proteins. We therefore found that ectopic overexpression in HeLa cells of human Sprouty2, or human Spred1 or 2, inhibits ERK1/2 and Elk-1 activation triggered by the cyclopentenone prostanoids PGA1 and 15d-PGJ2. Furthermore, we found that in HT cells that do not express Sprouty2 due to hypermethylation of its gene-promoter, PGA1-provoked ERK activation was more intense and sustained compared to other hematopoietic cell lines with unaltered Sprouty2 expression. Cyclopentenone prostanoids did not induce Sprouty2 tyrosine phosphorylation, in agreement with its incapability to activate tyrosine-kinase receptors. However, Sprouty2 Y55F, which acts as a defective mutant upon tyrosine-kinase receptor stimulation, did not inhibit cyclopentenone prostanoids-elicited ERK pathway activation. In addition, Sprouty2 did not affect the Ras-GTP levels promoted by cyclopentenone prostanoids. These results unveil both common and differential features in the activation of Ras-dependent pathways by cyclopentenone prostanoids and growth factors. Moreover, they provide the first evidence that Sprouty and Spred proteins are negative regulators of the ERK/Elk-1 pathway activation induced not only by growth-factors, but also by reactive lipidic mediators

Suppression of Sproutys Has a Therapeutic Effect for a Mouse Model of Ischemia by Enhancing Angiogenesis

Sprouty proteins (Sproutys) inhibit receptor tyrosine kinase signaling and control various aspects of branching morphogenesis. In this study, we examined the physiological function of Sproutys in angiogenesis, using gene targeting and short-hairpin RNA (shRNA) knockdown strategies. Sprouty2 and Sprouty4 double knockout (KO) (DKO) mice were embryonic-lethal around E12.5 due to cardiovascular defects. The number of peripheral blood vessels, but not that of lymphatic vessels, was increased in Sprouty4 KO mice compared with wild-type (WT) mice. Sprouty4 KO mice were more resistant to hind limb ischemia and soft tissue ischemia than WT mice were, because Sprouty4 deficiency causes accelerated neovascularization. Moreover, suppression of Sprouty2 and Sprouty4 expression in vivo by shRNA targeting accelerated angiogenesis and has a therapeutic effect in a mouse model of hind limb ischemia. These data suggest that Sproutys are physiologically important negative regulators of angiogenesis in vivo and novel therapeutic targets for treating peripheral ischemic diseases