Fibulin-4 and -5, but not Fibulin-2, are Associated with Tropoelastin Deposition in Elastin-Producing Cell Culture

Elastic system fibers consist of microfibrils and tropoelastin. During development, microfibrils act as a template on which tropoelastin is deposited. Fibrillin-1 is the major component of microfibrils. It is not clear whether elastic fiber-associated molecules, such as fibulins, contribute to tropoelastin deposition. Among the fibulin family, fibulin-2, -4 and -5 are capable of binding to tropoelastin and fibrillin-1. In the present study, we used the RNA interference (RNAi) technique to establish individual gene-specific knockdown of fibulin-2, -4 and -5 in elastin-producing cells (human gingival fibroblasts; HGF). We then examined the extracellular deposition of tropoelastin using immunofluorescence. RNAi-mediated down-regulation of fibulin-4 and -5 was responsible for the diminution of tropoelastin deposition. Suppression of fibulin-5 appeared to inhibit the formation of fibrillin-1 microfibrils, while that of fibulin-4 did not. Similar results to those for HGF were obtained with human dermal fibroblasts. These results suggest that fibulin-4 and -5 may be associated in different ways with the extracellular deposition of tropoelastin during elastic fiber formation in elastin-producing cells in culture

Feasibility study of high-resolution coherent diffraction microscopy using synchrotron x rays focused by Kirkpatrick-Baez mirrors

High-flux coherent x rays are necessary for the improvement of the spatial resolution in coherent x-ray diffraction microscopy (CXDM). In this study, high-resolution CXDM using Kirkpatrick-Baez (KB) mirrors is proposed, and the mirrors are designed for experiments of the transmission scheme at SPring-8. Both the photon density and spatial coherence of synchrotron x rays focused by the KB mirrors are investigated by wave optical simulation. The KB mirrors can produce nearly diffraction-limited two-dimensional focusing x rays of ∼1 μm in size at 8 keV. When the sample size is less than ∼1 μm, the sample can be illuminated with full coherent x rays by adjusting the cross-slit size set between the source and the mirrors. From the estimated photon density at the sample position, the feasibility of CXDM with a sub- 1-nm spatial resolution is suggested. The present ultraprecise figuring process enables us to fabricate mirrors for carrying out high-resolution CXDM experiments.Yukio Takahashi, Yoshinori Nishino, Hidekazu Mimura, Ryosuke Tsutsumi, Hideto Kubo, Tetsuya Ishikawa, and Kazuto Yamauchi, "Feasibility study of high-resolution coherent diffraction microscopy using synchrotron x rays focused by Kirkpatrick–Baez mirrors", Journal of Applied Physics 105, 083106 (2009) https://doi.org/10.1063/1.3108997

Development of incident X-ray flux monitor for coherent X-ray diffraction microscopy

An incident X-ray flux monitor for coherent X-ray diffraction microscopy was developed. The intensities of x-rays passing through the sample were measured using an X-ray photodiode, with the simultaneous measurement of the X-ray diffraction intensities of the sample. As a result of the normalization of the X-ray diffraction intensities by the incident X-ray flux determined from the monitor, the fluctuation of the speckle intensities was successfully suppressed. © 2009 IOP Publishing Ltd.Takahashi Y., Kubo H., Furukawa H., et al. Development of incident X-ray flux monitor for coherent X-ray diffraction microscopy. Journal of Physics: Conference Series, 186, , 012060. https://doi.org/10.1088/1742-6596/186/1/012060.9TH INTERNATIONAL CONFERENCE ON X-RAY MICROSCOPY 21–25 July 2008, Zürich, Switzerlan

High-resolution projection image reconstruction of thick objects by hard x-ray diffraction microscopy

Hard x-ray diffraction microscopy enables us to observe thick objects at high spatial resolution. The resolution of this method is limited, in principle, by only the x-ray wavelength and the largest scattering angle recorded. As the resolution approaches the wavelength, the thickness effect of objects plays a significant role in x-ray diffraction microscopy. In this paper, we report high-resolution hard x-ray diffraction microscopy for thick objects. We used highly focused coherent x rays with a wavelength of ∼0.1 nm as an incident beam and measured the diffraction patterns of a ∼150-nm -thick silver nanocube at the scattering angle of ∼3°. We observed a characteristic contrast of the coherent diffraction pattern due to only the thickness effect and collected the diffraction patterns at nine incident angles so as to obtain information on a cross section of Fourier space. We reconstructed a pure projection image by the iterative phasing method from the patched diffraction pattern. The edge resolution of the reconstructed image was ∼2 nm, which was the highest resolution so far achieved by x-ray microscopy. The present study provides us with a method for quantitatively observing thick samples at high resolution by hard x-ray diffraction microscopy. © 2010 The American Physical Society.Yukio Takahashi, Yoshinori Nishino, Ryosuke Tsutsumi, Nobuyuki Zettsu, Eiichiro Matsubara, Kazuto Yamauchi, and Tetsuya Ishikawa. Phys. Rev. B 82(21), 214102 (2010)

Fibulin-4 and -5, but not Fibulin-2, are Associated with Tropoelastin Deposition in Elastin-Producing Cell Culture.

Elastic system fibers consist of microfibrils and tropoelastin. During development, microfibrils act as a template on which tropoelastin is deposited. Fibrillin-1 is the major component of microfibrils. It is not clear whether elastic fiber-associated molecules, such as fibulins, contribute to tropoelastin deposition. Among the fibulin family, fibulin-2, -4 and -5 are capable of binding to tropoelastin and fibrillin-1. In the present study, we used the RNA interference (RNAi) technique to establish individual gene-specific knockdown of fibulin-2, -4 and -5 in elastin-producing cells (human gingival fibroblasts; HGF). We then examined the extracellular deposition of tropoelastin using immunofluorescence. RNAi-mediated down-regulation of fibulin-4 and -5 was responsible for the diminution of tropoelastin deposition. Suppression of fibulin-5 appeared to inhibit the formation of fibrillin-1 microfibrils, while that of fibulin-4 did not. Similar results to those for HGF were obtained with human dermal fibroblasts. These results suggest that fibulin-4 and -5 may be associated in different ways with the extracellular deposition of tropoelastin during elastic fiber formation in elastin-producing cells in culture.福岡歯科大学2013年

An experimental procedure for precise evaluation of electron density distribution of a nanostructured material by coherent x-ray diffraction microscopy

We developed a coherent x-ray diffraction microscopy (CXDM) system that enables us to precisely evaluate the electron density of an isolated sample. This system enables us to determine the dose per surface unit of x rays illuminated onto an isolated sample by combining incident x-ray intensity monitoring and the CXDM of a reference sample. By using this system, we determined the dose of x rays illuminated onto a nanostructured island fabricated by focused-ion-beam chemical vapor deposition and derived the electron density distribution of such a nanostructured island. A projection image of the nanostructured island with a spatial resolution of 24.1 nm and a contrast resolution higher than 2.3× 107 electrons/pixel was successfully reconstructed. © 2010 American Institute of Physics.Yukio Takahashi, Hideto Kubo, Yoshinori Nishino, Hayato Furukawa, Ryosuke Tsutsumi, Kazuto Yamauchi, Tetsuya Ishikawa, and Eiichiro Matsubara, "An experimental procedure for precise evaluation of electron density distribution of a nanostructured material by coherent x-ray diffraction microscopy", Review of Scientific Instruments 81(3), 033707 (2010) https://doi.org/10.1063/1.3361265

Quantitative ultrasound can assess the regeneration process of tissue-engineered cartilage using a complex between adherent bone marrow cells and a three-dimensional scaffold

Articular cartilage (hyaline cartilage) defects resulting from traumatic injury or degenerative joint disease do not repair themselves spontaneously. Therefore, such defects may require novel regenerative strategies to restore biologically and biomechanically functional tissue. Recently, tissue engineering using a complex of cells and scaffold has emerged as a new approach for repairing cartilage defects and restoring cartilage function. With the advent of this new technology, accurate methods for evaluating articular cartilage have become important. In particular, in vivo evaluation is essential for determining the best treatment. However, without a biopsy, which causes damage, articular cartilage cannot be accurately evaluated in a clinical context. We have developed a novel system for evaluating articular cartilage, in which the acoustic properties of the cartilage are measured by introducing an ultrasonic probe during arthroscopy of the knee joint. The purpose of the current study was to determine the efficacy of this ultrasound system for evaluating tissue-engineered cartilage in an experimental model involving implantation of a cell/scaffold complex into rabbit knee joint defects. Ultrasonic echoes from the articular cartilage were converted into a wavelet map by wavelet transformation. On the wavelet map, the percentage maximum magnitude (the maximum magnitude of the measurement area of the operated knee divided by that of the intact cartilage of the opposite, nonoperated knee; %MM) was used as a quantitative index of cartilage regeneration. Using this index, the tissue-engineered cartilage was examined to elucidate the relations between ultrasonic analysis and biochemical and histological analyses. The %MM increased over the time course of the implant and all the hyaline-like cartilage samples from the histological findings had a high %MM. Correlations were observed between the %MM and the semiquantitative histologic grading scale scores from the histological findings. In the biochemical findings, the chondroitin sulfate content increased over the time course of the implant, whereas the hydroxyproline content remained constant. The chondroitin sulfate content showed a similarity to the results of the %MM values. Ultrasonic measurements were found to predict the regeneration process of the tissue-engineered cartilage as a minimally invasive method. Therefore, ultrasonic evaluation using a wavelet map can support the evaluation of tissue-engineered cartilage using cell/scaffold complexes

At-wavelength figure metrology of hard x-ray focusing mirrors

We have developed an at-wavelength wave-front metrology of a grazing-incidence focusing optical systems in the hard x-ray region. The metrology is based on numerical retrieval from the intensity profile around the focal point. We demonstrated the at-wavelength metrology and estimated the surface figure error on a test mirror. An experiment for measuring the focusing intensity profile was performed at the 1-km-long beamline (BL29XUL) of SPring-8. The obtained results were compared with the profile measured using an optical interferometer and were confirmed to be in good agreement with it. This technique is a potential method of characterizing wave-front aberrations on elliptical mirrors for sub-10-nm focusing. © 2006 American Institute of Physics.Yumoto H., Mimura H., Matsuyama S., et al. At-wavelength figure metrology of hard x-ray focusing mirrors. Review of Scientific Instruments, 77, 6, 063712 (2006) https://doi.org/10.1063/1.221687

Direct determination of the wave field of an x-ray nanobeam

We present a remarkably accurate method for determining the wave field of an x-ray nanobeam. The intensity profile of a beam was directly measured over a range of three orders of magnitude while its phase distribution was successfully recovered using an iterative algorithm. The evolution of the wave field along the beam propagation direction was precisely simulated, and there was good agreement with the experimental results.Hidekazu Mimura, Hirokatsu Yumoto, Satoshi Matsuyama, Soichiro Handa, Takashi Kimura, Yasuhisa Sano, Makina Yabashi, Yoshinori Nishino, Kenji Tamasaku, Tetsuya Ishikawa, and Kazuto Yamauchi Phys. Rev. A 77, 015812 (2008)

Efficient focusing of hard x rays to 25 nm by a total reflection mirror

Nanofocused x rays are indispensable because they can provide high spatial resolution and high sensitivity for x-ray nanoscopy/spectroscopy. A focusing system using total reflection mirrors is one of the most promising methods for producing nanofocused x rays due to its high efficiency and energy-tunable focusing. The authors have developed a fabrication system for hard x-ray mirrors by developing elastic emission machining, microstitching interferometry, and relative angle determinable stitching interferometry. By using an ultraprecisely figured mirror, they realized hard x-ray line focusing with a beam width of 25 nm at 15 keV. The focusing test was performed at the 1-km -long beamline of SPring-8. © 2007 American Institute of Physics.Hidekazu Mimura, Hirokatsu Yumoto, et al. "Efficient focusing of hard x rays to 25nm by a total reflection mirror", Appl. Phys. Lett. 90(5), 051903 (2007) https://doi.org/10.1063/1.2436469