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Katsunori Yogo,1 Masaki Misawa,2 Morihito Shimizu,2 Hidetoshi Shimizu,3 Tomoki Kitagawa,3 Ryoichi Hirayama,4 Hiromichi Ishiyama,5 Takako Furukawa,1 Hiroshi Yasuda6 1Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan; 2Health and Medical Research Institute, National Institute of Advanced Industrial Science & Technology (AIST), Tsukuba, Ibaraki, Japan; 3Department of Radiation Oncology, Aichi Cancer Center Hospital, Nagoya, Aichi, Japan; 4National Institute of Radiological Sciences (NIRS), National Institutes for Quantum and Radiological Science and Technology (QST), Chiba-shi, Chiba, Japan; 5Graduate School of Medical Science, Kitasato University, Sagamihara, Kanagawa, Japan; 6Department of Radiation Biophysics, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, JapanCorrespondence: Katsunori YogoNagoya University Graduate School of Medicine, 1-1-20 Daiko-Minami, Higashi-Ku, Nagoya, Aichi 461-8673, JapanTel +81 52 719 1103Fax +81 52 719 3172Email yogo@met.nagoya-u.ac.jpPurpose: Gold nanoparticles (AuNPs) are candidate radiosensitizers for medium-energy photon treatment, such as γ-ray radiation in high-dose-rate (HDR) brachytherapy. However, high AuNP concentrations are required for sufficient dose enhancement for clinical applications. Here, we investigated the effect of positively (+) charged AuNP radiosensitization of plasmid DNA damage induced by 192Ir γ-rays, and compared it with that of negatively (−) charged AuNPs.Methods: We observed DNA breaks and reactive oxygen species (ROS) generation in the presence of AuNPs at low concentrations. pBR322 plasmid DNA exposed to 64 ng/mL AuNPs was irradiated with 192Ir γ-rays via HDR brachytherapy. DNA breaks were detected by observing the changes in the form of the plasmid and quantified by agarose gel electrophoresis. The ROS generated by the AuNPs were measured with the fluorescent probe sensitive to ROS. The effects of positively (+) and negatively (−) charged AuNPs were compared to study the effect of surface charge on dose enhancement.Results: +AuNPs at lower concentrations promoted a comparable level of radiosensitization by producing both single-stranded breaks (SSBs) and double-stranded breaks (DSBs) than those used in cell assays and Monte Carlo simulation experiments. The dose enhancement factor (DEF) for +AuNPs was 1.3 ± 0.2 for SSBs and 1.5 ± 0.4 for DSBs. The ability of +AuNPs to augment plasmid DNA damage is due to enhanced ROS generation. While −AuNPs generated similar ROS levels, they did not cause significant DNA damage. Thus, dose enhancement using low concentrations of +AuNPs presumably occurred via DNA binding or increasing local +AuNP concentration around the DNA.Conclusion: +AuNPs at low concentrations displayed stronger radiosensitization compared to −AuNPs. Combining +AuNPs with 192Ir γ-rays in HDR brachytherapy is a candidate method for improving clinical outcomes. Future development of cancer cell-specific +AuNPs would allow their wider application for HDR brachytherapy.Keywords: gold nanoparticles, high-dose-rate brachytherapy, DNA damage, radiosensitization, 192Ir γ-rays, positively charged nanoparticles

Gold nanoparticles (Au-NPs) have attracted attention as a promising sensitizer owing to their high atomic number (Z), and because they are considered fully multifunctional, they are preferred over other metal nanoparticles. Cold atmospheric plasma (CAP) has also recently gained attention, especially for cancer treatment, by inducing apoptosis through the formation of reactive oxygen species (ROS). In this study, the activity of different sized Au-NPs with helium-based CAP (He-CAP) was analyzed, and the underlying mechanism was investigated. Treating cells with only small Au-NPs (2 nm) significantly enhanced He-CAP-induced apoptosis. In comparison, 40 nm and 100 nm Au-NPs failed to enhance cell death. Mechanistically, the synergistic enhancement was due to 2 nm Au-NPs-induced decrease in intracellular glutathione, which led to the generation of intracellular ROS. He-CAP markedly induced ROS generation in an aqueous medium; however, treatment with He-CAP alone did not induce intracellular ROS formation. In contrast, the combined treatment significantly enhanced the intracellular formation of superoxide (O2• −) and hydroxyl radical (•OH). These findings indicate the potential therapeutic use of Au-NPs in combination with CAP and further clarify the role of Au-NPs in He-CAP-aided therapies.

Traditional tissue-engineered cartilage applied in clinical practice consists of cell suspensions or gel-form materials for which it is difficult to maintain their shapes. Although biodegradable polymer scaffolds are used for shape retention, deformation after transplantation can occur. Here, we showed that periostin (PN), which is abundantly expressed in fibrous tissues, contributes to the maturation and shape retention of tissue-engineered cartilage through conformational changes in collagen molecules. The tissue-engineered cartilage transplanted in an environment lacking PN exhibited irregular shapes, while transplants originating from chondrocytes lacking PN showed limited regeneration. In the in vitro assay, PN added to the culture medium of chondrocytes failed to show any effects, while the 3D culture embedded within the collagen gel premixed with PN (10 μg/mL) enhanced chondrogenesis. The PN-mediated collagen structure enhanced the mechanical strength of the surrounding fibrous tissues and activated chondrocyte extracellular signaling by interstitial fibrous tissues.

In order to achieve a nanometer-scale resolution in an x-ray microscopy system, a Gabor-type hologram was produced by eliminating the zero-order term of the object diffraction pattern. In this system, a Fresnel zone plate was used for strong illumination of an object, and the zero-order diffraction was physically eliminated by a center stop. An accurate phase plate of π / 2 in the Zernike method was numerically created, and the phase-contrast image was realized. The theoretical resolution was 19.8 nm. We proved that a gold nanocube with a size of 50 nm can be reconstructed with the predicted resolution.

In this work, the application of an undecimated wavelet transformation together with digital interferometric contrast to improve the resulting reconstructions in a digital hard X-ray Gabor holographic microscope is shown. Specifically, the starlet transform is used together with digital Zernike contrast. With this contrast, the results show that only a small set of scales from the hologram are, in effect, useful, and it is possible to enhance the details of the reconstruction.

We developed a facile one-pot fabrication process for magnetic iron oxide–calcium phosphate (IO–CaP) composite nanoparticles via coprecipitation in labile supersaturated CaP solutions containing IO nanocrystals. All the source solutions used were clinically approved for injection, including water and magnetic IO nanocrystals (ferucarbotran, used as a negative magnetic resonance imaging (MRI) contrast agent). This ensured that the resulting nanoparticles were pathogen- and endotoxin-free. The dispersants used were clinically approved heparin sodium (heparin) or adenosine triphosphate disodium hydrate (ATP), which were added to the IO-containing labile supersaturated CaP solutions. Both heparin and ATP coprecipitated with CaP and ferucarbotran to form heparin- and ATP-modified IO–CaP nanoparticles, respectively, with a hydrodynamic diameter of a few hundred nanometers. Both the resulting nanoparticles exhibited relatively large negative zeta potentials, caused by the negatively charged functional groups in heparin and ATP, which improved the particle dispersibility when compared to non-modified IO–CaP nanoparticles. The heparin-modified IO–CaP nanoparticles were effectively ingested by murine macrophages (RAW264.7) without showing significant cytotoxicity but barely ingested by non-phagocytotic human umbilical vein endothelial cells, indicating the potential of these nanoparticles for targeted delivery to macrophages. The heparin-modified IO–CaP nanoparticles exhibited a negative contrast enhancing ability for MRI. Our results show that IO–CaP nanoparticles have potential as delivery and MRI contrast agents for macrophages.

To reduce particulate matter (PM) including soot in diesel exhaust gas, a diesel particulate filter (DPF) has been developed. Since it is difficult to observe the phenomena in a DPF experimentally, we have conducted a lattice Boltzmann simulation. In this study, we simulated the flow in a metallic filter. An X-ray computed tomography (CT) technique was applied to obtain its inner structure. The processes of soot deposition and oxidation were included for a continuously regenerating diesel filter. By comparing experimental data, a parameter of soot deposition probability in the numerical model was determined.

We propose a differential interference contrast method for cells using hard x-ray Gabor holography and knife-edge filtering in the spatial frequency domain, without relying on beam shearing. A phase object is holographically recorded and reconstructed by computer. Interference between the wavefronts of zeroth order weighted by ejπ/2 in the positive frequency region produces a dark image. Similarly, interference between the wavefronts of the zeroth order weighted by ej3π/2 in the negative frequency region produces a bright image. By adding these two intensity distributions, good quality phase-contrast images of 8-μm-diameter polystyrene beads and human HeLa cells were obtained.

The interface area between the bubble and emulsion phases in a fluidized catalyst bed is one of the important parameters used to analyze and design the fluidized bed reactor. We used a fast-scanning X-ray CT system to observe the bubble shape and structure. We then obtained the transient 2-dimensional cross sectional gas-phase distribution in a fluidized catalyst bed. Using image-processing techniques, pseudo 3-dimensional images of the bubbles were reconstructed. The bubble structure was studied based on the 3-dimensional images and the previously obtained results in a 2-dimensional fluidized bed. It was found that the bubble shape was not spherical but complicated, and that the bubbles ascending in a fluidized catalyst bed consisted of some smaller bubbles.

Since diesel exhaust gas has more ambient air pollutants such as NOx and particulate matters (PM) including soot, the special treatment for exhaust emission standards is needed. Recently, a diesel particulate filter (DPF) has been developed to reduce PM in the after-treatment of exhaust gas. However, since the structure of the filter is small and complex, it is impossible to examine the phenomena inside the filter experimentally. In this study, we conduct fluid simulation in the diesel filter. We use the lattice Boltzmann method. The soot accumulation is considered to simulate the PM trap in the filter. For the wall-boundary in the simulation, the inner structure of the filter is obtained by a 3D-CT technique. Results show complex flow pattern in the diesel filter. Due to the soot accumulation, the velocity is changed and the pressure is increased. The pressure drop becomes larger as soot concentration at the inlet is higher.

In this study, we report on the potential use of platinum nanoparticles (Pt-NPs), a superoxide dismutase (SOD)/catalase mimetic antioxidant, in combination with 1MHz ultrasound (US) at an intensity of 0.4 W/cm(2), 10% duty factor, 100 Hz PRF, for 2 min. Apoptosis induction was assessed by DNA fragmentation assay, cell cycle analysis and Annexin V-FITC/PI staining. Cell killing was confirmed by cell counting and microscopic examination. The mitochondrial and Ca(2+)-dependent pathways were investigated. Caspase-8 expression and autophagy-related proteins were detected by spectrophotometry and western blot analysis, respectively. Intracellular reactive oxygen species (ROS) elevation was detected by flow cytometry, while extracellular free radical formation was assessed by electron paramagnetic resonance spin trapping spectrometry. The results showed that Pt-NPs exerted differential effects depending on their internalization. Pt-NPs functioned as potent free radical scavengers when added immediately before sonication while pre-treatment with Pt-NPs suppressed the induction of apoptosis as well as autophagy (AP), and resulted in enhanced cell killing. Dead cells displayed the features of pyknosis. The exact mode of cell death is still unclear. In conclusion, the results indicate that US-induced AP may contribute to cell survival post sonication. To our knowledge this is the first study to discuss autophagy as a pro-survival pathway in the context of US. The combination of Pt-NPs and US might be effective in cancer eradication.

In this study, using the lattice Boltzmann method, we simulate soot accumulation in porous media to examine the particle trap in a diesel particulate filter (DPF). The porous media are numerically formed for different inner structure with given porosity. The porous structure similar to the real sample, Ni-Cr metal, can be obtained, where the pressure fields in both cases are almost the same. In the simulation of soot accumulation, the velocity field is changed when the soot is attached to the porous wall. The pressure drop is largely increased. Interestingly, the friction factor is smaller than experimentally predicted value by Ergun equation.

Numerical simulation of gas-liquid two-phase flow is important for design of nuclear power systems. Conventional codes require experiments from laboratory scale to mock-up to obtain database for constitutive equations and verification. The aim of this study is to provide new simulation technique to substitute for a part of costly experiments. The extended two-fluid model developed by Hitachi, Ltd. was employed to treat inhomogeneous and intermittent two-phase flow behavior, which had been considered through constitutive equations. Validity of the new simulation code for complex two-phase flow was proved by comparison between calculated and experimental void fraction signals from two-phase flows in circular tubes, CCFL of a thick plate, and natural circulation of aerated pool in double cylinders. For measurement of instantaneous, multi-dimensional void fraction distributions, wire-mesh sensor techniques were employed for all test setups. It was found that the two-phase flow has higher orders of dynamics than initially expected, leading to development of robust analysis methods. The comparison was successful for well-structured flow patterns as slug flow, since the large diameter bubbles show low dimensional dynamics. It is also noted that an optimal delay time reconstruction method and a point correlation dimension method together were useful to increase the reliability of the analysis.

In order to detect the disease and degeneration of cartilage early, evaluations of acoustic properties are expected, but the physical meaning should be clarified. On the other hand, we have proposed a multimodal method using ultrasound (US) and magnetic resonance (MR) images for in vivo SOS measurement. In this study, the method was applied for the tissue-engineered cartilage, and the SOS was compared with T1, T2, apparent diffusion coefficient (ADC), attenuation coefficient (ATT), and Young’s modulus (YM). All procedures were approved by the Animal Care and Use Committee of our institution. 12-week-old male rats were used, and five different types of tissue-engineered cartilage was subcutaneously implanted in the back of rat (n = 30). Under anesthesia, MR and US images including same cross-sections were acquired separately, by using a MR imager of 2 Tesla and a US device of 13 MHz. At this time, T1, T2, ADC were also measured. The SOS was determined by the thickness measurement using the MR image and the...

5-Aminolevulinic acid (ALA) is a precursor of the photosensitizer used in photodynamic therapy. It accumulates in tumor cells and subsequently metabolizes to protoporphyrin IX (PpIX), which generates singlet oxygen after light irradiation. PpIX enhances the generation of reactive oxygen species following physicochemical interactions with X-rays. ALA-based treatment using fractionated doses of irradiation suppressed tumor growth in a mouse melanoma model. To study the transcriptomic effects of PpIX, microarray analyses were conducted using HeLa cells with limited proliferation capacity. Based on the p-values (p &lt, 0.01), we selected genes showing altered expression in each treatment group with reference to the non-treatment (NT) group. We detected 290, 196 and 28 upregulated genes, as well as 203, 146 and 36 downregulated genes after a 6 h-long PpIX treatment (1 μg/mL) prior to 3 Gy X-ray irradiation (PpIX-XT), 3 Gy X-ray irradiation alone (XT) and PpIX treatment alone (PpIXT), respectively. Functional analysis revealed that a majority of the regulated genes in the XT and PpIX-XT groups were related to cell-cycle arrest. The XT and PpIX-XT groups differed in the quantity, but not in the quality of their gene expression. The combined effect of PpIX and X-ray irradiation sensitized HeLa cells to X-ray treatment.

Microarray techniques allow to detect genome-wide perturbations during various treatments and to measure various responses by multitude of gene probes. Toxicogenomics, in which microarray techniques are specifically used in toxicology test, has been widely recognized as one of standard safety procedures for chemicals [1-3]. Gene expression microarrays have been used particularly for screening of genes involved in specific biological processes of interest, such as diseases or responses to environmental stimuli. Such experiments adopt the “healthy state” as a control, and identify highly expressed or suppressed genes. However, few studies deal with the features of gene expression and its variation at the “healthy state” to be influenced by species, age, sex, and individual variability. In measuring the state of disease and drug response, minimally invasive blood sampling, which allows for direct measurement of immune-responsive blood cells, excels other invasive biopsy techniques upon disease diagnostics and assessment of drug response, as well as health monitoring. Blood RNA contains an enormous amount of information on expression of messenger RNA and non coding functional RNA which remains without being translated into protein. Thus, blood RNA offers an opportunity to detect subtle change in physiological state. In this chapter, we discuss the potential of the RNA diagnosis using whole blood, showing a series of whole blood microarray experiments to evaluate variations of correlation among individuals and ages [4], dietary-induced hyperlipidemia, and other stresses using specific pathogen-free (SPF) miniature pigs.

In the temperature-pressure phase diagram of the organic superconductor (TMTSF)${}_{2}$PF${}_{6}$ (TMTSF: tetramethyltetraselenafulvalene), the superconducting phase and the spin-density-wave (SDW) phase are adjacent to each other. This salt exhibits non-Fermi-liquid (NFL) behavior and superconductivity under pressure. Its superconductive property does not exist at higher pressures where Fermi-liquid (FL) behavior is exhibited. In order to investigate the origin of NFL behavior, systematic $^{13}\mathrm{C}$-NMR measurement of this salt has been assessed under pressure in a wide temperature range. At low temperatures, $({T}_{1}T){}^{\ensuremath{-}1}$ increases, and this increase is suppressed by the increasing pressure. These results suggest that applying pressure alters the electron system from the NFL to the FL state, and that antiferromagnetic fluctuations contribute to the origin of NFL behavior. The connection between the emergence of FL behavior and the disappearance of superconductivity suggests that the SDW fluctuation relates to the mechanism of the superconductivity in (TMTSF)${}_{2}$PF${}_{6}$.

A fast-scanning X-ray CT system was used to obtain the transient 2-dimensional cross sectional gas-phase distribution in a fluidized catalyst bed with fine particles. The captured data was sent to a PC for image reconstruction, and the density distributions across the horizontal cross-section were visualized. Using image-processing techniques, pseudo 3-dimensional images of bubbles and the density distribution across the vertical cross-section were obtained. The bubble structure was studied based on these images and the results obtained previously in a 2-dimensional fluidized bed. It was found that bubble shape was not spherical, but that the bubble ascending in a fluidized catalyst bed consisted of some smaller bubbles.

In a fluidized catalyst bed, the reactant gas transfers from the bubble phase to the emulsion phase and reactions proceed in the emulsion phase. The catalyst particles around the bubbles should contact the gases containing a high concentration of the reactants. Therefore, the effect of the catalysts around the bubbles is very important for estimating the conversion and selectivity in the reactor. In order to study the role of these catalysts, the hydrogenation of carbon dioxide was carried out in a fluidized catalyst bed. Based on the results, the amount of the catalyst that was effective for the reaction was calculated. In addition, the shape of the bubbles ascending in the fluidized catalyst bed was observed using a fast X-ray computer tomography (CT) scanner. The structure of the bubbles in the fluidized catalyst bed was very complicated and the surface area of the bubbles was much greater than the obtained when assuming spherical shaped bubble. By assuming that effective catalysts existed around the bubbles, the thickness of catalyst layer was obtained. Finally, the 3-dimensional images of the catalyst layers around the bubbles were reconstructed.

To simulate the flow in a three-dimensional porous structure, we have used the Lattice Boltzmann method (LBM). The distribution functions for flow, temperature, and concentration fields are solved to simulate the regeneration process of diesel particulate filter (DPF). For a benchmark study, we have conducted the calculation in simple geometry. To change the flow characteristics such as porosity and tortuosity, obstacles of different number and size are selected, which are randomly placed in the duct. The flow pattern is changed with obstacle size even at the same porosity. To confirm the validity of our calculation, the results are compared with the empirical relationships of the Blake–Kozeny and Ergun equations, in the non-dimensional form using the Ergun coordinates of Reynolds number and friction factor. Numerical results show good agreements with the empirical predictions. Soot combustion is well simulated to show the effects of oxygen on the reaction rate. Finally, the real porous geometry has been tested. The porous structure of Ni–Cr metal has been obtained by the three-dimensional computer tomography technique. Results show that the inhomogeneous flow is observed, and there are regions where the temperature is locally higher. This information is indispensable for the better design of DPF. LBM can be a good tool for combustion simulation in porous media.

In hard X-ray phase imaging using interferometry, the spatial resolution is limited by the pixel size of digital sensors, inhibiting its use in magnifying observation of a sample. To solve this problem, we describe a digital phase contrast microscope that uses Zernike’s phase contrast method with a hard X-ray Gabor holography associated with numerical processing and spatial frequency domain filtering techniques. The hologram is reconstructed by a collimated beam in a computer. The hologram intensity distributions itself become the reconstructed wavefronts. For this transformation, the Rayleigh- Sommerfeld diffraction formula is used. The hard X-ray wavelength 0.1259 nm (an energy of 9.85 keV) was employed at the SPring-8 facility. We succeeded in obtaining high-resolution images by a CCD sensor with a pixel size of 3.14 μm, even while bound by the need to satisfy the sampling theorem and by the CCD pixel size. The test samples used here were polystyrene beads of 8 μm, and human HeLa cells. We thus proved that the resolution 0.951 μm smaller than the pixel size of CCD (3.14 μm) was achieved by the proposed reconstruction techniques and coherent image processing in the computer, suggesting even higher resolutions by adopting greater numerical apertures.

5-Aminolevulinic acid (ALA) is a photosensitizer used in photodynamic therapy (PDT) because it causes preferential accumulation of protoporphyrin IX (PpIX) in tumor cells, where it forms singlet oxygen upon light irradiation and kills the tumor cells. Our previous study demonstrated that PpIX enhances generation of reactive oxygen species by physicochemical interaction with X-rays. We investigated the effect of ALA administration with X-ray irradiation of mouse B16-BL6 melanoma cells in vitro and in vivo. ALA facilitates PpIX accumulation in tumor cells and enhances ROS generation in vitro. Tumor suppression significantly improved in animals treated with fractionated doses of radiation (3 Gy × 10; total, 30 Gy) with local administration of 50 mg/kg ALA at 24 h prior to fractional irradiation. These results suggest ALA may improve the efficacy of cancer radiotherapy by acting as a radiomediator.