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Intelligent Design of Nano-Scale Molecular Imaging Agents
Sung Bae Kim,Mitsuru Hattori,Takeaki Ozawa
International Journal of Molecular Sciences , 2012, DOI: 10.3390/ijms131216986
Abstract: Visual representation and quantification of biological processes at the cellular and subcellular levels within living subjects are gaining great interest in life science to address frontier issues in pathology and physiology. As intact living subjects do not emit any optical signature, visual representation usually exploits nano-scale imaging agents as the source of image contrast. Many imaging agents have been developed for this purpose, some of which exert nonspecific, passive, and physical interaction with a target. Current research interest in molecular imaging has mainly shifted to fabrication of smartly integrated, specific, and versatile agents that emit fluorescence or luminescence as an optical readout. These agents include luminescent quantum dots (QDs), biofunctional antibodies, and multifunctional nanoparticles. Furthermore, genetically encoded nano-imaging agents embedding fluorescent proteins or luciferases are now gaining popularity. These agents are generated by integrative design of the components, such as luciferase, flexible linker, and receptor to exert a specific on–off switching in the complex context of living subjects. In the present review, we provide an overview of the basic concepts, smart design, and practical contribution of recent nano-scale imaging agents, especially with respect to genetically encoded imaging agents.
In Vivo Monitoring of Liver Damage Using Caspase-3 Probe
Michitaka Ozaki, Sanae Haga, Takeaki Ozawa
Theranostics , 2012,
Abstract: Real-time monitoring of cellular and organ conditions improves our understanding of various physiopathological phenomena. Such monitoring is expected to provide important alternatives for clinical diagnosis and therapy. We have sought to show physiopathological changes of organs as well as cells. Here, we present an example of in vivo imaging of liver states using the luciferase-based caspase-3 optical probe. We examined dynamic changes of apoptosis (caspase-3 activity) of a mouse liver as well as those of liver cells, proving that the emitted signals reflected the biochemically evaluated apoptotic cell death. In live liver cell (AML 12) experiments, the optical probe for caspase-3 activity emitted signals in response to Fas-ligand, staurosporine and hypoxia/reoxygenation, demonstrating that the probe can measure cellular apoptosis quantitatively. We therefore applied this probe for mouse liver ischemia/reperfusion (I/R) and drug-toxicity to liver. By expressing the probe in a mouse liver adenovirally, we imaged liver caspase-3 activity (i.e. apoptotic damage) non-invasively and chronologically in the hepatic I/R model of mice. The duration of liver ischemia affected the post-ischemic caspase-dependent damage. Ischemia (up to 60 min) enhanced liver damage after reperfusion, but prolonged ischemia (90 min of ischemia) induced not apoptotic cell death but necrotic cell death. Direct observations of the changes of organ conditions elucidated the dynamism of organ function and damage. These technologies clearly possess clinical relevance. They are expected to provide a new diagnostic tool for various clinical settings in the future.
Synchronization of Circadian Per2 Rhythms and HSF1-BMAL1:CLOCK Interaction in Mouse Fibroblasts after Short-Term Heat Shock Pulse
Teruya Tamaru, Mitsuru Hattori, Kousuke Honda, Ivor Benjamin, Takeaki Ozawa, Ken Takamatsu
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0024521
Abstract: Circadian rhythms are the general physiological processes of adaptation to daily environmental changes, such as the temperature cycle. A change in temperature is a resetting cue for mammalian circadian oscillators, which are possibly regulated by the heat shock (HS) pathway. The HS response (HSR) is a universal process that provides protection against stressful conditions, which promote protein-denaturation. Heat shock factor 1 (HSF1) is essential for HSR. In the study presented here, we investigated whether a short-term HS pulse can reset circadian rhythms. Circadian Per2 rhythm and HSF1-mediated gene expression were monitored by a real-time bioluminescence assay for mPer2 promoter-driven luciferase and HS element (HSE; HSF1-binding site)-driven luciferase activity, respectively. By an optimal duration HS pulse (43°C for approximately 30 minutes), circadian Per2 rhythm was observed in the whole mouse fibroblast culture, probably indicating the synchronization of the phases of each cell. This rhythm was preceded by an acute elevation in mPer2 and HSF1-mediated gene expression. Mutations in the two predicted HSE sites adjacent (one of them proximally) to the E-box in the mPer2 promoter dramatically abolished circadian mPer2 rhythm. Circadian Per2 gene/protein expression was not observed in HSF1-deficient cells. These findings demonstrate that HSF1 is essential to the synchronization of circadian rhythms by the HS pulse. Importantly, the interaction between HSF1 and BMAL1:CLOCK heterodimer, a central circadian transcription factor, was observed after the HS pulse. These findings reveal that even a short-term HS pulse can reset circadian rhythms and cause the HSF1-BMAL1:CLOCK interaction, suggesting the pivotal role of crosstalk between the mammalian circadian and HSR systems.
Dual-Color Bioluminescence Analysis for Quantitatively Monitoring G-Protein-Coupled Receptor and β-Arrestin Interactions
A.K.M. Kafi,Mitsuru Hattori,Naomi Misawa,Takeaki Ozawa
Pharmaceuticals , 2011, DOI: 10.3390/ph4030457
Abstract: G protein-coupled receptors (GPCRs) are crucial elements in mammalian signal transduction, and are considered to represent potent drug targets. We have previously developed a GPCR assay system in cultured cells based on complementation of split fragments of click beetle ( Pyrearinus termitilluminans) luciferase. The interaction of GPCRs with its target, β-arrestin, resulted in strong emission of bioluminescence upon stimulation with its specific ligand. In this study, we improved precision of the GPCR assay system by using railroad worm ( Phrixothrix hirtus) luciferase as an internal control. We generated stable cell lines harboring the railroad worm luciferase and quantitatively evaluate the extent of GPCR-β-arrestin interactions. We showed concentration-dependent bioluminescence responses for four GPCRs: β2-adrenoceptor, endothelin receptor type A, α2-adrenoceptor and human μ-opioid receptor. We also demonstrated that the variation of responses was reduced significantly by normalizing the data with bioluminescence from railroad worm luciferase. This assay system represents a simple and reliable approach for screening drug candidates in a high throughput manner.
High-Sensitivity Real-Time Imaging of Dual Protein-Protein Interactions in Living Subjects Using Multicolor Luciferases
Naoki Hida, Muhammad Awais, Masaki Takeuchi, Naoto Ueno, Mayuri Tashiro, Chiyo Takagi, Tanuja Singh, Makoto Hayashi, Yoshihiro Ohmiya, Takeaki Ozawa
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0005868
Abstract: Networks of protein-protein interactions play key roles in numerous important biological processes in living subjects. An effective methodology to assess protein-protein interactions in living cells of interest is protein-fragment complement assay (PCA). Particularly the assays using fluorescent proteins are powerful techniques, but they do not directly track interactions because of its irreversibility or the time for chromophore formation. By contrast, PCAs using bioluminescent proteins can overcome these drawbacks. We herein describe an imaging method for real-time analysis of protein-protein interactions using multicolor luciferases with different spectral characteristics. The sensitivity and signal-to-background ratio were improved considerably by developing a carboxy-terminal fragment engineered from a click beetle luciferase. We demonstrate its utility in spatiotemporal characterization of Smad1–Smad4 and Smad2–Smad4 interactions in early developing stages of a single living Xenopus laevis embryo. We also describe the value of this method by application of specific protein-protein interactions in cell cultures and living mice. This technique supports quantitative analyses and imaging of versatile protein-protein interactions with a selective luminescence wavelength in opaque or strongly auto-fluorescent living subjects.
JVLA S and X-band Polarimetry of the Merging Cluster Abell 2256
Takeaki Ozawa,Hiroyuki Nakanishi,Takuya Akahori,Kenta Anraku,Motokazu Takizawa,Ikumi Takahashi,Sachiko Onodera,Yuya Tsuda,Yoshiaki Sofue
Physics , 2015, DOI: 10.1093/pasj/psv082
Abstract: We report polarimetry results of a merging cluster of galaxies Abell 2256 with Karl G. Jansky Very Large Array (JVLA). We performed new observations with JVLA at S-band (2051-3947 MHz) and X-band (8051-9947 MHz) in the C array configuration, and detected significant polarized emissions from the radio relic, Source A, and Source B in this cluster. We calculated the total magnetic field strengths toward the radio relic using revised equipartition formula, which is 1.8-5.0 microG. With dispersions of Faraday rotation measure, magnetic-field strengths toward Sources A and B are estimated to be 0.63-1.26 microG and 0.11-0.21 microG, respectively. An extremely high degree of linear polarization, as high as ~ 35 %, about a half of the maximum polarization, was detected toward the radio relic, which indicates highly ordered magnetic lines of force over the beam sizes (~ 52 kpc).The fractional polarization of the radio relic decreases from ~ 35 % to ~ 20 % around 3 GHz as the frequency decreases and is nearly constant between 1.37 and 3 GHz. Both analyses with depolarization models and Faraday tomography suggest multiple depolarization components toward the radio relic and imply the existence of turbulent magnetic fields.
Characteristic Properties of Advanced Coil-External Electrodes of Fluorescent Lamps  [PDF]
Lyuji Ozawa
Journal of Materials Science and Chemical Engineering (MSCE) , 2018, DOI: 10.4236/msce.2018.62004
Abstract: The coil-EEFL lamps, which are operated with DC driving circuit, have studied. The figure of the merit of the coil-EEFL lamps is the quantum efficiency (ηq) that is given by the emitted visible photons by one moving electron in the FL lamp. The electrons in the FL lamps move on in the superconductive vacuum (R = 0) in the positive column at above room temperature, giving rise to the astronomical ηq = 1013 visible photons (m3.s)-1. Miraculously, the developed coil-EEFL lamps in the parallel connections light up with WDC = 0, without the sacrifice of the illuminance (lm.m-2). Furthermore, the scraped 40 W-HCFL lamps revitalize to the coil-EEFL lamps, promising the operation life longer than 106 hours. The shallow gap between phosphor screen and positive column can be made by side by side arrangement of the low voltage CL phosphor particles and PL phosphor particles. The coil-EEFL lamps in parallel connections may contribute to the Green Energy Projects of UN.
HCFL Lamp Only Lights Up under Coexistence of Cathode and Anode in Ar Gas Space for Half Cycle of AC Driving Circuit  [PDF]
Lyuji Ozawa
Journal of Power and Energy Engineering (JPEE) , 2019, DOI: 10.4236/jpee.2019.72001
Abstract: Vacuum space between Ar atoms in unlighted HCFL lamps is an electric insulator, because vacuum fills up with strong negative electric field from orbital electrons in 3p6 electron shell of Ar atoms. Vacuum space in lighted FL lamps changes to the neutral vacuum that provides a superconductive vacuum for moving electrons at above room temperature. The complications of lighting mechanisms of HCFL lamps for more than 80 years have clearly solved with coexistence of disparate external and internal electric circuits for each half cycle. External electric circuit acts as two roles. One helps for formation of internal electric circuit in Ar gas space by electric field. Other picks up induced voltages from capacitor CFL. HCFL lamp only lights up with moving electrons in internal DC driving circuit. Electrons in HCFL lamp only move from cathode to anode, which respectively have negative and positive potentials against grand (V = 0), and which are formed with volumes of heated corona light (4G) at around W-filament metal electrodes with a help of heated BaO particles. The HCFL lamp that emits thermoelectrons is a false story. Here we have totally revised the fundamentals of the lighting mechanism of the established HCFL lamps for last 80 years.
ROS Stress Resets Circadian Clocks to Coordinate Pro-Survival Signals
Teruya Tamaru, Mitsuru Hattori, Yasuharu Ninomiya, Genki Kawamura, Guillaume Varès, Kousuke Honda, Durga Prasad Mishra, Bing Wang, Ivor Benjamin, Paolo Sassone-Corsi, Takeaki Ozawa, Ken Takamatsu
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0082006
Abstract: Dysfunction of circadian clocks exacerbates various diseases, in part likely due to impaired stress resistance. It is unclear how circadian clock system responds toward critical stresses, to evoke life-protective adaptation. We identified a reactive oxygen species (ROS), H2O2 -responsive circadian pathway in mammals. Near-lethal doses of ROS-induced critical oxidative stress (cOS) at the branch point of life and death resets circadian clocks, synergistically evoking protective responses for cell survival. The cOS-triggered clock resetting and pro-survival responses are mediated by transcription factor, central clock-regulatory BMAL1 and heat shock stress-responsive (HSR) HSF1. Casein kinase II (CK2) –mediated phosphorylation regulates dimerization and function of BMAL1 and HSF1 to control the cOS-evoked responses. The core cOS-responsive transcriptome includes CK2-regulated crosstalk between the circadian, HSR, NF-kappa-B-mediated anti-apoptotic, and Nrf2-mediated anti-oxidant pathways. This novel circadian-adaptive signaling system likely plays fundamental protective roles in various ROS-inducible disorders, diseases, and death.
Outer Rotation Curve of the Galaxy with VERA II: Annual Parallax and Proper Motion of the Star-Forming Region IRAS21379+5106
Hiroyuki Nakanishi,Nobuyuki Sakai,Tomoharu Kurayama,Mitsuhiro Matsuo,Hiroshi Imai,Ross A. Burns,Takeaki Ozawa,Mareki Honma,Katsunori Shibata,Noriyuki Kawaguchi
Physics , 2015, DOI: 10.1093/pasj/psv012
Abstract: We conducted astrometric VLBI observations of water-vapor maser emission in the massive star forming region IRAS 21379+5106 to measure the annual parallax and proper motion, using VERA. The annual parallax was measured to be $0.262 \pm 0.031$ mas corresponding to a trigonometric distance of $3.82^{+0.51}_{-0.41}$ kpc. The proper motion was $(\mu_\alpha\cos{\delta}, \mu_\delta)=(-2.74 \pm 0.08, -2.87 \pm 0.18)$ mas yr$^{-1}$. Using this result, the Galactic rotational velocity was estimated to be $V_\theta=218\pm 19$ km s$^{-1}$ at the Galactocentric distance $R=9.22\pm0.43$ kpc, when we adopted the Galactic constants $R_0=8.05\pm 0.45$ kpc and $V_0=238\pm 14$ km s$^{-1}$. With newly determined distance, {the bolometric luminosity of the central young stellar object was re-evaluated to $(2.15\pm 0.54)\times 10^3 L_\odot$, which corresponds to spectral type of} B2--B3. Maser features were found to be distributed along a straight line from south-west to north-east. In addition, a vector map of the internal motions constructed from the residual proper motions implies that maser features trace a bipolar flow and that it cannot be explained by simple ballistic motion.
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