Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.11249/jsbpjjpp.33.3_100

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Language：English   Publishing type：Research paper (scientific journal)  

It has become evident that somatic mutations in cancer-associated genes accumulate in the normal endometrium, but spatiotemporal understanding of the evolution and expansion of mutant clones is limited. To elucidate the timing and mechanism of the clonal expansion of somatic mutations in cancer-associated genes in the normal endometrium, we sequence 1311 endometrial glands from 37 women. By collecting endometrial glands from different parts of the endometrium, we show that multiple glands with the same somatic mutations occupy substantial areas of the endometrium. We demonstrate that "rhizome structures", in which the basal glands run horizontally along the muscular layer and multiple vertical glands rise from the basal gland, originate from the same ancestral clone. Moreover, mutant clones detected in the vertical glands diversify by acquiring additional mutations. These results suggest that clonal expansions through the rhizome structures are involved in the mechanism by which mutant clones extend their territories. Furthermore, we show clonal expansions and copy neutral loss-of-heterozygosity events occur early in life, suggesting such events can be tolerated many years in the normal endometrium. Our results of the evolutionary dynamics of mutant clones in the human endometrium will lead to a better understanding of the mechanisms of endometrial regeneration during the menstrual cycle and the development of therapies for the prevention and treatment of endometrium-related diseases.

DOI： 10.1038/s41467-022-28568-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

Abstract

Cerebral malaria (CM) is a life-threatening complication of the malaria disease caused by Plasmodium falciparum infection and is responsible for the death of half a million people annually. The molecular pathogenesis underlying CM in humans is not completely understood, although sequestration of infected erythrocytes in cerebral microvessels is thought to play a major role. In contrast, experimental cerebral malaria (ECM) models in mice have been thought to be distinct from human CM, and are mainly caused by inflammatory mediators. Here, to understand the spatial distribution and the potential sequestration of parasites in the whole-brain microvessels during a mouse model of ECM, we utilized the new tissue-clearing method CUBIC (Clear, Unobstructed, Brain/Body Imaging Cocktails and Computational analysis) with light-sheet fluorescent microscopy (LSFM), and reconstructed images in three dimensions (3D). We demonstrated significantly greater accumulation of Plasmodium berghei ANKA (PbANKA) parasites in the olfactory bulb (OB) of mice, compared with the other parts of the brain, including the cerebral cortex, cerebellum and brainstem. Furthermore, we show that PbANKA parasites preferentially accumulate in the brainstem when the OB is surgically removed. This study therefore not only highlights a successful application of CUBIC tissue-clearing technology to visualize the whole brain and its microvessels during ECM, but it also shows CUBIC’s future potential for visualizing pathological events in the whole ECM brain at the cellular level, an achievement that would greatly advance our understanding of human cerebral malaria.

DOI： 10.1093/intimm/dxab060

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Other Link： https://academic.oup.com/intimm/article-pdf/33/11/587/44558275/dxab060.pdf

Language：English   Publishing type：Research paper (scientific journal)  

Mechanical stimuli including loading after birth promote bone growth. However, little is known about how mechanical force triggers biochemical signals to regulate bone growth. Here, we identified a periosteal-osteoblast-derived secretory peptide, Osteocrin (OSTN), as a mechanotransducer involved in load-induced long bone growth. OSTN produced by periosteal osteoblasts regulates growth plate growth by enhancing C-type natriuretic peptide (CNP)-dependent proliferation and maturation of chondrocytes, leading to elongation of long bones. Additionally, OSTN cooperates with CNP to regulate bone formation. CNP stimulates osteogenic differentiation of periosteal osteoprogenitors to induce bone formation. OSTN binds to natriuretic peptide receptor 3 (NPR3) in periosteal osteoprogenitors, thereby preventing NPR3-mediated clearance of CNP and consequently facilitating CNP-signal-mediated bone growth. Importantly, physiological loading induces Ostn expression in periosteal osteoblasts by suppressing Forkhead box protein O1 (FoxO1) transcription factor. Thus, this study reveals a crucial role of OSTN as a mechanotransducer converting mechanical loading to CNP-dependent bone formation.

DOI： 10.1016/j.celrep.2021.109380

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.isci.2021.102258

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

<title>Abstract</title>Tissue clearing is one of the most powerful strategies for a comprehensive analysis of disease progression. Here, we established an integrated pipeline that combines tissue clearing, 3D imaging, and machine learning and applied to a mouse tumour model of experimental lung metastasis using human lung adenocarcinoma A549 cells. This pipeline provided the spatial information of the tumour microenvironment. We further explored the role of transforming growth factor-β (TGF-β) in cancer metastasis. TGF-β-stimulated cancer cells enhanced metastatic colonization of unstimulated-cancer cells in vivo when both cells were mixed. RNA-sequencing analysis showed that expression of the genes related to coagulation and inflammation were up-regulated in TGF-β-stimulated cancer cells. Further, whole-organ analysis revealed accumulation of platelets or macrophages with TGF-β-stimulated cancer cells, suggesting that TGF-β might promote remodelling of the tumour microenvironment, enhancing the colonization of cancer cells. Hence, our integrated pipeline for 3D profiling will help the understanding of the tumour microenvironment.

DOI： 10.1038/s42003-021-01786-y

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Other Link： http://www.nature.com/articles/s42003-021-01786-y

Language：English   Publishing type：Research paper (scientific journal)  

Dopaminergic neurotransmission is considered to play an important role not only in reward-based learning, but also in aversive learning. Here, we investigated the role of dopaminergic neurotransmission via dopamine D1 receptors (D1Rs) in aversive memory formation in a passive avoidance test using D1R knockdown (KD) mice, in which the expression of D1Rs can conditionally and reversibly be controlled by doxycycline (Dox) treatment. We also performed whole-brain imaging after aversive footshock stimulation in activity-regulated cytoskeleton protein (Arc)-dVenus D1RKD mice, which were crossbred from Arc-dVenus transgenic mice and D1RKD mice, to examine the distribution of Arc-controlled dVenus expression in the hippocampus and cerebral cortex during aversive memory formation. Knockdown of D1R expression following Dox treatment resulted in impaired performance in the passive avoidance test and was associated with a decrease in dVenus expression in the cerebral cortex (visual, somatosensory, and motor cortices), but not the hippocampus, compared with control mice without Dox treatment. These findings indicate that D1R-mediated dopaminergic transmission is critical for aversive memory formation, specifically by influencing Arc expression in the cerebral cortex.

DOI： 10.1016/j.neures.2020.04.006

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Language：English   Publishing type：Research paper (scientific journal)  

Whole-organ/body three-dimensional (3D) staining and imaging have been enduring challenges in histology. By dissecting the complex physicochemical environment of the staining system, we developed a highly optimized 3D staining imaging pipeline based on CUBIC. Based on our precise characterization of biological tissues as an electrolyte gel, we experimentally evaluated broad 3D staining conditions by using an artificial tissue-mimicking material. The combination of optimized conditions allows a bottom-up design of a superior 3D staining protocol that can uniformly label whole adult mouse brains, an adult marmoset brain hemisphere, an ~1 cm3 tissue block of a postmortem adult human cerebellum, and an entire infant marmoset body with dozens of antibodies and cell-impermeant nuclear stains. The whole-organ 3D images collected by light-sheet microscopy are used for computational analyses and whole-organ comparison analysis between species. This pipeline, named CUBIC-HistoVIsion, thus offers advanced opportunities for organ- and organism-scale histological analysis of multicellular systems.

DOI： 10.1038/s41467-020-15906-5

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

We describe a strategy for developing hydrophilic chemical cocktails for tissue delipidation, decoloring, refractive index (RI) matching, and decalcification, based on comprehensive chemical profiling. More than 1,600 chemicals were screened by a high-throughput evaluation system for each chemical process. The chemical profiling revealed important chemical factors: salt-free amine with high octanol/water partition-coefficient (logP) for delipidation, N-alkylimidazole for decoloring, aromatic amide for RI matching, and protonation of phosphate ion for decalcification. The strategic integration of optimal chemical cocktails provided a series of CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis) protocols, which efficiently clear mouse organs, mouse body including bone, and even large primate and human tissues. The updated CUBIC protocols are scalable and reproducible, and they enable three-dimensional imaging of the mammalian body and large primate and human tissues. This strategy represents a future paradigm for the rational design of hydrophilic clearing cocktails that can be used for large tissues.

DOI： 10.1016/j.celrep.2018.07.056

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

A three-dimensional single-cell-resolution mammalian brain atlas will accelerate systems-level identification and analysis of cellular circuits underlying various brain functions. However, its construction requires efficient subcellular-resolution imaging throughout the entire brain. To address this challenge, we developed a fluorescent-protein-compatible, whole-organ clearing and homogeneous expansion protocol based on an aqueous chemical solution (CUBIC-X). The expanded, well-cleared brain enabled us to construct a point-based mouse brain atlas with single-cell annotation (CUBIC-Atlas). CUBIC-Atlas reflects inhomogeneous whole-brain development, revealing a significant decrease in the cerebral visual and somatosensory cortical areas during postnatal development. Probabilistic activity mapping of pharmacologically stimulated Arc-dVenus reporter mouse brains onto CUBIC-Atlas revealed the existence of distinct functional structures in the hippocampal dentate gyrus. CUBIC-Atlas is shareable by an open-source web-based viewer, providing a new platform for whole-brain cell profiling.

DOI： 10.1038/s41593-018-0109-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Temperature compensation is a striking feature of the circadian clock. Here we investigate biochemical mechanisms underlying temperature-compensated, CKI delta-dependent multi-site phosphorylation in mammals. We identify two mechanisms for temperature-insensitive phosphorylation at higher temperature: lower substrate affinity to CKI delta-ATP complex and higher product affinity to CKI delta-ADP complex. Inhibitor screening of ADP-dependent phosphatase activity of CKI delta identified aurintricar-boxylic acid (ATA) as a temperature-sensitive kinase activator. Docking simulation of ATA and mutagenesis experiment revealed K224D/K224E mutations in CKI delta that impaired product binding and temperature-compensated primed phosphorylation. Importantly, K224D mutation shortens behavioral circadian rhythms and changes the temperature dependency of SCN's circadian period. Interestingly, temperature-compensated phosphorylation was evolutionary conserved in yeast. Molecular dynamics simulation and X-ray crystallography demonstrate that an evolutionally conserved CKI-specific domain around K224 can provide a structural basis for temperature-sensitive substrate and product binding. Surprisingly, this domain can confer temperature compensation on a temperature-sensitive TTBK1. These findings suggest the temperature-sensitive substrate-and product-binding mechanisms underlie temperature compensation.

DOI： 10.1016/j.molcel.2017.08.009

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Stochastic and proliferative events initiated from a single cell can disrupt homeostatic balance and lead to fatal disease processes such as cancer metastasis. To overcome metastasis, it is necessary to detect and quantify sparsely distributed metastatic cells throughout the body at early stages. Here, we demonstrate that clear, unobstructed brain/ body imaging cocktails and computational analysis (CUBIC)-based cancer (CUBIC-cancer) analysis with a refractive index (RI)=optimized protocol enables comprehensive cancer cell profiling of the whole body and organs. We applied CUBIC-cancer analysis to 13 mouse models using nine cancer cell lines and spatiotemporal quantification of metastatic cancer progression at single-cell resolution. CUBIC-cancer analysis suggests that the epithelial-mesenchymal transition promotes not only extravasation but also cell survival at metastatic sites. CUBIC-cancer analysis is also applicable to pharmacotherapeutic profiling of anti-tumor drugs. CUBIC-cancer analysis is compatible with in vivo bioluminescence imaging and 2D histology. We suggest that a scalable analytical pipeline with these three modalities may contribute to addressing currently incurable metastatic diseases.

DOI： 10.1016/j.celrep.2017.06.010

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Language：English   Publishing type：Part of collection (book)   Publisher：ANNUAL REVIEWS  

Mammalian bodies have more than a billion cells per cubic centimeter, which makes whole-body cell (WBC) profiling of an organism one of the ultimate challenges in biology and medicine. Recent advances in tissue-clearing technology have enabled rapid and comprehensive cellular analyses in whole organs and in the whole body by a combination of state-of-the-art technologies of optical imaging and image informatics. In this review, we focus mainly on the chemical principles in currently available techniques for tissue clearing and staining to facilitate our understanding of their underlying mechanisms. Tissue clearing is usually conducted by the following steps: (a) fixation, (b) permeabilization, (c) decolorizing, and (d) refractive index (RI) matching. To phenotype individual cells after tissue clearing, it is important to visualize genetically encoded fluorescent reporters and/or to stain tissues with fluorescent dyes, fluorescent labeled antibodies, or nucleic acid probes. Although some technical challenges remain, the chemical principles in tissue clearing and staining for WBC profiling will enable various applications, such as identifying cellular circuits across multiple organs and measuring their dynamics in stochastic and proliferative cellular processes, for example, autoimmune and malignant neoplastic diseases.

DOI： 10.1146/annurev-cellbio-111315-125001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Here we describe a protocol for advanced CUCUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis). The CUCUBIC protocol enables simple and efficient organ clearing, rapid imaging by light-sheet microscopy and quantitative imaging analysis of multiple samples. The organ or body is cleared by immersion for 1-14 d, with the exact time required dependent on the sample type and the experimental purposes. A single imaging set can be completed in 30-60 min. Image processing and analysis can take &lt;1 d, but it is dependent on the number of samples in the data set. The CUCUBIC clearing protocol can process multiple samples simultaneously. We previously used CUCUBIC to image whole-brain neural activities at single-cell resolution using Arc-dVenus transgenic (Tg) mice. CUCUBIC informatics calculated the Venus signal subtraction, comparing different brains at a whole-organ scale. These protocols provide a platform for organism-level systems biology by comprehensively detecting cells in a whole organ or body.

DOI： 10.1038/nprot.2015.085

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

The development of whole-body imaging at single-cell resolution enables system-level approaches to studying cellular circuits in organisms. Previous clearing methods focused on homogenizing mismatched refractive indices of individual tissues, enabling reductions in opacity but falling short of achieving transparency. Here, we show that an aminoalcohol decolorizes blood by efficiently eluting the heme chromophore from hemoglobin. Direct transcardial perfusion of an aminoalcohol-containing cocktail that we previously termed CUBIC coupled with a 10 day to 2 week clearing protocol decolorized and rendered nearly transparent almost all organs of adult mice as well as the entire body of infant and adult mice. This CUBIC-perfusion protocol enables rapid whole-body and whole-organ imaging at single-cell resolution by using light-sheet fluorescent microscopy. The CUBIC protocol is also applicable to 3D pathology, anatomy, and immunohistochemistry of various organs. These results suggest that whole-body imaging of colorless tissues at high resolution will contribute to organism-level systems biology.

DOI： 10.1016/j.cell.2014.10.034

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Systems-level identification and analysis of cellular circuits in the brain will require the development of whole-brain imaging with single-cell resolution. To this end, we performed comprehensive chemical screening to develop a whole-brain clearing and imaging method, termed CUBIC (clear, unobstructed brain imaging cocktails and computational analysis). CUBIC is a simple and efficient method involving the immersion of brain samples in chemical mixtures containing aminoalcohols, which enables rapid whole-brain imaging with single-photon excitation microscopy. CUBIC is applicable to multicolor imaging of fluorescent proteins or immunostained samples in adult brains and is scalable from a primate brain to subcellular structures. We also developed a whole-brain cell-nuclear counterstaining protocol and a computational image analysis pipeline that, together with CUBIC reagents, enable the visualization and quantification of neural activities induced by environmental stimulation. CUBIC enables time-course expression profiling of whole adult brains with single-cell resolution.

DOI： 10.1016/j.cell.2014.03.042

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

DNA can be concatenated by hybridization of DNA fragments with protruding single-stranded termini. DNA cleavage occurring at a nucleotide containing a DNA base analogue is a useful method to obtain DNA with designed protruding termini. Here, we report a novel non-enzymatic DNA cleavage reaction for DNA concatenation. We found that DNA is cleaved at a nucleotide containing 5-ethynyluracil in a methylamine aqueous solution to generate 59-phosphorylated DNA fragment as a cleavage product. We demonstrated that the reaction can be applied to DNA concatenation of PCR-amplified DNA fragments. This novel non-enzymatic DNA cleavage reaction is a simple practical approach for DNA concatenation.

DOI： 10.1371/journal.pone.0092369

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Hyperphosphorylated forms of tau protein are the main component of paired helical filaments (PHFs) of neurofibrillary tangles in the brain of Alzheimer's disease patients. To understand the effect of phosphorylation on the fibrillation of tau, we utilized tau-derived phosphorylated peptides. The V(306)QIVYK(311) sequence (PHF6) in the microtubule-binding domain is known to play a key role in the fibrillation of tau, and the short peptide corresponding to the PHF6 sequence forms amyloid-type fibrils similar to those generated by full-length tau. We focused on the amino acid residue located at the N-terminus of the PHF6 sequence, serine or lysine in the native isoform of tau, and synthesized the PHF6 derivative peptides with serine or lysine at the N-terminus of PHF6. Peptides phosphorylated at serine and/or tyrosine were synthesized to mimic the possible phosphorylation at these positions. The critical concentrations of the fibrillation of peptides were determined to quantitatively assess fibril stability. The peptide with the net charge of near zero tended to form stable fibrils. Interestingly, the peptide phosphorylated at the N-terminal serine residue exhibited remarkably low fibrillation propensity as compared to the peptide possessing the same net charge. Transmission electron microscopy measurements of the fibrils visualized the paired helical or straight fibers and segregated masses of the fibers or heterogeneous rodlike fibers depending on the phosphorylation status. Further analyses of the fibrils by the X-ray fiber diffraction method and Fourier transform infrared spectroscopic measurements indicated that all the peptides shared a common cross-beta structure. In addition, the phosphoserine-containing peptides showed the characteristics of beta-sandwiches that could interact with both faces of the beta-sheet. On the basis of these observations, possible protofilament models with four beta-sheets were constructed to consider the positional effects of the serine and/or tyrosine phosphorylations. The electrostatic intersheet interaction between phosphate groups and the amino group of lysine enhanced the lateral association between beta-sheets to compensate for the excess charge. In addition to the previously postulated net charge of the peptide, the position of the charged residue plays a critical role in the amyloid fibrillation of tau.

DOI： 10.1021/bi201451z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

DOI： 10.1002/chem.201101964

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Blackwell Publishing Inc.  

DNA methylation and demethylation significantly affect the deactivation and activation processes of gene expression, respectively. The determination of the location and frequency of DNA methylation is important for the elucidation of the mechanisms of cell differentiation and carcinogenesis and may be a useful and effective index for cancer diagnosis. We have developed an artificial DNA probe that induces a methylation detection reaction of a target cytosine in a long DNA sequence (ICON probe). This artificial DNA allows the rapid detection of a methyl group attached at the C5 position of the target cytosine. In addition, there is no nonspecific cleavage of genomic DNA in this reaction. The ICON probe also facilitates the quantification of methylation at the target cytosine using a small amount of genomicDNAsample. This unit provides a procedure for synthesizing bipyridine-modified adenosine phosphoramidite and preparation of ICON probes. Additionally, the protocol for the methylation quantification experiments by quantitative PCR utilizing ICON probes is also presented. © 2011 by John Wiley &amp
Sons, Inc.

DOI： 10.1002/0471142700.nc0807s47

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

DNA-mediated charge transfer has recently received a substantial attention because of its biological relevance in the DNA damage and DNA repair as well as the potential applications to nanoscale electronic devices In contrast to the numerous mechanistic studies on oxidative hole transfer (HT) through DNA our understanding of reductive electron transfer process still remains limited In this article, we demonstrate the results of direct observation of the excess electron transfer (EET) through DNA, which conjugated with aminopyrene ((A)Py) and diphenylacetylene (DPA) as a photosensitizing donor and an acceptor of excess electron, respectively By inserting dihydrothymine as a spacer between (A)Py and T or C, the yield of electron arrival to DPA was improved It was indicated that EET through DNA completed within a few or a few tens nanosecond time scale even for EET over 34 angstrom for both consecutive T and C sequences The various factors such as mismatch sequence and DNA length on the yield of electron arrival to DPA were examined

DOI： 10.1021/jp1024685

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MOLECULAR DIVERSITY PRESERVATION INTERNATIONAL-MDPI  

Fluorescent biosensors to detect the bona fide events of biologically important molecules in living cells are increasingly demanded in the field of molecular cell biology. Recent advances in the development of fluorescent biosensors have made an outstanding contribution to elucidating not only the roles of individual biomolecules, but also the dynamic intracellular relationships between these molecules. However, rational design strategies of fluorescent biosensors are not as mature as they look. An insatiable request for the establishment of a more universal and versatile strategy continues to provide an attractive alternative, so-called modular strategy, which permits facile preparation of biosensors with tailored characteristics by a simple combination of a receptor and a signal transducer. This review describes an overview of the progress in design strategies of fluorescent biosensors, such as auto-fluorescent protein-based biosensors, protein-based biosensors covalently modified with synthetic fluorophores, and signaling aptamers, and highlights the insight into how a given receptor is converted to a fluorescent biosensor. Furthermore, we will demonstrate a significance of the modular strategy for the sensor design.

DOI： 10.3390/s100201355

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Language：English   Publishing type：Research paper (scientific journal)  

We have recently introduced novel methods for constructing a wide variety of ligand-binding receptors and fluorescent biosensors based on a framework of ribonucleopeptide (RNP) composed of an RNA subunit and a peptide subunit. The modular RNP complex would be potentially applicable to the fabrication of the artificial enzyme because each subunit could be functionalized separately. In this study, we focused on the material conversion process in the photosynthesis and aimed at the development of the artificial photodriven enzymes based on the RNP complex. The strict control of the configuration and orientation of functionalized motifs is needed for the rational design of the RNP-based enzyme. To clarify the relationship between the structure and the function of RNP receptor, secondary structure of the ATP-binding RNP receptor was investigated. As a result, the model for the ATP-binding RNP complex could be proposed based on the chemical structure analyses. © Springer 2010.

DOI： 10.1007/978-4-431-99779-5_27

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

DOI： 10.1002/anie.200903951

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

A fluorescent sensor for the detection of inositol-1,3,4,5-tetrakisphosphate, Ins(1,3,4,5)P(4), was constructed from a split PH domain and a single circularly permuted GFP. A structure-based design was conducted to transduce a ligand-induced subtle structural perturbation of the split PH domain to an alteration in the population of the protonated and the deprotonated states of the GFP chromophore. Excitation of each distinct absorption band corresponding to the protonated or the deprotonated state of GFP resulted an increase and a decrease, respectively, in the intensity of emission spectra upon addition of Ins(1,3,4,5)P(4) to the split PH domain-based sensor. The Ins(1,3,4,5)P(4) sensor retained the ligand affinity and the selectivity of the parent PH domain, and realized the ratiometric fluorescence detection of Ins(1,3,4,5)P(4). (c) 2009 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmc.2009.08.015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The efficiency of osmium complex formation at 5-methylcytosine in mismatched DNA duplexes is a key point for the design of sequence-specific detection of DNA methylation. Osmium complexation was not observed in fully matched duplexes, whereas the complexation site and efficiency in mismatched duplexes changed depending on the type of 5&apos;-neighboring base of the 5-methylcytosine forming a mismatched base pair. In particular, when the base adjacent to the 5&apos; side of the mismatched base pair was thymine, a unique "side reaction" was observed. However, the nature of the mismatched base pairs in the reaction site did not influence the selectivity of osmium complex formation with methylated DNA.

DOI： 10.1021/bc800531z

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DOI： 10.1093/nass/nrp104

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Other Link： http://orcid.org/0000-0002-7418-6237

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.49.S39_5

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Language：English   Publisher：The Materials Research Society of Japan  

Post-translational phosphorylation of the microtubule-associated protein tau is believed to have close relevance to the pathogenesis of Alzheimers disease. The growth of amyloid fibrils of the short peptide segment VQIVY₃₁₀K (PHF6) corresponding to the core part of tau fibril formation is strongly affected by the phospholylation at Tyr310 via its electrostatic pairing with the neighboring charged lysine. Herein, we studied the propensity of resultant fibrils from PHF6 derivative peptides with charged aromatic amino acid residue at Tyr310. The results showed that the phosphorylation at Tyr310 contributed to significant enhancement of the amyloidogenicity and the stability of fibrillar aggregates. The physical origin of remarkable stabilization of fibrils by tyrosine phosphorylation is plausibly attributed to the charge pairing with the adjacent lysine residue in a similar fashion to the phosphorylation effect on the growth of amyloid fibril.

DOI： 10.14723/tmrsj.34.517

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Phosphorylation of a fibrillogenic protein, human tau, is believed to play crucial roles in the pathogenesis of Alzheimer&apos;s disease. For elucidating molecular mechanisms of the phosphorylation effect on tau fibrillation, we synthesized a peptide, VQTVY(310)K (PHF6) and its phosphorylated derivative (PHF(6)pY). PHF6 is a partial peptide surrounding a plausible in vivo phosphorylation site Tyr310 and forms amyloid-type fibrils similar to those generated by full-length tau. Fibrillation of PHF6 and PHF6pY were studied by spectroscopic and microscopic methods, and the critical concentration of the fibrillation was determined for comparing the fibril stability. The results showed that the phosphorylation strongly influenced the fibrillation propensity of PHF6 by changing its dependency on pH and ionic strength. On the basis of the observations, we suggested that charged sites on the phosphate group and its electrostatic pairing with the neighboring charged residues were physical origins of the phosphorylation effect. To verify this charge-pairing mechanism, we conducted experiments using a series of PHF6 derivatives with non-native charge distributions. The electrostatic interaction in an intermolecular mode was also demonstrated by the system composed of two different peptide species, which found that fibrillation of nonphosphorylated PHF6 was drastically enhanced when a trace amount of phosphorylated PHF6 molecules coexisted. A simulation analysis utilizing crystal coordinates of the PHF6 fibril was also performed for interpreting the experimental results in a molecular level. The present study using the model peptide system gave us a microscopically insightful view on the roles of tau phosphorylation in amyloid-related diseases.

DOI： 10.1021/bi8010994

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Nucleic acids often acquire new functions by forming a variety of complexes with metal ions. Osmium, in an oxidized state, also reacts with C5-methylated pyrimidines. However, control of the sequence specificity of osmium complexation with DNA is still immature, and the value of the resulting complexes is unknown. We have designed a bipyridine-attached adenine derivative for sequence-specific osmium complexation. Sequence-specific osmium complexation was achieved by hybridization of a short DNA molecule containing this functional nucleotide to a target DNA sequence and resulted in the formation of a cross-linked structure. The interstrand cross-link clearly distinguished methylated cytosines from unmethylated cytosines and was used to quantify the degree of methylation at a specific cytosine in the genome.

DOI： 10.1021/ja076140r

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Cytosine methylation is one of the most important epigenetic events, and much effort has been directed to develop a simple reaction for methylcytosine detection. In this paper, we describe the design of tag-attachable ligands for direct methylcytosine labeling and their application to fluorescent and electrochemical assays. The effect of the location of bipyridine substituents on the efficiency of osmium complexation at methylcytosine was initially investigated. As a result, a bipyridine derivative with a substituent at the C4 position showed efficient complexation at the methylcytosine residue of single-stranded DNA in a reaction mixture containing potassium osmate and potassium hexacyanoferrate(III). On the basis of this result, a bipyridine derivative with a tag-attachable amino linker at the C4 position was synthesized. The efficiency of metal complex formation in the presence of the osmate and the synthetic ligand was clearly changed by the presence/absence of a methyl group at the C5 position of cytosine. The succinimidyl esters of functional labeling units were then attached to the bipyridine ligand fixed on the methylcytosine. These labels attached to methylcytosine enabled us to detect the target methylcytosine in DNA both fluorometrically and electrochemically. For example, we were able to fluorometrically obtain information on the methylation status at a specific site by means of fluorescence resonance energy transfer from a hybridized fluorescent DNA probe to a fluorescent label on methylcytosine. In addition, by the combination of electrochemically labeled methylcytosine and an electrode modified by probe DNAs, a methylcytosine-selective characteristic current signal was observed. This direct labeling of methylcytosine is a conceptually new methylation detection assay with many merits different from conventional assays.

DOI： 10.1021/ja068660c

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

The design of probes for monitoring various structures and dynamics of DNA and its surroundings is an important step in understanding biological events accompanying interbiomolecular interaction. We have developed novel fluorescent nucleosides in which the uracil base and the fluorophore are tethered by rigid linkers. They show unique absorption and fluorescence emission spectra. Nucleoside 2 is a fluorophore with high CT character and the fluorescence is very sensitive to solvent polarity. Nucleoside 3 shows absorption and emission maxima with longer wavelength due to extension of the DAN-conjugate system. These fluorophore-deoxyuridine conjugates with unique fluorescence properties would work as reporter probes sensitive to the change in microenvironment around specific sites of DNA. (c) 2007 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.tet.2006.09.113

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

A solvatochromic fluorophore, PRODAN, has been used as a microenvironment-sensitive reporter. Based on the chemistry of PRODAN, we designed and synthesized four novel fluorescent nucleosides, X-PDN (X = U, C, A, and G), to which a PRODAN fluorophore was attached at pyrimidine C5 or purine C8. The fluorescent nucleosides sensitively varied the Stokes shift values depending on the orientational polarizability of the solvent. The X-PDN incorporated into DNA also changed the Stokes shift values depending on the DNA structure. In particular, the excitation spectrum of the X-PDN-containing duplex shifted to a longer wavelength and gave a smaller Stokes shift value when the base opposite X-PDN could form a Watson-Crick base pair with X-PDN. A lower energy excitation of X-PDN-containing DNA resulted in a strong fluorescence emission selective to the Watson-Crick pairing base. This unique photochemical character was applicable to the efficient typing of single-nucleotide polymorphisms of genes.

DOI： 10.1021/ja069156a

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

We report on the control of the emission from a fluorophore fixed on DNA using the methylcytosine-selective addition of an osmium-bipyridine complex. We have synthesized DNA modified by a microenvironment-sensitive fluorophore, 2-dimethylamino-6-acyl-naphthalene. The emission from the fluorophore tethered to a probe DNA was effectively quenched by a methylcytosine glycol-osmium-bipyridine triad, which was located in the immediate neighborhood of the fluorophore. The discrimination of the cytosine methylation status at a methylation hot spot in the p53 gene was also executed using a well-designed fluorescent DNA probe. (c) 2006 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmc.2006.12.023

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DOI： 10.1093/nass/nrm150

PubMed

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Other Link： http://orcid.org/0000-0002-7418-6237

DOI： 10.1093/nass/nrm088

PubMed

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Other Link： http://orcid.org/0000-0002-7418-6237

DOI： 10.1093/nass/nrm014

PubMed

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Other Link： http://orcid.org/0000-0002-7418-6237

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The fluorophore, Nile Red, effectively works as a polarity-sensitive fluorescence probe. We have designed a new nucleoside modified by Nile Red for examining the change in the polarity of the microenvironment surrounding DNA. We synthesized a Nile Red nucleoside (1), formed by replacing nucleobases with Nile Red, through the coupling of a 2-hydroxylated Nile Red derivative and 1,2-dideoxyglycan. This nucleoside showed a high solvatofluorochromicity. The fluorescence of 1 incorporated into DNA was greatly shifted to shorter wavelength by the addition of beta-cyclodextrin. The photophysical function of the Nile Red nucleoside will be a good optical indicator for monitoring the change in the micropolarity properties at a specific site on target sequences with interaction between DNA and DNA-binding molecules.

DOI： 10.1021/jo060168o

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

We have developed a new concept involving a single-step homogeneous method for single-nucleotide polymorphism (SNP) typing. in this method, a probe containing base-discriminating fluorescent (BDF) bases is added to a sample solution. BDF base-containing DNA usually shows only a weak fluorescence, but emits a strong blue fluorescence when it recognizes a target base at a specific site in a hybridized strand. By utilizing this feature, a simple mix-and-read SNP typing assay was achieved without any tedious probe-designing or washing processes for exclusion of hybridization error or any addition of DNA-modifying enzymes. This is very different from conventional methods. We simultaneously analyzed a number of samples with ease, with a high accuracy, using our BDF assay.

DOI： 10.1039/b515923g

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DOI： 10.1093/nass/nrl065

PubMed

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DOI： 10.1093/nass/nrl064

PubMed

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Other Link： http://orcid.org/0000-0002-7418-6237

DOI： 10.1093/nass/nrl068

PubMed

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Other Link： http://orcid.org/0000-0002-7418-6237

DOI： 10.1093/nass/nrl069

PubMed

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Other Link： http://orcid.org/0000-0002-7418-6237

DOI： 10.1093/nass/nrl066

PubMed

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Other Link： http://orcid.org/0000-0002-7418-6237

DOI： 10.1093/nass/nrl067

PubMed

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Other Link： http://orcid.org/0000-0002-7418-6237

DOI： 10.1093/nass/nrl070

PubMed

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Other Link： http://orcid.org/0000-0002-7418-6237

DOI： 10.1093/nass/nrl105

PubMed

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

5-Methylcytosine was distinguished from cytosine using the large difference of their osmium oxidation rates, and this reaction was applied to detection of the cytosine methylation status at a specific site of a long sequence using the formation of a bulge structure by hybridization with a guide DNA.

DOI： 10.1039/b600401f

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

We report a novel fluorescent nucleobase, U-DNC, for the mapping of the local dielectric properties around DNA. Fluorescence spectra of the nucleoside d U-DNC showed a significant shift depending on the solvent polarity. This property was used to determine the dielectric constants of the interior of DNA-binding proteins, such as DNA polymerases. We were able to demonstrate that the DNA-binding region of the Klenow fragment is a lower polarity site, as shown by the blue shift of the fluorescence peak originating from U-DNC.

DOI： 10.1021/bc050077i

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

DOI： 10.1021/ja053609e

Web of Science

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：ACAD SCI CZECH REPUBLIC, INST ORGANIC CHEM & BIOCHEMISTRY  

We designed a new type of pyrene-labeled base-discrimination fluorescent (BDF) nucleosides U-PY, C-PY, (8PY)A and (MePY)dA, which emitted strong florescence only when the bases opposite the BDF bases are A, G, T and C, respectively. The DNA probes containing four different BDF bases enabled us to distinguish single base alterations by simply mixing with a sample solution of target DNA. A example of SNP typing of c-Ha-ras SNP sequence has been demonstrated. Detection of base insertion in indel polymorphisms using pyrene excimer fluorescent probe has also been described.

Web of Science

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DOI： 10.1093/nass/49.1.201

PubMed

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Other Link： http://orcid.org/0000-0002-7418-6237

DOI： 10.1093/nass/49.1.155

PubMed

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Other Link： http://orcid.org/0000-0002-7418-6237

DOI： 10.1093/nass/49.1.45

PubMed

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Other Link： http://orcid.org/0000-0002-7418-6237

DOI： 10.1093/nass/48.1.31

PubMed

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Other Link： http://orcid.org/0000-0002-7418-6237

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

A new base-discriminating fluorescent nucleoside, NPP, that can sharply distinguish between A and G bases opposite NPP is described. The hybridization of an ODN probe containing NPP with a target DNA facilitates the judgment of the type of purine base located at a specific site on the target DNA. (C) 2003 Published by Elsevier Ltd.

DOI： 10.1016/S0040-4039(03)01740-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHEMICAL SOC JAPAN  

We report the fluorescence properties of oligodeoxynucleotides (ODNs) containing a base-discriminating fluorescent nucleoside, benzopyridopyrimidine (BPP), in hybridizing with RNAs possessing different bases opposite BPP. BPP-containing ODN selectively exhibited a strong fluorescence when the RNA base opposite BPP was A, whereas the fluorescence of BPP-containing ODN hybridized with RNA possessing G opposite BPP was very weak. BPP acts as a good base-discriminating fluorescent nucleoside for A/G single base alteration of RNA.

DOI： 10.1246/cl.2003.684

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

DOI： 10.1021/ja034090u

Web of Science

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

A modified nucleobase 7-vinyl-7-deazaguanine ((V)G) produced adducts with maleimides through Diels-Alder cycloaddition under very mild conditions. By this method, post-synthetic modification to oligonucleotides with diverse functionality (carboxylic acid, pyrene, benzophenone, succinimidyl ester, nitroxide and biotin) was accomplished. (C) 2002 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S0960-894X(02)00334-7

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Language：English   Publishing type：Research paper (scientific journal)  

The first facile incorporation of an aldehyde function into DNA without any protection/deprotection of the aldehyde was achieved by the use of 3-formylindole 2′-deoxynucleoside (1). This building block is very easily prepared and efficiently incorporated into oligonucleotides. In addition, 1 acted as a universal nucleoside in duplex. The post-synthetic modification of oligonucleotides bearing 1 with functionalized molecules was also effective. © 2002 Published by Elsevier Science Ltd.

DOI： 10.1016/S0040-4039(02)00888-2

Scopus

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Language：English   Publisher：(公社)日本生化学会  

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Language：Japanese  

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Language：Japanese  

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Language：Japanese  

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Language：Japanese   Publisher：公益財団法人 日本心臓財団  

DOI： 10.11281/shinzo.47.285

CiNii Article

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Language：Japanese   Publisher：公益社団法人 日本農芸化学会  

DOI： 10.1271/kagakutoseibutsu.53.737

CiNii Article

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Language：Japanese  

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Language：Japanese  

J-GLOBAL

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Language：Japanese  

J-GLOBAL

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Language：Japanese  

J-GLOBAL

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Language：Japanese  

J-GLOBAL

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Language：Japanese  

J-GLOBAL

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Language：English   Publishing type：Research paper, summary (international conference)  

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Language：Japanese  

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Language：Japanese  

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Language：English  

DOI： 10.1093/nass/nrn099

PubMed

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Language：English  

DOI： 10.1093/nass/nrn101

PubMed

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Language：English  

DOI： 10.1093/nass/nrn102

PubMed

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DOI： 10.1351/pac200678122305

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER CHEMICAL SOC  

Web of Science

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Language：Japanese  

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Language：Japanese   Presentation type：Oral presentation (keynote)  

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Language：English   Presentation type：Oral presentation (invited, special)  

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Language：English   Presentation type：Oral presentation (invited, special)  

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Language：Japanese   Presentation type：Oral presentation (invited, special)  

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Language：Japanese   Presentation type：Oral presentation (invited, special)  

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Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Language：Japanese   Presentation type：Oral presentation (invited, special)  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Language：English   Presentation type：Symposium, workshop panel (nominated)  

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Language：English   Presentation type：Oral presentation (invited, special)  

*Lecture of the Anne Heidenthal Prize for Fluorescence Research

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Application no：特願2005-335005  Date applied：2005.11

Announcement no：特開2006-169240  Date announced：2006.6

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Application no：US 11/135,368  Date applied：2005.5

Announcement no：US20060142311 A1  Date announced：2006.6

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Application no：特願2002-333326  Date applied：2002.11

Announcement no：特開2004-168672  Date announced：2004.6

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Application no：特願2007-520033 

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Application no：PCT/JP2014/070618 

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