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

In bacterial synthetic biology, whole genome transplantation has been achieved only in mycoplasmas that contain a small genome and are competent for foreign genome uptake. In this study, we developed Escherichia coli strains programmed by three 1-megabase (Mb) chromosomes by splitting the 3-Mb chromosome of a genome-reduced strain. The first split-chromosome retains the original replication origin (oriC) and partitioning (par) system. The second one has an oriC and the par locus from the F plasmid, while the third one has the ori and par locus of the Vibrio tubiashii secondary chromosome. The tripartite-genome cells maintained the rod-shaped form and grew only twice as slowly as their parent, allowing their further genetic engineering. A proportion of these 1-Mb chromosomes were purified as covalently closed supercoiled molecules with a conventional alkaline lysis method and anion exchange columns. Furthermore, the second and third chromosomes could be individually electroporated into competent cells. In contrast, the first split-chromosome was not able to coexist with another chromosome carrying the same origin region. However, it was exchangeable via conjugation between tripartite-genome strains by using different selection markers. We believe that this E. coli-based technology has the potential to greatly accelerate synthetic biology and synthetic genomics.

DOI： 10.1093/nar/gkab298

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

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

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

DOI： 10.1021/acssynbio.0c00008

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DOI： 10.1074/jbc.RA119.008219

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

Cell-free protein synthesis is useful for synthesizing difficult targets. The site-specific incorporation of non-natural amino acids into proteins is a powerful protein engineering method. In this study, we optimized the protocol for cell extract preparation from the Escherichia coli strain RFzero-iy, which is engineered to lack release factor 1 (RF-1). The BL21(DE3)-based RFzero-iy strain exhibited quite high cell-free protein productivity, and thus we established the protocols for its cell culture and extract preparation. In the presence of 3-iodo-l-tyrosine (IY), cell-free protein synthesis using the RFzero-iy-based S30 extract translated the UAG codon to IY at various sites with a high translation efficiency of >90%. In the absence of IY, the RFzero-iy-based cell-free system did not translate UAG to any amino acid, leaving UAG unassigned. Actually, UAG was readily reassigned to various non-natural amino acids, by supplementing them with their specific aminoacyl-tRNA synthetase variants (and their specific tRNAs) into the system. The high incorporation rate of our RFzero-iy-based cell-free system enables the incorporation of a variety of non-natural amino acids into multiple sites of proteins. The present strategy to create the RFzero strain is rapid, and thus promising for RF-1 deletions of various E. coli strains genomically engineered for specific requirements.

DOI： 10.3390/ijms20030492

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Language：English  

DOI： 10.1021/acssynbio.8b00157

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

DOI： 10.1080/15476286.2018.1474074

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

DOI： 10.1002/ange.201713215

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

DOI： 10.1002/anie.201713215

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

The diversity of the genetic code systems used by microbes on earth is yet to be elucidated. It is known that certain methanogenic archaea employ an alternative system for cysteine (Cys) biosynthesis and encoding; tRNACys is first acylated with phosphoserine (Sep) by O-phosphoseryl-tRNA synthetase (SepRS) and then converted to Cys-tRNACys by Sep-tRNA:Cys-tRNA synthase (SepCysS). In this study, we searched all genomic and metagenomic protein sequence data in the Integrated Microbial Genomes (IMG) system and at the NCBI to reveal new clades of SepRS and SepCysS proteins belonging to diverse archaea in the four major groups (DPANN, Euryarchaeota, TACK, and Asgard) and two groups of bacteria ("Candidatus Parcubacteria" and Chloroflexi). Bacterial SepRS and SepCysS charged bacterial tRNACys species with cysteine in vitro. Homologs of SepCysE, a scaffold protein facilitating SepRS.SepCysS complex assembly in Euryarchaeota class I methanogens, are found in a few groups of TACK and Asgard archaea, whereas the C-terminally truncated homologs exist fused or genetically coupled with diverse SepCysS species. Investigation of the selenocysteine (Sec)-and pyrrolysine (Pyl)-utilizing traits in SepRSutilizing archaea and bacteria revealed that the archaea carrying full-length SepCysE employ Sec and that SepRS is often found in Pyl-utilizing archaea and Chloroflexi bacteria. We discuss possible contributions of the SepRS-SepCysS system for sulfur assimilation, methanogenesis, and other metabolic processes requiring large amounts of iron-sulfur enzymes or Pyl-containing enzymes.
IMPORTANCE Comprehensive analyses of all genomic and metagenomic protein sequence data in public databases revealed the distribution and evolution of an alternative cysteine-encoding system in diverse archaea and bacteria. The finding that the SepRS-SepCysS-SepCysE-and the selenocysteine-encoding systems are shared by the Euryarchaeota class I methanogens, the Crenarchaeota AK8/W8A-19 group, and an Asgard archaeon suggests that ancient archaea may have used both systems. In contrast, bacteria may have obtained the SepRS-SepCysS system from archaea. The SepRS-SepCysS system sometimes coexists with a pyrrolysine-encoding system in both archaea and bacteria. Our results provide additional bioinformatic evidence for the contribution of the SepRS-SepCysS system for sulfur assimilation and diverse metabolisms which require vast amounts of iron-sulfur enzymes and proteins. Among these biological activities, methanogenesis, methylamine metabolism, and organohalide respiration may have local and global effects on earth. Taken together, uncultured bacteria and archaea provide an expanded record of the evolution of the genetic code.

DOI： 10.1128/mBio.00561-17

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

We report the identification of novel tRNA species with 12-base pair amino-acid acceptor branches composed of longer acceptor stem and shorter T-stem. While canonical tRNAs have a 7/5 configuration of the branch, the novel tRNAs have either 8/4 or 9/3 structure. They were found during the search for selenocysteine tRNAs in terabytes of genome, metagenome and metatranscriptome sequences. Certain bacteria and their phages employ the 8/4 structure for serine and histidine tRNAs, while minor cysteine and selenocysteine tRNA species may have a modified 8/4 structure with one bulge nucleotide. In Acidobacteria, tRNAs with 8/4 and 9/3 structures may function as missense and nonsense suppressor tRNAs and/or regulatory non-coding RNAs. In delta-proteobacteria, an additional cysteine tRNA with an 8/4 structure mimics selenocysteine tRNA and may function as opal suppressor. We examined the potential translation function of suppressor tRNA species in Escherichia coli; tRNAs with 8/4 or 9/3 structures efficiently inserted serine, alanine and cysteine in response to stop and sense codons, depending on the identity element and anticodon sequence of the tRNA. These findings expand our view of how tRNA, and possibly the genetic code, is diversified in nature.

DOI： 10.1093/nar/gkw898

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

DOI： 10.3390/life7010008

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

The genetic code-the language used by cells to translate their genomes into proteins that perform many cellular functions-is highly conserved throughout natural life. Rewriting the genetic code could lead to new biological functions such as expanding protein chemistries with noncanonical amino acids (ncAAs) and genetically isolating synthetic organisms from natural organisms and viruses. It has long been possible to transiently produce proteins bearing ncAAs, but stabilizing an expanded genetic code for sustained function in vivo requires an integrated approach: creating recoded genomes and introducing new translation machinery that function together without compromising viability or clashing with endogenous pathways. In this review, we discuss design considerations and technologies for expanding the genetic code. The knowledge obtained by rewriting the genetic code will deepen our understanding of how genomes are designed and how the canonical genetic code evolved.

DOI： 10.1146/annurev-micro-090816-093247

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

Selenocysteine (Sec or U) is encoded by UGA, a stop codon reassigned by a Sec-specific elongation factor and a distinctive RNA structure. To discover possible code variations in extant organisms we analyzed 6.4trillion base pairs of metagenomic sequences and 24903 microbial genomes for tRNA(Sec) species. As expected, UGA is the predominant Sec codon in use. We also found tRNA(Sec) species that recognize the stop codons UAG and UAA, and ten sense codons. Selenoprotein synthesis programmed by UAG in Geodermatophilus and Blastococcus, and by the Cys codon UGU in Aeromonas salmonicida was confirmed by metabolic labeling with Se-75 or mass spectrometry. Other tRNA(Sec) species with different anticodons enabled E.coli to synthesize active formate dehydrogenaseH, a selenoenzyme. This illustrates the ease by which the genetic code may evolve new coding schemes, possibly aiding organisms to adapt to changing environments, and show the genetic code is much more flexible than previously thought.

DOI： 10.1002/anie.201511657

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

DOI： 10.1002/ange.201511657

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Authorship：Lead author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

DOI： 10.1093/nar/gkv787

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

Escherichia coli is a widely used host organism for recombinant technology, and the bacterial incorporation of non-natural amino acids promises the efficient synthesis of proteins with novel structures and properties. In the present study, we developed E. coli strains in which the UAG codon was reserved for non-natural amino acids, without compromising the reproductive strength of the host cells. Ninety-five of the 273 UAG stop codons were replaced synonymously in the genome of E. coli BL21(DE3), by exploiting the oligonucleotide-mediated base-mismatch-repair mechanism. This genomic modification allowed the safe elimination of the UAG-recognizing cellular component (RF-1), thus leaving the remaining 178 UAG codons with no specific molecule recognizing them. The resulting strain B-95.Delta A grew as vigorously as BL21(DE3) in rich medium at 25-42 degrees C, and its derivative B-95.Delta A Delta fabR was better adapted to low temperatures and minimal media than B-95.Delta A. UAG was reassigned to synthetic amino acids by expressing the specific pairs of UAG-reading tRNA and aminoacyl-tRNA synthetase. Due to the preserved growth vigor, the B-95.Delta A strains showed superior productivities for hirudin molecules sulfonated on a particular tyrosine residue, and the Fab fragments of Herceptin containing multiple azido groups.

DOI： 10.1038/srep09699

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

Recent advances have fundamentally changed the ways in which synthetic amino acids are incorporated into proteins, enabling their efficient and multiple-site incorporation, in addition to the 20 canonical amino acids. This development provides opportunities for fresh approaches toward addressing fundamental problems in bioengineering. In the present study, we showed that the structural stability of proteins can be enhanced by integrating bulky halogenated amino acids at multiple selected sites. Glutathione S-transferase was thus stabilized significantly (by 5.2 and 5.6 kcal/mol) with 3-chloro- and 3-bromo-L-tyrosines, respectively, incorporated at seven selected sites. X-ray crystallographic analyses revealed that the bulky halogen moieties filled internal spaces within the molecules, and formed non-canonical stabilizing interactions with the neighboring residues. This new mechanism for protein stabilization is quite simple and applicable to a wide range of proteins, as demonstrated by the rapid stabilization of the industrially relevant azoreductase.

DOI： 10.1038/srep09762

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

Lysine methylation is one of the important post-translational modifications of histones, and produces an N-epsilon-mono-, di-, or trimethyllysine residues. Multiple and site-specific lysine methylations of histones are essential to define epigenetic statuses and control heterochromatin formation, DNA repair, and transcription regulation. A method was previously developed to build an analogue of N-epsilon-monomethyllysine, with cysteine substituting for lysine. Here, we have developed a new method of preparing histones bearing multiple N-epsilon-monomethyllysine residues at specified positions. Release factor 1-knockout (RFzero) Escherichia coli cells or a cell-free system based on the RFzero cell lysate was used for protein synthesis, as in RFzero cells UAG is redefined as a sense codon for non-canonical amino acids. During protein synthesis, a tert-butyloxycarbonyl- protected N-epsilon-monomethyllysine analogue is ligated to Methanosarcina mazei pyrrolysine tRNA (tRNAPyl) by M. mazei pyrrolysyl-tRNA synthetase mutants, and is translationally incorporated into one or more positions specified by the UAG codon. Protecting groups on the protein are then removed with trifluoroacetic acid to generate N-epsilon-monomethyllysine residues. We installed N-epsilon-monomethyllysine residues at positions 4, 9, 27, 36, and/or 79 of human histone H3. Each of the N-epsilon-monomethyllysine residues within the produced histone H3 was recognized by its specific antibody. Furthermore, the antibody recognized the authentic N-epsilon-monomethyllysine residue at position 27 better than the N-epsilon-monomethyllysine analogue built with cysteine. Mass spectrometry analyses also confirmed the lysine modifications on the produced histone H3. Thus, our method enables the installation of authentic N-epsilon-monomethyllysines at multiple positions within a protein for largescale production.

DOI： 10.1002/cbic.201402291

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

We previously reassigned the amber UAG stop triplet as a sense codon in Escherichia coli by expressing a UAG-decoding tRNA and knocking out the prfA gene, encoding release factor 1. UAG triplets were left at the ends of about 300 genes in the genome. In the present study, we showed that the detrimental effect of UAG reassignment could be alleviated by increasing the efficiency of UAG translation instead of reducing the number of UAGs in the genome. We isolated an amber suppressor tRNA(Gln) variant displaying enhanced suppression activity, and we introduced it into the prfA knockout strain, RFzero-q, in place of the original suppressor tRNA(Gln). The resulting strain, RFzero-q(3), translated UAG to glutamine almost as efficiently as the glutamine codons, and it proliferated faster than the parent RFzero-q strain. We identified two major factors in this growth enhancement. First, the sucB gene, which is involved in energy regeneration and has two successive UAG triplets at the end, was expressed at a higher level in RFzero-q3 than RFzero-q. Second, the ribosome stalling that occurred at UAG in RFzero-q was resolved in RFzero-q3. The results revealed the importance of "backup" stop triplets, UAA or UGA downstream of UAG, to avoid the deleterious impact of UAG reassignment on the proteome.

DOI： 10.1128/JB.00195-12

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

A derivative of N-benzyloxycarbonyl-L-lysine with a photo-reactive diazirinyl group, N-epsilon-[((4-(3-( trifluoromethyl)-3H-diazirin-3-yl)-benzyl)oxy)carbonyl]-L-lysine, was site-specifically incorporated into target proteins in mammalian cells. The incorporated photo-crosslinker is able to react not only with residues as distant as about 15 A but also with those in closer proximity, thus enabling "wide-range'' photo-crosslinking of proteins.

DOI： 10.1039/c2mb05321g

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The genetic encoding of synthetic or "non-natural" amino acids promises to diversify the functions and structures of proteins. We applied rapid codon-reassignment for creating Escherichia coli strains unable to terminate translation at the UAG "stop" triplet, but efficiently decoding it as various tyrosine and lysine derivatives. This complete change in the UAG meaning enabled protein synthesis with these non-natural molecules at multiple defined sites, in addition to the 20 canonical amino acids. UAG was also redefined in the E. coli BL21 strain, suitable for the large-scale production of recombinant proteins, and its cell extract served the cell-free synthesis of an epigenetic protein, histone H4, fully acetylated at four specific lysine sites. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2011.07.020

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Language：Japanese   Publishing type：Doctoral thesis  

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

Cell signaling pathways are essentially organized through the distribution of various types of binding domains in signaling proteins, with each domain binding to specific target molecules. Although identification of these targets is crucial for mapping the pathways, affinity-based or copurification methods are insufficient to distinguish between direct and indirect interactions in a cellular context. In the present study, we developed another approach involving the genetic encoding of a photo-crosslinkable amino acid. p-Trifluoromethyl-diazirinyl-L-phenylalanine was thus incorporated at a defined site in the Src homology 2 (SH2) domain of the adaptor protein GRB2 in human embryonic kidney cells. These cells were exposed to 365-nm light after an epidermal growth factor stimulus, and the crosslinkable GRB2-SH2 domain exclusively formed covalent bonds with directly interacting proteins. Proteomic mass spectrometry analysis identified these direct binders of GRB2-SH2 separately from the proteins noncovalently bound to the Src homology 3 domains of GRB2. In addition to two signaling-associated proteins (GIT1 and AF6), the heterogeneous nuclear ribonucleoproteins F, H1, and H2 were thus identified as novel direct binders. The results revealed a connection between the cell signaling protein and the nuclear machinery involved in mRNA processing, and demonstrated the usefulness of genetically encoded photo-crosslinkers for mapping protein protein interactions in cells. (C) 2010 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2010.12.022

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

Most organisms, from Escherichia coli to humans, use the 'universal' genetic code, which have been unchanged or 'frozen' for billions of years. It has been argued that codon reassignment causes mistranslation of genetic information, and must be lethal. In this study, we successfully reassigned the UAG triplet from a stop to a sense codon in the E. coli genome, by eliminating the UAG-recognizing release factor, an essential cellular component, from the bacterium. Only a few genetic modifications of E. coli were needed to circumvent the lethality of codon reassignment; erasing all UAG triplets from the genome was unnecessary. Thus, UAG was assigned unambiguously to a natural or non-natural amino acid, according to the specificity of the UAG-decoding tRNA. The result reveals the unexpected flexibility of the genetic code.

DOI： 10.1093/nar/gkq707

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS INC  

Insect cells are useful for the high-yield production of recombinant proteins including chemokines and membrane proteins. In this study, we developed an insect cell-based system for incorporating non-natural amino acids into proteins at specific sites. Three types of promoter systems were constructed, and their efficiencies were compared for the expression of the prokaryotic amber suppressor tRNA(Tyr) in Drosophila melanogaster Schneider 2 cells. When paired with a variant of Escherichia coli tyrosyl-tRNA synthetase specific for 3-iodo-L-tyrosine, the suppressor tRNA transcribed from the U6 promoter most efficiently incorporated the amino acid into proteins in the cells. The transient and stable introductions of these prokaryotic molecules into the insect cells were then compared in terms of the yield of proteins containing non-natural amino acids, and the "transient" method generated a sevenfold higher yield. By this method, 4-azido-L-phenylalanine was incorporated into human interleukin-8 at a specific site. The yield of the azido-containing IL-8 was 1 mu g/1 mL cell culture, and the recombinant protein was successfully labeled with a fluorescent probe by the Staudinger-Bertozzi reaction.

DOI： 10.1002/pro.322

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

We report a method for site-specifically incorporating L-lysine derivatives into proteins in mammalian cells, based on the expression of the pyrrolysyl-tRNA synthetase (PyIRS)-tRNA(PYl) pair from Methanosarcino mazei. Different types of external promoters were tested for the expression of tRNA(PYl) in Chinese hamster ovary cells. When tRNA(PYl) was expressed from a gene cluster under the control of the U6 promoter, the wild-type PylRS-tRNA(PYl) pair facilitated the most efficient incorporation of a pyrrolysine analog, N-epsilon-tert-butyloxycarbonyl-L-lysine (Boc-lysine), into proteins at the amber position. This PyIRS-tRNA(PYl) system yielded the Boc-lysine-containing protein in an amount accounting for 1% of the total protein in human embryonic kidney (HEK) 293 cells. We also created a PyIRS variant specific to N-epsilon-benzyloxycarbonyl-L-lysine, to incorporate this long, bulky, non-natural lysine derivative into proteins in HEK293. The recently reported variant specific to N-epsilon-acetyllysine was also expressed, resulting in the genetic encoding of this naturally-occurring lysine modification in mammalian cells. (c) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2008.04.164

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：名古屋  

新型コロナウイルス感染症対策の基本方針に基づき、年会を中止し、発表が行われたと認定

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Language：Japanese  

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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：English   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Applicant：独立行政法人理化学研究所

Application no：特願2009-542600  Date applied：21 11 2008

Patent/Registration no：特許第5419220号  Date issued：29 11 2013

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Applicant：独立行政法人理化学研究所

Application no：特願2008-502740  Date applied：22 2 2007

Patent/Registration no：特許第5196378号  Date issued：15 2 2013

J-GLOBAL

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Patent/Registration no：特許EP1992698B1  Date issued：5 9 2018

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Patent/Registration no：特許US8785152B2  Date issued：22 7 2014

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Patent/Registration no：特許EP2221370B1  Date issued：16 4 2014

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Patent/Registration no：特許US8293512B2  Date issued：23 10 2012

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Patent/Registration no：特許US9340790B2  Date issued：17 5 2016

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Patent/Registration no：特許EP2584037B1  Date issued：10 8 2016

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Patent/Registration no：特許US8822180B2  Date issued：2 9 2014

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Patent/Registration no：特許US8168407B2  Date issued：1 5 2012

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