Mitsubishi Research Institute (Japan)

Numerous microbes inhabit the human intestine, many of which are uncharacterized or uncultivable. They form a complex microbial community that deeply affects human physiology. To identify the genomic features common to all human gut microbiomes as well as those variable among them, we performed a large-scale comparative metagenomic analysis of fecal samples from 13 healthy individuals of various ages, including unweaned infants. We found that, while the gut microbiota from unweaned infants were simple and showed a high inter-individual variation in taxonomic and gene composition, those from adults and weaned children were more complex but showed a high functional uniformity regardless of age or sex. In searching for the genes over-represented in gut microbiomes, we identified 237 gene families commonly enriched in adult-type and 136 families in infant-type microbiomes, with a small overlap. An analysis of their predicted functions revealed various strategies employed by each type of microbiota to adapt to its intestinal environment, suggesting that these gene sets encode the core functions of adult and infant-type gut microbiota. By analysing the orphan genes, 647 new gene families were identified to be exclusively present in human intestinal microbiomes. In addition, we discovered a conjugative transposon family explosively amplified in human gut microbiomes, which strongly suggests that the intestine is a 'hot spot' for horizontal gene transfer between microbes.

Recent advances in DNA sequencers are accelerating genome sequencing, especially in microbes, and complete and draft genomes from various species have been sequenced in rapid succession. Here, we present a comprehensive gene prediction tool, the MetaGeneAnnotator (MGA), which precisely predicts all kinds of prokaryotic genes from a single or a set of anonymous genomic sequences having a variety of lengths. The MGA integrates statistical models of prophage genes, in addition to those of bacterial and archaeal genes, and also uses a self-training model from input sequences for predictions. As a result, the MGA sensitively detects not only typical genes but also atypical genes, such as horizontally transferred and prophage genes in a prokaryotic genome. In this paper, we also propose a novel approach for analyzing the ribosomal binding site (RBS), which enables us to detect species-specific patterns of the RBSs. The MGA has the ingenious RBS model based on this approach, and precisely predicts translation starts of genes. The MGA also succeeds in improving prediction accuracies for short sequences by using the adapted RBS models (96% sensitivity and 93% specificity for 700 bp fragments). These features of the MGA expedite wide ranges of microbial genome studies, such as genome annotations and metagenome analyses.

BACKGROUND: Conditional knockout mice and transgenic mice expressing recombinases, reporters, and inducible transcriptional activators are key for many genetic studies and comprise over 90% of mouse models created. Conditional knockout mice are generated using labor-intensive methods of homologous recombination in embryonic stem cells and are available for only ~25% of all mouse genes. Transgenic mice generated by random genomic insertion approaches pose problems of unreliable expression, and thus there is a need for targeted-insertion models. Although CRISPR-based strategies were reported to create conditional and targeted-insertion alleles via one-step delivery of targeting components directly to zygotes, these strategies are quite inefficient. RESULTS: Here we describe Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR), a targeting strategy in which long single-stranded DNA donors are injected with pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complexes into mouse zygotes. We show for over a dozen loci that Easi-CRISPR generates correctly targeted conditional and insertion alleles in 8.5-100% of the resulting live offspring. CONCLUSIONS: Easi-CRISPR solves the major problem of animal genome engineering, namely the inefficiency of targeted DNA cassette insertion. The approach is robust, succeeding for all tested loci. It is versatile, generating both conditional and targeted insertion alleles. Finally, it is highly efficient, as treating an average of only 50 zygotes is sufficient to produce a correctly targeted allele in up to 100% of live offspring. Thus, Easi-CRISPR offers a comprehensive means of building large-scale Cre-LoxP animal resources.

Includes bibliographical references.

Eukaryotic chromosomes reach their stable rod-shaped appearance in mitosis in a reaction dependent on the evolutionarily conserved condensin complex. Little is known about how and where condensin associates with chromosomes. Here, we analyze condensin binding to budding yeast chromosomes using high-resolution oligonucleotide tiling arrays. Condensin-binding sites coincide with those of the loading factor Scc2/4 of the related cohesin complex. The sites map to tRNA and other genes bound by the RNA polymerase III transcription factor TFIIIC, and ribosomal protein and SNR genes. An ectopic B-box element, recognized by TFIIIC, constitutes a minimal condensin-binding site, and TFIIIC and the Scc2/4 complex promote functional condensin association with chromosomes. A similar pattern of condensin binding is conserved along fission yeast chromosomes. This reveals that TFIIIC-binding sites, including tRNA genes, constitute a hitherto unknown chromosomal feature with important implications for chromosome architecture during both interphase and mitosis.

This work presents the genome sequencing of the lager brewing yeast (Saccharomyces pastorianus) Weihenstephan 34/70, a strain widely used in lager beer brewing. The 25 Mb genome comprises two nuclear sub-genomes originating from Saccharomyces cerevisiae and Saccharomyces bayanus and one circular mitochondrial genome originating from S. bayanus. Thirty-six different types of chromosomes were found including eight chromosomes with translocations between the two sub-genomes, whose breakpoints are within the orthologous open reading frames. Several gene loci responsible for typical lager brewing yeast characteristics such as maltotriose uptake and sulfite production have been increased in number by chromosomal rearrangements. Despite an overall high degree of conservation of the synteny with S. cerevisiae and S. bayanus, the syntenies were not well conserved in the sub-telomeric regions that contain lager brewing yeast characteristic and specific genes. Deletion of larger chromosomal regions, a massive unilateral decrease of the ribosomal DNA cluster and bilateral truncations of over 60 genes reflect a post-hybridization evolution process. Truncations and deletions of less efficient maltose and maltotriose uptake genes may indicate the result of adaptation to brewing. The genome sequence of this interspecies hybrid yeast provides a new tool for better understanding of lager brewing yeast behavior in industrial beer production.

PURPOSE: A comprehensive haplotype analysis of UGT1A1 in the Japanese population was conducted, and the effects of these haplotypes were investigated with respect to UGT1A1-related phenotypic parameters in patients with cancer who received irinotecan. METHODS: The UGT1A1 gene, including the enhancer, the promoter, and all 5 exons and their flanking regions, was sequenced from 195 Japanese subjects. The gene was divided into 2 blocks, and the haplotypes of each block were assigned. The association of these haplotypes with area under the concentration-time curve (AUC) ratios (7-ethyl-10-hydroxycamptothecin glucuronide [SN-38G]/7-ethyl-10-hydroxycamptothecin [SN-38]) and pretreatment levels of serum total bilirubin was investigated in 85 cancer patients who received irinotecan. RESULTS: Four haplotype groups (*1, *60, *28, and *6) were assigned in block 1, and 2 haplotype groups (*IA and *IB) were in block 2. The majority of the *IB haplotypes in block 2 were linked to either the *1 or the *60 haplotype but not to *28 in block 1. Highly significant associations were obtained between the *28 haplotypes and both a reduced AUC ratio (P =.0014, Jonckheere-Terpstra [JT] test) and an increased total bilirubin level (P =.0007, JT test). Increased total bilirubin levels in the *60 (P =.0048, JT test) and *IB groups (P =.0224, JT test) were also observed. The reduction in the AUC ratio by the *6 group was moderate (P =.0372, JT test) but was remarkable in combination with *60 (*6/*60) or *28 (*6/*28) as compared with the *1 group (*1/*1) (P =.049 and P =.0071, respectively; nonparametric Dunnett test). CONCLUSION: This study identified several UGT1A1 haplotypes significantly associated with the reduced AUC ratio (*28 and *6) and with the increased total bilirubin level (*28, *60, and *IB) and suggested that the novel haplotype *IB might be functionally important. These findings will be useful for further pharmacogenetic studies on adverse reactions to irinotecan.

Bacteriophages are major genetic factors promoting horizontal gene transfer (HGT) between bacteria. Their roles in dynamic bacterial genome evolution have been increasingly highlighted by the fact that many sequenced bacterial genomes contain multiple prophages carrying a wide range of genes. Enterohemorrhagic Escherichia coli O157 is the most striking case. A sequenced strain (O157 Sakai) possesses 18 prophages (Sp1-Sp18) that encode numerous genes related to O157 virulence, including those for two potent cytotoxins, Shiga toxins (Stx) 1 and 2. However, most of these prophages appeared to contain multiple genetic defects. To understand whether these defective prophages have the potential to act as mobile genetic elements to spread virulence determinants, we looked closely at the Sp1-Sp18 sequences, defined the genetic defects of each Sp, and then systematically analyzed all Sps for their biological activities. We show that many of the defective prophages, including the Stx1 phage, are inducible and released from O157 cells as particulate DNA. In fact, some prophages can even be transferred to other E. coli strains. We also show that new Stx1 phages are generated by recombination between the Stx1 and Stx2 phage genomes. The results indicate that these defective prophages are not simply genetic remnants generated in the course of O157 evolution, but rather genetic elements with a high potential for disseminating virulence-related genes and other genetic traits to other bacteria. We speculate that recombination and various other types of inter-prophage interactions in the O157 prophage pool potentiate such activities. Our data provide new insights into the potential activities of the defective prophages embedded in bacterial genomes and lead to the formulation of a novel concept of inter-prophage interactions in defective prophage communities.

The recently released human genome sequences provide us with reference data to conduct comparative genomic research on primates, which will be important to understand what genetic information makes us human. Here we present a first-generation human-chimpanzee comparative genome map and its initial analysis. The map was constructed through paired alignment of 77,461 chimpanzee bacterial artificial chromosome end sequences with publicly available human genome sequences. We detected candidate positions, including two clusters on human chromosome 21 that suggest large, nonrandom regions of difference between the two genomes.

In this paper, two important factors which affect the pedestrian outflow at a bottleneck significantly are studied in detail to analyze the effect of an obstacle setup in front of an exit. One is a conflict at an exit when pedestrians evacuate from a room. We use floor field model for simulating such behavior, which is a well-studied pedestrian model using cellular automata. The conflicts have been taken into account by the friction parameter. However, the friction parameter so far is a constant and does not depend on the number of the pedestrians conflicting at the same time. Thus, we have improved the friction parameter by the frictional function, which is a function of the number of the pedestrians involved in the conflict. Second, we have presented the cost of turning of pedestrians at the exit. Since pedestrians have inertia, their walking speeds decrease when they turn and the pedestrian outflow decreases. The validity of the extended model, which includes the frictional function and the turning function, is supported by the comparison of a mean-field theory and real experiments. We have observed that the pedestrian flow increases when we put an obstacle in front of an exit in our real experiments. The analytical results clearly explains the mechanism of the effect of the obstacle, i.e., the obstacle blocks pedestrians moving to the exit and decreases the average number of pedestrians involved in the conflict. We have also found that an obstacle works more effectively when we shift it from the center since pedestrians go through the exit with less turning.

This paper reports on a project for collection of the sound scene data.The sound scene data is necessary for studies such as sound source localization, sound retrieval, sound recognition and hands-free speech recognition in real acoustical environments.There are many kinds of sound scenes in real environments.The sound scene is denoted by sound sources and room acoustics.The number of combination of the sound sources, source positions and rooms is huge in real acoustical environments.However, the sound in the environments can be simulated by convolution of the isolated sound sources and impulse responses.As an isolated sound source, a hundred kinds of non-speech sounds and speech sounds are collected.The impulse responses are collected in various acoustical environments.In this paper, progress of our sound scene database project and application to environment sound recognition are described.

Presents a methodology for applying Quality Function Deployment (QFD) for environmentally conscious design in the early stage of product development. This methodology has been developed by incorporating environmental aspects into QFD to handle the environmental and traditional product quality requirements simultaneously. The “QFD for environment (QFDE)” proposed consists of four phases. Designers can find out which parts are the most important in enhancing environmental consciousness of their products by executing QFDE phase I and phase II. Further, a methodology is developed to evaluate the effects of design improvement on environmental quality requirements as phases III and IV. The results obtained from the case study of IC package show that QFDE could be applicable in the early stage of assembled product design, because the most important component from the viewpoint of the environment is clearly identified and multiple options for design improvement are effectively evaluated.

Oleaginous photosynthetic organisms such as microalgae are promising sources for biofuel production through the generation of carbon-neutral sustainable energy. However, the metabolic mechanisms driving high-rate lipid production in these oleaginous organisms remain unclear, thus impeding efforts to improve productivity through genetic modifications. We analyzed the genome and transcriptome of the oleaginous diatom Fistulifera solaris JPCC DA0580. Next-generation sequencing technology provided evidence of an allodiploid genome structure, suggesting unorthodox molecular evolutionary and genetic regulatory systems for reinforcing metabolic efficiencies. Although major metabolic pathways were shared with nonoleaginous diatoms, transcriptome analysis revealed unique expression patterns, such as concomitant upregulation of fatty acid/triacylglycerol biosynthesis and fatty acid degradation (β-oxidation) in concert with ATP production. This peculiar pattern of gene expression may account for the simultaneous growth and oil accumulation phenotype and may inspire novel biofuel production technology based on this oleaginous microalga.

In order to identify single nucleotide polymorphisms (SNPs) and haplotype frequencies of CYP3A4 in a Japanese population, the distal enhancer and proximal promoter regions, all exons, and the surrounding introns were sequenced from genomic DNA of 416 Japanese subjects. We found 24 SNPs, including 17 novel ones: two in the distal enhancer, four in the proximal promoter, one in the 5'-untranslated region (UTR), seven in the introns, and three in the 3'-UTR. The most common SNP was c.1026+12G>A (IVS10+12G>A), with a 0.249 frequency. Four non-synonymous SNPs, c.554C>G (p.T185S, CYP3A4(*)16), c.830_831insA (p.E277fsX8, (*)6), c.878T>C (p.L293P, (*)18), and c.1088 C>T (p.T363M, (*)11) were found with frequencies of 0.014, 0.001, 0.028, and 0.002, respectively. No SNP was found in the known nuclear transcriptional factor-binding sites in the enhancer and promoter regions. Using these 24 SNPs, 16 haplotypes were unambiguously identified, and nine haplotypes were inferred by aid of an expectation-maximization-based program. In addition, using data from 186 subjects enabled a close linkage to be found between CYP3A4 and CYP3A5 SNPs, especially among the SNPs at c.1026+12 in CYP3A4 and c.219-237 (IVS3-237, a key SNP site for CYP3A5(*)3), c.865+77 (IVS9+77) and c.1523 in CYP3A5. This result suggested that CYP3A4 and CYP3A5 are within the same gene block. Haplotype analysis between CYP3A4 and CYP3A5 revealed several major haplotype combinations in the CYP3A4-CYP3A5 block. Our findings provide fundamental and useful information for genotyping CYP3A4 (and CYP3A5) in the Japanese, and probably Asian populations.

There are abundance of transcripts that code for no particular protein and that remain functionally uncharacterized. Some of these transcripts may have novel functions while others might be junk transcripts. Unfortunately, the experimental validation of such transcripts to find functional non-coding RNA candidates is very costly. Therefore, our primary interest is to computationally mine candidate functional transcripts from a pool of uncharacterized transcripts. We introduce fRNAdb: a novel database service that hosts a large collection of non-coding transcripts including annotated/non-annotated sequences from the H-inv database, NONCODE and RNAdb. A set of computational analyses have been performed on the included sequences. These analyses include RNA secondary structure motif discovery, EST support evaluation, cis-regulatory element search, protein homology search, etc. fRNAdb provides an efficient interface to help users filter out particular transcripts under their own criteria to sort out functional RNA candidates. fRNAdb is available at http://www.ncrna.org/

Abstract Global R&D management, the management of company R&D effort distributed over different countries, a task that concerns multinational firms, has not been widely studied. The authors have therefore carried out in‐depth studies of global R&D conducted by 7 European and 15 Japanese companies. Their objective was to identify ‘best practice’ and so construct a framework for future research. The authors' conclusions are as follows. Globalisation, that is decentralisation of R&D, has become a necessity for multinationals as a result of the localisation of competition, of product life becoming shorter than development time, and the need to locate laboratories near sources of new technological know‐how. Because foreign acquisitions often lead to the acquisition of laboratories, questions are raised about how best to integrate them with the administrative practices of the ‘home’ organization, whether to reorganize them or to close them down. When it is necessary to set up a new foreign‐based laboratory deciding its exact location will require the weighing of factors such as whether the activities are to be market or process oriented, where on the R to D scale the activities will be placed, and how far direction of the laboratory's programmes and work will be decentralised. Global management also demands special attention to the building of an open communication network among the laboratories, the best form of which has yet to be determined. The main concern in human resource management will be how to select and develop an internationally oriented management corps and how to train R&D professionals to communicate across sites. If globally dispersed R&D laboratories are to be most effectively used then a new framework for their management needs to be developed.

We performed comprehensive haplotyping of ABCB1/MDR1 gene blocks using 49 genetic polymorphisms, including seven novel ones, obtained from 145 Japanese subjects. The ABCB1/MDR1 gene was divided into four blocks (Blocks -1, 1, 2, and 3) based on linkage disequilibrium analysis of polymorphisms. Using an expectation-maximization based program, 1, 2, 8, and 3 haplotype groups (3, 12, 32, and 18 haplotypes) were identified in Blocks -1, 1, 2, and 3, respectively. Within Block 2, haplotype groups *1, *2, *4, *6, and *8 reported by Kim and colleagues (Clin Pharmacol Ther 2001; 70:189-199) were found, and additional three groups (*9 to *11) were newly defined. We analyzed the association of haplotypes with the renal clearance of irinotecan and its metabolites in 49 Japanese cancer patients given irinotecan intravenously. There was a significant association of the *2 haplotype in Block 2, which includes 1236C>T, 2677G>T and 3435C>T, with a reduced renal clearance of those compounds. Moreover, tendencies of reduced and increased renal clearance were also observed with *1f in Block 2 and *1b in Block 3, respectively. These findings suggest that the P-glycoprotein encoded by ABCB1/MDR1 in the proximal tubules plays a substantial role in renal exclusion of drugs and, moreover, that block-haplotyping is valuable for pharmacogenetic studies.

This paper introduces the RoboCup-Rescue Simulation Project, a contribution to the disaster mitigation, search and rescue problem. A comprehensive urban disaster simulator is constructed on distributed computers. Heterogeneous intelligent agents such as fire fighters, victims and volunteers conduct search and rescue activities in this virtual disaster world. A real world interface integrates various sensor systems and controllers of infrastructures in the real cities with the virtual world. Real-time simulation is synchronized with actual disasters, computing complex relationship between various damage factors and agent behaviors. A mission-critical man-machine interface provides portability and robustness of disaster mitigation centers, and augmented-reality interfaces for rescue parties in real disasters. It also provides a virtual reality training function for the public. This diverse spectrum of RoboCup-Rescue contributes to the creation of the safer social system.

In 2008, Nuclear and Industrial Safety Agency (NISA) (currently integrated to the Nuclear Regulatory Authority) launched a project to develop a soundness assessment method for concrete members subject to a radiation environment. Presently, the soundness of concrete members subject to radiation is evaluated based on whether the predicted fast neutron fluence and gamma-ray dose values are lower than specific reference values in Japan, which are 1×1020 n/cm2 and 2×105 kGy, respectively. These reference values were determined based on report by Hilsdorf et al. This project begins by reviewing Hilsdorf et al.’s report, and we find that the scientific evidence for the current reference values is weak. We thus conclude that new experimental research is required to assess the current reference values and to propose a new alternative soundness assessment procedure if needed. We quantitatively evaluated the influence of neutrons, gamma-rays, and the resultant heating and drying processes on the strength of concrete as well as their underlying mechanisms. The irradiation experiments confirmed the degradation mechanism of concrete due to neutron irradiation. The main reason for this degradation is the metamictization of rock-forming minerals, which, in turn, leads to aggregate expansion. Due to aggregate expansion, cracks around aggregates form, which reduce the compressive strength and Young’s modulus of concrete. Among the rock-forming minerals, α-quartz is the most sensitive to neutron radiation. 60Co gamma-ray irradiation experiments demonstrated that concrete strength increased as the gamma-ray dose and gamma-ray flux does not have a dose-rate impact on the first radiolysis of evaporable water in cement paste within the present study. The effect of gamma-ray irradiation on the properties of concrete is equivalent to that of heating and drying. Concrete strength alteration due to heating and drying is attributed to the colloidal and porous nature of hardened cement paste and crack formation around the aggregate due to a mismatch in the volume changes of the mortar and aggregate. In addition, a numerical analysis code called DEVICE (Damage EValuation for Irradiated ConcretE) is developed to harness knowledge obtained from concrete samples to predict the distribution of the physical properties in concrete members and their changes over time. From these fundamental studies, we propose a new soundness assessment procedure for concrete members subject to radiation. We also recommend a new radiation-induced strength-degradation reference value of 1×1019 n/cm2 for fast neutron.

Routes to enhance superconducting instability are explored for doped Mott insulators. With the help of insight for criticalities of metal-insulator transitions, geometrical design of lattice structure is proposed to control the instability. A guideline is to explicitly make flat band dispersions near the Fermi level without suppressing two-particle channels. In a one-dimensional model, numerical studies show that our prescription with finite-ranged hoppings realizes large enhancement of spin-gap and pairing dominant regions. We also propose several multiband systems, where the pairing is driven by intersite Coulomb repulsion.