Updated on 2024/02/28

写真a

 
HOSOKAWA, Masahito
 
Affiliation
Faculty of Science and Engineering, Graduate School of Advanced Science and Engineering
Job title
Associate Professor(non-tenure-track)
Degree
博士(工学) ( 東京農工大学 )

Research Experience

  • 2022.04
    -
    Now

    Japan Science and Technology Agency

  • 2021.04
    -
    Now

    Waseda University

  • 2018.11
    -
    Now

    bitBiome, Inc.   Director, CSO

  • 2018.09
    -
    2020.03

    Waseda University   Research Institute for Science and Engineering

  • 2015.10
    -
    2019.03

    Japan Science and Technology Agency

  • 2014.04
    -
    2016.03

    Waseda University   Institute for Nanoscience and Nanotechnology

  • 2013.06
    -
    2014.03

    Waseda University   Faculty of Science and Engineering

  • 2011.04
    -
    2013.05

    Shizuoka Cancer Center, Research Institute

  • 2010.04
    -
    2011.03

    Tokyo University of Agriculture and Technology

▼display all

Education Background

  • 2008.04
    -
    2010.03

    Tokyo University of Agriculture and Technology  

Research Areas

  • Applied biochemistry

Awards

  • 経済産業大臣賞

    2022.09   大学発ベンチャー表彰2022  

  • 第5回「バイオインダストリー奨励賞」

    2021.07   (一財)バイオインダストリー協会  

  • The Commendation for Science and Technology by the Minister of Education, Culture, Sports, Science and Technology, The Young Scientists’ Prize

    2019.04  

    Winner: Masahito Hosokawa

  • イノベーション賞

    2019.03   科学技術振興機構さきがけ(統合1細胞領域)  

    Winner: 細川 正人

  • 第96春季年会 優秀講演賞(学術)

    2016.05   日本化学会  

    Winner: 細川 正人

 

Papers

  • A combination of two-enzyme system and enzyme engineering improved the activity of a new PET hydrolase identified from soil bacterial genome

    Hideaki Mabashi-Asazuma, Makoto Hirai, Shigeru Sakurai, Keigo Ide, Masato Kogawa, Ai Matsushita, Masahito Hosokawa, Soichiro Tsuda

    bioRxiv    2024.02

    DOI

  • Single Amplified Genome Catalog Reveals the Dynamics of Mobilome and Resistome in the Human Microbiome

    Tetsuro Kawano-Sugaya, Koji Arikawa, Tatsuya Saeki, Taruho Endoh, Kazuma Kamata, Ayumi Matsuhashi, Masahito Hosokawa

       2023.12

    Authorship:Corresponding author

     View Summary

    Abstract

    The increase in metagenome-assembled genomes (MAGs) has significantly advanced our understanding of the functional characterization and taxonomic assignment within the human microbiome. However, MAGs, as population consensus genomes, often mask heterogeneity among species and strains, thereby obfuscating the precise relationships between microbial hosts and mobile genetic elements (MGEs). In contrast, single amplified genomes (SAGs) derived via single-cell genome sequencing can capture individual genomic content, including MGEs. We present the bbsag20 dataset, which encompasses 17,202 human-associated prokaryotic SAGs and 869 MAGs, spanning 647 gut and 312 oral bacterial species. The SAGs revealed diverse bacterial lineages and MGEs with a broad host range that were absent in the MAGs and traced the translocation of oral bacteria to the gut. Importantly, our SAGs linked individual mobilomes to resistomes and meticulously charted a dynamic network of antibiotic resistance genes (ARGs) on MGEs, pinpointing potential ARG reservoirs in the microbial community.

    DOI

  • Tools for microbial single-cell genomics for obtaining uncultured microbial genomes

    Masahito Hosokawa, Yohei Nishikawa

    Biophysical Reviews    2023.09  [Refereed]

    Authorship:Lead author, Corresponding author

     View Summary

    Abstract

    The advent of next-generation sequencing technologies has facilitated the acquisition of large amounts of DNA sequence data at a relatively low cost, leading to numerous breakthroughs in decoding microbial genomes. Among the various genome sequencing activities, metagenomic analysis, which entails the direct analysis of uncultured microbial DNA, has had a profound impact on microbiome research and has emerged as an indispensable technology in this field. Despite its valuable contributions, metagenomic analysis is a “bulk analysis” technique that analyzes samples containing a wide diversity of microbes, such as bacteria, yielding information that is averaged across the entire microbial population. In order to gain a deeper understanding of the heterogeneous nature of the microbial world, there is a growing need for single-cell analysis, similar to its use in human cell biology. With this paradigm shift in mind, comprehensive single-cell genomics technology has become a much-anticipated innovation that is now poised to revolutionize microbiome research. It has the potential to enable the discovery of differences at the strain level and to facilitate a more comprehensive examination of microbial ecosystems. In this review, we summarize the current state-of-the-art in microbial single-cell genomics, highlighting the potential impact of this technology on our understanding of the microbial world. The successful implementation of this technology is expected to have a profound impact in the field, leading to new discoveries and insights into the diversity and evolution of microbes.

    DOI

    Scopus

    4
    Citation
    (Scopus)
  • Target enrichment of uncultured human oral bacteria with phage-derived molecules found by single-cell genomics.

    Masahito Hosokawa, Naoya Iwai, Koji Arikawa, Tatsuya Saeki, Taruho Endoh, Kazuma Kamata, Takuya Yoda, Soichiro Tsuda, Haruko Takeyama

    Journal of bioscience and bioengineering   136 ( 1 ) 58 - 66  2023.07  [Refereed]  [Domestic journal]

    Authorship:Lead author, Corresponding author

     View Summary

    Advances in culture-independent microbial analysis, such as metagenomics and single-cell genomics, have significantly increased our understanding of microbial lineages. While these methods have uncovered a large number of novel microbial taxa, many remain uncultured, and their function and mode of existence in the environment are still unknown. This study aims to explore the use of bacteriophage-derived molecules as probes for detecting and isolating uncultured bacteria. Here, we proposed multiplex single-cell sequencing to obtain massive uncultured oral bacterial genomes and searched prophage sequences from over 450 obtained human oral bacterial single-amplified genomes (SAGs). The focus was on the cell wall binding domain (CBD) in phage endolysin, and fluorescent protein-fused CBDs were generated based on several CBD gene sequences predicted from Streptococcus SAGs. The ability of the Streptococcus prophage-derived CBDs to detect and enrich specific Streptococcus species from human saliva while maintaining cell viability was confirmed by magnetic separation and flow cytometry. The approach to phage-derived molecule generation based on uncultured bacterial SAG is expected to improve the process of designing molecules that selectively capture or detect specific bacteria, notably from uncultured gram-positive bacteria, and will have applications in isolation and in situ detection of beneficial or pathogenic bacteria.

    DOI PubMed

    Scopus

  • Combined actions of bacteriophage-encoded genes in Wolbachia-induced male lethality.

    Hiroshi Arai, Hisashi Anbutsu, Yohei Nishikawa, Masato Kogawa, Kazuo Ishii, Masahito Hosokawa, Shiou-Ruei Lin, Masatoshi Ueda, Madoka Nakai, Yasuhisa Kunimi, Toshiyuki Harumoto, Daisuke Kageyama, Haruko Takeyama, Maki N Inoue

    iScience   26 ( 6 ) 106842 - 106842  2023.06  [Refereed]  [International journal]

     View Summary

    Some Wolbachia endosymbionts induce male killing, whereby male offspring of infected females are killed during development; however, the origin and diversity of the underlying mechanisms remain unclear. In this study, we identified a 76 kbp prophage region specific to male-killing Wolbachia hosted by the moth Homona magnanima. The prophage encoded a homolog of the male-killing gene oscar in Ostrinia moths and the wmk gene that induces various toxicities in Drosophila melanogaster. Upon overexpressing these genes in D. melanogaster, wmk-1 and wmk-3 killed all males and most females, whereas Hm-oscar, wmk-2, and wmk-4 had no impact on insect survival. Strikingly, co-expression of tandemly arrayed wmk-3 and wmk-4 killed 90% of males and restored 70% of females, suggesting their conjugated functions for male-specific lethality. While the male-killing gene in the native host remains unknown, our findings highlight the role of bacteriophages in male-killing evolution and differences in male-killing mechanisms among insects.

    DOI PubMed

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    5
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    (Scopus)
  • Enhancing the sensitivity of bacterial single-cell RNA sequencing using RamDA-seq and Cas9-based rRNA depletion.

    Mika Nishimura, Haruko Takeyama, Masahito Hosokawa

    Journal of bioscience and bioengineering    2023.06  [Refereed]  [Domestic journal]

    Authorship:Last author, Corresponding author

     View Summary

    Bacterial populations exhibit heterogeneity in gene expression, which facilitates their survival and adaptation to unstable and unpredictable environments through the bet-hedging strategy. However, unraveling the rare subpopulations and heterogeneity in gene expression using population-level gene expression analysis remains a challenging task. Single-cell RNA sequencing (scRNA-seq) has the potential to identify rare subpopulations and capture heterogeneity in bacterial populations, but standard methods for scRNA-seq in bacteria are still under development, mainly due to differences in mRNA abundance and structure between eukaryotic and prokaryotic organisms. In this study, we present a hybrid approach that combines random displacement amplification sequencing (RamDA-seq) with Cas9-based rRNA depletion for scRNA-seq in bacteria. This approach allows cDNA amplification and subsequent sequencing library preparation from low-abundance bacterial RNAs. We evaluated its sequenced read proportion, gene detection sensitivity, and gene expression patterns from the dilution series of total RNA or the sorted single Escherichia coli cells. Our results demonstrated the detection of more than 1000 genes, about 24% of the genes in the E. coli genome, from single cells with less sequencing effort compared to conventional methods. We observed gene expression clusters between different cellular proliferation states or heat shock treatment. The approach demonstrated high detection sensitivity in gene expression analysis compared to current bacterial scRNA-seq methods and proved to be an invaluable tool for understanding the ecology of bacterial populations and capturing the heterogeneity of bacterial gene expression.

    DOI PubMed

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    2
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    (Scopus)
  • Linking antigen specific T-cell dynamics in a microfluidic chip to single cell transcription patterns.

    Hiroki Ide, Taiki Aoshi, Masato Saito, Wilfred Villariza Espulgar, Jonathan Campos Briones, Masahito Hosokawa, Hiroko Matsunaga, Koji Arikawa, Haruko Takeyama, Shohei Koyama, Hyota Takamatsu, Eiichi Tamiya

    Biochemical and biophysical research communications   657   8 - 15  2023.03  [Refereed]  [International journal]

     View Summary

    A new non-invasive screening profile has been realized that can aid in determining T-cell activation state at single-cell level. Production of activated T-cells with good specificity and stable proliferation is greatly beneficial for advancing adoptive immunotherapy as innate immunological cells are not effective in recognizing and eliminating cancer as expected. The screening method is realized by relating intracellular Ca2+ intensity and motility of T-cells interacting with APC (Antigen Presenting Cells) in a microfluidic chip. The system is tested using APC pulsed with OVA257-264 peptide and its modified affinities (N4, Q4, T4 and V4), and the T-cells from OT-1 mice. In addition, single cell RNA sequencing reveals the activation states of the cells and the clusters from the derived profiles can be indicative of the T-cell activation state. The presented system here can be versatile for a comprehensive application to proceed with T-cell-based immunotherapy and screen the antigen-specific T-cells with excellent efficiency and high proliferation.

    DOI PubMed

    Scopus

    1
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    (Scopus)
  • Uncultured prokaryotic genomes in the spotlight: An examination of publicly available data from metagenomics and single-cell genomics.

    Koji Arikawa, Masahito Hosokawa

    Computational and structural biotechnology journal   21   4508 - 4518  2023  [International journal]

    Authorship:Last author, Corresponding author

     View Summary

    Owing to the ineffectiveness of traditional culture techniques for the vast majority of microbial species, culture-independent analyses utilizing next-generation sequencing and bioinformatics have become essential for gaining insight into microbial ecology and function. This mini-review focuses on two essential methods for obtaining genetic information from uncultured prokaryotes, metagenomics and single-cell genomics. We analyzed the registration status of uncultured prokaryotic genome data from major public databases and assessed the advantages and limitations of both the methods. Metagenomics generates a significant quantity of sequence data and multiple prokaryotic genomes using straightforward experimental procedures. However, in ecosystems with high microbial diversity, such as soil, most genes are presented as brief, disconnected contigs, and lack association of highly conserved genes and mobile genetic elements with individual species genomes. Although technically more challenging, single-cell genomics offers valuable insights into complex ecosystems by providing strain-resolved genomes, addressing issues in metagenomics. Recent technological advancements, such as long-read sequencing, machine learning algorithms, and in silico protein structure prediction, in combination with vast genomic data, have the potential to overcome the current technical challenges and facilitate a deeper understanding of uncultured microbial ecosystems and microbial dark matter genes and proteins. In light of this, it is imperative that continued innovation in both methods and technologies take place to create high-quality reference genome databases that will support future microbial research and industrial applications.

    DOI PubMed

    Scopus

  • Revealing within-species diversity in uncultured human gut bacteria with single-cell long-read sequencing.

    Masato Kogawa, Yohei Nishikawa, Tatsuya Saeki, Takuya Yoda, Koji Arikawa, Haruko Takeyama, Masahito Hosokawa

    Frontiers in microbiology   14   1133917 - 1133917  2023  [Refereed]  [International journal]

    Authorship:Last author, Corresponding author

     View Summary

    Abstract

    Bacterial genome structure changes dynamically, and structural variants can change bacterial phenotype; However, obtaining the complete genome and analyzing genome structure of uncultured bacteria has been challenging. We aimed to develop a single-cell amplified genome long-read assembly (scALA) workflow to construct circular single-cell amplified genomes (cSAGs) from long-read single-cell sequencing data of targeted uncultured bacteria. In particular, scALA generated cSAGs from nanopore long-read sequencing data of SAGs by producing contiguous sequences with repeated bias reduction and assembly processes. From 12 human fecal samples, scALA generated 16 cSAGs of three specifically targeted bacterial species, Anaerostipes hadrus, Agathobacter rectalis, and Ruminococcus gnavus. A. hadrus cSAGs exhibited large, ten kbp-long, phage insertions, saccharide metabolic capacity, and frequent genomic recombination with related strains from cohabitant hosts. Noteworthy, cSAGs constructed using this method could expand bacterial genome databases and our understanding of within-species diversities in uncultured bacteria.

    DOI PubMed

    Scopus

    4
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  • Targeted single-cell genomics reveals novel host adaptation strategies of the symbiotic bacteria Endozoicomonas in Acropora tenuis coral.

    Keigo Ide, Yohei Nishikawa, Toru Maruyama, Yuko Tsukada, Masato Kogawa, Hiroki Takeda, Haruka Ito, Ryota Wagatsuma, Rimi Miyaoka, Yoshikatsu Nakano, Koji Kinjo, Michihiro Ito, Masahito Hosokawa, Kei Yura, Shoichiro Suda, Haruko Takeyama

    Microbiome   10 ( 1 ) 220 - 220  2022.12  [Refereed]  [International journal]

     View Summary

    Abstract

    Endozoicomonas bacteria symbiose with various marine organisms and are known to be beneficial for coral health. However, genome analysis of coral-associated Endozoicomonas has been limited owing to the difficulty in cultivation and metagenomic approach by contamination of host-derived sequences. In this study, we applied a novel single-cell genomics technique using droplet microfluidics to obtain single-cell amplified genome (SAGs) for coral-associated Endozoicomonas spp. genome. We obtained seven novel Endozoicomonas genomes from Acropora tenuis coral. These genomes revealed that Endozoicomonas bacteria played host-associated functions in host corals and had undergone independent host-adaptive evolution in different clades. These adaptive evolutions were mediated by host-derived eukaryotic-like genes, some of which were speculated to influence host immune mechanisms. These genes are speculated to enhance coral tolerance to environmental stresses. This study suggests the possibility of host adaptation of Endozoicomonas spp. in symbiosis with corals and their contribution to coral bleaching tolerance.

    DOI PubMed

    Scopus

    2
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  • Reproducible and sensitive micro-tissue RNA sequencing from formalin-fixed paraffin-embedded tissues for spatial gene expression analysis.

    Hiroko Matsunaga, Koji Arikawa, Miki Yamazaki, Ryota Wagatsuma, Keigo Ide, Ashok Zachariah Samuel, Kazuya Takamochi, Kenji Suzuki, Takuo Hayashi, Masahito Hosokawa, Hideki Kambara, Haruko Takeyama

    Scientific reports   12 ( 1 ) 19511 - 19511  2022.11  [Refereed]  [International journal]

     View Summary

    Abstract

    Spatial transcriptome analysis of formalin-fixed paraffin-embedded (FFPE) tissues using RNA-sequencing (RNA-seq) provides interactive information on morphology and gene expression, which is useful for clinical applications. However, despite the advantages of long-term storage at room temperature, FFPE tissues may be severely damaged by methylene crosslinking and provide less gene information than fresh-frozen tissues. In this study, we proposed a sensitive FFPE micro-tissue RNA-seq method that combines the punching of tissue sections (diameter: 100 μm) and the direct construction of RNA-seq libraries. We evaluated a method using mouse liver tissues at 2 years after fixation and embedding and detected approximately 7,000 genes in micro-punched tissue-spots (thickness: 10 μm), similar to that detected with purified total RNA (2.5 ng) equivalent to the several dozen cells in the spot. We applied this method to clinical FFPE specimens of lung cancer that had been fixed and embedded 6 years prior, and found that it was possible to determine characteristic gene expression in the microenvironment containing tumor and non-tumor cells of different morphologies. This result indicates that spatial gene expression analysis of the tumor microenvironment is feasible using FFPE tissue sections stored for extensive periods in medical facilities.

    DOI PubMed

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    5
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  • Cancer Cachexia among Patients with Advanced Non-Small-Cell Lung Cancer on Immunotherapy: An Observational Study with Exploratory Gut Microbiota Analysis.

    Taiki Hakozaki, Alexis Nolin-Lapalme, Masato Kogawa, Yusuke Okuma, Shohei Nakamura, Danielle Moreau-Amaru, Taichi Tamura, Yukio Hosomi, Haruko Takeyama, Corentin Richard, Masahito Hosokawa, Bertrand Routy

    Cancers   14 ( 21 )  2022.11  [Refereed]  [International journal]

     View Summary

    Cancer cachexia exerts a negative clinical influence on patients with advanced non-small-cell lung cancer (NSCLC) treated with immune checkpoint inhibitors (ICI). The prognostic impact of body weight change during ICI treatment remains unknown. The gut microbiota (GM) is a key contributor to the response to ICI therapy in cancer patients. However, the association between cancer cachexia and GM and their association with the response to ICIs remains unexplored. This study examined the association of cancer cachexia with GM composition and assessed the impact of GM on clinical outcomes in patients with NSCLC treated with ICIs. In this observational, prospective study, which included 113 Japanese patients with advanced NSCLC treated with ICIs, the prevalence of cachexia was 50.4% (57/113). The median progression-free survival (PFS) and overall survival (OS) were significantly shorter in the cachexia group than in the non-cachexia group (4.3 vs. 11.6 months (p = 0.003) and 12.0 months vs. not reached (p = 0.02), respectively). A multivariable analysis revealed that baseline cachexia was independently associated with a shorter PFS. Moreover, a gain in body weight from the baseline (reversible cachexia) was associated with a significantly longer PFS and OS compared to irreversible cachexia. Microbiome profiling with 16S rRNA analysis revealed that the cachexia group presented an overrepresentation of the commensal bacteria, Escherichia-Shigella and Hungatella, while the non-cachexia group had a preponderance of Anaerostipes, Blautia, and Eubacterium ventriosum. Anaerostipes and E. ventriosum were associated with longer PFS and OS. Moreover, a cachexia status correlated with the systemic inflammatory marker-derived-neutrophil-to-lymphocytes ratio (dNLR) and Lung Immune Prognostic Index (LIPI) indexes. Our study demonstrates that cachexia and longitudinal bodyweight change have a prognostic impact on patients with advanced NSCLC treated with ICI therapy. Moreover, our study demonstrates that bacteria associated with ICI resistance are also linked to cachexia. Targeted microbiota interventions may represent a new type of treatment to overcome cachexia in patients with NSCLC.

    DOI PubMed

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    11
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  • Validation of the application of gel beads-based single-cell genome sequencing platform to soil and seawater

    Yohei Nishikawa, Masato Kogawa, Masahito Hosokawa, Ryota Wagatsuma, Katsuhiko Mineta, Kai Takahashi, Keigo Ide, Kei Yura, Hayedeh Behzad, Takashi Gojobori, Haruko Takeyama

    ISME Communications   2 ( 1 )  2022.09  [Refereed]

    Authorship:Lead author, Corresponding author

     View Summary

    Abstract

    Single-cell genomics is applied to environmental samples as a method to solve the problems of current metagenomics. However, in the fluorescence-activated cell sorting-based cell isolation and subsequent whole genome amplification, the sorting efficiency and the sequence quality are greatly affected by the type of target environment, limiting its adaptability. Here, we developed an improved single-cell genomics platform, named SAG-gel, which utilizes gel beads for single-cell isolation, lysis, and whole genome amplification. To validate the versatility of SAG-gel, single-cell genome sequencing was performed with model bacteria and microbial samples collected from eight environmental sites, including soil and seawater. Gel beads enabled multiple lysis treatments. The genome coverage with model bacteria was improved by 9.1–25%. A total of 734 single amplified genomes were collected from the diverse environmental samples, and almost full-length 16S rRNA genes were recovered from 57.8% of them. We also revealed two marine Rhodobacter strains harboring nearly identical 16S rRNA genes but having different genome contents. In addition, searching for viral sequences elucidated the virus-host linkage over the sampling sites, revealing the geographic distribution and diverse host range of viruses.

    DOI

  • Identification of lipolytic enzymes using high-throughput single-cell screening and sorting of a metagenomic library.

    Amani Alma'abadi, Hayedeh Behzad, Mohammed Alarawi, David Conchouso, Yoshimoto Saito, Masahito Hosokawa, Yohei Nishikawa, Masato Kogawa, Haruko Takeyama, Katsuhiko Mineta, Takashi Gojobori

    New biotechnology   70   102 - 108  2022.09  [Refereed]  [International journal]

     View Summary

    The demand for novel, robust microbial biocatalysts for use in industrial and pharmaceutical applications continues to increase rapidly. As a result, there is a need to develop advanced tools and technologies to exploit the vast metabolic potential of unculturable microorganisms found in various environments. Single-cell and functional metagenomics studies can explore the enzymatic potential of entire microbial communities in a given environment without the need to culture the microorganisms. This approach has contributed substantially to the discovery of unique microbial genes for industrial and medical applications. Functional metagenomics involves the extraction of microbial DNA directly from environmental samples, constructing expression libraries comprising the entire microbial genome, and screening of the libraries for the presence of desired phenotypes. In this study, lipolytic enzymes from the Red Sea were targeted. A high-throughput single-cell microfluidic platform combined with a laser-based fluorescent screening bioassay was employed to discover new genes encoding lipolytic enzymes. Analysis of the metagenomic library led to the identification of three microbial genes encoding lipases based on their functional similarity and sequence homology to known lipases. The results demonstrated that microfluidics is a robust technology that can be used for screening in functional metagenomics. The results also indicate that the Red Sea is a promising, under-investigated source of new genes and gene products.

    DOI PubMed

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    6
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  • Identification of two cancer stem cell-like populations in triple-negative breast cancer xenografts.

    Jun Nakayama, Hiroko Matsunaga, Koji Arikawa, Takuya Yoda, Masahito Hosokawa, Haruko Takeyama, Yusuke Yamamoto, Kentaro Semba

    Disease models & mechanisms   15 ( 6 )  2022.05  [Refereed]  [International journal]

     View Summary

    Gene expression analysis at the single-cell level by next generation sequencing has revealed the existence of clonal dissemination and microheterogeneity in cancer metastasis. The current spatial analysis technologies can elucidate the heterogeneity of cell-cell interactions in situ. To reveal the regional and expressional heterogeneity in primary tumors and metastases, we performed transcriptomic analysis of microtissues dissected from a triple-negative breast cancer (TNBC) cell line MDA-MB-231 xenograft model with our automated tissue microdissection punching technology. This multiple-microtissue transcriptome analysis revealed three cancer cell-type clusters in the primary tumor and axillary lymph node metastasis, two of which were cancer stem cell (CSC)-like clusters (CD44/MYC-high, HMGA1-high). Reanalysis of public single-cell RNA-seq (scRNA-seq) datasets confirmed that the two CSC-like populations existed both in TNBC xenograft models and TNBC patients. The diversity of these multiple CSC-like populations may cause differential anticancer drug resistance, increasing the difficulty of curing this cancer.

    DOI PubMed

  • Strain-level profiling of viable microbial community by selective single-cell genome sequencing.

    Masahito Hosokawa, Taruho Endoh, Kazuma Kamata, Koji Arikawa, Yohei Nishikawa, Masato Kogawa, Tatsuya Saeki, Takuya Yoda, Haruko Takeyama

    Scientific reports   12 ( 1 ) 4443 - 4443  2022.03  [Refereed]  [International journal]

    Authorship:Lead author, Corresponding author

     View Summary

    Culture-independent analysis with high-throughput sequencing has been widely used to characterize bacterial communities. However, signals derived from non-viable bacteria and non-cell DNA may inhibit its characterization. Here, we present a method for viable bacteria-targeted single-cell genome sequencing, called PMA-SAG-gel, to obtain comprehensive whole-genome sequences of surviving uncultured bacteria from microbial communities. PMA-SAG-gel uses gel matrixes that enable sequential enzymatic reactions for cell lysis and genome amplification of viable single cells from the microbial communities. PMA-SAG-gel removed the single-amplified genomes (SAGs) derived from dead bacteria and enabled selective sequencing of viable bacteria in the model samples of Escherichia coli and Bacillus subtilis. Next, we demonstrated the recovery of near-complete SAGs of eight oxygen-tolerant bacteria, including Bacteroides spp. and Phocaeicola spp., from 1331 human feces SAGs. We found the presence of two different strains in each species and identified their specific genes to investigate the metabolic functions. The survival profile of an entire population at the strain level will provide the information for understanding the characteristics of the surviving bacteria under the specific environments or sample processing and insights for quality assessment of live bacterial products or fecal microbiota transplantation and for understanding the effect of antimicrobial treatments.

    DOI PubMed

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  • Single-cell metabolite detection and genomics reveals uncultivated talented producer

    Masato Kogawa, Rimi Miyaoka, Franziska Hemmerling, Masahiro Ando, Kei Yura, Keigo Ide, Yohei Nishikawa, Masahito Hosokawa, Yuji Ise, Jackson K B Cahn, Kentaro Takada, Shigeki Matsunaga, Tetsushi Mori, Jörn Piel, Haruko Takeyama

    PNAS Nexus   1 ( 1 ) pgab007  2022.03  [Refereed]  [International journal]

     View Summary

    Abstract

    The production of bioactive metabolites is increasingly recognized as an important function of host-associated bacteria. An example is defensive symbiosis that might account for much of the chemical richness of marine invertebrates including sponges (Porifera), 1 of the oldest metazoans. However, most bacterial members of sponge microbiomes have not been cultivated or sequenced, and therefore, remain unrecognized. Unequivocally linking metabolic functions to a cellular source in sponge microbiomes is, therefore, a challenge. Here, we report an analysis pipeline of microfluidic encapsulation, Raman microscopy, and integrated digital genomics (MERMAID) for an efficient identification of uncultivated producers. We applied this method to the chemically rich bacteriosponge (sponge that hosts a rich bacterial community) Theonella swinhoei, previously shown to contain ‘Entotheonella’ symbionts that produce most of the bioactive substances isolated from the sponge. As an exception, the antifungal aurantosides had remained unassigned to a source. Raman-guided single-bacterial analysis and sequencing revealed a cryptic, distinct multiproducer, ‘Candidatus Poriflexus aureus’ from a new Chloroflexi lineage as the aurantoside producer. Its exceptionally large genome contains numerous biosynthetic loci and suggested an even higher chemical richness of this sponge than previously appreciated. This study highlights the importance of complementary technologies to uncover microbiome functions, reveals remarkable parallels between distantly related symbionts of the same host, and adds functional support for diverse chemically prolific lineages being present in microbial dark matter.

    DOI PubMed

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  • Massively parallel single-cell genomics of microbiomes in rice paddies.

    Wataru Aoki, Masato Kogawa, Shuhei Matsuda, Keisuke Matsubara, Shintaro Hirata, Yohei Nishikawa, Masahito Hosokawa, Haruko Takeyama, Toru Matoh, Mitsuyoshi Ueda

    Frontiers in microbiology   13   1024640 - 1024640  2022  [Refereed]  [International journal]

     View Summary

    Plant growth-promoting microbes (PGPMs) have attracted increasing attention because they may be useful in increasing crop yield in a low-input and sustainable manner to ensure food security. Previous studies have attempted to understand the principles underlying the rhizosphere ecology and interactions between plants and PGPMs using ribosomal RNA sequencing, metagenomic sequencing, and genome-resolved metagenomics; however, these approaches do not provide comprehensive genomic information for individual species and do not facilitate detailed analyses of plant-microbe interactions. In the present study, we developed a pipeline to analyze the genomic diversity of the rice rhizosphere microbiome at single-cell resolution. We isolated microbial cells from paddy soil and determined their genomic sequences by using massively parallel whole-genome amplification in microfluidic-generated gel capsules. We successfully obtained 3,237 single-amplified genomes in a single experiment, and these genomic sequences provided insights into microbial functions in the paddy ecosystem. Our approach offers a promising platform for gaining novel insights into the roles of microbes in the rice rhizomicrobiome and to develop microbial technologies for improved and sustainable rice production.

    DOI PubMed

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    3
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  • Integrated spatial analysis of gene mutation and gene expression for understanding tumor diversity in formalin-fixed paraffin-embedded lung adenocarcinoma.

    Miki Yamazaki, Masahito Hosokawa, Hiroko Matsunaga, Koji Arikawa, Kazuya Takamochi, Kenji Suzuki, Takuo Hayashi, Hideki Kambara, Haruko Takeyama

    Frontiers in oncology   12   936190 - 936190  2022  [Refereed]  [International journal]

     View Summary

    INTRODUCTION: A deeper understanding of intratumoral heterogeneity is essential for prognosis prediction or accurate treatment plan decisions in clinical practice. However, due to the cross-links and degradation of biomolecules within formalin-fixed paraffin-embedded (FFPE) specimens, it is challenging to analyze them. In this study, we aimed to optimize the simultaneous extraction of mRNA and DNA from microdissected FFPE tissues (φ = 100 µm) and apply the method to analyze tumor diversity in lung adenocarcinoma before and after erlotinib administration. METHOD: Two magnetic beads were used for the simultaneous extraction of mRNA and DNA. The decross-linking conditions were evaluated for gene mutation and gene expression analyses of microdissected FFPE tissues. Lung lymph nodes before treatment and lung adenocarcinoma after erlotinib administration were collected from the same patient and were preserved as FFPE specimens for 4 years. Gene expression and gene mutations between histologically classified regions of lung adenocarcinoma (pre-treatment tumor in lung lymph node biopsies and post-treatment tumor, normal lung, tumor stroma, and remission stroma, in resected lung tissue) were compared in a microdissection-based approach. RESULTS: Using the optimized simultaneous extraction of DNA and mRNA and whole-genome amplification, we detected approximately 4,000-10,000 expressed genes and the epidermal growth factor receptor (EGFR) driver gene mutations from microdissected FFPE tissues. We found the differences in the highly expressed cancer-associated genes and the positive rate of EGFR exon 19 deletions among the tumor before and after treatment and tumor stroma, even though they were collected from tumors of the same patient or close regions of the same specimen. CONCLUSION: Our integrated spatial analysis method would be applied to various FFPE pathology specimens providing area-specific gene expression and gene mutation information.

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  • Exploring strain diversity of dominant human skin bacterial species using single-cell genome sequencing.

    Keigo Ide, Tatsuya Saeki, Koji Arikawa, Takuya Yoda, Taruho Endoh, Ayumi Matsuhashi, Haruko Takeyama, Masahito Hosokawa

    Frontiers in microbiology   13   955404 - 955404  2022  [Refereed]  [International journal]

    Authorship:Last author, Corresponding author

     View Summary

    To understand the role of the skin commensal bacterial community in skin health and the spread of pathogens, it is crucial to identify genetic differences in the bacterial strains corresponding to human individuals. A culture-independent genomics approach is an effective tool for obtaining massive high-quality bacterial genomes. Here we present a single-cell genome sequencing to obtain comprehensive whole-genome sequences of uncultured skin bacteria from skin swabs. We recovered 281 high-quality (HQ) and 244 medium-quality single-amplified genomes (SAGs) of multiple skin bacterial species from eight individuals, including cohabiting group. Single-cell sequencing outperformed in the genome recovery from the same skin swabs, showing 10-fold non-redundant strain genomes compared to the shotgun metagenomic sequencing and binning approach. We then focused on the abundant skin bacteria and identified intra-species diversity, especially in 47 Moraxella osloensis derived HQ SAGs, characterizing the strain-level heterogeneity at mobile genetic element profiles, including plasmids and prophages. Even between the cohabiting individual hosts, they have unique skin bacterial strains in the same species, which shows microdiversity in each host. Genetic and functional differences between skin bacterial strains are predictive of in vivo competition to adapt bacterial genome to utilize the sparse nutrients available on the skin or produce molecules that inhibit the colonization of other microbes or alter their behavior. Thus, single-cell sequencing provides a large number of genomes of higher resolution and quality than conventional metagenomic analysis and helps explore the skin commensal bacteria at the strain level, linking taxonomic and functional information.

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  • Recovery of strain-resolved genomes from human microbiome through an integration framework of single-cell genomics and metagenomics.

    Koji Arikawa, Keigo Ide, Masato Kogawa, Tatsuya Saeki, Takuya Yoda, Taruho Endoh, Ayumi Matsuhashi, Haruko Takeyama, Masahito Hosokawa

    Microbiome   9 ( 1 ) 202 - 202  2021.10  [Refereed]  [International journal]

    Authorship:Last author, Corresponding author

     View Summary

    BACKGROUND: Obtaining high-quality (HQ) reference genomes from microbial communities is crucial for understanding the phylogeny and function of uncultured microbes in complex microbial ecosystems. Despite improvements in bioinformatic approaches to generate curated metagenome-assembled genomes (MAGs), existing metagenome binners obtain population consensus genomes but they are nowhere comparable to genomes sequenced from isolates in terms of strain level resolution. Here, we present a framework for the integration of single-cell genomics and metagenomics, referred to as single-cell (sc) metagenomics, to reconstruct strain-resolved genomes from microbial communities at once. RESULTS: Our sc-metagenomics integration framework, termed SMAGLinker, uses single-cell amplified genomes (SAGs) generated using microfluidic technology as binning guides and integrates them with metagenome-assembled genomes (MAGs) to recover improved draft genomes. We compared sc-metagenomics with the metagenomics-alone approach using conventional metagenome binners. The sc-metagenomics approach showed precise contig binning and higher recovery rates (>97%) of rRNA and plasmids than conventional metagenomics in genome reconstruction from the cell mock community. In human microbiota samples, sc-metagenomics recovered the largest number of genomes with a total of 103 gut microbial genomes (21 HQ, with 65 showing >90% completeness) and 45 skin microbial genomes (10 HQ, with 40 showing >90% completeness), respectively. Conventional metagenomics recovered one Staphylococcus hominis genome, whereas sc-metagenomics recovered two S. hominis genomes from identical skin microbiota sample. Single-cell sequencing revealed that these S. hominis genomes were derived from two distinct strains harboring specifically different plasmids. We found that all conventional S. hominis MAGs had a substantial lack or excess of genome sequences and contamination from other Staphylococcus species (S. epidermidis). CONCLUSIONS: SMAGLinker enabled us to obtain strain-resolved genomes in the mock community and human microbiota samples by assigning metagenomic sequences correctly and covering both highly conserved genes such as rRNA genes and unique extrachromosomal elements, including plasmids. SMAGLinker will provide HQ genomes that are difficult to obtain using metagenomics alone and will facilitate the understanding of microbial ecosystems by elucidating detailed metabolic pathways and horizontal gene transfer networks. SMAGLinker is available at https://github.com/kojiari/smaglinker . Video abstract.

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  • Cortical transcriptome analysis after spinal cord injury reveals the regenerative mechanism of central nervous system in CRMP2 knock-in mice.

    Ayaka Sugeno, Wenhui Piao, Miki Yamazaki, Kiyofumi Takahashi, Koji Arikawa, Hiroko Matsunaga, Masahito Hosokawa, Daisuke Tominaga, Yoshio Goshima, Haruko Takeyama, Toshio Ohshima

    Neural regeneration research   16 ( 7 ) 1258 - 1265  2021.07  [Refereed]  [International journal]

     View Summary

    Recent studies have shown that mutation at Ser522 causes inhibition of collapsin response mediator protein 2 (CRMP2) phosphorylation and induces axon elongation and partial recovery of the lost sensorimotor function after spinal cord injury (SCI). We aimed to reveal the intracellular mechanism in axotomized neurons in the CRMP2 knock-in (CRMP2KI) mouse model by performing transcriptome analysis in mouse sensorimotor cortex using micro-dissection punching system. Prior to that, we analyzed the structural pathophysiology in axotomized or neighboring neurons after SCI and found that somatic atrophy and dendritic spine reduction in sensorimotor cortex were suppressed in CRMP2KI mice. Further analysis of the transcriptome has aided in the identification of four hemoglobin genes Hba-a1, Hba-a2, Hbb-bs, and Hbb-bt that are significantly upregulated in wild-type mice with concomitant upregulation of genes involved in the oxidative phosphorylation and ribosomal pathways after SCI. However, we observed substantial upregulation in channel activity genes and downregulation of genes regulating vesicles, synaptic function, glial cell differentiation in CRMP2KI mice. Moreover, the transcriptome profile of CRMP2KI mice has been discussed wherein energy metabolism and neuronal pathways were found to be differentially regulated. Our results showed that CRMP2KI mice displayed improved SCI pathophysiology not only via microtubule stabilization in neurons, but also possibly via the whole metabolic system in the central nervous system, response changes in glial cells, and synapses. Taken together, we reveal new insights on SCI pathophysiology and the regenerative mechanism of central nervous system by the inhibition of CRMP2 phosphorylation at Ser522. All these experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee at Waseda University, Japan (2017-A027 approved on March 21, 2017; 2018-A003 approved on March 25, 2018; 2019-A026 approved on March 25, 2019).

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  • Integration of Droplet Microfluidic Tools for Single-Cell Functional Metagenomics: An Engineering Head Start.

    David Conchouso, Amani Al-Ma'abadi, Hayedeh Behzad, Mohammed Alarawi, Masahito Hosokawa, Yohei Nishikawa, Haruko Takeyama, Katsuhiko Mineta, Takashi Gojobori

    Genomics, proteomics & bioinformatics   19 ( 3 ) 504 - 518  2021.06  [Refereed]  [International journal]

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    Droplet microfluidic techniques have shown promising outcome to study single cells at high throughput. However, their adoption in laboratories studying "-omics" sciences is still irrelevant due to the complex and multidisciplinary nature of the field. To facilitate their use, here we provide engineering details and organized protocols for integrating three droplet-based microfluidic technologies into the metagenomic pipeline to enable functional screening of bioproducts at high throughput. First, a device encapsulating single cells in droplets at a rate of ∼250 Hz is described considering droplet size and cell growth. Then, we expand on previously reported fluorescence-activated droplet sorting systems to integrate the use of 4 independent fluorescence-exciting lasers (i.e., 405, 488, 561, and 637 nm) in a single platform to make it compatible with different fluorescence-emitting biosensors. For this sorter, both hardware and software are provided and optimized for effortlessly sorting droplets at 60 Hz. Then, a passive droplet merger is also integrated into our pipeline to enable adding new reagents to already-made droplets at a rate of 200 Hz. Finally, we provide an optimized recipe for manufacturing these chips using silicon dry-etching tools. Because of the overall integration and the technical details presented here, our approach allows biologists to quickly use microfluidic technologies and achieve both single-cell resolution and high-throughput capability (>50,000 cells/day) for mining and bioprospecting metagenomic data.

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  • Distinctive Regulation of Emotional Behaviors and Fear-Related Gene Expression Responses in Two Extended Amygdala Subnuclei With Similar Molecular Profiles.

    Shuhei Ueda, Masahito Hosokawa, Koji Arikawa, Kiyofumi Takahashi, Mao Fujiwara, Manami Kakita, Taro Fukada, Hiroaki Koyama, Shin-Ichiro Horigane, Keiichi Itoi, Masaki Kakeyama, Hiroko Matsunaga, Haruko Takeyama, Haruhiko Bito, Sayaka Takemoto-Kimura

    Frontiers in molecular neuroscience   14   741895 - 741895  2021  [Refereed]  [International journal]

     View Summary

    The central nucleus of the amygdala (CeA) and the lateral division of the bed nucleus of the stria terminalis (BNST) are the two major nuclei of the central extended amygdala that plays essential roles in threat processing, responsible for emotional states such as fear and anxiety. While some studies suggested functional differences between these nuclei, others showed anatomical and neurochemical similarities. Despite their complex subnuclear organization, subnuclei-specific functional impact on behavior and their underlying molecular profiles remain obscure. We here constitutively inhibited neurotransmission of protein kinase C-δ-positive (PKCδ+) neurons-a major cell type of the lateral subdivision of the CeA (CeL) and the oval nucleus of the BNST (BNSTov)-and found striking subnuclei-specific effects on fear- and anxiety-related behaviors, respectively. To obtain molecular clues for this dissociation, we conducted RNA sequencing in subnuclei-targeted micropunch samples. The CeL and the BNSTov displayed similar gene expression profiles at the basal level; however, both displayed differential gene expression when animals were exposed to fear-related stimuli, with a more robust expression change in the CeL. These findings provide novel insights into the molecular makeup and differential engagement of distinct subnuclei of the extended amygdala, critical for regulation of threat processing.

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  • Draft Genome Sequence of Okeania sp. Strain KiyG1, Assembled from Single-Amplified Genomes Collected from Cape Kiyan, Okinawa, Japan.

    Muhammad Wahyudin Lewaru, Yohei Nishikawa, Keigo Ide, Masato Kogawa, Masahito Hosokawa, Ashok Zachariah Samuel, Shinpei Sumimoto, Handung Nuryadi, Shoichiro Suda, Haruko Takeyama

    Microbiology resource announcements   9 ( 46 )  2020.11  [Refereed]  [International journal]

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    The genus Okeania is a globally distributed group of microorganisms that live in shallow seabed regions. These organisms play several environmentally important roles and are also known producers of several active secondary metabolites with potential human applications. Here, we present a draft genome of Okeania sp. strain KiyG1 (92.7% completeness) that was assembled from four single-amplified genomes.

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  • Slow-Cycling Cancer Stem Cells Regulate Progression and Chemoresistance in Colon Cancer.

    Daisuke Shiokawa, Hiroaki Sakai, Hirokazu Ohata, Toshiaki Miyazaki, Yusuke Kanda, Shigeki Sekine, Daichi Narushima, Masahito Hosokawa, Mamoru Kato, Yutaka Suzuki, Haruko Takeyama, Hideki Kambara, Hitoshi Nakagama, Koji Okamoto

    Cancer research   80 ( 20 ) 4451 - 4464  2020.10  [Refereed]  [International journal]

     View Summary

    Cancer chemoresistance is often attributed to the presence of cancer stem cell (CSC)-like cells, but whether they are homogeneously chemoresistant remains unclear. We previously showed that in colon tumors, a subpopulation of LGR5+ CSC-like cells driven by TCF1 (TCF7), a Wnt-responsive transcription factor, were responsible for tumorigenicity. Here we demonstrate that the tumorigenic subpopulation of mouse LGR5+ cells exists in a slow-cycling state and identify a unique 22-gene signature that characterizes these slow-cycling CSC. Seven of the signature genes are specifically expressed in slow-cycling LGR5+ cells from xenografted human colon tumors and are upregulated in colon cancer clinical specimens. Among these seven, four genes (APCDD1, NOTUM, PROX1, and SP5) are known to be direct Wnt target genes, and PROX1 was expressed in the invasive fronts of colon tumors. PROX1 was activated by TCF1 to induce CDKN1C and maintain a slow-cycling state in colon cancer organoids. Strikingly, PROX1 was required for recurrent growth after chemotherapeutic treatment, suggesting that inhibition of slow-cycling CSC by targeting the TCF1-PROX1-CDKN1C pathway is an effective strategy to combat refractory colon cancer in combination with conventional chemotherapy. SIGNIFICANCE: These findings illustrate the importance of a slow-cycling CSC subpopulation in colon cancer development and chemoresistance, with potential implications for the identified slow-cycling CSC signatures and the TCF1-PROX1-CDKN1C pathway as therapeutic targets.

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  • High-Quality Draft Single-Cell Genome Sequences of Two Gammaproteobacteria Strains Sampled from Soil in a Strawberry Farm.

    Takuya Yoda, Koji Arikawa, Tatsuya Saeki, Ayumi Matsuhashi, Masahito Hosokawa

    Microbiology resource announcements   9 ( 35 )  2020.08  [Refereed]  [International journal]

    Authorship:Last author, Corresponding author

     View Summary

    Here, we present high-quality draft single-cell genome sequences of Gammaproteobacteria strains BBSC-SA01 and BBSC-SA02, obtained from uncultivated cells of soil in a strawberry farm using the single-cell sequencing platform bit-MAP. These draft genomes putatively represent novel species within Gammaproteobacteria and allow further investigation into the soil microbiome.

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  • Rapid inspection method for investigating the heat processing conditions employed for chicken meat using Raman spectroscopy.

    Rimi Miyaoka, Masahiro Ando, Rieko Harada, Hiroyuki Osaka, Ashok Zachariah Samuel, Masahito Hosokawa, Haruko Takeyama

    Journal of bioscience and bioengineering   129 ( 6 ) 700 - 705  2020.06  [Refereed]  [Domestic journal]

     View Summary

    In Japan, the imports of meat products have been increasing every year. Heat processing of meat is the current standard method for ensuring domestic animal health, particularly in case of meat products from areas where infectious diseases are known to have occurred in domestic animals. The Animal Quarantine Service needs to establish a method that detects the temperature at which the meat has been heat-processed (endpoint temperature) to ensure that the standard protocol is followed at the production location. Here, we developed a Raman spectroscopy and multivariate statistics (viz. multivariate curve resolution (MCR))-based simple and rapid method for accurately estimating the end point temperature. We showed that the temperature-dependent secondary structure modification of proteins can serve as an accurate indicator of the temperature of heat processing. This methodology can be easily automated for effective utilization by someone who is not an expert in spectroscopy. We envisage a wider application of this method in food analysis, although the present research investigated the application of this method in chicken meat heat processing analysis.

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  • Evaluation of the effects of cell-dispensing using an inkjet-based bioprinter on cell integrity by RNA-seq analysis.

    Masayuki Yumoto, Natsuko Hemmi, Naoki Sato, Yudai Kawashima, Koji Arikawa, Keigo Ide, Masahito Hosokawa, Manabu Seo, Haruko Takeyama

    Scientific reports   10 ( 1 ) 7158 - 7158  2020.04  [Refereed]  [International journal]

     View Summary

    Bioprinting technology is expected to be applied in the fields of regenerative medicine and drug discovery. There are several types of bioprinters, especially inkjet-based bioprinter, which can be used not only as a printer for arranging cells but also as a precision cell-dispensing device with controlled cell numbers similar to a fluorescence activated cell sorter (FACS). Precise cell dispensers are expected to be useful in the fields of drug discovery and single-cell analysis. However, there are enduring concerns about the impacts of cell dispensers on cell integrity, particularly on sensitive cells, such as stem cells. In response to the concerns stated above, we developed a stress-free and media-direct-dispensing inkjet bioprinter. In the present study, in addition to conventional viability assessments, we evaluated the gene expression using RNA-seq to investigate whether the developed bioprinter influenced cell integrity in mouse embryonic stem cells. We evaluated the developed bioprinter based on three dispensing methods: manual operation using a micropipette, FACS and the developed inkjet bioprinter. According to the results, the developed inkjet bioprinter exhibited cell-friendly dispensing performance, which was similar to the manual dispensing operation, based not only on cell viability but also on gene expression levels.

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  • Effective microtissue RNA extraction coupled with Smart-seq2 for reproducible and robust spatial transcriptome analysis.

    Miki Yamazaki, Masahito Hosokawa, Koji Arikawa, Kiyofumi Takahashi, Chikako Sakanashi, Takuya Yoda, Hiroko Matsunaga, Haruko Takeyama

    Scientific reports   10 ( 1 ) 7083 - 7083  2020.04  [Refereed]  [International journal]

    Authorship:Lead author

     View Summary

    Spatial transcriptomics is useful for understanding the molecular organization of a tissue and providing insights into cellular function in a morphological context. In order to obtain reproducible results in spatial transcriptomics, we have to maintain tissue morphology and RNA molecule stability during the image acquisition and biomolecule collection processes. Here, we developed a tissue processing method for robust and reproducible RNA-seq from tissue microdissection samples. In this method, we suppressed RNA degradation in fresh-frozen tissue specimens by dehydration fixation and effectively collected a small amount of RNA molecules from microdissection samples by magnetic beads. We demonstrated the spatial transcriptome analysis of the mouse liver and brain in serial microdissection samples (100 μm in a diameter and 10 μm in thickness) produced by a microdissection punching system. Using our method, we could prevent RNA degradation at room temperature and effectively produce a sequencing library with Smart-seq2. This resulted in reproducible sequence read mapping in exon regions and the detection of more than 2000 genes compared to non-fixed samples in the RNA-seq analysis. Our method would be applied to various transcriptome analyses, providing the information for region specific gene expression in tissue specimens.

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  • Time-lapse single-cell transcriptomics reveals modulation of histone H3 for dormancy breaking in fission yeast.

    Hayato Tsuyuzaki, Masahito Hosokawa, Koji Arikawa, Takuya Yoda, Naoyuki Okada, Haruko Takeyama, Masamitsu Sato

    Nature communications   11 ( 1 ) 1265 - 1265  2020.03  [Refereed]  [International journal]

     View Summary

    How quiescent cells break dormancy is a key issue in eukaryotic cells including cancer. Fungal spores, for example, remain quiescent for long periods until nourished, although the mechanisms by which dormancy is broken remain enigmatic. Transcriptome analysis could provide a clue, but methods to synchronously germinate large numbers of spores are lacking, and thus it remains a challenge to analyse gene expression upon germination. Hence, we develop methods to assemble transcriptomes from individual, asynchronous spore cells of fission yeast undergoing germination to assess transcriptomic changes over time. The virtual time-lapse analyses highlights one of three copies of histone H3 genes whose transcription fluctuates during the initial stage of germination. Disruption of this temporal fluctuation causes defects in spore germination despite no visible defects in other stages of the life cycle. We conclude that modulation of histone H3 expression is a crucial 'wake-up' trigger at dormancy breaking.

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  • Massively parallel single-cell genome sequencing enables high-resolution analysis of soil and marine microbiome

    Nishikawa Y, Kogawa M, Hosokawa M, Mineta K, Takahashi K, Sakanashi C, Behzad H, Gojobori T, Takeyama H

       2020.03

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  • Single-cell genomics of uncultured bacteria reveals dietary fiber responders in the mouse gut microbiota.

    Rieka Chijiiwa, Masahito Hosokawa, Masato Kogawa, Yohei Nishikawa, Keigo Ide, Chikako Sakanashi, Kai Takahashi, Haruko Takeyama

    Microbiome   8 ( 1 ) 5 - 5  2020.01  [Refereed]  [International journal]

    Authorship:Lead author, Corresponding author

     View Summary

    BACKGROUND: The gut microbiota can have dramatic effects on host metabolism; however, current genomic strategies for uncultured bacteria have several limitations that hinder their ability to identify responders to metabolic changes in the microbiota. In this study, we describe a novel single-cell genomic sequencing technique that can identify metabolic responders at the species level without the need for reference genomes, and apply this method to identify bacterial responders to an inulin-based diet in the mouse gut microbiota. RESULTS: Inulin-feeding changed the mouse fecal microbiome composition to increase Bacteroides spp., resulting in the production of abundant succinate in the mouse intestine. Using our massively parallel single-cell genome sequencing technique, named SAG-gel platform, we obtained 346 single-amplified genomes (SAGs) from mouse gut microbes before and after dietary inulin supplementation. After quality control, the SAGs were classified as 267 bacteria, spanning 2 phyla, 4 classes, 7 orders, and 14 families, and 31 different strains of SAGs were graded as high- and medium-quality draft genomes. From these, we have successfully obtained the genomes of the dominant inulin-responders, Bacteroides spp., and identified their polysaccharide utilization loci and their specific metabolic pathways for succinate production. CONCLUSIONS: Our single-cell genomics approach generated a massive amount of SAGs, enabling a functional analysis of uncultured bacteria in the intestinal microbiome. This enabled us to estimate metabolic lineages involved in the bacterial fermentation of dietary fiber and metabolic outcomes such as short-chain fatty acid production in the intestinal environment based on the fibers ingested. The technique allows the in-depth isolation and characterization of uncultured bacteria with specific functions in the microbiota and could be exploited to improve human and animal health. Video abstract.

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  • High-throughput identification of peptide agonists against GPCRs by co-culture of mammalian reporter cells and peptide-secreting yeast cells using droplet microfluidics.

    Kenshi Yaginuma, Wataru Aoki, Natsuko Miura, Yuta Ohtani, Shunsuke Aburaya, Masato Kogawa, Yohei Nishikawa, Masahito Hosokawa, Haruko Takeyama, Mitsuyoshi Ueda

    Scientific reports   9 ( 1 ) 10920 - 10920  2019.07  [Refereed]  [International journal]

     View Summary

    Since G-protein coupled receptors (GPCRs) are linked to various diseases, screening of functional ligands against GPCRs is vital for drug discovery. In the present study, we developed a high-throughput functional cell-based assay by combining human culture cells producing a GPCR, yeast cells secreting randomized peptide ligands, and a droplet microfluidic device. We constructed a reporter human cell line that emits fluorescence in response to the activation of human glucagon-like peptide-1 receptor (hGLP1R). We then constructed a yeast library secreting an agonist of hGLP1R or randomized peptide ligands. We demonstrated that high-throughput identification of functional ligands against hGLP1R could be performed by co-culturing the reporter cells and the yeast cells in droplets. We identified functional ligands, one of which had higher activity than that of an original sequence. The result suggests that our system could facilitate the discovery of functional peptide ligands of GPCRs.

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  • Correction to: A CCR5+ memory subset within HIV-1-infected primary resting CD4+ T cells is permissive for replication-competent, latently infected viruses in vitro.

    Terahara K, Iwabuchi R, Hosokawa M, Nishikawa Y, Takeyama H, Takahashi Y, Tsunetsugu-Yokota Y

    BMC research notes   12 ( 1 ) 322  2019.06  [Refereed]

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  • A CCR5+ memory subset within HIV-1-infected primary resting CD4+ T cells is permissive for replication-competent, latently infected viruses in vitro.

    Kazutaka Terahara, Ryutaro Iwabuchi, Masahito Hosokawa, Yohei Nishikawa, Haruko Takeyama, Yoshimasa Takahashi, Yasuko Tsunetsugu-Yokota

    BMC research notes   12 ( 1 ) 242 - 242  2019.04  [Refereed]  [International journal]

     View Summary

    OBJECTIVE: Resting CD4+ T cells are major reservoirs of latent HIV-1 infection, and may be formed during the early phase of the infection. Although CCR5-tropic (R5) HIV-1 is highly transmissible during the early phase, newly infected individuals have usually been exposed to a mixture of R5 and CXCR4-tropic (X4) viruses, and X4 viral DNA is also detectable in the host. Our aim was to identify which subsets of resting CD4+ T cells contribute to forming the latent reservoir in the presence of both X4 and R5 viruses. RESULTS: Primary resting CD4+ naïve T (TN) cells, CCR5- memory T (TM) cells, and CCR5+ TM cells isolated by flow cytometry were infected simultaneously with X4 and R5 HIV-1, which harbored different reporter genes, and were cultured in the resting condition. Flow cytometry at 3 days post-infection demonstrated that X4 HIV-1+ cells were present in all three subsets of cells, whereas R5 HIV-1+ cells were present preferentially in CCR5+ TM cells, but not in TN cells. Following CD3/CD28-mediated activation at 3 days post-infection, numbers of R5 HIV-1+ cells and X4 HIV-1+ cells increased significantly only in the CCR5+ TM subset, suggesting that it provides a major reservoir of replication-competent, latently infected viruses.

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  • Sequential Sensing by TLR2 and Mincle Directs Immature Myeloid Cells to Protect against Invasive Group A Streptococcal Infection in Mice.

    Takayuki Matsumura, Tadayoshi Ikebe, Koji Arikawa, Masahito Hosokawa, Michio Aiko, Aoi Iguchi, Ikuko Togashi, Sayaka Kai, Sakiko Ohara, Naoya Ohara, Makoto Ohnishi, Haruo Watanabe, Kazuo Kobayashi, Haruko Takeyama, Sho Yamasaki, Yoshimasa Takahashi, Manabu Ato

    Cell reports   27 ( 2 ) 561 - 571  2019.04  [Refereed]  [International journal]

     View Summary

    Severe invasive group A Streptococcus (GAS) infection evades anti-bacterial immunity by attenuating the cellular components of innate immune responses. However, this loss of protection is compensated for by interferon (IFN)-γ-producing immature myeloid cells (γIMCs), which are selectively recruited upon severe invasive GAS infection in mice. Here, we demonstrate that γIMCs provide this IFN-γ-mediated protection by sequentially sensing GAS through two distinct pattern recognition receptors. In a mouse model, GAS is initially recognized by Toll-like receptor 2 (TLR2), which promptly induces interleukin (IL)-6 production in γIMCs. γIMC-derived IL-6 promotes the upregulation of a recently identified GAS-sensing receptor, macrophage-inducible C-type lectin (Mincle), in an autocrine or paracrine manner. Notably, blockade of γIMC-derived IL-6 abrogates Mincle expression, downstream IFN-γ production, and γIMC-mediated protection against severe invasive GAS infection. Thus, γIMCs regulate host protective immunity against severe invasive GAS infection via a TLR2-IL-6-Mincle axis.

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  • Effects of short-term endurance exercise on gut microbiota in elderly men.

    Hirokazu Taniguchi, Kumpei Tanisawa, Xiaomin Sun, Takafumi Kubo, Yuri Hoshino, Masahito Hosokawa, Haruko Takeyama, Mitsuru Higuchi

    Physiological reports   6 ( 23 ) e13935  2018.12  [Refereed]  [International journal]

     View Summary

    Regular exercise reduces the risks for cardiovascular diseases. Although the gut microbiota has been associated with fitness level and cardiometabolic risk factors, the effects of exercise-induced gut microbiota changes in elderly individuals are unclear. This study evaluated whether endurance exercise modulates the gut microbiota in elderly subjects, and whether these changes are associated with host cardiometabolic phenotypes. In a randomized crossover trial, 33 elderly Japanese men participated in a 5-week endurance exercise program. 16S rRNA gene-based metagenomic analyses revealed that the effect of endurance exercise on gut microbiota diversity was not greater than interindividual differences, whereas changes in α-diversity indices during intervention were negatively correlated with changes in systolic and diastolic blood pressure, especially during exercise. Microbial composition analyses showed that the relative abundance of Clostridium difficile significantly decreased, whereas that of Oscillospira significantly increased during exercise as compared to the control period. The changes in these taxa were correlated with the changes in several cardiometabolic risk factors. The findings indicate that short-term endurance exercise has little effect on gut microbiota in elderly individuals, and that the changes in gut microbiota were associated with cardiometabolic risk factors, such as systolic and diastolic blood pressure, providing preliminary insight into the associations between the gut microbiota and cardiometabolic phenotypes.

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  • Combinatory use of distinct single-cell RNA-seq analytical platforms reveals the heterogeneous transcriptome response.

    Yukie Kashima, Ayako Suzuki, Ying Liu, Masahito Hosokawa, Hiroko Matsunaga, Masataka Shirai, Kohji Arikawa, Sumio Sugano, Takashi Kohno, Haruko Takeyama, Katsuya Tsuchihara, Yutaka Suzuki

    Scientific reports   8 ( 1 ) 3482 - 3482  2018.02  [Refereed]  [International journal]

     View Summary

    Single-cell RNA-seq is a powerful tool for revealing heterogeneity in cancer cells. However, each of the current single-cell RNA-seq platforms has inherent advantages and disadvantages. Here, we show that combining the different single-cell RNA-seq platforms can be an effective approach to obtaining complete information about expression differences and a sufficient cellular population to understand transcriptional heterogeneity in cancers. We demonstrate that it is possible to estimate missing expression information. We further demonstrate that even in the cases where precise information for an individual gene cannot be inferred, the activity of given transcriptional modules can be analyzed. Interestingly, we found that two distinct transcriptional modules, one associated with the Aurora kinase gene and the other with the DUSP gene, are aberrantly regulated in a minor population of cells and may thus contribute to the possible emergence of dormancy or eventual drug resistance within the population.

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    15
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  • Obtaining high-quality draft genomes from uncultured microbes by cleaning and co-assembly of single-cell amplified genomes.

    Masato Kogawa, Masahito Hosokawa, Yohei Nishikawa, Kazuki Mori, Haruko Takeyama

    Scientific reports   8 ( 1 ) 2059 - 2059  2018.02  [Refereed]  [International journal]

    Authorship:Lead author

     View Summary

    Single-cell genomics is a straightforward approach to obtain genomes from uncultured microbes. However, sequence reads from a single-cell amplified genome (SAG) contain significant bias and chimeric sequences. Here, we describe Cleaning and Co-assembly of a Single-Cell Amplified Genome (ccSAG), a novel analytical workflow to obtain composite single-cell genomes with elimination of sequence errors. By the integration of ccSAG with a massively parallel single-cell genome amplification platform based on droplet microfluidics, we can generate multiple SAGs and effectively integrate them into the composite genomes quality equivalent to the data obtained from bulk DNA. We obtained two novel draft genomes from single gut microbial cells with high completeness (>96.6%) and extremely low contamination (<1.25%). Moreover, we revealed the presence of single nucleotide polymorphisms in the specific gene by sequence comparison at the single-cell level. Thus, the workflow yields near-complete genomes from uncultured microbes, and enables analyses of genetic heterogeneity within identical strains.

    DOI PubMed

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    37
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  • Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics.

    Masahito Hosokawa, Yohei Nishikawa, Masato Kogawa, Haruko Takeyama

    Scientific reports   7 ( 1 ) 5199 - 5199  2017.07  [Refereed]  [International journal]

    Authorship:Lead author

     View Summary

    Massively parallel single-cell genome sequencing is required to further understand genetic diversities in complex biological systems. Whole genome amplification (WGA) is the first step for single-cell sequencing, but its throughput and accuracy are insufficient in conventional reaction platforms. Here, we introduce single droplet multiple displacement amplification (sd-MDA), a method that enables massively parallel amplification of single cell genomes while maintaining sequence accuracy and specificity. Tens of thousands of single cells are compartmentalized in millions of picoliter droplets and then subjected to lysis and WGA by passive droplet fusion in microfluidic channels. Because single cells are isolated in compartments, their genomes are amplified to saturation without contamination. This enables the high-throughput acquisition of contamination-free and cell specific sequence reads from single cells (21,000 single-cells/h), resulting in enhancement of the sequence data quality compared to conventional methods. This method allowed WGA of both single bacterial cells and human cancer cells. The obtained sequencing coverage rivals those of conventional techniques with superior sequence quality. In addition, we also demonstrate de novo assembly of uncultured soil bacteria and obtain draft genomes from single cell sequencing. This sd-MDA is promising for flexible and scalable use in single-cell sequencing.

    DOI PubMed

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    89
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  • Site-specific gene expression analysis using an automated tissue micro-dissection punching system.

    Takuya Yoda, Masahito Hosokawa, Kiyofumi Takahashi, Chikako Sakanashi, Haruko Takeyama, Hideki Kambara

    Scientific reports   7 ( 1 ) 4325 - 4325  2017.06  [Refereed]  [International journal]

     View Summary

    Site-specific gene expression analyses are important for understanding tissue functions. Despite rapid developments in DNA-related technologies, the site-specific analysis of whole genome expression for a tissue remains challenging. Thus, a new tool is required for capturing multiple tissue micro-dissections or single cells while retaining the positional information. Here, we describe the development of such a system, which can pick up micro-dissections by punching a tissue repeatedly in a very short period, e.g., 5 s/sampling cycle. A photo of the punched tissue provides information on the dissected positions, allowing site-specific gene expression analysis. We demonstrate the site-specific analysis of a frozen tissue slice of mouse brain by analyzing many micro-dissections produced from the tissue at a 300-μm pitch. The site-specific analysis provided new insights into the gene expression profiles in a tissue and on tissue functions. The analysis of site-specific whole genome expression may therefore, open new avenues in life science.

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    13
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  • Evaluation of cancer cell deformability by microcavity array.

    Tomoko Yoshino, Tsuyoshi Tanaka, Seita Nakamura, Ryo Negishi, Nozomi Shionoiri, Masahito Hosokawa, Tadashi Matsunaga

    Analytical biochemistry   520   16 - 21  2017.03  [Refereed]  [International journal]

     View Summary

    A cell entrapment device consisting of a microcavity array was used to analyze the deformability of MCF-10 human breast epithelial and MCF-7 human breast cancer cell lines by confocal laser scanning microscopy. Entrapment of up to 8 × 103 cells was achieved within 3 min. Protrusions were formed at the bottom surface of the array with a pore size of 3 μm. Protrusion length increased at higher filtration pressures and could be used to distinguish between MCF-7 and MCF-10 cells. These results indicate that our system is useful for high-throughput deformability analysis of cancer cells, which can provide insight into the mechanisms underlying tumor cell malignancy.

    DOI PubMed

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    8
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  • Manipulation of a Single Circulating Tumor Cell Using Visualization of Hydrogel Encapsulation toward Single-Cell Whole-Genome Amplification.

    Tomoko Yoshino, Tsuyoshi Tanaka, Seita Nakamura, Ryo Negishi, Masahito Hosokawa, Tadashi Matsunaga

    Analytical chemistry   88 ( 14 ) 7230 - 7  2016.07  [Refereed]  [International journal]

     View Summary

    Genetic characterization of circulating tumor cells (CTCs) could guide the choice of therapies for individual patients and also facilitate the development of new drugs. We previously developed a CTC recovery system using a microcavity array, which demonstrated highly efficient CTC recovery based on differences in cell size and deformability. However, the CTC recovery system lacked an efficient cell manipulation tool suitable for subsequent genetic analysis. Here, we resolve this issue and present a simple and rapid manipulation method for single CTCs using a photopolymerized hydrogel, polyethylene glycol diacrylate (PEGDA), which is useful for subsequent genetic analysis. First, PEGDA was introduced into the cells entrapped on the microcavity array. Then, excitation light was projected onto the target single cells for encapsulation of each CTC by confocal laser-scanning microscopy. The encapsulated single CTCs could be visualized by the naked eye and easily handled with tweezers. The single CTCs were only partially encapsulated on the PEGDA hydrogel, which allowed for sufficient whole-genome amplification and accurate genotyping. Our proposed methodology is a valuable tool for the rapid and simple manipulation of single CTCs and is expected to become widely utilized for analyses of mammalian cells and microorganisms in addition to CTCs.

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    26
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  • Bacterial Inactivation by Applying an Alternating Electromagnetic Field Using PAMAM Dendron-modified Magnetic Nanoparticles

    Atsushi Arakaki, Mami Takahashi, Masahito Hosokawa, Tadashi Matsunaga, Tsuyoshi Tanaka

    ELECTROCHEMISTRY   84 ( 5 ) 324 - 327  2016.05  [Refereed]

     View Summary

    In this study, a method involving polyamidoamine dendron-modified magnetic nanoparticles (PAMAM-MNPs) along with application of an alternating magnetic field (AMF) was developed for inactivation of bacteria in water samples. The PAMAM-MNPs efficiently bound to Escherichia coli cells, resulting in magnetic recovery of cells from aqueous solutions. By applying the AMF (5 kW, 250 kHz) to the cell suspension, E. coli cells were successfully inactivated within 10 min in the presence of the MNPs, while no effect was observed in the absence of the MNPs. The use of PAMAM-MNPs could increase the inactivation rate of E. coli under the applied AMF. E. coli cells with PAMAM-MNPs stained by propidium iodide (PI) exhibited apparent fluorescence after exposure to the AMF, suggesting the occurrence of membrane damage in the cells because of direct heat transfer from the PAMAM-MNPs. Our technique can be used to address bacterial contamination with wide varieties of microorganisms in water samples. (C) The Electrochemical Society of Japan, All rights reserved.

    DOI

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    5
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  • Droplet microfluidics for precise and high throughput whole genome amplification toward single-cell genome sequencing

    Hosokawa M, Nishikawa Y, Kogawa M, Takeyama H

    20th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2016     178 - 179  2016  [Refereed]

    Authorship:Lead author

  • Simple and rapid CD4 testing based on large-field imaging system composed of microcavity array and two-dimensional photosensor.

    Tatsuya Saeki, Yuriko Sugamura, Masahito Hosokawa, Tomoko Yoshino, Tae-Kyu Lim, Manabu Harada, Tadashi Matsunaga, Tsuyoshi Tanaka

    Biosensors & bioelectronics   67   350 - 5  2015.05  [Refereed]  [International journal]

     View Summary

    This study presents a novel method for CD4 testing based on one-shot large-field imaging. The large-field imaging system was fabricated by a microcavity array and a two-dimensional (2D) photosensor within the desk-top-sized instrument. The microcavity array was employed to separate leukocytes from whole blood based on differences in the size of leukocytes and other blood cells. The large-field imaging system with lower side irradiation enabled acquisition of cell signatures with high signal-to-noise ratio, because the metallic substrate of the microcavity array obstructed excessive excitation light. In this setting, dual-color imaging of CD4(+) and CD8(+) T cells was achieved within the entire image area (64 mm(2)) in 2s. The practical performance of the large-field imaging system was demonstrated by determining the CD4/CD8 ratio in a few microliter of control whole blood as small as those obtained by a finger prick. The CD4/CD8 ratios measured using the large-field imaging system correlated well with those measured by microscopic analysis. These results indicate that our proposed system provides a simple and rapid CD4 testing for the application of HIV/AIDS treatment.

    DOI PubMed

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    7
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  • Droplet-based microfluidics for high-throughput screening of a metagenomic library for isolation of microbial enzymes.

    Masahito Hosokawa, Yuri Hoshino, Yohei Nishikawa, Tomotada Hirose, Dong Hyun Yoon, Tetsushi Mori, Tetsushi Sekiguchi, Shuichi Shoji, Haruko Takeyama

    Biosensors & bioelectronics   67   379 - 85  2015.05  [Refereed]  [International journal]

    Authorship:Lead author

     View Summary

    This paper proposes a high-throughput, function-based screening approach of a metagenomic library for isolating novel microbial enzymes by droplet-based microfluidics. We used gel microdroplets (GMDs) dispersed in oil as picoliter-volume reaction vessels for lipolytic enzyme by encapsulating cells in individual GMDs. Using this approach, we monitored the growth of individual cells encapsulated in GMDs and assessed the enzyme reaction activities at the level of an individual GMD. We then applied this method to screen lipolytic enzyme genes from the metagenomic library constructed from soil collected from a quercus serrate forest of Mount Tsukuba, Ibaraki, Japan. In the workflow presented in this study, metagenomic library clones were encapsulated in 100-pL GMDs with a fluorogenic reporter substrate. A total of 67,000 metagenomic library clones can be screened in only 24 h with reduced consumption of reagents (i.e., <10 μL). As a result, we identified a novel lipolytic enzyme, EstT1, belonging to the EstD2 family of esterases and containing a putative signal peptide, which facilitates enzyme export and catalyzation of substrates in the periplasm. Our study demonstrates the potential of microfluidic GMDs as an efficient tool for metagenomic library screening of industrially relevant enzymes with the potential of significantly reducing the cost and time factors involved in successful practical application of microbial enzymes.

    DOI PubMed

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    88
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  • Development of the automated circulating tumor cell recovery system with microcavity array.

    Ryo Negishi, Masahito Hosokawa, Seita Nakamura, Hisashige Kanbara, Masafumi Kanetomo, Yoshihito Kikuhara, Tsuyoshi Tanaka, Tadashi Matsunaga, Tomoko Yoshino

    Biosensors & bioelectronics   67   438 - 42  2015.05  [Refereed]  [International journal]

     View Summary

    Circulating tumor cells (CTCs) are well recognized as useful biomarker for cancer diagnosis and potential target of drug discovery for metastatic cancer. Efficient and precise recovery of extremely low concentrations of CTCs from blood has been required to increase the detection sensitivity. Here, an automated system equipped with a microcavity array (MCA) was demonstrated for highly efficient and reproducible CTC recovery. The use of MCA allows selective recovery of cancer cells from whole blood on the basis of differences in size between tumor and blood cells. Intra- and inter-assays revealed that the automated system achieved high efficiency and reproducibility equal to the assay manually performed by well-trained operator. Under optimized assay workflow, the automated system allows efficient and precise cell recovery for non-small cell lung cancer cells spiked in whole blood. The automated CTC recovery system will contribute to high-throughput analysis in the further clinical studies on large cohort of cancer patients.

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    22
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  • Monodisperse Picoliter Droplets for Low-Bias and Contamination-Free Reactions in Single-Cell Whole Genome Amplification.

    Yohei Nishikawa, Masahito Hosokawa, Toru Maruyama, Keisuke Yamagishi, Tetsushi Mori, Haruko Takeyama

    PloS one   10 ( 9 ) e0138733  2015  [Refereed]  [International journal]

    Authorship:Lead author

     View Summary

    Whole genome amplification (WGA) is essential for obtaining genome sequences from single bacterial cells because the quantity of template DNA contained in a single cell is very low. Multiple displacement amplification (MDA), using Phi29 DNA polymerase and random primers, is the most widely used method for single-cell WGA. However, single-cell MDA usually results in uneven genome coverage because of amplification bias, background amplification of contaminating DNA, and formation of chimeras by linking of non-contiguous chromosomal regions. Here, we present a novel MDA method, termed droplet MDA, that minimizes amplification bias and amplification of contaminants by using picoliter-sized droplets for compartmentalized WGA reactions. Extracted DNA fragments from a lysed cell in MDA mixture are divided into 105 droplets (67 pL) within minutes via flow through simple microfluidic channels. Compartmentalized genome fragments can be individually amplified in these droplets without the risk of encounter with reagent-borne or environmental contaminants. Following quality assessment of WGA products from single Escherichia coli cells, we showed that droplet MDA minimized unexpected amplification and improved the percentage of genome recovery from 59% to 89%. Our results demonstrate that microfluidic-generated droplets show potential as an efficient tool for effective amplification of low-input DNA for single-cell genomics and greatly reduce the cost and labor investment required for determination of nearly complete genome sequences of uncultured bacteria from environmental samples.

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    50
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  • Evaluation of a Microbial Sensor as a Tool for Antimicrobial Activity Test of Cosmetic Preservatives.

    Hideyuki Gomyo, Masaki Ookawa, Kota Oshibuchi, Yuriko Sugamura, Masahito Hosokawa, Nozomi Shionoiri, Yoshiaki Maeda, Tadashi Matsunaga, Tsuyoshi Tanaka

    Biocontrol science   20 ( 4 ) 247 - 53  2015  [Refereed]  [Domestic journal]

     View Summary

    For high-throughput screening of novel cosmetic preservatives, a rapid and simple assay to evaluate the antimicrobial activities should be developed because the conventional agar dilution method is time-consuming and labor-intensive. To address this issue, we evaluated a microbial sensor as a tool for rapid antimicrobial activity testing. The sensor consists of an oxygen electrode and a filter membrane that holds the test microorganisms, Staphylococcus aureus and Candida albicans. The antimicrobial activity of the tested cosmetic preservative was evaluated by measuring the current increases corresponding to the decreases in oxygen consumption in the microbial respiration. The current increases detected by the sensor showed positive correlation to the concentrations of two commercially used preservatives, chlorphenesin and 2-phenoxyethanol. The same tendency was also observed when a model cosmetic product was used as a preservative solvent, indicating the feasibility in practical use. Furthermore, the microbial sensor and microfluidic flow-cell was assembled to achieve sequential measurements. The sensor system presented in this study could be useful in large-scale screening experiments.

    DOI PubMed

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    2
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  • In vivo live cell imaging for the quantitative monitoring of lipids by using Raman microspectroscopy.

    Masahito Hosokawa, Masahiro Ando, Shoichiro Mukai, Kyoko Osada, Tomoko Yoshino, Hiro-o Hamaguchi, Tsuyoshi Tanaka

    Analytical chemistry   86 ( 16 ) 8224 - 30  2014.08  [Refereed]  [International journal]

    Authorship:Lead author

     View Summary

    A straightforward in vivo monitoring technique for biomolecules would be an advantageous approach for understanding their spatiotemporal dynamics in living cells. However, the lack of adequate probes has hampered the quantitative determination of the chemical composition and metabolomics of cellular lipids at single-cell resolution. Here, we describe a method for the rapid, direct, and quantitative determination of lipid molecules from living cells using single-cell Raman imaging. In vivo localization of lipids in the form of triacylglycerol (TAG) within oleaginous microalga and their molecular compositions are monitored with high spatial resolution in a nondestructive and label-free manner. This method can provide quantitative and real-time information on compositions, chain lengths, and degree of unsaturation of fatty acids in living cells for improving the cultivating parameters or for determining the harvest timing during large-scale cultivations for microalgal lipid accumulation toward biodiesel production. Therefore, this technique is a potential tool for in vivo lipidomics for understanding the dynamics of lipid metabolisms in various organisms.

    DOI PubMed

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    37
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  • In situ detection of antibiotic amphotericin B produced in Streptomyces nodosus using Raman microspectroscopy.

    Rimi Miyaoka, Masahito Hosokawa, Masahiro Ando, Tetsushi Mori, Hiro-O Hamaguchi, Haruko Takeyama

    Marine drugs   12 ( 5 ) 2827 - 39  2014.05  [Refereed]  [International journal]

     View Summary

    The study of spatial distribution of secondary metabolites within microbial cells facilitates the screening of candidate strains from marine environments for functional metabolites and allows for the subsequent assessment of the production of metabolites, such as antibiotics. This paper demonstrates the first application of Raman microspectroscopy for in situ detection of the antifungal antibiotic amphotericin B (AmB) produced by actinomycetes-Streptomyces nodosus. Raman spectra measured from hyphae of S. nodosus show the specific Raman bands, caused by resonance enhancement, corresponding to the polyene chain of AmB. In addition, Raman microspectroscopy enabled us to monitor the time-dependent change of AmB production corresponding to the growth of mycelia. The Raman images of S. nodosus reveal the heterogeneous distribution of AmB within the mycelia and individual hyphae. Moreover, the molecular association state of AmB in the mycelia was directly identified by observed Raman spectral shifts. These findings suggest that Raman microspectroscopy could be used for in situ monitoring of antibiotic production directly in marine microorganisms with a method that is non-destructive and does not require labeling.

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    29
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  • Monitoring of cellular behaviors by microcavity array-based single-cell patterning.

    Kyoko Osada, Masahito Hosokawa, Tomoko Yoshino, Tsuyoshi Tanaka

    The Analyst   139 ( 2 ) 425 - 30  2014.01  [Refereed]  [International journal]

     View Summary

    In this study, we describe a less invasive and rapid single-cell patterning technique for monitoring of cellular behaviors. To form a high-density grid pattern of living cells, single cells were firstly captured on a geometry-controlled array pattern of 100,000 microcavities by applying negative pressure. The captured cells on the microcavities were immersed in an agarose solution and embedded in agarose gels. The high efficiency transfer of individual yeast cells (Saccharomyces cerevisiae) and diatom cells (Fistulifera sp.) onto agarose gels was successfully achieved in 20 min. The patterning process had no effect on the cell proliferation or division. These results indicate that this technique shows a dramatic increase in patterning efficiency compared to previous patterning technologies. Furthermore, it allows the long-term monitoring of diatom cell divisions for 24 h. Continuous long-term observation of single cells provides technological advantages for the successful acquisition of information to better understand cellular activities.

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    15
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  • Digital cell counting device integrated with a single-cell array.

    Tatsuya Saeki, Masahito Hosokawa, Tae-kyu Lim, Manabu Harada, Tadashi Matsunaga, Tsuyoshi Tanaka

    PloS one   9 ( 2 ) e89011  2014  [Refereed]  [International journal]

     View Summary

    In this paper, we present a novel cell counting method accomplished using a single-cell array fabricated on an image sensor, complementary metal oxide semiconductor sensor. The single-cell array was constructed using a microcavity array, which can trap up to 7,500 single cells on microcavities periodically arranged on a plane metallic substrate via the application of a negative pressure. The proposed method for cell counting is based on shadow imaging, which uses a light diffraction pattern generated by the microcavity array and trapped cells. Under illumination, the cell-occupied microcavities are visualized as shadow patterns in an image recorded by the complementary metal oxide semiconductor sensor due to light attenuation. The cell count is determined by enumerating the uniform shadow patterns created from one-on-one relationships with single cells trapped on the microcavities in digital format. In the experiment, all cell counting processes including entrapment of non-labeled HeLa cells from suspensions on the array and image acquisition of a wide-field-of-view of 30 mm(2) in 1/60 seconds were implemented in a single integrated device. As a result, the results from the digital cell counting had a linear relationship with those obtained from microscopic observation (r(2)  = 0.99). This platform could be used at extremely low cell concentrations, i.e., 25-15,000 cells/mL. Our proposed system provides a simple and rapid miniaturized cell counting device for routine laboratory use.

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    17
    Citation
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  • Electrochemical disinfection of fish pathogens in seawater without the production of a lethal concentration of chlorine using a flow reactor.

    Tsuyoshi Tanaka, Mari Shimoda, Nozomi Shionoiri, Masahito Hosokawa, Tomoyuki Taguchi, Hitoshi Wake, Tadashi Matsunaga

    Journal of bioscience and bioengineering   116 ( 4 ) 480 - 4  2013.10  [Refereed]  [Domestic journal]

     View Summary

    An electrochemical disinfection system employing a honeycombed platinum coated titanium electrode was developed for the disinfection of seawater. Cell suspensions (2 l, 10³ cells/ml) of the fish pathogens, Vibrio alginolyticus, Edwardsiella tarda, Lactococcus garvieae and Vibrio anguillarum were circulated in a reactor equipped with 10 sets of these electrodes at a flow rate of 200 ml/min with an applied potential of 1.0 V vs. Ag/AgCl reference electrode. The circulated cells were completely disinfected after 3 h of treatment, whereas free residual chlorine generated due to seawater electrolysis was below 0.1 ppm. In addition, a diphenyl-1-pyrenylphosphine fluorescent assay revealed that lipid peroxidation in the cell membranes of disinfected bacteria was induced probably by reactive oxygen species generated during electrochemical treatment.

    DOI PubMed

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    21
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  • Microcavity array system for size-based enrichment of circulating tumor cells from the blood of patients with small-cell lung cancer.

    Masahito Hosokawa, Takayuki Yoshikawa, Ryo Negishi, Tomoko Yoshino, Yasuhiro Koh, Hirotsugu Kenmotsu, Tateaki Naito, Toshiaki Takahashi, Nobuyuki Yamamoto, Yoshihito Kikuhara, Hisashige Kanbara, Tsuyoshi Tanaka, Ken Yamaguchi, Tadashi Matsunaga

    Analytical chemistry   85 ( 12 ) 5692 - 8  2013.06  [Refereed]  [International journal]

    Authorship:Lead author

     View Summary

    In this study, we present a method for efficient enrichment of small-sized circulating tumor cells (CTCs) such as those found in the blood of small-cell lung cancer (SCLC) patients using a microcavity array (MCA) system. To enrich CTCs from whole blood, a microfabricated nickel filter with a rectangular MCA (10(4) cavities/filter) was integrated with a miniaturized device, allowing for the isolation of tumor cells based on differences in size and deformability between tumor and blood cells. The shape and porosity of the MCA were optimized to efficiently capture small tumor cells on the microcavities under low flow resistance conditions, while allowing other blood cells to effectively pass through. Under optimized conditions, approximately 80% of SCLC (NCI-H69 and NCI-H82) cells spiked in 1 mL of whole blood were successfully recovered. In clinical samples, CTCs were detectable in 16 of 16 SCLC patients. In addition, the number of leukocytes captured on the rectangular MCA was significantly lower than that on the circular MCA (p < 0.001), suggesting that the use of the rectangular MCA diminishes a considerable number of carryover leukocytes. Therefore, our system has potential as a tool for the detection of CTCs in small cell-type tumors and detailed molecular analyses of CTCs.

    DOI PubMed

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    88
    Citation
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  • Monitoring of benzene-induced hematotoxicity in mice by serial leukocyte counting using a microcavity array.

    Masahito Hosokawa, Marie Asami, Tomoko Yoshino, Noriyuki Tsujimura, Masayuki Takahashi, Satoshi Nakasono, Tsuyoshi Tanaka, Tadashi Matsunaga

    Biosensors & bioelectronics   40 ( 1 ) 110 - 4  2013.02  [Refereed]  [International journal]

    Authorship:Lead author

     View Summary

    Monitoring of hematotoxicity, which requires serial blood collection, is difficult to carry out in small animals due to a lack of non-invasive, individual animal-appropriate techniques that enable enumeration of leukocyte subsets from limited amounts of whole blood. In this study, a microfluidic device equipped with a microcavity array that enables highly efficient separation of leukocytes from submicroliters of whole blood was applied for hematotoxicity monitoring in mice. The microcavity array can specifically separate leukocytes from whole blood based on differences in the size and deformability between leukocytes and other blood cells. Mouse leukocytes recovered on aligned microcavities were continuously processed for image-based immunophenotypic analysis. Our device successfully recovered almost 100% of mouse leukocytes in 0.1 μL of whole blood without the effect of serial blood collection such as changes in body weight and total leukocyte count. We assessed benzene-associated hematotoxicity in mice using this system. Mice were administered with benzene once daily and the depression of leukocyte numbers induced in individual mice was successfully monitored from tail vein blood collected every other day for 2 weeks. Serial monitoring of the leukocyte number in individual mice will contribute to the understanding of hematotoxicity and reduction of the number of animal experiment trials.

    DOI PubMed

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    7
    Citation
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  • Size-based isolation of circulating tumor cells in lung cancer patients using a microcavity array system.

    Masahito Hosokawa, Hirotsugu Kenmotsu, Yasuhiro Koh, Tomoko Yoshino, Takayuki Yoshikawa, Tateaki Naito, Toshiaki Takahashi, Haruyasu Murakami, Yukiko Nakamura, Asuka Tsuya, Takehito Shukuya, Akira Ono, Hiroaki Akamatsu, Reiko Watanabe, Sachiyo Ono, Keita Mori, Hisashige Kanbara, Ken Yamaguchi, Tsuyoshi Tanaka, Tadashi Matsunaga, Nobuyuki Yamamoto

    PloS one   8 ( 6 ) e67466  2013  [Refereed]  [International journal]

    Authorship:Lead author

     View Summary

    BACKGROUND: Epithelial cell adhesion molecule (EpCAM)-based enumeration of circulating tumor cells (CTC) has prognostic value in patients with solid tumors, such as advanced breast, colon, and prostate cancer. However, poor sensitivity has been reported for non-small cell lung cancer (NSCLC). To address this problem, we developed a microcavity array (MCA) system integrated with a miniaturized device for CTC isolation without relying on EpCAM expression. Here, we report the results of a clinical study on CTCs of advanced lung cancer patients in which we compared the MCA system with the CellSearch system, which employs the conventional EpCAM-based method. METHODS: Paired peripheral blood samples were collected from 43 metastatic lung cancer patients to enumerate CTCs using the CellSearch system according to the manufacturer's protocol and the MCA system by immunolabeling and cytomorphological analysis. The presence of CTCs was assessed blindly and independently by both systems. RESULTS: CTCs were detected in 17 of 22 NSCLC patients using the MCA system versus 7 of 22 patients using the CellSearch system. On the other hand, CTCs were detected in 20 of 21 small cell lung cancer (SCLC) patients using the MCA system versus 12 of 21 patients using the CellSearch system. Significantly more CTCs in NSCLC patients were detected by the MCA system (median 13, range 0-291 cells/7.5 mL) than by the CellSearch system (median 0, range 0-37 cells/7.5 ml) demonstrating statistical superiority (p = 0.0015). Statistical significance was not reached in SCLC though the trend favoring the MCA system over the CellSearch system was observed (p = 0.2888). The MCA system also isolated CTC clusters from patients who had been identified as CTC negative using the CellSearch system. CONCLUSIONS: The MCA system has a potential to isolate significantly more CTCs and CTC clusters in advanced lung cancer patients compared to the CellSearch system.

    DOI PubMed

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    148
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  • Leukocyte counting from a small amount of whole blood using a size-controlled microcavity array.

    Masahito Hosokawa, Marie Asami, Seita Nakamura, Tomoko Yoshino, Noriyuki Tsujimura, Masayuki Takahashi, Satoshi Nakasono, Tsuyoshi Tanaka, Tadashi Matsunaga

    Biotechnology and bioengineering   109 ( 8 ) 2017 - 24  2012.08  [Refereed]  [International journal]

    Authorship:Lead author

     View Summary

    Absolute counting of total leukocytes or specific subsets within small amounts of whole blood is difficult due to a lack of techniques that enable separation of all leukocytes from limited amounts of whole blood. In this study, a microfluidic device equipped with a size-controlled microcavity array for highly efficient separation of leukocytes from submicroliters of whole blood was developed. The microcavity array can separate leukocytes from whole blood based on differences in the size and deformability between leukocytes and other blood cells. Leukocytes recovered on aligned microcavities were continuously processed for image-based immunophenotypic analysis. Our device successfully recovered over 90% of leukocytes in 1 µL of whole blood without pretreatment such as density gradient centrifugation or erythrocyte lysis. In addition, the proposed system successfully performed absolute enumeration of human CD4(+) and CD8(+) leukocytes from 1 µL of whole blood, and the obtained data showed good correlation with conventional flow cytometric analysis. Our microfluidic device has great potential as a tool for a point-of-care leukocyte analysis system.

    DOI PubMed

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    33
    Citation
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  • Characterization of magnetic nanoparticles modified with thiol functionalized PAMAM dendron for DNA recovery.

    Tsuyoshi Tanaka, Keiyu Shibata, Masahito Hosokawa, Keiichi Hatakeyama, Atsushi Arakaki, Hideyuki Gomyo, Takeyuki Mogi, Tomoyuki Taguchi, Hitoshi Wake, Takeo Tanaami, Tadashi Matsunaga

    Journal of colloid and interface science   377 ( 1 ) 469 - 75  2012.07  [Refereed]  [International journal]

     View Summary

    Magnetic nanoparticles (MNPs) modified with the thiol functionalized polyamidoamine (PAMAM) dendron were synthesized to estimate their DNA recovery capabilities. Aminosilane-modified MNPs and MNPs surrounded by a phospholipid (distearoylphosphatidylethanolamine (DSPE)) bilayer were used as core particles. Cystamine-core PAMAM dendrimers were reduced by dithiothreitol to dendron thiols and chemically conjugated to the core particles. Characterization of the synthesis revealed an increase of the surface amine charge from generation 1 (G1) to G6, starting with an aminosilane initiator. Particle size distribution analysis indicated that G6 PAMAM-modified MNPs exhibited monodispersity in an aqueous solution. G6 PAMAM-MNPs and G6 PAMAM-PE-MNPs synthesized by the proposed method have equivalent DNA recovery abilities to PAMAM-MNPs prepared by the conventional divergent synthesis method. In optimized conditions, 96% of λDNA was recovered using G6 PAMAM-PE-MNPs. Therefore, the method for preparing PAMAM-MNPs and PAMAM-PE-MNPs proposed in this study will be a novel approach for producing DNA carriers for efficient DNA purification by magnetic separation.

    DOI PubMed

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    26
    Citation
    (Scopus)
  • Efficient DNA release from PAMAM dendrimer-modified superparamagnetic nanoparticles for DNA recovery

    Atsushi Arakaki, Keiyu Shibata, Takeyuki Mogi, Masahito Hosokawa, Keiichi Hatakeyama, Hideyuki Gomyo, Tomoyuki Taguchi, Hitoshi Wake, Takeo Tanaami, Tadashi Matsunaga, Tsuyoshi Tanaka

    POLYMER JOURNAL   44 ( 6 ) 672 - 677  2012.06  [Refereed]

     View Summary

    DNA recovery using solid-phase extraction is a fundamental technique in molecular biology and biotechnology. Our research group developed a novel DNA recovery method using amine-modified magnetic nanoparticles (MNPs) as a solid support. The use of MNPs simplifies the DNA recovery processes and permits their use in automated systems. In this study, we prepared polyamidoamine-modified superparamagnetic particles (PAMAM-SpMNPs) with 10-nm magnetite cores and used them for DNA recovery. To improve the DNA-release efficiency, the surface amine numbers on the particles were evaluated to modify each generation of PAMAM. With this optimization, the PAMAM-SpMNPs maintained a high DNA adsorption capacity and high dispersivity in solution. As a result, the DNA release from the PAMAM-SpMNPs of every generation was highly efficient. In particular, the release of DNA from the G4 to G6 PAMAM-SpMNPs was greater than 95%. Furthermore, an alternating magnetic field (AMF) was applied to expedite the DNA release from the PAMAM-SpMNPs. Complete DNA release was achieved using AMF treatment for 10 min. The DNA recovery method using the PAMAM-SpMNPs will permit various types of testing using DNA from a low volume sample, such as in a micro total analytical system. Polymer Journal (2012) 44, 672-677; doi:10.1038/pj.2012.32; published online 4 April 2012

    DOI

    Scopus

    18
    Citation
    (Scopus)
  • Assessment of benzene-induced hematotoxicity using a human-like hematopoietic lineage in NOD/Shi-scid/IL-2Rγnull mice.

    Masayuki Takahashi, Noriyuki Tsujimura, Tomoko Yoshino, Masahito Hosokawa, Kensuke Otsuka, Tadashi Matsunaga, Satoshi Nakasono

    PloS one   7 ( 12 ) e50448  2012  [Refereed]  [International journal]

     View Summary

    Despite recent advancements, it is still difficult to evaluate in vivo responses to toxicants in humans. Development of a system that can mimic the in vivo responses of human cells will enable more accurate health risk assessments. A surrogate human hematopoietic lineage can be established in NOD/Shi-scid/IL-2Rγ(null) (NOG) mice by transplanting human hematopoietic stem/progenitor cells (Hu-NOG mice). Here, we first evaluated the toxic response of human-like hematopoietic lineage in NOG mice to a representative toxic agent, benzene. Flow cytometric analysis showed that benzene caused a significant decrease in the number of human hematopoietic stem/progenitor cells in the bone marrow and the number of human leukocytes in the peripheral blood and hematopoietic organs. Next, we established chimeric mice by transplanting C57BL/6 mouse-derived bone marrow cells into NOG mice (Mo-NOG mice). A comparison of the degree of benzene-induced hematotoxicity in donor-derived hematopoietic lineage cells within Mo-NOG mice indicated that the toxic response of Hu-NOG mice reflected interspecies differences in susceptibilities to benzene. Responses to the toxic effects of benzene were greater in lymphoid cells than in myeloid cells in Mo-NOG and Hu-NOG mice. These findings suggested that Hu-NOG mice may be a powerful in vivo tool for assessing hematotoxicity in humans, while accounting for interspecies differences.

    DOI PubMed

    Scopus

    6
    Citation
    (Scopus)
  • Microfluidic device with chemical gradient for single-cell cytotoxicity assays.

    Masahito Hosokawa, Takuma Hayashi, Tetsushi Mori, Tomoko Yoshino, Satoshi Nakasono, Tadashi Matsunaga

    Analytical chemistry   83 ( 10 ) 3648 - 54  2011.05  [Refereed]  [International journal]

    Authorship:Lead author

     View Summary

    Here, we report the fabrication of a chemical gradient microfluidic device for single-cell cytotoxicity assays. This device consists of a microfluidic chemical gradient generator and a microcavity array that enables entrapment of cells with high efficiency at 88 ± 6% of the loaded cells. A 2-fold logarithmic chemical gradient generator that is capable of generating a serial 2-fold gradient was designed and then integrated with the microcavity array. High density single-cell entrapment was demonstrated in the device without cell damage, which was performed in 30 s. Finally, we validated the feasibility of this device to perform cytotoxicity assays by exposing cells to potassium cyanide (0-100 μM KCN). The device captured images of 4000 single cells affected by 6 concentrations of KCN and determined cell viability by counting the effected cells. Image scanning of the microcavity array was completed within 10 min using a 10× objective lens and a motorized stage. Aligning cells on the microcavity array eases cell counting, observation, imaging, and evaluation of singular cells. Thus, this platform was able to determine the cytotoxicity of chemicals at a single-cell level, as well as trace the cytotoxicity over time. This device and method will be useful for cytotoxicity analysis and basic biomedical research.

    DOI PubMed

    Scopus

    45
    Citation
    (Scopus)
  • Real-time detection of DNA hybridization on microarray using a CCD-based imaging system equipped with a rotated microlens array disk.

    Takeyuki Mogi, Keiichi Hatakeyama, Tomoyuki Taguchi, Hitoshi Wake, Takeo Tanaami, Masahito Hosokawa, Tsuyoshi Tanaka, Tadashi Matsunaga

    Biosensors & bioelectronics   26 ( 5 ) 1942 - 6  2011.01  [Refereed]  [International journal]

     View Summary

    This work describes a novel charge-coupled device (CCD)-based imaging system (MB Biochip Reader™) for real-time detection of DNA hybridization to DNA microarrays. The MB Biochip Reader™ consisted of a laser light source (532 nm), a microlens array for generation of a multi-beam laser, and a CCD for 2-D signal imaging. The MB Biochip Reader™ with a rotated microlens array, allowed large-field imaging (6.2 mm × 7.6 mm with 6.45 μm resolution) with fast time-resolution at 0.2 s without speckle noise. Furthermore, real-time detection of DNA hybridization, which is sufficient to obtain accurate data from tens of thousands of array element per field, was successfully performed without the need for laser scanning. The performance of the MB Biochip Reader™ for DNA microarray imaging was similar to the commercially available photomultiplier tube (PMT)-based microarray scanner, ScanArray Lite. The system potentially could be applied toward real-time analysis in many other fluorescent techniques in addition to real-time DNA microarray analysis.

    DOI PubMed

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    22
    Citation
    (Scopus)
  • Size-selective microcavity array for rapid and efficient detection of circulating tumor cells.

    Masahito Hosokawa, Taishi Hayata, Yorikane Fukuda, Atsushi Arakaki, Tomoko Yoshino, Tsuyoshi Tanaka, Tadashi Matsunaga

    Analytical chemistry   82 ( 15 ) 6629 - 35  2010.08  [Refereed]  [International journal]

    Authorship:Lead author

     View Summary

    Circulating tumor cells (CTCs) are tumor cells circulating in the peripheral blood of patients with metastatic cancer. Detection of CTCs has clinical significance in cancer therapy because it would enable earlier diagnosis of metastasis. In this research, a microfluidic device equipped with a size-selective microcavity array for highly efficient and rapid detection of tumor cells from whole blood was developed. The microcavity array can specifically separate tumor cells from whole blood on the basis of differences in the size and deformability between tumor and hematologic cells. Furthermore, the cells recovered on the microcavity array were continuously processed for image-based immunophenotypic analysis using a fluorescence microscope. Our device successfully detected approximately 97% of lung carcinoma NCI-H358 cells in 1 mL whole blood spiked with 10-100 NCI-H358 cells. In addition, breast, gastric, and colon tumor cells lines that include EpCAM-negative tumor cells, which cannot be isolated by conventional immunomagnetic separation, were successfully recovered on the microcavity array with high efficiency (more than 80%). On an average, approximately 98% of recovered cells were viable. Our microfluidic device has high potential as a tool for the rapid detection of CTCs and can be used to study CTCs in detail.

    DOI PubMed

    Scopus

    300
    Citation
    (Scopus)
  • TCR-beta repertoire analysis of antigen-specific single T cells using a high-density microcavity array.

    Atsushi Arakaki, Kaori Ooya, Yasuto Akiyama, Masahito Hosokawa, Masaru Komiyama, Akira Iizuka, Ken Yamaguchi, Tadashi Matsunaga

    Biotechnology and bioengineering   106 ( 2 ) 311 - 8  2010.06  [Refereed]  [International journal]

     View Summary

    The antigen specificity of cytotoxic T cells, provided by T-cell receptors (TCRs), plays a central role in human autoimmune diseases, infection, and cancer. As the TCR repertoire is unique in individual cytotoxic T cells, a strategy to analyze its gene rearrangement at the single-cell level is required. In this study, we applied a high-density microcavity array enabling target cell screening of several thousands of single cells for identification of functional TCR-beta gene repertoires specific to melanoma (gp100) and cytomegalovirus (CMV) antigens. T cells expressing TCRs with the ability to recognize fluorescent-labeled antigen peptide tetramers were isolated by using a micromanipulator under microscopy. Regularly arranged cells on the microcavity array eased detection and isolation of target single cells from a polyclonal T-cell population. The isolated single cells were then directly utilized for RT-PCR. By sequencing the amplified PCR products, antigen-specific TCR-beta repertoires for gp100 and human cytomegalovirus antigens were successfully identified at the single-cell level. This simple, accurate, and cost-effective technique for single-cell analysis has further potential as a valuable and widely applicable tool for studies on gene screening and expression analyses of various kinds of cells.

    DOI PubMed

    Scopus

    13
    Citation
    (Scopus)
  • Preparation of genomic DNA from a single species of uncultured magnetotactic bacterium by multiple-displacement amplification.

    Atsushi Arakaki, Mie Shibusawa, Masahito Hosokawa, Tadashi Matsunaga

    Applied and environmental microbiology   76 ( 5 ) 1480 - 5  2010.03  [Refereed]  [International journal]

     View Summary

    Magnetotactic bacteria comprise a phylogenetically diverse group that is capable of synthesizing intracellular magnetic particles. Although various morphotypes of magnetotactic bacteria have been observed in the environment, bacterial strains available in pure culture are currently limited to a few genera due to difficulties in their enrichment and cultivation. In order to obtain genetic information from uncultured magnetotactic bacteria, a genome preparation method that involves magnetic separation of cells, flow cytometry, and multiple displacement amplification (MDA) using phi29 polymerase was used in this study. The conditions for the MDA reaction using samples containing 1 to 100 cells were evaluated using a pure-culture magnetotactic bacterium, "Magnetospirillum magneticum AMB-1," whose complete genome sequence is available. Uniform gene amplification was confirmed by quantitative PCR (Q-PCR) when 100 cells were used as a template. This method was then applied for genome preparation of uncultured magnetotactic bacteria from complex bacterial communities in an aquatic environment. A sample containing 100 cells of the uncultured magnetotactic coccus was prepared by magnetic cell separation and flow cytometry and used as an MDA template. 16S rRNA sequence analysis of the MDA product from these 100 cells revealed that the amplified genomic DNA was from a single species of magnetotactic bacterium that was phylogenetically affiliated with magnetotactic cocci in the Alphaproteobacteria. The combined use of magnetic separation, flow cytometry, and MDA provides a new strategy to access individual genetic information from magnetotactic bacteria in environmental samples.

    DOI PubMed

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    24
    Citation
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  • A single-cell based biosensing device directed for lipophilic chemical screening and evaluation

    Mori Tetsushi, Hayashi Takuma, Hosokawa Masahito, Yoshino Tomoko, Nakasono Satoshi, Takeyama Haruko, Matsunaga Tadashi

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   108   S150 - S151  2009.11  [Refereed]

    DOI

  • High-density microcavity array for cell detection: single-cell analysis of hematopoietic stem cells in peripheral blood mononuclear cells.

    Masahito Hosokawa, Atsushi Arakaki, Masayuki Takahashi, Tetsushi Mori, Haruko Takeyama, Tadashi Matsunaga

    Analytical chemistry   81 ( 13 ) 5308 - 13  2009.07  [Refereed]  [International journal]

    Authorship:Lead author

     View Summary

    Detection and isolation of specific cell types from limited biological samples have become a major challenge in clinical diagnosis and cell biology research. Here, we report a high-density microcavity array for target cell detection in which thousands of single cells were neatly arrayed onto 10,000 microcavities with high efficiency at approximately 90% of the loaded cells. Cell-specific immunophenotypes were exclusively identified at the single-cell level by measuring fluorescence intensities of cells labeled with antibodies targeting cell surface markers, and the purity of hematopoietic stem cells (HSCs) within human peripheral blood analyzed by this system was correlated with those obtained by conventional flow cytometry. Furthermore, gene expression of the stem cell marker, CD34, was determined from HSCs by isolating single cells using a micromanipulator. This technology has proven to be an effective tool for target cell detection and subsequent cellular analytical research at the single-cell level.

    DOI PubMed

    Scopus

    67
    Citation
    (Scopus)
  • High-efficiency single-cell entrapment and fluorescence in situ hybridization analysis using a poly(dimethylsiloxane) microfluidic device integrated with a black poly(ethylene terephthalate) micromesh.

    Tadashi Matsunaga, Masahito Hosokawa, Atsushi Arakaki, Tomoyuki Taguchi, Tetsushi Mori, Tsuyoshi Tanaka, Haruko Takeyama

    Analytical chemistry   80 ( 13 ) 5139 - 45  2008.07  [Refereed]  [International journal]

     View Summary

    Here, we report a high-efficiency single-cell entrapment system with a poly(dimethylsiloxane) (PDMS) microfluidic device integrated with a micromesh, and its application to single-cell fluorescence in situ hybridization (FISH) analysis. A micromesh comprising of 10 x 10 microcavities was fabricated on a black poly(ethylene terephthalate) (PET) substrate by laser ablation. The cavity was approximately 2 microm in diameter. Mammalian cells were driven and trapped onto the microcavities by applying negative pressure. Trapped cells were uniformly arrayed on the micromesh, enabling high-throughput microscopic analysis. Furthermore, we developed a method of PDMS surface modification by using air plasma and the copolymer Pluronic F-127 to prevent nonspecific adsorption on the PDMS microchannel. This method decreased the nonspecific adsorption of cells onto the microchannel to less than 1%. When cells were introduced into the microfluidic device integrated with the black PET micromesh, approximately 70-80% of the introduced cells were successfully trapped. Moreover, for mRNA expression analysis, on-chip fluorescence in situ hybridization (e.g., membrane permeabilization, hybridization, washing) can be performed in a microfluidic assay on an integrated device. This microfluidic device has been employed for the detection of beta-actin mRNA expression in individual Raji cells. Differences in the levels of beta-actin mRNA expression were observed in serum-supplied or serum-starved cell populations.

    DOI PubMed

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    51
    Citation
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▼display all

Books and Other Publications

  • 未培養微生物研究の最新動向

    青柳, 秀紀( Part: Contributor, 第 10 章 未培養微生物を活用するための シングルセルゲノム解析)

    シーエムシー出版  2023.04 ISBN: 9784781317328

  • シングルセル解析でなにがわかるか

    竹山, 春子, 細川, 正人

    化学同人  2020.07 ISBN: 9784759817348

Presentations

  • 1細胞/微小組織マルチオミックスのオールインワン解析による生命科学研究支援

    細川正人  [Invited]

    臨床薬理学会 

    Presentation date: 2023.12

  • 次世代バイオものづくりのための未培養微生物ゲノムデータベース

    細川正人  [Invited]

    第61回日本生物物理学会年会 2SKP 超越分子シンポジウム: 基礎研究を超越し社会実装へつなげる 

    Presentation date: 2023.11

  • 土壌微生物を知り理解するためのゲノム解析技術

    細川正人  [Invited]

    バイオインダストリー協会 植物バイオ研究会・Food Bio Plus研究会 公開講演会 

    Presentation date: 2023.10

  • 未培養微生物の遺伝子獲得に向けた シングルセルゲノミクスの活用

    細川正人  [Invited]

    第75回日本生物工学会大会 シンポジウム 合成生物学が切り拓く次世代型天然物創薬 

    Presentation date: 2023.09

  • 微生物ビッグデータ・AI・ロボティクスを統合した 次世代のバイオものづくり

    細川正人

    エタンセル オンラインフォーラム 

    Presentation date: 2023.08

  • Unlock the full potential of microbes

    Masahito Hosokawa  [Invited]

    Presentation date: 2023.07

  • 微生物遺伝子データで実現するバイオものづくり

    細川正人  [Invited]

    JBA バイオリーダーズ研修 2023 

    Presentation date: 2023.07

  • Microbial gene big data for biomanufacturing

    Masahito Hosokawa  [Invited]

    Presentation date: 2023.06

  • ヒト共生・未培養微生物におけるシングルセルゲノム解析の活用法

    細川 正人  [Invited]

    第27回腸内細菌学会学術集会 シンポジウム1 

    Presentation date: 2023.06

  • バクテリアのシングルセルRNA-seqで何ができるか

    細川 正人  [Invited]

    大隅基礎科学創成財団 微生物コンソーシアム定例会 

    Presentation date: 2023.06

  • はじめてのbit-MAP®: 微生物シングルセルゲノム解析の活用ガイド

    細川 正人  [Invited]

    QIAGENウェビナー 

    Presentation date: 2023.05

  • 微生物シングルセルゲノミクス:進歩と将来の展望

    細川正人  [Invited]

    第96回日本細菌学会総会 

    Presentation date: 2023.03

  • 微生物の大規模ゲノムデータを生かした バイオものづくりへの展開

    細川 正人  [Invited]

    ABMESモーニングセミナー 

    Presentation date: 2023.02

  • 高解像度ゲノム解析による腸内細菌叢機能の理解と活用

    細川 正人  [Invited]

    バイオ共創コンソーシアム 第3回会議 

    Presentation date: 2022.11

  • Obtaining complete genomes from uncultured human gut bacteria with single-cell long-read sequencing

    Masahito Hosokawa  [Invited]

    International Human Microbiome Consortium 9th Congress 2022 

    Presentation date: 2022.11

  • 腸内細菌のシングルセル解析と微生物遺伝子の活用

    細川正人  [Invited]

    第22回 日本抗加齢医学会総会 シンポジウム11「腸内細菌x新テクノロジー」 

    Presentation date: 2022.06

  • 環境細菌の大規模シングルセルゲノミクスから その先へ

    細川正人  [Invited]

    Visionary 農芸化学100 シンポジウム 第48回 農芸化学「化学と生物」シンポジウム 微生物・バイオマス利用研究領域 第3回シンポジウム 微生物の共生・生態 ~世界は微生物で溢れている~ 

    Presentation date: 2022.05

  • Single-cell genomics for uncultured animal gut microbes

    Masahito Hosokawa  [Invited]

    Presentation date: 2022.04

  • 大規模ゲノムデータによる未培養微生物資源の利用への道

    細川正人  [Invited]

    バイオインダストリー奨励賞受賞者企画講演会「レッドバイオの新たな息吹 ~次世代創薬はじめ産業基盤の革新的変化をもたらすバイオ技術研究の最前線~」 

    Presentation date: 2022.04

  • 微生物シングルセル解析技術による 植物・土壌微生物の理解と利用への展望

    細川正人  [Invited]

    第二回植物微生物シンバイオロジー協議会シンポジウム 

    Presentation date: 2022.01

  • 微生物シングルセル解析技術 bit-MAP®と 大規模ゲノムデータが拓く革新的バイオ生産への道

    細川正人  [Invited]

    KISTEC先端科学技術セミナー2021 AIと融合するバイオテクノロジー|越境と共創がもたらす革新的シングルセル解析 

    Presentation date: 2021.12

  • 微生物のシングルセルゲノム解析を実現するツールたち

    細川正人  [Invited]

    QIAGEN ユーザーウェビナー 

    Presentation date: 2021.11

  • 未培養腸内細菌の1細胞ロングリードシーケンスによる完全長ゲノムの獲得

    細川正人, 小川雅人, 西川洋平, 佐伯達也, 依田卓也, 有川浩司, 竹山春子

    第73回 日本生物工学会大会 

    Presentation date: 2021.10

  • Development of technologies for single-cell genome sequencing of uncultured microbes

    Masahito Hosokawa  [Invited]

    The 11th International Conference on Post-Genomic Technologies 

    Presentation date: 2021.10

  • シングルセルゲノミクスで未培養細菌ゲノムを網羅解析

    細川正人  [Invited]

    蛋白研セミナー 

    Presentation date: 2021.01

▼display all

Research Projects

  • Elucidation of mucosal barrier regulation by intestinal microbiota

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research

    Project Year :

    2023.04
    -
    2028.03
     

  • Exploration of the limits and adaptations of life in subseafloor biosphere

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research

    Project Year :

    2023.04
    -
    2027.03
     

  • 肺炎球菌の細胞壁分解能が病態形成と薬剤耐性獲得に果たす役割の解明

    日本学術振興会  科学研究費助成事業

    Project Year :

    2023.04
    -
    2027.03
     

    山口 雅也, 川端 重忠, 住友 倫子, 細川 正人, 山下 隼人

  • 1細胞解像度で紐解く不均質な細菌群集からの亜集団発生

    日本学術振興会  科学研究費助成事業

    Project Year :

    2023.06
    -
    2026.03
     

    細川 正人

  • 腸内細菌による粘膜バリア制御機構の解明

    日本学術振興会  科学研究費助成事業

    Project Year :

    2023.04
    -
    2026.03
     

    長谷 耕二, 新藏 礼子, 細川 正人

  • シン・パレオゲノミクスが創る博物館資料群活用の新展開

    日本学術振興会  科学研究費助成事業

    Project Year :

    2021.04
    -
    2024.03
     

    覚張 隆史, 松前 ひろみ, 金原 正明, 本橋 慶一, 岡崎 健治, 中込 滋樹, 石谷 孔司, 細川 正人, 和久 大介

  • シングルセルゲノムデータに基づく未培養微生物の戦略的資源化プロセスの開発

    日本学術振興会  科学研究費助成事業

    Project Year :

    2021.04
    -
    2024.03
     

    細川 正人, 西川 洋平

     View Summary

    本研究では、1細胞ゲノミクスとマイクロ流体技術を活用して、環境試料に存在する微生物の素性を明らかにし、最適な培養法を設計する「ゲノムデータに基づく戦略的微生物培養プロセス」を開発する。「単離培養を繰り返し、新規・有用微生物のヒットを期待する」という古典的・労働集約的な方法に対して、我々が提唱する新法では、「新規・有用種の存在・特性を事前に検知し、培養条件を事前に最適化して対象物を釣り上げる」という合理的なアプローチを微生物培養に採用することを目的としている。対象試料中の微生物種の推定と培養法の最適化には、未培養微生物の全ゲノム情報の網羅的取得が求められる。そこで、代表者が開発した網羅的なシングルセルゲノム解析技術(SAG-gel)を基盤技術として用いる(Chijiiwa et al. Microbiome 2020)。
    初年度は、シングルセルゲノム解析技術(SAG-gel)を改良して、一度に千個を超える微生物シングルセルゲノムデータを取得する手法を確立し、さらに生菌特異的なシーケンス技術(PMA-SAG-gel, Hosokawa et al. Sci. Rep. 2022)を開発し、目標を達成した。第2年度は、大規模シングルセルシーケンス技術の論文報告に向けて、手法の最適化とデータ拡充を進める。このほかに、シングルセルトランスクリプトーム解析技術へも展開する予定である。また、ここまでに確立したデータ取得技術を用いて、特定の微生物から単離・培養条件を推定し、微生物集団からの単離培養を試みる。これには標準的なプレート培養のほか、マイクロ流体デバイスを用いた培養も検討する。対象はおもに土壌細菌やヒト常在菌を予定している。

  • 大規模1細胞ゲノムから設計する微生物叢の戦略的制御

    科学技術振興機構  創発的研究支援事業

    Project Year :

    2022
    -
    2024
     

  • Elucidation of the molecular mechanism of immunotherapy treatment resistance by microtissue multidisciplinary sequence

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research

    Project Year :

    2019.04
    -
    2022.03
     

    Takuo Hayashi

     View Summary

    In the present study, 550 differential expression genes (DEGs) were identified in the cancerous areas with therapeutic and non-therapeutic effects. Among them, GO terms such as ’extracellular matrix organization’ and ’extracellular structure organization' was predominantly concentrated in the cancerous area where the therapeutic effect was observed. In KEGG pathway analysis, 'focal adhesion' and 'PIK3-AKT signaling pathway' were predominantly enriched. In addition, it was shown that MT-ATP8 expression was low and MZB1 expression was high in patients in complete remission with immunotherapy.

  • Development of platform for ultra high-throughput screening of novel bioactive compound producers

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research

    Project Year :

    2017.05
    -
    2022.03
     

    takeyama haruko

     View Summary

    High-throughput screening of biologically active substance-producing bacteria and their metabolic gene clusters is expected to lead to the acquisition and production of new lead compounds for drug discovery. In this project, we have developed a screening method for bioactive substance-producing strains using raman micro-spectroscopy at the single-cell level and a high-throughput single-cell genome analysis technique with droplet microfluidics. We have established a new platform for the efficient screening for microbes producing novel bioactive substances.

  • Obtaining of complete genomes of uncultured microbes by sequencing of single-cell genomes without whole genome amplification

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research

    Project Year :

    2019.06
    -
    2021.03
     

    Hosokawa Masahito

     View Summary

    More than 99% of the microbes in the environment cannot be cultured in the laboratory. To understand these uncultured microbes, direct analysis of their genomes is practical. In particular, "single-cell genomics," which decodes the genome sequence from a single cell, effectively obtains individual genome information from diverse microbial populations, but the accuracy of genome decoding has been an issue with conventional methods. In this study, we developed a technology to decode the genome from a single cell and reconstruct the sequence information into a complete genome, which was challenging to do with conventional methods and demonstrated its effectiveness in human intestinal bacteria.

  • Integrated imaging and sequencing analysis to unravel the genome and transcriptional activation at the single-cell level

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research

    Project Year :

    2018.04
    -
    2021.03
     

    Hosokawa Masahito

     View Summary

    As the technology to analyze gene expression at the single-cell levels becomes widespread, and data accumulation advances dramatically, what is needed as the next-generation analysis method is the technology to analyze multi-level information in an integrated manner to understand the cause and effect of cellular diversity. In this study, we developed a technology to analyze genomic mutations at the single-cell level and technology to extract micro-regions from tissue and analyze site-specific gene expression and genome sequence integrated with tissue regional information in the tissue.

  • 組織内の細胞多様性を明らかにする超並列ゲノム解析技術の創成

    科学技術振興機構  戦略的創造研究推進事業さきがけ 統合1細胞解析のための革新的技術基盤

    Project Year :

    2015.10
    -
    2019.03
     

    細川 正人

  • Development of single-cell whole genome amplification method for uncultivable bacteria using droplet microfluidics

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research

    Project Year :

    2014.04
    -
    2016.03
     

    Masahito Hosokawa

     View Summary

    In this study, we aimed to develop a microfluidic droplet-based whole genome amplification method for single bacterial cells. We developed a microfluidic device for generation of picoliter-sized droplet for compartmentalized reaction environment. Single bacterial cells or their genome DNA fragments were encapsulated into each droplet. By compartmenting genome fragments into individual droplets, DNA fragments are amplified uniformly, that minimizes amplification bias and the effect of contaminating DNA. Our results demonstrated the potential of microfluidic generated droplets as an efficient tool for the analysis of single cell genome along with great reduction in the cost and labor investment required for the investigation of genome diversity of uncultivable bacteria.

  • Advanced Micro Fluidic Engineering and Its Applications for High Sensitive Quantitative Measurements of Biomolecules

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research

    Project Year :

    2011.04
    -
    2016.03
     

    SHOJI Shuichi, TAKEYAMA Haruko, MIZUNO Jun, SEKIGUCHI Tetsushi, HOSOKAWA Masato, YOON Dong Hyun, SUZUKI Miho, FUKUDA Takeshi, FUNATSU Takashi, TAKEDA Naoya, MORI Tetsushi, EDAGAWA Yoshikuni

     View Summary

    In this study, the noble microfluidic device technology was developed in order to enable the optical high sensitive quantitative measurement of the micro luminescent sample as follows. 1) The micro droplet manufacturing technology of the various size. 2) The control technology of the micro flow. 3) The passive sorting technology of the micro droplet. Next, the measurement of the micro luminescent samples by systematizing microfluidic device was realized. 1) Insert a living cell to a droplet one by one, and analyze the gene of the environmental microbe. 2) Observation cellular growth and evaluate enzymatic reaction activity. Optical quantitative measurement was realized by this study.

  • Development of technique for digital counting of DNA copy number at the single-cell level

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research

    Project Year :

    2012.04
    -
    2015.03
     

    TAKEYAMA Haruko, YOKOTA Yasuko, HOSOKAWA Masahito

     View Summary

    In this study, we aimed to develop a technique for digital counting of low copy viral DNA within infected cells. We developed a microfluidic device for generation of microdroplets for droplet-based digital PCR. In this method, the target genes were separately amplified within picoliter droplets and detected with fluorescent microscopy. The number of fluorescent droplets correlated with the copy numbers of target genes. In addition, we constructed lentivirus vector modeled after HIV. After optimization of the reaction conditions of droplet digital PCR, we demonstrated its highly sensitive detection of proviral DNA within a small number of infected cells during early infection process.

  • セルクロマトグラフィーによる循環腫瘍細胞の量的・質的評価法の開発

    日本学術振興会  科学研究費助成事業

    Project Year :

    2011
    -
    2013
     

    細川 正人

     View Summary

    本研究では、細胞の機械的強度や流動性などの物理的特性に基づいて、がん患者血液から循環腫瘍細胞(CTC)を分離・検出するセルクロマトグラフィー技術を開発することを目的とした。また、回収したCTCの二次解析から、CTCの量的評価のみならず質的評価についても検討することを目指した。
    第2年度までに、セルクロマトグラフィーデバイスの開発とCTCの高感度検出のための基礎検討を行い、静岡がんセンターにて臨床検体を対象とした試験を行った。この結果、本デバイスは従来の抗原抗体反応に基づくCTC回収法よりも優れた回収性能を有している事が実証された。本成果を元に、本年度は回収した細胞の遺伝子変異解析による細胞の質的評価を検討した。
    具体的には、単一細胞からの全ゲノム増幅法を用いた特定遺伝子の変異検出のためのプロトコルを検討した。肺癌細胞株をモデルとしてEGFR遺伝子などの変異の検出系を立ち上げた。はじめに、増幅バイアスを低減させた全ゲノム増幅反応条件を検討した。PCRや次世代シーケンサーを用いた解析により、上記遺伝子の変異の検出可否を指標として評価を進め、モデル癌細胞から単一細胞レベルで塩基欠失や増幅等の変異を検出可能であることを確認した。また、CTCの純度は目的の遺伝子の変異検出の精度に関わるため、CTCの回収純度を向上させるアプローチについても平行して改良を進めた。前年度までの検討に加えて、再度フィルター構造の検討を進めたことと、回収プロセスを自動化した装置を開発したことにより、CTC回収の再現性が向上した。以上、3年間の研究実施により、臨床試験を始めCTC回収に関わる諸検討を実施し、得られた結果を元に技術向上のためのフィードバックをかけて本手法の精度向上を実現することができた。また、単一細胞からの遺伝子変異検出にかかる基本プロトコルの構築をすすめ、CTCの量的・質的な判定につなげる基礎技術を確立することができた。

  • 多孔質担体集積基板を利用した単一細胞の網羅的な遺伝子発現解析法の開発

    日本学術振興会  科学研究費助成事業

    Project Year :

    2009
    -
    2010
     

    細川 正人

     View Summary

    本研究では、動物細胞を対象とした非侵襲的なイメージング解析から侵襲的な遺伝子発現解析までを同時・多平行に行える網羅的細胞解析技術の確立を目指し、多孔質担体集積基板を利用した細胞の高密度集積技術、mRNA回収技術、及び遺伝子発現解析技術を開発・統合することを目的とした。昨年度までの研究から、基板上に網羅的に集積した細胞集団の中から、キャピラリーを用いて一細胞を回収し遺伝子発現を解析する手法を開発している。本研究期間ではこの手法を応用してイヌ血液等の臨床サンプルを用いた研究活動を実施し、血中腫瘍細胞の同定法の開発を試みた。この結果、イヌ腫瘍同定用の候補遺伝子を選別することができた。本手法とは別のアプローチとして、多孔質担体モノリスシリカを用いた細胞からのmRNA精製技術の開発も並行して進めた。これまでに、このモノリスシリカを集積化した基板を作製し、細胞からmRNAを抽出できることを実証している。本研究期間では、基板上に捉えた細胞のイメージング解析を行った後に、基板上で細胞を溶解し、モノリスシリカを介してmRNAを回収して遺伝子発現解析を行う技術までを確立した。また、本研究課題を進める中で、厳密に制御された微細貫通孔を介して細胞をフィルトレーションすることにより、細胞の大きさや変形能の違いに基づいて細胞集団を分離出来ることを発見した。このサイズ選択的な細胞回収法を応用して、全血から白血球や腫瘍細胞を特異的に分離回収できる可能性が示唆されている。これらの技術を組み合わせることにより、様々な細胞を含む血液などのサンプルから特定の細胞を選別し、イメージング解析により細胞表面の表現型を解析した後に、遺伝子発現解析によって個々の細胞の表現型をより詳細に評価する技術を確立することができると考えられる。

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Misc

  • バイオものづくり 成功のためのコンパス

    細川 正人

    生物工学会誌   101 ( 7 ) 378 - 379  2023.07

    DOI

  • 未培養微生物遺伝子の大規模データを活用した酵素探索

    細川正人, 細川正人, 津田宗一郎

    酵素工学ニュ-ス   ( 90 )  2023

    J-GLOBAL

  • 未培養微生物群集からの網羅的1細胞ゲノム解析法の開発

    細川 正人

    バイオサイエンスとインダストリー   80 ( 4 ) 354 - 355  2022.07

  • 【臨床実装が進む 次世代がんバイオマーカー 新規の検出技術、AIが加速するリキッドバイオプシーとその先の診断モダリティ】(第2章)新たながん診断モダリティと検出技術 マイクロバイオームを利用したがん診断技術の開発動向

    佐伯 達也, 笹倉 由貴江, 細川 正人

    実験医学   40 ( 10 ) 1529 - 1536  2022.06

     View Summary

    がん患者のヒト腸内細菌叢等を対象としたメタゲノム解析から、がんの病態や免疫チェックポイント阻害薬(ICI)の治療効果に相関するマイクロバイオームが捉えられ、細菌を対象としたバイオマーカー候補が見出されつつある。また、特定のがんにおいては、がん発症や進行に関与する細菌種に関する知見も集積されている。今後、シングルセルゲノム解析などの新たな計測技術や腸内細菌叢の制御技術がマイクロバイオーム研究に活用され、がんに関与する細菌株・遺伝子が絞り込まれることで、新規バイオマーカーとしての確立が期待される。(著者抄録)

  • 【若手研究者が拓くこれからの生物工学(後編)】細菌叢のシングルセル解析

    細川 正人

    生物工学会誌   100 ( 6 ) 298 - 301  2022.06

  • 経済産業大臣賞 膨大な微生物ゲノムデータベースを活用してバイオものづくり産業を革新する

    細川 正人

    産学官連携ジャーナル   18 ( 10 ) 9 - 10  2022

     View Summary

    ※本記事に抄録はありません。

    DOI

  • Interview 2

    細川 正人

    生物工学会誌   99 ( 8 ) 444 - 445  2021.08

    DOI CiNii

  • 【1細胞解析-技術と応用】技術 1細胞ゲノム解析で多様な細菌叢を捉える

    細川 正人, 小川 雅人

    医学のあゆみ   276 ( 10 ) 947 - 954  2021.03

     View Summary

    次世代シーケンサー(NGS)の活躍によって大量のDNA配列情報を低コストに取得可能となり、さまざまな細菌ゲノムが解読され、新しい発見が相次いでいる。その活躍のなかでも、未培養細菌を直接解析するメタゲノム解析が微生物研究に与えたインパクトは大きく、マイクロバイオーム研究に欠かせない技術となっている。しかし、メタゲノム解析は多種多様な細菌が混在する試料をまとめて解析する"バルク解析"であり、そこから得られるのは全体で平均化された情報である。ヒト細胞の不均質性理解のために"1細胞解析"が用いられるのが今では当たり前となったように、微生物研究においても"1細胞解析"が必要とされ、個々の細胞の個性から細菌株レベルでの差異を見出し、細菌叢全体を議論するという方向性へ研究トレンドが移行するであろう。このパラダイムシフトには、網羅的な1細胞ゲノム解析技術の登場が必要であり、長らく技術革新が待たれていたが、今それが実現しようとしている。(著者抄録)

  • シングルセルRNA-seq解析から明らかになった分裂酵母の目覚め機構

    露崎 隼, 細川 正人, 竹山 春子, 佐藤 政充

    バイオサイエンスとインダストリー   78 ( 6 ) 507 - 509  2020.11

  • 環境細菌のシングルセルゲノム解析 微小液滴を用いたゲノム解析手法とその応用例

    西川 洋平, 細川 正人, 小川 雅人, 竹山 春子

    日本乳酸菌学会誌   31 ( 1 ) 17 - 24  2020.03

     View Summary

    環境中に生息する多様な難培養性細菌の機能解明に向けて、培養に依存しない手法として、ゲノム情報に基づいた解析が注目を集めている。次世代シークエンサーの発達によって多量の配列情報を一度に取得することが可能になったことにより、多様な環境を対象としたゲノム解析が現在進行形で進められている。また、蓄積されたゲノム情報は様々な解析に利用され、日々新たな知見が報告されている。本総説においては近年の筆者らの研究成果を交え、メタゲノムおよびシングルセルゲノムを用いた環境細菌のゲノム解析における最新の研究トピックを概説する。特に、微小液滴を用いたシングルセルゲノム解析技術について、その応用例と有用性を紹介したい。(著者抄録)

  • 【シングルセルゲノミクス 組織の機能、病態が1細胞レベルで見えてきた!】(第3章)技術開発 マイクロバイオームのシングルセル解析

    細川 正人, 小川 雅人, 竹山 春子

    実験医学   37 ( 20 ) 3521 - 3526  2019.12

     View Summary

    マイクロバイオームのメタゲノム解析では、種組成や全遺伝子組成などの微生物集団の全体像を捉えることができる。シングルセルゲノム解析は、微生物の個別ゲノムを明らかにし、その注目すべき微生物の役割、遺伝子の特徴を整理して理解することができる。現行のシングルセルゲノム解析は、動物細胞を対象とするものが主流であるが、近年では微生物に適合した技術が開発されている。本稿では、最近のメタゲノム解析の動向に触れながら、マイクロバイオーム研究におけるシングルセルゲノム解析の活用の可能性について考察する。(著者抄録)

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Sub-affiliation

  • Affiliated organization   Global Education Center

Research Institute

  • 2022
    -
    2024

    Waseda Research Institute for Science and Engineering   Concurrent Researcher