Biology subjects

Takeyama, H.

Publications and source records attributed to Takeyama, H..

22 records · Page 2Linked to original sources

bioRxiv2020

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

To improve our understanding of the environmental microbiome, we developed a single-cell genome sequencing platform, named SAG-gel, which utilizes gel beads for single-cell isolation, cell lysis, and whole genome amplification (WGA) for sequencing. SAG-gel enables serial, parallel and independent reactions of > 100,000 single cells in a single tube, delivering high-quality genome recovery with storable randomized single-cell genome libraries. From soil and marine environmental sources, we acquired 734 partial genomes that are recapitulated in 231 species, 35% of which were assigned as high-to-medium qualities. We found that each genome to be almost unique and 98.7% of them were newly identified, implying the complex genetic diversities across 44 phyla. The various metabolic capabilities including virulence factors and biosynthetic gene clusters were found across the lineages at single-cell resolution. This technology will accelerate the accumulation of reference genomes of uncharacterized environmental microbes and provide us new insights for their roles.

genomics↗

bioRxiv2020

Molecular profiling of lipid droplets inside HuH7 Cells with Raman micro-spectroscopy

Raman imaging has become an attractive technology in molecular biology because of its ability to detect multiple molecular components simultaneously without labeling. Two major limitations in accurately accounting for spectral features, viz. background removal and spectral unmixing, have been overcome by employing a modified and effective routine in multivariate curve resolution (MCR). With our improved strategy, we have spectrally isolated seven structurally specific biomolecules without any post-acquisition spectral treatments. Consequently, the isolated intensity profiles reflected concentrations of corresponding biomolecules with high statistical accuracy. Our study reveals the changes in the molecular composition of lipid droplets (LD) inside HuH7 cells and its relation to the physiological state of the cell. Further, we show that the accurate separation of spectral components permits analysis of in vivo structural modification of molecules after cellular uptake. A detailed discussion is presented to highlight the potential of Raman spectroscopy with MCR in semi-quantitative molecular profiling of living cells.

bioengineering↗

bioRxiv2019

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

BackgroundThe 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. ResultsInulin 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 two phyla, four classes, seven 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. ConclusionsOur 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.

microbiology↗

bioRxiv2019

Time-lapse single-cell transcriptomics reveals modulation of histone H3 for dormancy breaking

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 developed 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 highlighted one of three copies of histone H3 genes for which transcription fluctuates during the initial stage of germination. Disruption of this temporal fluctuation caused 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.

cell biology↗