Biology subjects

Sakurai, S.

Publications and source records attributed to Sakurai, S..

5 recordsLinked to original sources

bioRxiv2024

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

We here report a novel PET hydrolase originating from a soil microbial genome sequence. This enzyme, bbPET0069, exhibits characteristics resembling a cutinase-like Type I PET-degrading enzyme but lacks disulfide bonds. Notably, bbPET0069 displayed remarkable synergy with Candida antarctica lipase B (CALB), demonstrating rapid and efficient PET degradation. To improve the PET degradation activity of bbPET0069, we employed a three-dimensional (3D) structural modeling to identify mutation sites around its substrate binding domain combined with a protein language model for effective mutation prediction. Through three initial rounds of directed evolution, we achieved a significant enhancement in PET degradation with CALB, resulting in a 12.6-fold increase compared to wild-type bbPET0069 without CALB. We confirmed its PET degradation activity in PET nanoparticles and films, and our proposed approach enabled efficient PET degradation to terephthalic acid monomers up to 95.5%. Our approach, which integrates a two-enzyme system with protein engineering, demonstrates the potential for enhancing the activity of emerging PET-degradation enzymes, which may possess unique attributes. Graphical AbstractA novel PET hydrolase, bbPET0069, was identified from a soil microbial genome. bbPET0069 and CALB showed remarkable synergy in PET degradation. Using surface feature analysis, PET degradation activity of bbPET0069 was significantly improved. This combination of a two-enzyme system and surface feature analysis holds promise for enhancing emerging PET-degradation enzymes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/578500v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@4c1c08org.highwire.dtl.DTLVardef@c47c74org.highwire.dtl.DTLVardef@1c6cd19org.highwire.dtl.DTLVardef@c32e73_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

Hit-and-run silencing of endogenous DUX4 by targeting DNA hypomethylation on D4Z4 repeats in facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD), a progressive skeletal muscle disorder, is epigenetically characterized by DNA hypomethylation of the D4Z4 repeats in the 4q35 region, which enables aberrant DUX4 expression. Sustainable DUX4 suppression is thus a promising therapeutic strategy by which to prevent disease progression, but most of the supposed methods to achieve this depend on the expression of a mediator biochemical entity that would potentially narrow the quality of life of individuals with FSHD in the clinical context. In this study, we report that by applying hit-and-run silencing with dCas9-mediated epigenetic editing targeting DNA hypomethylation on D4Z4 repeats, we could achieve the suppression of endogenous DUX4 in our FSHD patient-derived iPSC model. Notably, DNA methylation was significantly upregulated in FSHD cells and suppression effects were observed for at least two weeks after intervention, which was not the case with transient treatments of typical dCas9-KRAB alone. Off-target analysis showed that despite the potential genome-wide risk for DNA methylation, the impact on the transcriptome was limited. We propose that hit-and-run silencing could be a promising option to prevent disease progression with minimum intervention for individuals with FSHD, motivating further study for clinical development. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/487997v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@19e85dforg.highwire.dtl.DTLVardef@672798org.highwire.dtl.DTLVardef@58717dorg.highwire.dtl.DTLVardef@83044b_HPS_FORMAT_FIGEXP M_FIG C_FIG

pathology↗

bioRxiv2021

Resistance to chemical carcinogenesis induction via a dampened inflammatory response in naked mole-rats

Naked mole-rats (NMRs) have a very low spontaneous carcinogenesis rate, which has prompted scientists to study their cancer resistance mechanisms in order to provide clues for human cancer prevention. Although cancer resistance in NMRs has been intensively investigated at the cellular level, it is still unknown how strongly resistant NMR individuals are to carcinogenesis and how NMR tissues respond to experimental carcinogenesis induction. Here, we show that NMRs exhibit extraordinary resistance against potent chemical carcinogenesis induction through a dampened inflammatory response. Although carcinogenic insults damaged skin cells of both NMRs and mice, NMR skin showed markedly lower immune cell infiltration and reduced induction of inflammatory genes. NMRs harbor loss-of-function mutations in receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) genes, which are essential for necroptosis, a type of necrotic cell death that activates strong inflammation. A necroptosis-inducing stimulus did not increase death of NMR cells. After carcinogenic insults, leakage of the HMGB1, a marker of necrotic cell death, was not increased in NMR skin. In mice, inhibition or knockout of RIPK3 reduced immune cell infiltration and delayed the onset of chemical carcinogenesis. Therefore, necroptosis deficiency may serve as a cancer resistance mechanism via attenuating the inflammatory response in NMRs. Our study sheds light on the importance of a dampened inflammatory response as a non-cell-autonomous cancer resistance mechanism in NMRs. Further in vivo study of the unusual tissue immune system and carcinogenesis resistance of NMRs may lead to the development of new strategies to prevent carcinogenesis in humans. Significance StatementIn contrast with intensive studies of cancer resistance mechanisms in naked mole-rats (NMRs) at the cellular level, little is known about how NMR individuals respond to carcinogenesis induction, despite the fact that cell-to-cell interactions in tissues regulate carcinogenesis in vivo. Here, we demonstrate that NMRs are remarkably resistant to chemical carcinogenesis induction and characteristically have attenuated tissue inflammatory responses to carcinogenic insults. NMRs have loss-of-function mutations in RIPK3 and MLKL genes and thus cannot activate necroptosis, a type of inflammation-inducing cell death. RIPK3 inhibition in mice reduced immune cell infiltration in response to carcinogenic insults and delayed the onset of chemical-induced carcinogenesis. Our results highlight the importance of studies on dampened tissue inflammatory responses to understand cancer resistance of NMRs.

cancer biology↗

bioRxiv2019

Modeling the Human Segmentation Clock with Pluripotent Stem Cells

Pluripotent stem cells (PSCs) have increasingly been used to model different aspects of embryogenesis and organ formation1. Despite recent advances in the in vitro induction of major mesodermal lineages and mesoderm-derived cell types2,3, experimental model systems that can recapitulate more complex biological features of human mesoderm development and patterning are largely missing. Here, we utilized induced pluripotent stem cells (iPSCs) for the stepwise in vitro induction of presomitic mesoderm (PSM) and its derivatives to model distinct aspects of human somitogenesis. We focused initially on modeling the human segmentation clock, a major biological concept believed to underlie the rhythmic and controlled emergence of somites, which give rise to the segmental pattern of the vertebrate axial skeleton. We succeeded to observe oscillatory expression of core segmentation clock genes, including HES7 and DKK1, and identified novel oscillatory genes in human iPSC-derived PSM. We furthermore determined the period of the human segmentation clock to be around five hours and showed the presence of dynamic traveling wave-like gene expression within in vitro induced human PSM. Utilizing CRISPR/Cas9-based genome editing technology, we then targeted genes, for which mutations in patients with abnormal axial skeletal development such as spondylocostal dysostosis (SCD) (HES7, LFNG and DLL3) or spondylothoracic dysostosis (STD) (MESP2) have been reported. Subsequent analysis of patient-like iPSC knock-out lines as well as patient-derived iPSCs together with their genetically corrected isogenic controls revealed gene-specific alterations in oscillation, synchronization or differentiation properties, validating the overall utility of our model system, to recapitulate not only key features of human somitogenesis but also to provide novel insights into diseases associated with the formation and patterning of the human axial skeleton.

developmental biology↗

bioRxiv2017

Quantitative monitoring of multispecies fish environmental DNA using high-throughput sequencing

Effective ecosystem conservation and resource management require quantitative monitoring of biodiversity, including accurate descriptions of species composition and temporal variations of species abundance. Therefore, quantitative monitoring of biodiversity has been performed for many ecosystems, but it is often time- and effort-consuming and costly. Recent studies have shown that environmental DNA (eDNA), which is released to the environment from macro-organisms living in a habitat, contains information about species identity and abundance. Thus, analyzing eDNA would be a promising approach for more efficient biodiversity monitoring. In the present study, we added internal standard DNAs (i.e., known amounts of short DNA fragments from fish species that have never been observed in a sampling area) to eDNA samples, which were collected weekly from a coastal marine ecosystem in Maizuru-Bay, Kyoto, Japan (from April 2015 to March 2016), and performed metabarcoding analysis using Illumina MiSeq to simultaneously identify fish species and quantify fish eDNA copy numbers. A correction equation was obtained for each sample using the relationship between the number of sequence reads and the added amount of the standard DNAs, and this equation was used to estimate the copy numbers from the sequence reads of non-standard fish eDNA. The calculated copy numbers showed significant positive correlation with those determined by quantitative PCR, suggesting that eDNA metabarcoding with standard DNA enabled useful quantification of eDNA. Furthermore, for samples that show a high level of PCR inhibition, our method might allow more accurate quantification than qPCR because the correction equations generated using internal standard DNAs would include the effect of PCR inhibition. A single run of Illumina MiSeq produced > 70 quantitative fish eDNA time series in our study, showing that our method could contribute to more efficient and quantitative monitoring of biodiversity.

ecology↗