Biology subjects

Kasahara, M.

Publications and source records attributed to Kasahara, M..

17 recordsLinked to original sources

bioRxiv2025

Positional identification of a candidate gene for MALE STERILITY 2 (MS2) by linkage mapping and transcriptomic data in Cryptomeria japonica D. Don

BackgroundJapanese cedar (Cryptomeria japonica D. Don) is a major plantation species in Japan, but its abundant pollen production is a primary cause of seasonal allergic rhinitis (pollinosis). To mitigate this public health issue, the use of male-sterile cultivars has been promoted. Five types of recessive male-sterile mutations (ms1-ms5) have been identified, and the causal genes and mutations for MS1 and MS4 have been elucidated. However, the gene responsible for MS2-type male sterility remains unknown. ResultsWe aimed to identify the candidate gene responsible for MS2-type male sterility using a map-based cloning strategy. High-resolution linkage mapping localized the MS2 locus to a 1.56 cM interval on linkage group 5, corresponding to an 8.64 Mb region of the reference genome. Ninety-one genes in this region were subjected to functional annotation, gene expression analysis, and mutation screening. Among these, a single gene, SUGI_0493010, encoding a GDSL-type esterase/lipase protein (GELP), fulfilled all three criteria: it showed homology to pollen development genes in Arabidopsis thaliana, was specifically expressed in male strobili, and carried a deleterious amino acid substitution (S40F) within the predicted catalytic domain in ms2 mutant. The same mutation was also detected in a heterozygous individual (Ms2/ms2) from a separate breeding population, whose genotype was confirmed through progeny testing. Structural annotation revealed that the affected serine residue lies within the conserved GDSL motif, suggesting a functional disruption of enzymatic activity. ConclusionsOur results strongly suggest that SUGI_0493010 (GELP) is the causal gene for MS2-type male sterility in C. japonica. This finding enhances our understanding of male sterility mechanisms in conifers and provides a valuable genetic resource for breeding pollen-free trees. The study also demonstrates the effectiveness of combining genetic mapping with transcriptomic and mutational data in forest tree genomics.

genomics↗

bioRxiv2023

A chromosome-level genome assembly of amodel conifer plant, the Japanese cedar,Cryptomeria japonica D. Don

Japanese cedar (Cryptomeria japonica D. Don) is the most important Japanese forest tree, occupying about 44% of artificial forests in Japan, and planted in East Asia, Azores Archipelago, and some islands in the Indian Ocean. Although the huge genome of the species (ca. 11 Gb) with abundant repeat elements might have been an obstacle for genetic analysis, the species is easily propagated by cutting, flowered by plant hormones like gibberellic acid, transformed by agrobacterium, and edited by CRISPR/Cas9. These characteristics of C. japonica are preferable to make the species a model conifer for which reference genome sequences are necessary. In this study, we report the first chromosome-level assembly for C. japonica (2n = 22) using a third generation selfed progeny with an estimated homozygosity of 0.96. Young leaf tissue was used to extract high-molecular-weight DNA (>50 kb) for HiFi PacBio long read sequencing and to construct Hi-C/Omni-C library for Illumina short read sequencing. Using the 29x and 26x genome coverage of HiFi and Illumina reads, respectively, de novo assembly resulted in 2,650 contigs (9.1 Gb in total) with N50 contig size of 12.0 Mb. The Hi-C analysis mapped 97% of the nucleotides on the 11 chromosomes. The assembly was verified by comparing with a consensus linkage map of 7,785 markers. The BUSCO analysis confirmed ~91% of conserved genes. Annotations of genes, repeat elements and synteny with other Cupressaceae and Pinaceae species were performed, providing fundamental resources for genomic research of conifers.

genomics↗

bioRxiv2023

Drug metabolic activity as a selection factor for pluripotent stem cell-derived hepatic progenitor cells

As a metabolic organ, the liver plays a variety of roles, including detoxification. It has been difficult to obtain stable supplies of hepatocytes for transplantation and for accurate hepatotoxicity determination in drug discovery research. Human pluripotent stem cells, capable of unlimited self-renewal, may be a promising source of hepatocytes. In order to develop a stable supply of embryonic stem cell (ESC)-derived hepatocytes, we have purified human ESC-derived hepatic progenitor cells with exposure to cytocidal puromycin by using their ability to metabolize drugs. Hepatic progenitor cells stably proliferated at least 2^20-fold over 120 days, maintaining hepatic progenitor cell-like properties. High drug-metabolizing hepatic progenitor cells can be matured into liver cells by suppressing hepatic proliferative signals. The method we developed enables the isolation and proliferation of functional hepatic progenitors from human ESCs, thereby providing a stable supply of high-quality cell resources at high efficiency. Cells produced by this method may facilitate cell therapy for hepatic diseases and reliable drug discovery research.

cell biology↗

bioRxiv2023

The molecular clock in long-lived tropical trees is independent of growth rate

The rates of appearance of new mutations play a central role in evolution. However, mutational processes in natural environments and their relationship with growth rates are largely unknown, particular in tropical ecosystems with high biodiversity. Here, we examined the somatic mutation landscapes of two tropical trees, Shorea laevis (slow-growing) and S. leprosula (fast-growing), in central Borneo, Indonesia. Using newly-constructed genomes, we identified a greater number of somatic mutations in tropical trees than in temperate trees. In both species, we observed a linear increase in the number of somatic mutations with physical distance between branches. However, we found that the rate of somatic mutation accumulation per meter of growth was 3.7-fold higher in S. laevis than in S. leprosula. This difference in the somatic mutation rate was scaled with the slower growth rate of S. laevis compared to S. leprosula, resulting in a constant somatic mutation rate per year between the two species. We also found that somatic mutations are neutral within an individual, but those mutations transmitted to the next generation are subject to purifying selection. These findings suggest that somatic mutations accumulate with absolute time and older trees have a greater contribution towards generating genetic variation. Significance StatementThe significance of our study lies in the discovery of an absolute time-dependent accumulation of somatic mutations in long-lived tropical trees, independent of growth rate. Through a comparative analysis of somatic mutation landscapes in slow- and fast-growing species, we observed a clock-like accumulation of somatic mutations in both species, regardless of their growth rates. Although the majority of somatic mutations were restricted to a single branch, we also identified mutations present in multiple branches, likely transmitted during growth. Our findings suggest that older trees make a greater contribution towards generating genetic variation.

evolutionary biology↗

bioRxiv2022

Single-nucleotide substitution determines pollen production in Japanese cedar

Pollinosis, also known as pollen allergy or hay fever, is a global problem caused by pollen produced by various plant species1-6. The wind-pollinated Japanese cedar (Cryptomeria japonica) is the largest contributor to severe pollinosis in Japan, where increasing proportions of people have been affected in recent decades7. The MS4 (MALE STERILITY 4) locus of Japanese cedar controls pollen production, and its homozygous mutants (ms4/ms4) show abnormal pollen development after the tetrad stage and produce no mature pollen. In this study, we narrowed down the MS4 locus by fine mapping in Japanese cedar and found TKPR1 (TETRAKETIDE -PYRONE REDUCTASE) gene in this region. Transformation experiments using Arabidopsis thaliana showed that single-nucleotide substitution of CjTKPR1 determines pollen production. Broad conservation of TKPR1 beyond plant division could lead to the creation of pollen-free plant not only for Japanese cedar but also for broader plant species.

plant biology↗

bioRxiv2021

Hepatocytes with a phenotype of substantial CYP3A4 induction generated from drug-associated fulminant hepatitis-derived induced pluripotent stem cells

Hepatocyte-like cells (HLCs) generated from human pluripotent stem cells (PSCs) exhibit hepatocytic properties in vitro; however, their engraftment and functionality in vivo remain unsatisfactory. Despite optimization of differentiation protocols, HLCs did not engraft in a mouse model of liver injury. In contrast, organ-derived hepatocytes reproducibly formed colonies in the liver injury mouse model. As an extension of the phenomenon observed in hematopoietic stem cells giving rise to colonies within the spleen, commonly referred to as "colony-forming units in spleen (CFU-s", we hypothesize that "colony-forming units in liver (CFU-L)" serves as a reliable indicator of stemness, engraftment, and functionality of hepatocytes. The uniform expression of the randomly inactivated gene in a single colony, as reported by Sugahara et al. 2022, suggests that the colonies generated by isolated hepatocytes likely originate from a single cell. We, therefore, propose that CFU-L can be used to quantify the number of "hepatocytes that engraft and proliferate in vivo" as a quantitative assay for stem cells that utilize colony-forming ability, similar to that observed in hematopoietic stem cells.

cell biology↗

bioRxiv2021

LPMX: A pure rootless composable container system

SummaryDelivering tools for genome analysis to users is often difficult given their complex dependencies and conflicts. Container virtualization systems such as Singularity isolate environments, helping developers avoid conflicts between tools. However, they lack composability, an easy way to integrate multiple tools in different containers or multiple tools both in a container and a host, which compromises the use of container systems in genome research. Another issue is that one may not be able to use a single container system of the same version at all sites they use, which discourages the use of container systems. To this end, we present a pure rootless composable container system, LPMX, that provides composability for letting developers easily integrate tools in different existing containers or on host, allowing researchers to compose existing containers. LPMX is pure rootless, so it does not require root privilege neither during installation nor at runtime, allowing researchers to use LPMX across sites without asking permissions from administrators. LPMX provides a pure userspace layered filesystem with at least an order of magnitude lower overhead for launching a new process than existing container systems. LPMX can import Docker and Singularity images. Availability and ImplementationThe source code of LPMX is available at https://github.com/jasonyangshadow/lpmx under Apache 2.0 License. Contactmkasa@k.u-tokyo.ac.jp Supplementary informationSupplementary data are available at Bioinformaticsonline.

bioinformatics↗

bioRxiv2021

Drug metabolism activity is a Critical Intrinsic Selector for Hepatocytes to elongate cell lifespan by using a cell-killing antibiotic

Hepatocytes can be used to study the pathogenesis of liver diseases and drug discovery research. Human hepatocytes are, however, hardly expandable in vitro making it difficult to secure large numbers of cells from one donor. In this study, we aimed to establish an in vitro long-term culture system that enables stable proliferation and maintenance of human hepatocytes to ensure a constant supply. We purified human hepatocytes by selection with cytocidal puromycin, and cultured them for more than 60 population doublings over a span of more than 350 days. These results show that this simple culture system with usage of the cytocidal antibiotics enables efficient hepatocyte proliferation and is an effective method for generating a stable supply of hepatocytes for drug discovery research at a significant cost reduction.

cell biology↗

bioRxiv2020

Development of diagnostic PCR and LAMP markers for MALE STERILITY 1 (MS1) in Cryptomeria japonica D. Don

ObjectiveDue to the allergic nature of the pollen of Cryptomeria japonica, the most important Japanese forestry conifer, a pollen-free cultivar is preferred. Mutant trees detected in nature have been used for the production of a pollen-free cultivar. In order to reduce the time and cost needed for the production and breeding, we aimed to develop simple diagnostic molecular markers for mutant alleles of the causative gene MALE STERILITY 1 (MS1) in C. japonica. The expected function of this gene, its two dysfunctional mutations, and genetic diversity were described recently in a related study. ResultsWe have developed PCR and LAMP markers to detect mutant alleles and to present experimental options depending on available laboratory equipment. At field stations, where PCR machines are not available, LAMP markers were developed. LAMP only needs heat-blocks or a water bath to perform the isothermal amplification and assay results can be easily seen by eye. Because the causative mutations were deletions, two kinds of PCR markers, amplified length polymorphism (ALP) and allele specific PCR (ASP) markers, were developed. These assays can be carried out by capillary or agarose gel electrophoresis.

genetics↗

bioRxiv2020

Identification and genetic diversity analysis of a male-sterile gene (MS1) in Japanese cedar (Cryptomeria japonica D. Don)

Identifying causative genes for a target trait in conifer reproduction is challenging for species lacking whole-genome sequences. In this study, we searched for the male-sterility gene (MS1) in Cryptomeria japonica, aiming to promote marker-assisted selection (MAS) of male-sterile C. japonica to reduce the pollinosis caused by pollen dispersal from artificial C. japonica forests in Japan. We searched for mRNA sequences expressed in male strobili and found the gene CJt020762, coding for a lipid transfer protein containing a 4-bp deletion specific to male-sterile individuals. We also found a 30-bp deletion by sequencing the entire gene of another individual with the ms1. All nine breeding materials with the allele ms1 had either a 4-bp or 30-bp deletion in gene CJt020762, both of which are expected to result in faulty gene transcription and function. Furthermore, the 30-bp deletion was detected from three of five individuals in the Ishinomaki natural forest. From our findings, CJt020762 was considered to be the causative gene of MS1. Thus, by performing MAS using two deletion mutations as a DNA marker, it will be possible to find novel breeding materials of C. japonica with the allele ms1 adapted to the unique environment of each region of the Japanese archipelago.

genetics↗

bioRxiv2020

Puromycin selection of cells with a high expression of the cytochrome P450 CYP3A4 gene activity from a patient with drug-induced liver injury (DILI) and their lifespan prolongation using a combination of CDK4R24C, cyclin D1 and TERT

Many drugs have the potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in hepatocytes. Hepatocytes can accurately evaluate drug-mediated CYP3A4 induction as the gold standard for in vitro hepatic toxicology test, but their lot variation is an issue to be solved. Only a limited number of immortalized hepatocyte cells have been reported. In this study, we generated an immortalized cell expressing CYP3A4 from a patient with drug-induced liver injury (DILI). To generate DILI-derived cells with a high expression of CYP3A4, we employed a three-step approach: 1. Differentiation of DILI-induced pluripotent stem cells (DILI-iPSCs); 2. Immortalization of the differentiated cells; 3. Selection of the cells with puromycin. We hypothesize that cells with a high expression of cytochrome P450 genes can survive even after exposure to cytotoxic antibiotics because of high drug-metabolism activity. Puromycin, one of the cytotoxic antibiotics, was used in this study because of its rapid cytocidal effect at a low concentration. Phenotypic studies in vitro revealed that the puromycin-selected cells (HepaSM or SI cells) constitutively expressed the CYP3A4 gene at an extremely high level, and continued to proliferate at least up to 34 population doublings for more than 250 days. The expression profiles were independent of population doublings. Drug-mediated induction test revealed that the cells significantly increased CYP3A4 after exposure to rifampicin, suggesting that the immortalized cells would serve as another useful source for in vitro examination of drug metabolism and CYP3A4 induction.

cell biology↗

bioRxiv2020

Ammonia-based enrichment and long-term propagation of zone I hepatocyte-like cells

Zone I and zone III hepatocytes metabolize ammonia through urea cycle and drug by cytochrome P450, respectively. Ammonia has a cytotoxic effect, and can therefore be used as a selection agent for enrichment of hepatocytes. Besides, isolated hepatocytes from livers can be propagated ex vivo under appropriate condition. However, it has not been investigated so far whether ammonia-treated hepatocyte-like cells are able to proliferate in vitro. In this study, we employed the ammonia selection strategy to purify hepatocyte-like cells that were differentiated from human pluripotent stem cells (PSCs) that are embryonic stem cells (ESCs) and induced pluripotent stem cells. Hepatocyte-like cells after exposure to ammonia highly expressed the CPS1 gene that metabolizes ammonia to carbamoyl phosphate. The resistance to cytotoxicity or cell death by ammonia is probably attributed to the metabolic activity of ammonia in the cells. In addition to the ammonia metabolism-related genes, ammonia-selected PSC-derived hepatocytes increased expression of the CYP3A4 gene, one of the cytochrome P450 genes, that is mainly expressed in zone III hepatocytes. Ammonia-selected hepatocyte-like cells derived from both ESCs and iPSCs can be propagated in vitro up to 30 population doublings for more than 190 days without affecting expression of the liver-associated genes, implying that the ammonia-selected cells have immortality or equivalent life span on the appropriate feeder cells like ESCs and iPSCs. The long-term cultivation of ammonia-selected hepatocyte-like cells resulted in the increased expression of hepatocyte-associated genes such as the CPS1 and CYP3A4 genes. The ammonia selection method to enrich a hepatocyte population was also applicable to immortalized cells from the liver. Ammonia treatment in combination with in vitro propagation will be used to obtain large amounts of hepatocytes or hepatocyte-like cells for pharmacology, toxicology and regenerative medicine.

cell biology↗

bioRxiv2019

Proteolytic Cleavage by Matriptase Exacerbating Kidney Injury: a Novel Therapeutic Target

Chronic kidney disease (CKD) is a progressive disease, and podocyte injury is a potential mechanism. We found that Matriptase was activated at podocytes in CKD patients and mice while a Matriptase inhibitor slowed the progression of mouse kidney disease. The mechanism could be accounted for by an imbalance favoring Matriptase over its cognate inhibitor, hepatocyte growth factor activator inhibitor type 1 (HAI-1), as conditional depletion of HAI-1 in podocytes accelerates podocyte injury. Intriguingly, the N-terminal of Podocin (Podocin-N), as a consequence of Matriptase cleavage of Podocin, translocates to nucleoli. These results suggest that aberrant activation of Matriptase would cause podocyte injury, and a targeting Matriptase could be a novel therapeutic strategy for CKD patients. Significant statementChronic kidney disease (CKD) is a progressive disease. If podocytes, which are specialized cells of the kidney glomerulus that wrap around capillaries, are injured, kidney injury is exacerbated. Thus, a therapeutic strategy to addressing CKD would be to block podocyte injury. The present study provides evidence that Matriptase cleaves Podocin, a component of podocyte slit membrane, and the N-terminal of podocin translocates to nucleoli, causing kidney injury. Our findings show that the N-terminal of Podocin plays an efficient role for cell fate in podocytes. In addition, the inhibition of Matriptase would be a potential therapeutic target for CKD.

biochemistry↗

bioRxiv2019

Immortalization of human zone I hepatocytes from biliary atresia with CDK4R24C, cyclin D1, and TERT for cytochrome P450 induction testing

BackgroundHepatocytes are an important tool for in vitro toxicology testing. In addition to primary cultures, a limited number of immortalized cell lines have been developed. We here describe a new cell line, designated as HepaMN, which has been established from a liver associated with biliary atresia.\n\nMethodsHepatocytes were isolated from a liver of 4-year-old girl with biliary atresia and immortalized by inoculation with CSII-CMV-TERT, CSII-CMV-Tet-Off, CSII-TRE-Tight-cyclin D1 and CSII-TRE-Tight-CDK4R24C (mutant CDK4: an INK4a-resistant form of CDK4) lentiviruses at the multiplicity of infection of 3 to 10.\n\nResultsHepaMN cells exhibited morphological homogeneity, displaying hepatocyte-like phenotypes. Phenotypic studies in vivo and in vitro revealed that HepaMN cells showed polarized and functional hepatocyte features along with a canalicular cell phenotype under defined conditions, and constitutively expressed albumin and carbamoyl phosphate synthetase I in addition to epithelial markers. Since HepaMN cells are immortal and subcloned, kinetics and expression profiles were independent of population doublings.\n\nConclusionsHepaMN cells showed increased CYP3A4 expression after exposure to rifampicin, implying that their close resemblance to normal human hepatocytes makes them suitable for research applications including drug metabolism studies.

cell biology↗

bioRxiv2019

Long read sequencing reveals a novel class of structural aberrations in cancers: identification and characterization of cancerous local amplifications

Here we report identification of a new class of local structural aberrations in lung cancers. The whole-genome sequencing of cell lines using a long read sequencer, PromethION, demonstrated that typical cancerous mutations, such as point mutations, large deletions and gene fusions can be detected also on this platform. Unexpectedly, we revealed unique structural aberrations consisting of complex combinations of local duplications, inversions and micro deletions. We further analyzed and found that these mutations also occur in vivo, even in key cancer-related genes. These mutations may elucidate the molecular etiology of patients for whom causative cancerous events and therapeutic strategies remain elusive.

genomics↗

bioRxiv2019

MoMI-G: Modular Multi-scale Integrated Genome Graph Browser

Long-read sequencing allows more sensitive and accurate discovery of structural variants (SVs). While more and more SVs are being identified, a number of them are difficult to visualize using existing SV visualization tools. Therefore, methods to visualize SVs such as nested or large SVs of over a megabase pair need to be developed. To this end, we developed MOdular Multi-scale Integrated Genome graph browser, MoMI-G, a web-based genome browser to visualize SVs, genes, repeats, and other annotations as a variation graph with paths. This browser allows more intuitive recognition of large, nested, and potentially more complex SVs. MoMI-G has view modules for different scales, which allow users to view the whole genome down to nucleotide-level alignments of long reads. Alignments spanning reference alleles and those spanning alternative alleles are shown in the same view. Users can customize the view, if they are not satisfied with the preset views. In addition, MoMI-G has Interval Card Deck, a feature for rapid manual inspection of hundreds of SVs. Herein, we describe the utility of MoMI-G by using representative examples of large and nested SVs found in two cell lines, LC-2/ad and CHM1. MoMI-G is freely available at https://github.com/MoMI-G/MoMI-G under the MIT license.

bioinformatics↗

bioRxiv2017

Acceleration Of Nucleotide Semi-Global Alignment With Adaptive Banded Dynamic Programming

MotivationPairwise alignment of nucleotide sequences has previously been carried out using the seed- and-extend strategy, where we enumerate seeds (shared patterns) between sequences and then extend the seeds by Smith-Waterman-like semi-global dynamic programming to obtain full pairwise alignments. With the advent of massively parallel short read sequencers, algorithms and data structures for efficiently finding seeds have been extensively explored. However, recent advances in single-molecule sequencing technologies have enabled us to obtain millions of reads, each of which is orders of magnitude longer than those output by the short-read sequencers, demanding a faster algorithm for the extension step that accounts for most of the computation time required for pairwise local alignment. Our goal is to design a faster extension algorithm suitable for single-molecule sequencers with high sequencing error rates (e.g., 10-15%) and with more frequent insertions and deletions than substitutions.\n\nResultsWe propose an adaptive banded dynamic programming algorithm for calculating pairwise semi-global alignment of nucleotide sequences that allows a relatively high insertion or deletion rate while keeping band width relatively low (e.g., 32 or 64 cells) regardless of sequence lengths. Our new algorithm eliminated mutual dependences between elements in a vector, allowing an efficient Single-Instruction-Multiple-Data parallelization. We experimentally demonstrate that our algorithm runs approximately 5x faster than the extension alignment algorithm in NCBI BLAST+ while retaining similar sensitivity (recall).\n\nWe also show that our extension algorithm is more sensitive than the extension alignment routine in DALIGNER, while the computation time is comparable.\n\nAvailabilityThe implementation of the algorithm and the benchmarking scripts are available at https://github.com/ocxtal/adaptivebandbench.\n\nContactmkasa@edu.k.u-tokyo.ac.jp

bioinformatics↗