Biology subjects

Takagi, R.

Publications and source records attributed to Takagi, R..

5 recordsLinked to original sources

bioRxiv2026

Transcriptome-based genome-wide analysis reveals hybridization dynamics and genetic structure of Japanese giant salamanders

AbstractsThe Japanese giant salamander (Andrias japonicus), an apex predator and a Special Natural Monument in Japan, is threatened by hybridization with introduced Chinese giant salamanders (Andrias davidianus). This hybridization has caused genetic introgression and expansion of hybrid populations, posing a serious conservation risk. Because morphological identification of hybrids is occasionally unreliable and current genetic methods rely on limited markers, a genome-wide approach is required. However, the extremely large genome ([~]50 Gb) of giant salamanders has hindered whole-genome analyses. In this study, we conducted transcriptome-based analyses of Japanese giant salamanders, Chinese giant salamanders, and their hybrids, generating RNA-seq data from 34 individuals. A total of over 419,000 SNP candidates were identified, from which 4,457 high-confidence SNPs in highly expressed genes were selected for analysis. Population structure analyses for Nabari colony revealed that hybrid individuals form two major groups, corresponding to different degrees of genetic contribution from Japanese and Chinese lineages. Most hybrids were inferred to be F2 or backcross individuals, while F1 hybrids were rare. Mitochondrial analysis indicated that all hybrids possessed Japanese-type mitochondrial genome, suggesting male-mediated introgression from Chinese salamanders. Differential expression analysis revealed enhanced stress-response pathways in hybrids and stronger antiviral responses in Japanese individuals. Using the axolotl genome as a reference, we constructed a virtual chromosomal map, identifying large haplotype blocks and supporting recent hybridization with limited recombination. This study provides a genome-wide framework for understanding hybridization dynamics and supports future conservation and evolutionary studies.

ecology↗

bioRxiv2024

Comprehensive analysis of end-modified long dsDNA donors in CRISPR-mediated endogenous tagging

CRISPR-mediated endogenous tagging is a powerful gene editing technique for studying protein dynamics and function in their native cellular environment. While the use of 5 modified DNA donors has emerged as a promising strategy to improve the typically low efficiency of knock-in gene editing, the underlying mechanisms remain poorly understood. In this study, we conducted a comprehensive analysis of end-modified long linear dsDNA donors in CRISPR-mediated endogenous tagging in human non-transformed cells. In-depth analysis of repair patterns reveals that 5 biotinylation of dsDNA donors significantly reduces imprecise insertions, thereby enhancing homology-directed repair (HDR)-mediated precise insertion efficiency. Notably, the impact of biotinylation on repair patterns resembles that of non-homologous end joining (NHEJ) pathway inhibition, suggesting its role in preventing NHEJ-mediated mis-integration. Moreover, combining biotin modification with NHEJ inhibitor treatment further improves bi-allelic knock-in efficiency. Overall, this study provides novel insights into the mechanisms by which 5 modifications enhance precise knock-ins and demonstrates their potential for achieving high-efficient, prercise endogenous tagging in human cells.

cell biology↗

bioRxiv2023

Functional and long-lived melanocytes from human pluripotent stem cells with transient ectopic expression of JMJD3

BackgroundMelanocytes are an essential part of the epidermis, and their regeneration has received much attention because propagation of human adult melanocytes in vitro is too slow for clinical use. Differentiation from human pluripotent stem cells to melanocytes has been reported, but the protocols to produce them require multiple and complex differentiation steps. MethodWe differentiated human embryonic stem cells (hESCs) that transiently express JMJD3 to pigmented cells. We investigated whether the pigmented cells have melanocytic characteristics and functions by qRT-PCR, immunocytochemical analysis and flow cytometry. We also investigated their biocompatibility by injecting the cells into immunodeficient mice for clinical use. ResultWe successfully differentiated and established a pure culture of melanocytes. The melanocytes maintained their growth rate for a long time, approximately 200 days, and were functional. They exhibited melanogenesis and transfer of melanin to peripheral keratinocytes. Moreover, melanocytes simulated the developmental processes from melanoblasts to melanocytes. The melanocytes had high engraftability and biocompatibility in the immunodeficient mice. ConclusionThe robust generation of functional and long-lived melanocytes are key to developing clinical applications for the treatment of pigmentary skin disorders.

cell biology↗

bioRxiv2021

Istradefylline, an adenosine A2a receptor antagonist, ameliorates neutrophilic airway inflammation and psoriasis in mice

ObjectiveExtracellular adenosine is produced from secreted ATP by cluster of differentiation (CD)39 and CD73. Both are critical nucleotide metabolizing enzymes of the adenosine generating pathway and are secreted by neuronal or immune cells. Adenosine plays a role in energy processes, neurotransmission, and endogenous regulation of inflammatory responses. Istradefylline is a selective adenosine A2a receptor (A2aR) antagonist used for the treatment of Parkinsons disease. We have reported that adenosine primes hypersecretion of interleukin (IL)-17A via A2aR. Istradefylline, as well as an inhibitor of CD39 (ARL67156) and an inhibitor of CD73 (AMP-CP), suppressed IL-17A production, and the administration of istradefylline to mice with experimental autoimmune encephalomyelitis (EAE) led to the marked amelioration of the disease. These previous results suggest that adenosine is an endogenous modulator of neutrophilic inflammation. We investigated the effect of istradefylline, ARL67156 and AMP-CP on other mouse models of neutrophilic inflammation. MethodsWe tested the effect of istradefylline, ARL67156 and AMP-CP on OVA-induced neutrophilic airway inflammation or imiquimod (IMQ)-induced psoriasis in mice. These two model mice received these drugs orally or percutaneously, respectively. The production of IL-17A in the lung and ear thickness were used as an index of the effects. ResultsWe show that istradefylline, ARL67156 and AMP-CP suppressed the OVA-induced IL-17A production in the lung and IMQ-induced psoriasis. ConclusionThese results indicate that adenosine-mediated IL-17A production plays a role in neutrophilic inflammation models, and moreover, istradefylline, ARL67156, and AMP-CP are effective in animal models of neutrophilic inflammation. Some clinical relevancies in COVID-19 are discussed.

immunology↗

bioRxiv2021

Extracellular adenosine induces hypersecretion of IL-17A by T-helper 17 cells through the adenosine A2a receptor to promote neutrophilic inflammation

Extracellular adenosine, produced from ATP secreted by neuronal or immune cells, may play a role in endogenous regulation of inflammatory responses. However, the underlying molecular mechanisms are largely unknown. Here, we show that adenosine primes hypersecretion of interleukin (IL)-17A by CD4+ T cells via T cell receptor activation. This hypersecretion was also induced by an adenosine A2a receptor (A2aR) agonist, PSB0777. In addition, an A2aR antagonist (Istradefylline), and inhibitors of adenyl cyclase, protein kinase A, and cAMP response element binding protein (which are signaling molecules downstream of the Gs protein coupled with the A2aR), suppressed IL-17A production, suggesting that activation of A2aR induces IL-17A production by CD4+ T cells. Furthermore, immune subset studies revealed that adenosine induced hypersecretion of IL-17A by T-helper (Th)17 cells. These results indicate that adenosine is an endogenous modulator of neutrophilic inflammation. Administration of an A2aR antagonist to mice with experimental autoimmune encephalomyelitis led to marked amelioration of symptoms, suggesting that suppression of adenosine-mediated IL-17A production is an effective treatment for Th17-related autoimmune diseases.

immunology↗