Biology subjects

Nagashima, H.

Publications and source records attributed to Nagashima, H..

8 recordsLinked to original sources

bioRxiv2025

The generation of viable, structurally integrated human-mouse chimaeras through enhanced hPSCs proliferation

The generation of human organs in animals through blastocyst complementation offers a promising solution to the shortage of transplantable organs. While human pluripotent stem cells (hPSCs) can contribute to interspecies chimaeric embryos when injected into preimplantation embryos of mice, pigs, or monkeys, their integration is often limited due to low chimaerism and segregation from host tissues, significantly impeding progress toward exogenic organ generation. Here, we demonstrate that co-overexpression of the anti-apoptotic gene BCL2 and the proto-oncogene MYCL, along with various cell cycle regulators significantly enhances human cell chimaerism by promoting cell proliferation. This strategy facilitates the generation of viable mouse pups containing hPSC-derived tissues without tumorigenesis. scRNA-seq analysis revealed that hPSCs already exit pluripotent by early post-implantation stage yet hPSCs with enhanced proliferation were able to integrate effectively into the cardiomyocytes and vasculature of both embryonic and extraembryonic tissues with gene expression profiles reflecting their structural integration. These findings highlight the critical role of cell cycle regulation in overcoming xenogeneic barriers. Our findings offer new insights into strategies for enhancing interspecies organogenesis and advancing the field of regenerative medicine. HighlightO_LIEnhancing hPSC proliferation increases human cell contribution in interspecies chimaeras C_LIO_LIAchieving the generation of viable human-mouse chimaeras without tumour formation C_LIO_LIhPSCs in post-implantation mouse epiblast exit pluripotency but retain the capacity to integrate into mouse embryogenesis C_LIO_LIhPSCs derivatives integrate into vasculature and cardiac tissues with lineage-matched transcriptional profiles C_LI

developmental biology↗

bioRxiv2024

A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4+ T helper cell signaling, differentiation and epigenetic remodeling

While inputs regulating CD4+ T helper cell (Th) differentiation are well-defined, the integration of downstream signaling with transcriptional and epigenetic programs that define Th-lineage identity remain unresolved. PI3K signaling is a critical regulator of T cell function; activating mutations affecting PI3K{delta} result in an immunodeficiency with multiple T cell defects. Using mice expressing activated-PI3K{delta}, we found aberrant expression of proinflammatory Th1-signature genes under Th2-inducing conditions, both in vivo and in vitro. This dysregulation was driven by a robust PI3K{delta}-IL-2-Foxo1 signaling loop, fueling Foxo1-inactivation, loss of Th2-lineage restriction, altered chromatin accessibility and global impairment of CTCF-DNA interactions. Surprisingly, ablation of Fasl, a Foxo1-repressed gene, restored normal Th2 differentiation, TCR signaling and CTCF expression. BioID revealed Fas interactions with TCR- signaling components, which were supported by Fas-mediated potentiation of TCR signaling. Our results highlight Fas-FasL signaling as a critical intermediate in phenotypes driven by activated-PI3K{delta}, thereby linking two key pathways of immune dysregulation.

immunology↗

bioRxiv2023

Post-fertilization transcription initiation in an ancestral LTR retrotransposon drives lineage-specific genomic imprinting of ZDBF2

The imprinted ZDBF2 gene is controlled by oocyte-derived DNA methylation, but its epigenetic regulation is quite different from that of other canonically imprinted genes that are dependent on DNA methylation deposited in the gametes. At the ZDBF2 locus, maternal DNA methylation in the imprinted differentially methylated region (DMR) does not persist after implantation. Instead, a transient transcript expressed in the early embryo exclusively from the unmethylated paternal allele of the DMR, known as GPR1-AS in humans and Liz in mice, contributes to establishing secondary DMRs that maintain paternal expression of ZDBF2 in the somatic lineage. While the imprinting of ZDBF2 is evident in humans and mice, whether this process is conserved in other mammals has not been addressed. Here, we show that the first exon of human GPR1-AS overlaps with that of a long terminal repeat (LTR) belonging to the MER21C subfamily of retrotransposons. Although this LTR family appears and is amplified in Boroeutherians, the magnorder of placental mammals that includes the Euarchontoglires and Laurasiatheria superorders, the MER21C insertion into the GPR1-AS orthologous region occurred specifically in the common ancestor of Euarchontoglires, a clade that includes extant primates, rodents, and rabbits. The first exon of mouse Liz does not overlap with an annotated LTR in standard repeat annotation; however, promoter activity assay and multiple sequence alignment suggests that it retains a functionally conserved relationship with the MER21C-overlapping first exon of GPR1-AS. Furthermore, directional RNA sequencing of placental tissues from rabbits and nonhuman primates also revealed GPR1-AS orthologs, with their first exon embedded within the same ancestral LTR. In contrast, allele-specific expression profiling of cow and tammar wallaby, mammals outside the Euarchontoglires group, revealed expression from both alleles in all tissues analyzed. Taken together, these observations suggest that imprinting of ZDBF2 in Euarchontoglires had its genesis in the insertion of a MER21C element in their common ancestor. Our previous studies showed that LTRs reactivated in oocytes contribute to lineage-specific imprinting during mammalian evolution. The data presented here suggest that post-fertilization activation of an ancestral LTR-derived sequence can also contribute to the lineage-specific establishment of imprinted genes.

genomics↗

bioRxiv2022

Remodeling of Il4-Il13-Il5 locus underlies selective gene expression

The type 2 cytokines, interleukin (IL)-4, IL-5 and IL-13 reside within a tandem multi-gene cluster in mammals. These cytokines represent the hallmark of type 2 immune responses controlling parasites, promoting tissue repair as well as causing allergic diseases. Both innate and adaptive lymphocytes secrete type 2 cytokines with discordant production spectra. We took a holistic structural and functional view of the type 2 cytokine locus before and after activation, comparing innate (ILC2) and adaptive (Th2) lymphocytes to understand mechanisms underlying their distinctive programs. Rapid induction of IL-5 dominates in ILC2, whereas IL-4 does so in Th2 cells. Using high-resolution chromatin conformation capture we found that global cellular chromatin architecture remained constant, whereas the type 2 cytokine locus rapidly remodeled. In ILC2, Il13 and Il5 loci were aligned in proximity whereas Il4 locus was insulated. In Th2 cells, Il4 and Il13 positioned in proximity while the Il5 locus remained distal. Select REs were separately deleted in mice to confirm cell-type specific and activation-dependent roles in type 2 responses in vivo. Thus, contrary to the premise that chromatin architecture plays a minimal role in steady-state gene induction, signal-dependent remodeling of 3D configuration underlies the discordant cytokine outputs in ILC2s versus Th2 cells.

immunology↗

bioRxiv2021

A scalable, clinically severe pig model for Duchenne muscular dystrophy

Large animal models for Duchenne muscular dystrophy (DMD) are crucial for preclinical evaluation of novel diagnostic procedures and treatment strategies. Pigs cloned from male cells lacking DMD exon 52 (DMD{Delta}52) resemble molecular, clinical and pathological hallmarks of DMD, but cannot be propagated by breeding due to death before sexual maturity. Therefore, female DMD+/- carriers were generated. A single founder animal had 11 litters with 29 DMDY/-, 34 DMD+/- as well as 36 male and 29 female wild-type (WT) offspring. Breeding with F1 and F2 DMD+/- carriers resulted in additional 114 DMDY/- piglets. The majority of them survived for 3-4 months, providing large cohorts for experimental studies. Pathological investigations and proteome studies of skeletal muscles and myocardium confirmed the resemblance of human disease mechanisms. Importantly, DMDY/- pigs reveal progressive fibrosis of myocardium and increased expression of connexin-43, associated with significantly reduced left ventricular fractional shortening and ejection fraction already at age 3 months. Furthermore, behavioral tests provided evidence for impaired cognitive ability of DMDY/- pigs. Our breeding cohort of DMD{Delta}52 pigs and standardized tissue repositories from DMDY/- pigs, DMD+/- carriers, and WT littermate controls provide important resources for studying DMD disease mechanisms and for testing novel diagnostic procedures and treatment strategies.

pathology↗

bioRxiv2019

Handling and Packaging of Medical Bags at the Acute Disaster Site Under High Temperature Conditions

The handling of medical bags in high temperature disaster sites has not been fully evaluated. In this study, medical bags were assessed in the closed, semi-opened, and opened state, with and without heat insulating material (HIM) and a coolant. The bags were heated for 300 minutes at 35 or 40{degrees}C, and the internal temperature was measured. closed and semi-opened bags were able to limit temperature increases in the central part of the bag at both 35 and 40{degrees}C to a greater extent than opened bags. When coolant and HIM were used in closed and semi-opened bags, the internal temperature was significantly lower than that seen in the opened state at 40{degrees}C (P < 0.01 and P < 0.001, respectively). In conclusion, in high temperature disaster sites, medical bags should be maintained in a semi-opened or closed state using a HIM and coolant.

scientific communication and education↗

bioRxiv2019

RNF144A shapes the hierarchy of cytokine signaling to provide protective immunity against influenza

Cytokine-induced signaling pathways are tightly regulated and self-limiting, as their dysregulation causes immune disorders and cancer. The precise mechanisms that fine-tune these responses are incompletely understood. We show that the E3 ubiquitin ligase RNF144A is an IL-2/STAT5-induced gene in T cells and critically orchestrates the hierarchy of IL-2R signaling to promote STAT5 activation and limit RAF-ERK-MAPK output from the IL-2R. Mechanistically, RNF144A increased the interaction between IL-2R{beta} and STAT5 and polyubiquitinated RAF1, enhancing its proteasomal degradation and preventing the formation of the potent RAF1/BRAF kinase complex. CD8+ T cells from Rnf144a-/- mice had impaired IL-2-induction of effector genes, including Tnf and granzymes, and these mice demonstrated increased susceptibility to influenza. Reduced RNF144A expression was associated with more severe influenza in humans and its expression in patients was a biomarker distinguishing moderate from severe disease. These studies reveal a vital physiological role for RNF144A in maintaining the fidelity of IL-2R signaling in CTLs to prevent severe inflammation in response to infection.\n\nOne Sentence SummaryRNF144A promotes anti-viral immunity by regulating the hierarchy of cytokine signal output

immunology↗

bioRxiv2018

Homologous expression and characterization of gassericin T and gassericin S, a novel class IIb bacteriocin produced by Lactobacillus gasseri LA327

Lactobacillus gasseri LA327 isolated from the large intestine tissue in humans is a bacteriocinogenic strain and is predicted to produce two kinds of class IIb bacteriocins, i.e. gassericin T (GT) and acidocin LF221A (Acd LF221A). In this study, DNA sequencing of the genes for GT and Acd LF221A on Lb. gasseri LA327 revealed that the amino acid sequences for GT completely corresponded with those of gat except for GatK (histidine kinase). However, those for the Acd LF221A had analogues which differed in at least one amino acid residue to be a putative class IIb bacteriocin designated as gassericin S (GS). By deletion test of GT structural genes (gatAX), the LA327 strain retained the bacteriocin activity, and the LA327 mutant strain lacking the ABC-type transporter gene (gatT) completely lost the bacteriocin activity. This indicates that LA327 strain is a GS producer, and GS production is performed via gat with the inclusion of gatT. Homologous expression using deletion mutants for GS and GT containing each single peptide elucidated that GS (GasAX) and GT (GatAX) showed synergistic activity as class IIb bacteriocins, respectively, and no synergistic activity was observed between each peptide of GS and GT. The molecular mass of GS was estimated to be theoretical ca. 5,400 Da by in situ activity assay after SDS-PAGE, clarifying that GS was actually expressed as an active class IIb bacteriocin. Furthermore, stability of GS expressed against pH, heat and protease was determined.\n\nImportanceWe determined the complete DNA sequence for GS, a novel class IIb bacteriocin of Lb. gasseri, and succeeded to express GS as active bacteriocins. Our results clarified the interaction of each class IIb component peptide for GT in addition to GS via construction of homologous mutants which were not dependent on the purification. These data may demonstrate the characteristics of class IIb bacteriocins for Lb. gasseri.

microbiology↗