Biology subjects

Kimura, T.

Publications and source records attributed to Kimura, T..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Characterization of the placenta-specific EnvV2-Fca soluble protein derived from endogenous retrovirus

Endogenous retroviruses (ERVs) are remnants of ancestral viruses in the host genome. Here, we identified the expression of a defective retroviral env gene belonging to the ERV group V member 2 Env (EnvV2) in Felis catus (EnvV2-Fca), which was specifically detected in the placental trophoblast syncytiotrophobic layer. EnV2-Fca was expressed as a secreted protein in cultured cells. Genetic analyses indicated that EnvV2 genes are widely present in vertebrate, and are under purifying selection among carnivores, suggesting a potential benefit for the host. Notably, this study suggests that birds, bats, and rodents carrying EnvV2 may play significant roles as intermediate vectors in spreading or cross-transmitting viruses among species. Overall, our findings provide valuable insights into the evolution of ERV in vertebrate hosts.

microbiology↗

bioRxiv2023

A single-cell atlas of transcribed cis-regulatory elements in the human genome

Transcribed cis-regulatory elements (tCREs), such as promoters and enhancers, are fundamental to modulate gene expression and define cell identity. The detailed mapping of tCREs at single-cell resolution is essential for understanding the regulatory mechanisms that govern cellular functions. Prior tCRE catalogs, limited by bulk analysis, have often overlooked cellular heterogeneity. We have constructed a tCRE atlas using single-cell 5-RNA-seq, capturing over 340,000 single-cells from 23 human tissues and annotating more than 175,000 tCREs, substantially enhancing the scope and granularity of existing cis-regulatory element annotations in the human genome. This atlas unveils patterns of gene regulation, revealing connections between broadly expressed promoters and cell type-specific distal tCREs. Assessing trait heritability at single-cell resolution with a novel tCRE module-based approach, we uncovered the nuanced trait-gene regulatory relationships across a continuum of cell populations, offering insights beyond traditional gene-level and bulk-sample analyses. Our study bridges the gap between gene regulation and trait heritability, underscoring the potential of single-cell analysis to elucidate the genetic foundations of complex traits. These insights set the stage for future research to investigate the impact of genetic variations on diseases at the individual level, advancing the understanding of cellular and molecular basis of trait heritability.

genomics↗

bioRxiv2023

FeLIX is a restriction factor for mammalian retrovirus infection

Endogenous retroviruses (ERVs) are remnants of ancestral viral infections. Feline leukemia virus (FeLV) is an exogenous and endogenous retrovirus in domestic cats. It is classified into several subgroups (A, B, C, D, E, and T) based on viral receptor interference properties or receptor usage. ERV-derived molecules benefit animals, conferring resistance to infectious diseases. However, the soluble protein encoded by the defective envelope (env) gene of endogenous FeLV (enFeLV) functions as a co-factor in FeLV subgroup T infections. Thus, whether the gene emerged to facilitate viral infection is unclear. Based on the properties of ERV-derived molecules, we hypothesized that the defective env genes possess antiviral activity that would be advantageous to the host because FeLV subgroup B (FeLV-B), a recombinant virus derived from enFeLV env, is restricted to viral transmission among domestic cats. When soluble truncated Env proteins from enFeLV were tested for their inhibitory effects against enFeLV and FeLV- B, they inhibited viral infection. Notably, this antiviral machinery was extended to infection with the Gibbon ape leukemia virus, Koala retrovirus-A, and Hervey pteropid gammaretrovirus. Although these viruses used feline phosphate transporter1 (fePit1) or fePit1 and phosphate transporter2 (fePit2) as receptors, the inhibitory mechanism involved competitive receptor binding in a fePit1-dependent manner. The shift of receptor usage may have occurred to avoid the inhibitory effect. Overall, these findings highlight the possible emergence of soluble truncated Env proteins from enFeLV as a restriction factor against retroviral infection, and might help in the control of retroviral spread for host immunity and antiviral defense. ImportanceRetroviruses are unique in using reverse transcriptase to convert RNA genomes into DNA, infecting germ cells, and transmitting to offspring. A large amount of ancient retroviral sequences are known as endogenous retroviruses (ERVs). Soluble Env protein derived from ERVs have been identified to function as a co-factor that assists in FeLV-T infection. However, herein, we show that the soluble Env protein exhibits antiviral activity and provides resistance to mammalian retrovirus infection through competitive receptor binding. In particular, this finding may explain why FeLV-B transmission is not observed among domestic cats. ERV-derived molecules can benefit animals in an evolutionary arm race, highlighting the double-edged sword nature of ERVs.

microbiology↗

bioRxiv2023

Human intestinal organoid-derived PDGFRα+ mesenchymal stroma empowers LGR4+ epithelial stem cells

The columnar epithelial cells comprising the intestinal tract, stomach, and uterus can be cultured in vitro as organoids or in adherent culture. However, the proliferation of these columnar epithelial cells in adherent culture is limited. Likewise, human pluripotent stem cell (hPSC)-derived intestinal epithelial cells do not show extensive or clonal propagation in vitro. In this study, we induced proliferation of hPSC-derived small intestinal epithelium for a longer time by utilizing mesenchymal stromal cells derived from self-organized intestinal organoids as feeders. The proliferating cells exhibited columnar form, microvilli and glycocalyx formation, and cell polarity, as well as expression of drug-metabolizing enzymes and transporters. It is noteworthy that small intestinal epithelial stem cells cannot be cultured in adherent culture alone, and the stromal cells cannot be replaced by other feeders. Organoid-derived mesenchymal stromal cells resemble the trophocytes essential for maintaining small intestinal epithelial stem cells, and play a crucial role in adherent culture. The high proliferative expansion, productivity, and functionality of hPSC-derived small intestinal epithelial stem cells could have potential applications in pharmacokinetic and toxicity studies and regenerative medicine.

cell biology↗

bioRxiv2023

A novel in silico scaffold-hopping method for drug repositioning in rare and intractable diseases

In the field of rare and intractable diseases, new drug development is difficult and drug repositioning (DR) is a key method to improve this situation. In this study, we present a new method for finding DR candidates utilizing virtual screening, which integrates amino acid interaction mapping into scaffold-hopping (AI-AAM). At first, we used a spleen associated tyrosine kinase (SYK) inhibitor as a reference to evaluate the technique, and succeeded in scaffold-hopping maintaining the pharmacological activity. Then we applied this method to five drugs and obtained 144 compounds with diverse structures. Among these, 31 compounds were known to target the same proteins as their reference compounds and 113 compounds were known to target different proteins. We found that AI-AAM dominantly selected functionally similar compounds; thus, these selected compounds may represent improved alternatives to their reference compounds. Moreover, the latter compounds were presumed to bind to the targets of their references as well. This new "compound-target" information provided DR candidates that could be utilized for future drug development.

pharmacology and toxicology↗

bioRxiv2023

Pax3 and Pax7 function in combination with Mitf to generate melanophores and xanthophores in medaka and zebrafish

Neural crest cells generate numerous derivatives, including pigment cells, and are a model for studying how fate specification from multipotent progenitors is controlled. In mammals, the core gene regulatory network (GRN) for melanocytes (their only pigment cell-type) contains three transcription factors Sox10, Pax3 and Mitf, with the latter considered a master regulator of melanocyte development. In teleosts, which have three to four pigment cell types (melanophores, iridophores and xanthophores, plus leucophores e.g. in medaka), GRNs governing fate specification are poorly understood, although Mitf function is considered conserved. Here, we show that the regulatory relationships between Sox10, Pax3 and Mitf are conserved in zebrafish, but the role for Mitf is more complex than previously emphasised, affecting xanthophore development too. Similarly, medaka Mitf is necessary for melanophore, xanthophore and leucophore formation. Furthermore, expression patterns and mutant phenotypes of pax3 and pax7 suggest that Pax3 and Pax7 act sequentially, activating mitf expression. Pax7 modulates Mitf function, driving co-expressing cells to differentiate as xanthophores and leucophores rather than melanophores. We propose that pigment cell fate specification is better considered as resulting from the combinatorial activity of Mitf with other transcription factors.

developmental biology↗

bioRxiv2023

Thrombospondin 2 is a key determinant of fibrogenesis in NAFLD

Hepatic overexpression of the thrombospondin 2 gene (THBS2) and elevated levels of circulating thrombospondin 2 (TSP2) have been observed in patients with chronic liver disease. The current study aimed to identify the specific cells expressing THBS2/TSP2 in non-alcoholic fatty liver disease (NAFLD) and investigate the underlying mechanism behind THBS2/TSP2 up-regulation. Comprehensive NAFLD liver gene datasets, including single-cell RNA sequencing (scRNA-seq), in-house NAFLD liver tissue, and LX-2 cells derived from human hepatic stellate cells (HSCs), were analyzed using a combination of computational biology, genetic, immunological, and pharmacological approaches. Analysis of the genetic dataset revealed the presence of 1433 variable genes in patients with advanced fibrosis NAFLD, with THBS2 ranked among the top 2 genes. Quantitative polymerase chain reaction examination of NAFLD livers showed a significant correlation between THBS2 expression and fibrosis stage (r=0.349, p<0.001). In support of this, scRNA-seq data and in situ hybridization demonstrated that the THBS2 gene was highly expressed in HSCs of NAFLD patients with advanced fibrosis. Pathway analysis of the gene dataset revealed THBS2 expression to be associated with the transforming growth factor beta (TGF{beta}) pathway and collagen gene activation. Moreover, the activation of LX-2 cells with TGF{beta} increased THBS2/TSP2 and collagen expression independently of the TGF{beta}-SMAD2/3 pathway. THBS2 gene knockdown significantly decreased collagen expression in LX-2 cells. In conclusion, THBS2/TSP2 is highly expressed in HSCs and plays a role in regulating fibrogenesis in NAFLD patients. THBS2/TSP2 may therefore represent a potential target for anti-fibrotic therapy in NAFLD. (241 words) One-sentence summariesThrombospondin 2 represent a potential target for anti-fibrotic therapy in NAFLD.

molecular biology↗

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↗

bioRxiv2022

Analysis of splicing abnormalities in the white matter of myotonic dystrophy type 1 brain using RNA-sequence

Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by the genomic expansions of CTG repeats, in which RNA-binding proteins, such as muscleblind-like protein, are sequestered in the nucleus, and abnormal splicing is observed in various genes. Although abnormal splicing reportedly occurs in the brains of patients with DM1, it is relation to the central nervous system symptoms is unknown. Several imaging studies have indicated substantial white matter (WM) defects in patients with DM1. Here, we performed RNA-sequencing and analysis of CTG repeat lengths in the frontal lobe of patients with DM1, separating the grey matter (GM) and WM, to investigate the splicing abnormalities in the DM1 brain, especially in the WM. The results demonstrated the number of repeats in the GM tended to be increase, with several genes showing similar levels of splicing abnormalities in the GM and WM, suggesting that the WM defects in DM1 are not only caused by aberrant splicing of GM RNA but also of WM RNA, which could be attributed to abnormal splicing of glial cell RNAs.

molecular biology↗

bioRxiv2022

The gradient-based parameter optimization to determine membrane ionic current composition of human induced pluripotent stem cell-derived cardiomyocytes

1.Premature cardiac myocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) show heterogeneous action potentials (APs), most probably because of different expression patterns of membrane ionic currents. We aim to develop a method of determining expression patterns of functional channels in terms of the whole-cell ionic conductances (Gx) using individual spontaneous AP configurations. However, it has been suggested that apparently identical AP configurations were obtained by different sets of ionic currents in a mathematical model of cardiac membrane excitation. If so, the inverse problem of Gx estimation might not be solved. We computationally tested the feasibility of the gradient-based optimization method. For realistic examination, conventional cell-specific models were prepared by superimposing the model output of AP on each experimental AP record by the conventional manual adjustment of Gxs of the baseline model. Then, Gxs of 4 ~ 6 major ionic currents of the cell-specific models were randomized within a range of {+/-}5 ~ 15% and were used as initial parameter sets for the gradient-based automatic Gxs recovery by decreasing the mean square error (MSE) between the target and model output. When plotted all data points of MSE - Gx relation during the optimization, we found that the randomized population of Gxs progressively converged to the original value of the cell-specific model with decreasing MSE. To confirm the absence of any other local minimum in the global search space, we mapped the MSE by randomizing Gxs over a range of 0.1 ~ 10 times the control. No additional local minimum of MSE was obvious in the whole parameter space besides the global minimum of MSE at the default model parameter.

physiology↗

bioRxiv2022

The swan genome and transcriptome: its not all black and white

The Australian black swan (Cygnus atratus) is an iconic species with contrasting plumage to that of the closely related Northern Hemisphere white swans. The relative geographic isolation of the black swan may have resulted in a limited immune repertoire and increased susceptibility to infectious disease, notably infectious diseases from which Australia has been largely shielded. Indeed, unlike Mallard ducks and the mute swan (Cygnus olor), the black swan is extremely sensitive to severe highly pathogenic avian influenza (HPAI). Understanding this susceptibility has been impaired by the absence of any available swan genome and transcriptome information. Here, we generate the first chromosome-length annotated black and mute swan genomes annotated with transcriptome data, all using long-read based pipelines generated for vertebrate species. We used these genomes and transcriptomes, to show that unlike other wild waterfowl, black swans lack an expanded immune gene repertoire, lack a key viral pattern-recognition receptor in endothelial cells and mount a poorly controlled inflammatory response to HPAI. We also implicate genetic differences in SLC45A2 in the iconic plumage of the Australian black swan. Together, these data suggest that the immune system of the black swan is such that should any avian viral infection become established in its native habitat the survival of the black swan would be in significant peril.

genomics↗

bioRxiv2022

A Single Cell Cloning Platform for Gene Edited Functional Murine Hematopoietic Stem Cells

Gene editing using engineered nucleases frequently produces on- and off-target indels in hematopoietic stem cells (HSCs). Gene-edited HSC cultures thus contain genetically heterogenous populations, the majority of which either do not carry the desired edit or harbor unwanted mutations. In consequence, transplanting edited HSCs carries the risks of suboptimal efficiency and of unwanted mutations in the graft. Here, we present an approach for expanding gene-edited HSCs at clonal density, allowing for genetic profiling of individual clones before transplantation. We achieved this by developing a defined, polymer-based expansion system and identifying long-term expanding clones within the CD201+CD150+CD48-c-Kit+Sca-1+Lin- population of pre-cultured HSCs. Using the Prkdcscid immunodeficiency model, we demonstrate that we can expand and profile edited HSC clones to check for desired and unintended modifications. Transplantation of Prkdc-corrected HSCs rescued the immunodeficient phenotype. Our ex vivo-manipulation platform establishes a novel paradigm to control genetic heterogeneity in HSC gene editing and therapy.

cell biology↗

bioRxiv2022

Self-organization, quality control, and preclinical studies of human iPSC-derived retinal sheets for tissue-transplantation therapy

Three-dimensional retinal organoids (3D-retinas) are a promising graft source for transplantation therapy. We previously developed self-organizing culture for 3D-retina generation from human pluripotent stem cells (hPSCs). Here we present a quality control method and preclinical studies for tissue-sheet transplantation. Self-organizing hPSCs differentiated into both retinal and off-target tissues. Gene expression analyses identified the major off-target tissues as eye-related, cortex-like, and spinal cord-like tissues. For quality control, we developed a qPCR-based test in which each hPSC-derived neuroepithelium was dissected into two tissue-sheets: inner-central sheet for transplantation and outer-peripheral sheet for qPCR to ensure retinal tissue selection. During qPCR, tissue-sheets were stored for 3-4 days using a newly developed preservation method. In a rat tumorigenicity study, no transplant-related adverse events were observed. In retinal degeneration model rats, retinal transplants differentiated into mature photoreceptors and exhibited light responses in electrophysiology assays. These results demonstrate our rationale toward self-organizing retinal sheet transplantation therapy.

neuroscience↗

bioRxiv2021

Manipulating Histone Acetylation Leads to Adverse Effects in Hemangiosarcoma Cells

Canine hemangiosarcoma (HSA) is a malignant tumour derived from endothelial cells. No effective treatment has yet been developed because of the lack of understanding of its pathogenesis. Histone acetylation, an epigenetic modification, is highly associated with cancer pathogenesis. Manipulating histone acetylation by histone deacetylase inhibitors (HDACi) or bromodomain and extraterminal domain inhibitors (BETi) is one approach to treat various cancers. However, the role of histone acetylation in HSA remains unknown. This study aimed to investigate how histone acetylation functions in HSA pathogenesis using two HDACi, suberanilohydroxamic acid (SAHA) and valproic acid (VPA), and one BETi, JQ1, in vitro and in vivo. Histone acetylation levels were high in cell lines and heterogeneous in clinical cases. SAHA and JQ1 induced apoptosis in HSA cell lines. SAHA and VPA treatment in HSA cell lines upregulated inflammatory-related genes, thereby attracting macrophages. This implies that SAHA and VPA can induce anti-tumour immunity. JQ1 stimulated autophagy and inhibited the cell cycle. Finally, JQ1 suppressed HSA tumour cell proliferation in vivo. These results suggest that HDACi and BETi can be alternative drugs for HSA treatment. Although further research is required, this study provides useful insights for developing new treatments for HSA.

cancer biology↗

bioRxiv2021

Myosin phosphatase target subunit 1 governs integrity of the embryonic gut epithelium to circumvent atresia development in medaka, Oryzias latipes

Congenital intestinal atresia (IA) is a birth defect characterised by the absence or closure of part of the intestine. Although genetic factors are implicated, mechanistic understanding has been hindered by the lack of suitable animal models. Here, we describe a medaka (Oryzias latipes) mutant, generated by N-ethyl-N-nitrosourea (ENU) mutagenesis, that develops IA during embryogenesis. Positional cloning identified a nonsense mutation in mypt1, encoding myosin phosphatase target subunit 1. Mutant embryos exhibited ectopic accumulation of F-actin and phosphorylated myosin regulatory light chain (Mrlc) in the intestinal epithelium, consistent with disrupted actomyosin regulation. These cytoskeletal abnormalities were accompanied by epithelial disorganisation without notable alterations in cell proliferation, motility, or apoptosis. Inhibition of myh11a, encoding smooth muscle (SM) myosin heavy chain, ameliorated the IA phenotype but Blebbistatin treatment completely rescued the defect, suggesting a non-contractile role prior to SM maturation. Together, these findings demonstrate that mypt1 loss disrupts intestinal morphogenesis through actomyosin dysregulation. Given the recent clinical identification of IA associated with MYPT1 mutations, this medaka model offers a valuable platform to investigate the developmental and molecular basis of MYPT1-associated IA in human.

developmental 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

Hemangiosarcoma cells induce M2 polarization and PD-L1 expression in macrophages

Hemangiosarcoma (HSA) is a malignant tumor derived from endothelial cells. Tumor-associated macrophages are one of the major components of tumor microenvironment and crucial for cancer development. The presence and function of macrophages in HSA have not been studied because there is no syngeneic model for HSA. In this study, we evaluated two mouse HSA cell lines and one immortalized mouse endothelial cells for their usefulness as syngeneic models for canine HSA. Our results show that the ISOS-1 cell line develops tumors with similar morphology to canine HSA. ISOS-1 cells highly express KDM2B and have similar KDM2B target expression patterns with canine HSA. Moreover, we determine that in both ISOS-1 and canine HSA tumors, macrophages are present as a major constituent of the tumor microenvironment. These macrophages are positive for CD204, an M2 macrophage marker, and express PD-L1. ISOS-1-conditioned medium can induce M2 polarization and PD-L1 expression in RAW264.7 mouse macrophage cell line. These results show that ISOS-1 can be used as a syngenic model for canine HSA and suggest that macrophages play an important role in immune evasion in HSA. Using the syngeneic mouse model for canine HSA, we can further study the role of immune cells in the pathology of HSA.

cancer biology↗

bioRxiv2021

Tracheal motile cilia require CAMSAP3 for formation of central microtubule pair and coordinated beating

Motile cilia of multiciliated epithelial cells undergo synchronized beating to produce fluid flow along the luminal surface of various organs. Each motile cilium consists of an axoneme and a basal body, which are linked by a transition zone. The axoneme exhibits a characteristic 9+2 microtubule arrangement important for ciliary motion, but how this microtubule system is generated is not yet fully understood. Here we show that CAMSAP3, a protein that can stabilize the minus end of a microtubule, concentrates at multiple sites of the cilium-basal body complex, including the upper region of the transition zone or the axonemal basal plate where the central pair of microtubules (CP) initiates. CAMSAP3 dysfunction resulted in loss of the CP and partial distortion of the basal plate, as well as the failure of multicilia to undergo synchronized beating. These findings suggest that CAMSAP3 plays pivotal roles in the formation or stabilization of the CP by localizing at the basal region of the axoneme, and thereby supports the coordinated motion of multicilia in airway epithelial cells.

cell biology↗