Biology subjects

Naito, Y.

Publications and source records attributed to Naito, Y..

15 recordsLinked to original sources

bioRxiv2026

NAT10 supports CAG repeat-associated mRNA stabilization and is associated with hepatic metabolic reprogramming during spaceflight

The regulatory factors underlying posttranscriptional adaptation to spaceflight are incompletely understood. We identified NAT10, an RNA acetyltransferase, among RNA modification enzymes significantly upregulated in spaceflight-exposed mouse liver. Transcriptome-wide correlation analysis linked NAT10 expression to an RNA-metabolism module enriched for CAG repeat density in coding sequences. Actinomycin D chase assays showed that NAT10 knockdown destabilized CAG repeat-containing transcripts, with DDX17 serving as the primary validated target; DCP1A showed a directionally consistent pattern. Wild-type NAT10 re-expression fully rescued DDX17 mRNA stability, the acetyltransferase-dead G641E mutant rescued mRNA stability to a comparable extent, and the K290A helicase-domain mutant failed to rescue, consistent with a requirement for helicase domain integrity, whereas acetyltransferase activity was not strictly required under these experimental conditions. RNA immunoprecipitation further supported preferential NAT10 association with CAG repeat-containing mRNAs, with reduced association after single-site CAGCAG deletion in DDX17 and DCP1A reporters. Gene set enrichment analysis associated NAT10 upregulation in spaceflight liver with suppressed fatty acid beta-oxidation and induced cholesterol biosynthesis; NAT10 depletion in hepatocellular carcinoma cells selectively opposed the cholesterol-associated, but not the broader metabolic, component of this signature. These findings support a model linking NAT10 to CAG repeat-associated mRNA stabilization in the context of hepatic metabolic and posttranscriptional adaptation to spaceflight.

molecular biology↗

bioRxiv2026

Stepwise bladder carcinogenesis reveals transcriptional reprogramming involving MMP1 and COL7A1

Bladder cancer develops through multistep molecular alterations, yet the events underlying tumor initiation and progression remain poorly understood. Here, we reconstructed stepwise bladder carcinogenesis using canine bladder organoids combined with chemical carcinogenesis and serial xenotransplantation. Carcinogen-exposed normal organoids generated benign tumors that subsequently progressed to invasive carcinomas. Despite a low tumor mutational burden and limited acquisition of cancer-associated mutations, malignant progression was accompanied by extensive transcriptional reprogramming, including enrichment of epithelial-to-mesenchymal transition programs. COL7A1 and MMP1 were progressively upregulated during progression, and knockdown of either gene suppressed organoid proliferation and invasion. These functional dependencies were reproduced in independently established spontaneous canine bladder cancer organoids, where silencing of either gene also reduced xenograft growth and mitosis-related gene programs. MMP1 knockdown decreased COL7A1 expression, suggesting a potential regulatory relationship. MMP1 was also elevated in basal/squamous human bladder cancers, and its depletion suppressed human bladder cancer cell proliferation. These findings identify transcriptional reprogramming and MMP1/COL7A1 dependencies as prominent features of malignant progression.

cancer biology↗

bioRxiv2026

EV-Tracer enables lineage-resolved detection and molecular profiling of extracellular vesicle-associated signals in cancer-fibroblast co-culture

Extracellular vesicles (EVs) facilitate intercellular communication by transferring diverse bioactive molecules from donor to recipient cells. However, EVs released by distinct cellular lineages become difficult to distinguish when mixed in multicellular experimental models, limiting the analysis of how cell-cell interactions affect EV-associated molecular profiles. To address this, EV-Tracer, a CD63-based dual-fluorescence tracing and capture system for detecting, isolating, and profiling lineage-associated EV fractions, was developed. Achilles or mScarlet was inserted into the small extracellular loop of CD63, enabling tracer-specific EV detection by digital counting, antibody-based isolation, and live-cell visualisation. Exploratory EV RNA sequencing suggested that physical cell-cell contact was associated with distinct EV RNA profiles, including interferon-related signals, which were supported by targeted cellular and EV-associated RNA analyses. EV-Tracer provides a practical framework for investigating lineage-associated EV dynamics and molecular signals in mixed-cell systems.

cell biology↗

bioRxiv2025

Cell migration sculpts evolutionary dynamics favouring therapy resistance in lung cancer

The evolution of cancer undermines the long-term efficacy of therapy. In this study, we combine experimental, computational, and clinical data analysis to investigate factors influencing the competition of subclones in lung cancer. Lineage tracing reveals unexpected variation in the long-term fate of neutral subclones, with subclones arising near the edge of tumours being favoured. Low levels of cell migration and cell mixing lead to high cell densities in the interior of the tumour that suppress proliferation. Using agent-based modelling and in silico analysis we inferred the extent of cell mixing in human tumours from the TRACERx lung cancer study. This reveals correlations between Epithelial to Mesenchymal Transition (EMT), stromal fibroblasts and levels of cell mixing. Experimental analysis confirms that both TGF{beta}-driven EMT and stromal fibroblasts reduce the variability in subclone fate and promote subclone mixing. Moreover, mixing favours clonal sweeps by subclones resistant to therapy-induced cell killing. Together, these analyses demonstrate that EMT and stromal fibroblasts sculpt tumour evolution by promoting cell mixing and thereby favour the rapid dominance of therapy resistant subclones.

cancer biology↗

bioRxiv2025

Oral antigen exposure under co-stimulation blockade generates Treg cells to establish immune tolerance despite prior sensitization

Antigen-specific oral tolerance is effective in preventing harmful immune responses in antigen-non-sensitized animals but difficult to be induced in those already antigen-primed. Here, we show in mice that feeding of antigen-containing diet generates peripherally derived regulatory T (pTreg) cells that exhibit a tissue-adapted effector phenotype. The antigen feeding also enables the generated pTreg cells to acquire Treg-specific epigenomic changes in Treg signature genes including Foxp3, hence the stability of Treg-specific function. Cessation of antigen feeding, however, results in decline in oral tolerance with diminution of pTreg cells. Transcriptomic analysis has revealed that the induced pTreg cells predominantly express CD101. CD101+ Treg cells with a similar phenotype and epigenetic alterations can also be generated in vitro from antigen-primed naive CD4+ T cells by blockade of CD28-mediated co-stimulation during TGF-{beta}-dependent Treg induction. Furthermore, in vivo CD28 signal blockade by CTLA-4-immunoglobulin fusion protein (CTLA4-Ig) before antigen feeding can establish oral tolerance in the mice precedingly antigen-sensitized by a non-oral route. The blockade facilitates differentiation of antigen-specific conventional T cells into CD101+ pTreg cells. Thus, continuous oral antigen administration combined with CD28 co-stimulation blockade generates CD101+ antigen-specific functionally stable pTreg cells, which can establish long-term systemic antigen-specific immune tolerance even in antigen-pre-sensitized animals.

immunology↗

bioRxiv2025

Risk Prediction of RNA Off-Targets of CRISPR Base Editors in Tissue-Specific Transcriptomes Using Language Models

Base-editing technologies, particularly cytosine base editors (CBEs), allow precise gene modification without introducing double-strand breaks; however, unintended RNA off-target effects remain a critical concern and are under-studied. To address this gap, we developed PiCTURE, a standardized computational pipeline for detecting and quantifying transcriptome-wide CBE-induced RNA off-target events. PiCTURE identifies both canonical ACW (W = A or T/U) motif-dependent and non-canonical RNA off-targets, revealing a broader WCW motif that underlies many unanticipated edits. Additionally, we developed two machine learning models based on the DNABERT-2 language model, termed STL and SNL, which outperformed motif-only approaches in terms of accuracy, precision, recall, and F1 score. To demonstrate the practical application of our predictive model for CBE-induced RNA off-target risk, we integrated PiCTURE outputs with the PROTECTiO pipeline and estimated RNA off-target risk for each transcript showing tissue-specific expression. The analysis revealed differences among tissues: while the brain and ovaries exhibited relatively low off-target burden, the colon and lungs displayed relatively high risks. Our study provides a comprehensive frame-work for RNA off-target profiling, emphasizing the importance of advanced machine learning-based classifiers in CBE safety evaluations and offering valuable insights for the development of safer genome-editing therapies.

genomics↗

bioRxiv2024

Ancient biomolecules suggest learned foraging strategy in extinct cave bears

Studying the behavioural ecology of long-extinct species is challenging due to the difficulty in measuring the behavioural phenotype and correlating this with genetic and environmental factors. However, a multidisciplinary approach integrating isotope analysis of diet and ancient DNA analysis of genetic relationships offers a potential framework to test the proximate causes of dietary preferences. Our study focuses on Late Pleistocene cave bears from the Romanian Carpathians. Stable isotope analysis of bone collagen reveals substantial lifetime variation in food plant preferences among individuals. We find that bears with similar diets do not cluster according to their population structure, sex, time period, climatic conditions, or location. This disconnect suggests that diet preference in cave bears is not genetically inherited, and instead that individuals adapted their diets based on foraging experience. This integrative approach opens new avenues for understanding Pleistocene animal behaviour, leveraging ancient biomolecules synergistically to reveal insights otherwise inaccessible.

evolutionary biology↗

bioRxiv2024

Isotonic and minimally invasive optical clearing media for live cell imaging ex vivo and in vivo

Tissue clearing has been widely used for fluorescence imaging of fixed tissues, but not for live tissues due to its toxicity. Here we develop minimally invasive optical clearing media for fluorescence imaging of live mammalian tissues. Light scattering is minimized by adding spherical polymers with low osmolarity to the extracellular medium. A clearing medium containing bovine serum albumin (SeeDB-Live) is minimally invasive to live cells, allowing for structural and functional imaging of live tissues, such as spheroids, organoids, acute brain slices, and the mouse brain in vivo. SeeDB-Live minimally affects the electrophysiological properties and sensory responses of neurons. We demonstrate its utility for widefield imaging of subcellular voltage dynamics, such as backpropagating action potentials, in acute brain slices. We also utilize SeeDB-Live for widefield voltage imaging of dozens of dendrites in vivo, demonstrating population dynamics. Thus, SeeDB-Live expands the scale and modalities of fluorescence imaging of live mammalian tissues.

neuroscience↗

bioRxiv2024

A novel and potent MICA/B antibody is therapeutically effective in KRAS LKB1 mutant lung cancer models

Concurrent KRAS LKB1 (STK11, KL) mutant Non-Small Cell Lung Cancers (NSCLC) is particularly difficult to treat and does not respond well to current immune checkpoint blockade (ICB) therapies. This is due to numerous mechanisms including low antigen presentation limiting T cell mediated killing. To activate anti-tumor immunity, we targeted tumor cell - natural killer (NK) cell interactions. We tested whether a novel antibody based therapeutic strategy that predominantly activates natural killer (NK) cells demonstrates efficacy in pre-clinical mouse models of KL NSCLC. NK cells rely on binding of ligands, such as Major Histocompatibility Complex (MHC) class I-related chain A or B (MICA/B), to the activating receptor NKG2D. Importantly MICA and MICB are widely expressed in elevated levels across NSCLC subtypes including KL lung cancers. Proteases with the tumor microenvironment (TME) can cleave these proteins rendering tumor cells less visible to NK cells. We therefore developed a MICA monoclonal antibody, AHA-1031, which utilizes two NK cell activating receptors. AHA1031 prevents ligand shedding without interfering with binding to NKG2D while targeting cancer cells to antibody mediated cell dependent cytotoxicity (ADCC). Our therapeutic novel antibody has significant monotherapy activity in KL cancer models including xenografts of human cell lines and patient derived xenografts. Activating NK cells through MICA/B stabilization and inducing ADCC offers an alternative and potent therapy option in KL tumors. MICA/B are shed across different tumors making this therapeutic strategy universally applicable.

immunology↗

bioRxiv2023

Structure and function of a YeeE-YeeD complex for sophisticated thiosulfate uptake

Uptake of thiosulfate ions as an inorganic sulfur source from the environment is important for bacterial sulfur assimilation. Recently, a selective thiosulfate uptake pathway involving a membrane protein YeeE (TsuA) in Escherichia coli was characterized. YeeE-like proteins are conserved in some bacteria, archaea, and eukaryotes. However, the precise function of YeeE, along with its potential partner protein in the thiosulfate ion uptake pathway, remained unclear. Here, we assessed selective thiosulfate transport via Spirochaeta thermophila YeeE in vitro and characterized E. coli YeeD (TsuB) as an adjacent and essential protein for YeeE-mediated thiosulfate uptake in vivo. We further showed that S. thermophila YeeD possesses thiosulfate decomposition activity and that a conserved cysteine in YeeD was modified to several forms in the presence of thiosulfate. Finally, the crystal structures of S. thermophila YeeE-YeeD fusion proteins at 3.34-[A] and 2.60-[A] resolutions revealed their interactions. The association was evaluated by a binding assay using purified S. thermophila YeeE and YeeD. Based on these results, a model of the sophisticated uptake of thiosulfate ions by YeeE and YeeD is proposed. 125-character sentenceThe structural study of the YeeE-YeeD membrane protein complex provides insight into the thiosulfate uptake and degradation mechanism.

biochemistry↗

bioRxiv2023

Cancer cell - fibroblast crosstalk signature promotes macrophage recruitment via HB-EGF and stromal EGFR/MEK signalling

Interactions between cells in the tumor microenvironment (TME) shape cancer progression and patient outcomes. To gain new insights into how the TME influences cancer outcomes, we derive gene expression signatures indicative of signaling between stromal fibroblasts and cancer cells, and demonstrate their prognostic significance in multiple and independent squamous cell carcinoma cohorts. By leveraging information within the signatures, we discover that the HB-EGF/EGFR/MEK axis represents a hub of tumor - stroma crosstalk, promoting the expression of CSF2 and LIF and favoring the recruitment of macrophages. Together these analyses demonstrate the utility of our approach for interrogating the extent and consequences of TME crosstalk.

cancer biology↗

bioRxiv2023

mRNA vaccine induces protective immunity against the type III secretory virulence of Pseudomonas aeruginosa

Few studies have addressed mRNA vaccination against bacteria despite the desperate need to control antimicrobial resistance. Herein, we developed mRNA vaccines targeting the type III secretion system of Pseudomonas aeruginosa, which plays a critical role in the pathogenesis. Vaccination with a nucleoside-modified mRNA encapsulated in lipid nanoparticles improved mouse survival, reduced the bacterial load in the lung, and alleviated pathological changes in the lungs after bacterial challenge.

immunology↗

bioRxiv2022

Border-associated macrophages transventricularly infiltrate the early embryonic cerebral wall to differentiate into microglia

The relationships between microglia and macrophages, especially their lineage segregation outside the yolk sac, have been recently explored, providing a model in which a conversion from macrophages seeds microglia during brain development. However, spatiotemporal evidence to support such microglial seeding and to explain how it occurs has not been obtained. By cell tracking via slice culture, intravital imaging, and Flash tag-mediated labeling, we found that a group of intraventricular macrophages belonging to border-associated macrophages (BAMs), which were abundantly observed along the inner surface of the mouse cerebral wall at embryonic day 12, frequently entered the brain wall. Immunohistochemistry of the tracked cells showed that postinfiltrative BAMs acquired microglial properties while losing a macrophage phenotype. We also found that the intraventricular BAMs were supplied transepithelially from the roof plate. Thus, this study demonstrates that the "roof plate[->]ventricle[->]cerebral wall" route is an essential path for microglial colonization into the embryonic mouse brain.

neuroscience↗

bioRxiv2022

Iron Deficiency Induces Heart Failure with Ectopic Cardiac Calcification in Mice with Metabolic Syndrome

Iron deficiency is linked to worse clinical status and outcomes in heart failure. Although metabolic syndrome contributes to the development of heart failure, the impact of iron deficiency in heart failure complicated with metabolic syndrome remains obscure. KKAy mice were used as a model of metabolic syndrome. Four-week-old male C57BL/6J and KKAy mice were fed either a normal diet or iron-restricted (IR) diet for 12 weeks. During the experiment, 40% of mice died due to pulmonary congestion in KKAy mice with IR diet (KKAy-IR), while no mice died in other groups. Necropsy showed the presence of multiple white lesions on the cardiac surface in those KKAy-IR mice. Echocardiography and histological analyses revealed that KKAy-IR mice exhibited cardiac hypertrophy and cardiac dysfunction with cardiac calcification. Cardiac mRNA of ectonucleotide pyrophosphatase/phosphodiesterase-1 (Enpp1), a key enzyme for bone mineralization, was highly abundant in KKAy-IR mice. Of note, iron restriction-induced cardiac calcification and dysfunction were attenuated by etidronate, an inhibitor of bone mineralization, with decreased cardiac Enpp1 mRNA abundance in KKAy-IR mice. In conclusion, iron deficiency leads to ectopic cardiac calcification and dysfunction with the increase of Enpp1 in metabolic syndrome model mice.

pathology↗

bioRxiv2020

Full-length 16S rRNA gene amplicon analysis of human gut microbiota using MinION™ nanopore sequencing confers species-level resolution

BackgroundSpecies-level genetic characterization of complex bacterial communities has important clinical applications in both diagnosis and treatment. Amplicon sequencing of the 16S ribosomal RNA (rRNA) gene has proven to be a powerful strategy for the taxonomic classification of bacteria. This study aims to improve the method for full-length 16S rRNA gene analysis using the nanopore long-read sequencer MinION. We compared it to the conventional short-read sequencing method in both a mock bacterial community and human fecal samples. ResultsWe modified our existing protocol for full-length 16S amplicon sequencing by MinION. A new strategy for library construction with an optimized primer set overcame PCR-associated bias and enabled taxonomic classification across a broad range of bacterial species. We compared the performance of full-length and short-read 16S amplicon sequencing for the characterization of human gut microbiota with a complex bacterial composition. The relative abundance of dominant bacterial genera was highly similar between full-length and short-read sequencing. At the species level, MinION long-read sequencing had better resolution for discriminating between members of particular taxa such as Bifidobacterium, allowing an accurate representation of the sample bacterial composition. ConclusionsOur present microbiome study, comparing the discriminatory power of full-length and short-read sequencing, clearly illustrated the analytical advantage of sequencing the full-length 16S rRNA gene, which provided the requisite species-level resolution and accuracy in clinical settings.

microbiology↗