Biology subjects

Tamura, T.

Publications and source records attributed to Tamura, T..

At least 19 recordsLinked to original sources

bioRxiv2026

A novel recombinant BCG vaccine using a mycobacteriophage promoter shows improved protection against tuberculosis

The limited efficacy of conventional bacillus Calmette-Guerin (BCG) vaccination against adult pulmonary tuberculosis highlights the need for improved vaccine strategies. Genome integration can overcome the plasmid instability of recombinant BCG, but we hypothesized that this approach would be constrained by reduced transcriptional activity of conventional promoters when present as a single genomic copy. Here we identified P79, a mycobacteriophage-derived promoter that sustains high-level expression from a single genomic locus and used it to construct a genome-integrated recombinant BCG, termed LRC-BCG. LRC-BCG secretes the HSP70-MMPII fusion antigen flanked by the PEST sequences, maintained stable antigen expression over serial passages, and induced strong activation of macrophages and dendritic cells in vitro. In vivo, LRC-BCG inhibited Mycobacterium tuberculosis multiplication more effectively than conventional BCG, even at low doses. These findings demonstrate the feasibility of using P79 for stable genome-integrated recombinant BCG and support LRC-BCG as a promising tuberculosis vaccine candidate.

microbiology↗

bioRxiv2026

Developmental Gfi1 Dynamics Define Hematopoietic Emergenceand Adult Hematopoietic Stem Cell Potency

Gfi1 regulates endothelial-to-hematopoietic transition (EHT) and hematopoietic stem cell (HSC) maintenance; however, its expression dynamics remain unclear. We generated a non-disruptive Gfi1-T2A-mScarlet reporter to track Gfi1 expression. At E10.5, reporter selectively marked EHT in the dorsal aorta, umbilical artery, and vitelline artery. At E9.5, mScarlet-positive endothelial cells were present in the umbilical and vitelline arteries but were scarce in the dorsal aorta, indicating vascular-bed-specific differences in hemogenic activation. Gfi1 was broadly expressed in fetal liver HSCs, while higher expression levels identified HSCs with enhanced multilineage reconstitution and preferential T-cell output. Although Gfi1 expression declined during fetal-to-adult maturation, a subset of adult bone marrow HSCs retained expression and exhibited superior repopulating and self-renewal capacity. Bulk and single-cell transcriptomic analyses linked this subset to dormant and fetal-associated programs, including imprinted genes. Reduced chromatin accessibility at a conserved Gfi1 +26.5kb putative enhancer correlated with developmental Gfi1 downregulation. Thus, Gfi1 dynamics define EHT onset and functionally distinct HSC stemness.

cell biology↗

bioRxiv2026

DNA barcoding-based paired daughter cell analysis reveals division preferences of hematopoietic stem cells

Hematopoietic stem cells(HSCs) maintain their pools by stem-stem division and produce mature blood cells through stem-progenitor or progenitor-progenitor division. A paired daughter cell(PDC) assay combined with single cell transplantation is a powerful method to compare the lineage outputs of two HSC daughter cells. However, single-cell transplantation precludes large-scale analysis of daughter-cell pairs, as only one cell can be transplanted per recipient. Here, we developed a DNA barcoding-based PDC assay to overcome this limitation, enabling simultaneous analysis of 476 daughter pairs from individual HSC divisions and revealing that daughter-cell fates are coordinated and that HSC division patterns are biased toward stem-stem and progenitor-progenitor divisions rather than stem-progenitor divisions. These findings indicate that HSC fate outcomes are directed toward symmetric division outcomes. Integration of single-cell RNA sequencing with DNA barcoding revealed a continuum of HSC states--from balanced HSCs to myeloid-biased HSCs and ultimately to a low-output HSC subset--in which progressively reduced production of mature blood cells relative to stem cell expansion, a proxy for stem-stem division bias, exhibits distinct activities of transcription factors and signaling pathways. Overall, our analysis uncovers characteristic patterns of HSC division and links stem maintenance with distinct molecular features.

cell biology↗

bioRxiv2026

Patterned photocatalytic proximity labelling for spatial interactomics

Mapping protein-protein interactions and proximity within their native spatial context is essential for elucidating molecular mechanisms underlying biological processes; however, methods for spatial interactomics with subanatomical resolution are limited. Here, we introduce digital micromirror device (DMD)-PhoxID, a method that combines photocatalytic proximity labelling with patterned irradiation using a DMD to map the proximal molecular environment of proteins of interest within defined subregions of fixed brain tissue.

neuroscience↗

bioRxiv2026

RIPK3-RNASE1 axis as a potential therapeutic and clinical monitoring target in VEXAS syndrome

Vacuole, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) syndrome (VS) is a severe autoinflammatory disease driven by somatic mutations in ubiquitin-like modifier activating enzyme 1 (UBA1), for which disease activity biomarkers and therapeutic targets are needed. We conducted longitudinal deep phenotyping of patients with VS and found that ribonuclease 1 (RNASE1) expression is most strongly correlated with the disease activity score measured by the VEXAS Current Activity Form (VEXASCAF). Single-cell RNA-sequencing showed that RNASE1 was upregulated in VS monocytes. We generated human monocytic cell lines harboring UBA1 mutations (p.Met41Val, p.Met41Leu, and p.Met41Thr). These cells exhibited varying degrees of impaired ubiquitination and subsequent pathological features such as the unfolded protein response, increased pro-inflammatory cytokine production, cell death, and RNASE1 expression, mirroring the genotype-phenotype associations observed in patients with VS. Multi-omics analyses revealed that genotypes linked to greater clinical severity were enriched in pro-inflammatory, interferon, and necroptosis signatures. Notably, functional interrogation demonstrated that inhibition of receptor-interacting protein kinase 3 (RIPK3), a key regulator of necroptosis, markedly suppressed all these pathological features, including the elevated RNASE1 expression. These results identify the RIPK3-RNASE1 axis in VS, highlighting RNASE1 as a potential biomarker and RIPK3 as an attractive therapeutic target.

immunology↗

bioRxiv2026

Transmembrane PhoxID: Photo-proximity labelling across the plasma membrane

Transmembrane proteins perform essential roles in cellular transport, signalling, and communication. The function and dynamics of these proteins are precisely regulated by interactions on both sides of the plasma membrane; thus, mapping the composition of these interactomes is a fundamental challenge in molecular biology. Proximity labelling methods are powerful tools for this purpose; however, existing approaches that rely on membrane-impermeable reactive species are limited by the ability to detect only one side, either the extracellular or the intracellular region, of transmembrane proteins. Here, we capitalized on the cell permeability of singlet oxygen to carry out proximity labelling of the cytoplasmic side of transmembrane proteins using an extracellularly anchored photosensitizer. We applied this method, termed transmembrane PhoxID (tmPhoxID), to several receptors (GRID2, GABAAR, and GRM1) in the living mouse brain and successfully determined their specific intracellular interactomes. Notably, network analysis of the identified proteins revealed that this method can characterize the native components of transsynaptic nanocolumns formed at parallel fibre-Purkinje cell synapses. Furthermore, our study revealed a previously uncharacterized GABAAR-CAMKV interaction in mice and human brains. Our results provide a proof of concept for transmembrane and transcellular proximity labelling, providing a powerful platform for analysing the interactome of transmembrane proteins.

neuroscience↗

bioRxiv2025

Graph topology reframes the coherence of cell-state manifold inference under heterogeneous single-cell observations

Manifold-based single-cell omics analyses assume that high-dimensional observations can fall into a low-dimensional space encoding biological constraints. In practice, per-cell observation is highly heterogeneous: shallowly- and deeply-observed cells coexist. In an empirical scRNA-seq dataset, shallowly-observed cells cluster together to generate spurious hubs that can give rise to illusory loops in low-dimensional manifold skeletons in graph abstraction. Several imputation methods leave these artifacts largely intact, whereas graph abstraction restricted to homogeneously-observed cells alone recovers tree-like structures locally representing constrained cell state transitions. Simulations further demonstrate that realistic heterogeneous observation can create spurious subclusters and false branching. Grounded by these observations, we propose topological stability descriptors of low-dimensional manifold skeletons to delineate a regime in which manifold-based inference is trustworthy despite realistic heterogeneous observations. Our findings underscore that the heterogeneity of observations is not merely noise but a source of systemic distortion in manifold-based inference that must be addressed.

bioinformatics↗

bioRxiv2025

Discovery of a Selective Inhibitor of ZIP14 with Therapeutic Potential for Cancer-associated Cachexia

ZIP14/SLC39A14, a membrane-bound metal transporter, is essential for systemic metal homeostasis and has been implicated in inflammatory and metabolic disorders, including cancer-associated cachexia. Despite its biological and therapeutic significance, no selective inhibitors have been identified. Here, we identify 1-phenyl-8-(2-phenylethyl)-1,3,8-triazaspiro[4.5]decan-4-one (PPTD) as the first selective small-molecule inhibitor of ZIP14. PPTD efficiently blocks ZIP14-mediated uptake of zinc, iron, manganese, and cadmium, while sparing the closely related transporter ZIP8/SLC39A8. Mechanistically, PPTD binds specifically to a pocket formed at the dimer interface of ZIP14, as revealed by AlphaFold3 structural prediction, ligand-interaction profiling, structure-activity analyses, and site-directed mutagenesis, providing direct evidence for a targeted inhibition mechanism. ZIP14-driven metal influx promotes reactive oxygen species and lipid peroxidation, leading to cytotoxicity, which PPTD effectively reverses. In vivo, PPTD ameliorates major features of cancer cachexia in mice, including weight loss, reduced survival, muscle wasting, impaired locomotor activity, and disease progression. PPTD thus provides both a chemical probe to dissect ZIP14 function and a potential therapeutic candidate for cancer cachexia, establishing a foundation for the development of therapies targeting ZIP14-mediated metal dysregulation.

biochemistry↗

bioRxiv2025

Plcl1 Regulates Hematopoietic Stem Cell Function During Aging and Stress by Modulating Calcium Dynamics

Long-term hematopoietic stem cells (HSCs) can generate all blood lineages but typically remain quiescent, becoming activated only in response to acute stress. We previously demonstrated that quiescent HSCs exhibit heterogeneity in intracellular calcium levels. However, the mechanisms underlying this heterogeneity and its physiological relevance remain unclear. Herein, we identify phospholipase C-like 1 (Plcl1), a noncatalytic protein that binds inositol 1,4,5-trisphosphate (IP3), as being selectively enriched in the most quiescent HSC subset. Loss-of-function studies revealed that Plcl1 deficiency at steady state reduced basal intracellular calcium levels and skewed the HSC compartment toward CD41 subsets while preserving overall HSC numbers and long-term reconstitution capacity. Under acute hematopoietic stress, Plcl1 loss accelerated and amplified platelet rebound and the expansion of non-canonical megakaryocyte progenitors (ncMkPs), indicating activation of the thrombopoietic bypass pathway. In aged HSCs, Plcl1 deficiency exacerbated aging-related features, including expansion of the HSC pool, accumulation of CD41 HSCs and ncMkPs, and myeloid-skewed differentiation with impaired competitive reconstitution. These changes were accompanied by diminished induction of calcium-responsive immediate-early genes. Collectively, we identified Plcl1 as an intrinsic regulator that stabilizes calcium dynamics in HSCs, thereby restraining stress- and aging-associated megakaryocytic priming and preserving stem cell function.

cell biology↗

bioRxiv2025

A non-spike nucleocapsid R204P mutation in SARS-CoV-2 Omicron XEC enhances inflammation and pathogenicity

The global circulation of SARS-CoV-2 in human populations has driven the emergence of Omicron subvariants, which have become highly diversified through recombination. In late 2024, SARS-CoV-2 Omicron XEC variant emerged from the recombination of two JN.1 progeny, KS.1.1 and KP.3.3, and became predominant worldwide. Here, we investigated virological features of the XEC variant. Epidemic dynamics modeling suggested that spike substitutions in XEC mainly contribute to its increased viral fitness. Additionally, four licensed antivirals were effective against XEC. Although the fusogenicity of XEC spike is comparable to that of the JN.1 spike, the intrinsic pathogenicity of XEC in hamsters was significantly higher than that of JN.1. Notably, we found that the nucleocapsid R204P mutation of XEC enhanced inflammation through NF-{kappa}B activation. Recent studies suggest that the evolutionary potential of spike protein is reaching its limit. Indeed, our findings highlight the critical role of non-spike mutations in the future evolution of SARS-CoV-2.

microbiology↗

bioRxiv2025

Longitudinal analysis of antibody titers after primary and booster mRNA COVID-19 vaccination can identify individuals at risk for breakthrough infection

A key issue in the post-COVID-19 era is the ongoing administration of COVID-19 vaccines. Repeated vaccination is essential for preparing against currently circulating and newly emerging SARS-CoV-2 variants while enabling people to continue with daily life. Optimizing vaccination strategies is crucial to efficiently manage medical resources and establish an effective vaccination framework. Therefore, it is important to quantitatively understand vaccine-induced immunity dynamics and to be able to identify poor responders with lower sustained antibody titers as potential priorities for revaccination. We investigated longitudinal antibody titer data in a cohort of 2,526 people in Fukushima, Japan, from April 2021 to November 2022 for whom basic demographic and health information was available. Using mathematical modeling and machine learning, we stratified the time-course patterns of antibody titers after 2 primary doses and 1 booster dose of mRNA COVID-19 vaccines. We identified 3 notable populations, which we refer to as the durable, the vulnerable, and the rapid-decliner populations, approximately half of which remained in the same population after the booster dose. Notably, the rapid-decliner population experienced earlier infections than the others. Furthermore, when comparing IgG(S) titers, IgA(S) titers, and T-spot counts between participants who experienced breakthrough infections after booster vaccination and those who did not, we found that IgA(S) titers were significantly lower in breakthrough infected participants during the early stage after booster vaccination. Our computational approach is adaptable to various types of vaccinations. This flexibility can inform policy decisions on vaccine distribution to enhance immunity both in future pandemics and in the post-COVID-19 era.

immunology↗

bioRxiv2025

Evolution of BA.2.86 to JN.1 reveals functional changes in non-structural viral proteins are required for fitness of SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19), is still circulating among humans, leading to the continuous evolution. SARS-CoV-2 Omicron JN.1 evolved from a distinct SARS-CoV-2 lineage, BA.2.86, spread rapidly worldwide. It is unclear why BA.2.86 did not become dominant and was quickly replaced by JN.1, which possesses one amino acid substitution in the spike protein (S:L455S) and two in the non-spike proteins NSP6 and ORF7b (NSP6:R252K and ORF7b:F19L) compared to BA.2.86. Here, we utilized recombinant viruses to elucidate the impact of these mutations on the virological characteristics of JN.1. We found that the mutation in the spike attenuated viral replication, but the non-spike mutations enhanced replication, suggesting the mutations in the non-spike proteins compensate for the one in the spike to improve viral fitness, as the mutations in the spike contribute to further immune evasion. Our findings suggest that functional changes in both the spike and non-spike proteins are necessary in the evolution of SARS-CoV-2 to enable evasion of adaptive immunity within the human population while sustaining replication. IMPORTANCEBecause the spike protein is strongly associated with certain virological properties of SARS-CoV-2, such as immune evasion and infectivity, most previous studies on SARS-CoV-2 variants have focused on spike protein mutations. However, the non-spike proteins also contribute to infectivity, as observed throughout the evolution of Omicron subvariants. In this study, we demonstrate a "trade-off" strategy in SARS-CoV-2 Omicron JN.1 in which the reduced infectivity caused by spike mutation is compensated by non-spike mutations. Our results provide insight into the evolutionary scenario of the emerging virus in the human population.

microbiology↗

bioRxiv2024

The crucial role of intercellular calcium wave propagation triggered by influenza A virus in promoting infection

Influenza A viruses (IAVs) initially infect a few host cells, and the infection subsequently spreads to neighboring cells. However, the molecular mechanisms underlying this expansion remain unclear. Here, we show that IAV infection upregulates the frequency of intercellular calcium wave propagations (iCWPs) that mediate the spread of IAVs. ADP released from initially infected cells mediated iCWPs via the P2Y1 receptor. The generation of iCWPs and spread of viral infection were inhibited by a P2Y1 antagonist. Enhanced endocytosis in the surrounding cells that received ADP signaling upregulated viral entry. Expression of IAV matrix protein 2 (M2) in initially infected cells triggered iCWPs through ADP diffusion, thereby increasing infection, whereas an ion permeability-deficient mutation of M2 or inhibition of its ion channel activity suppressed iCWPs. Therefore, intercellular calcium signaling is essential for early expansion and potential establishment of IAV infection, which offers a promising target for IAV prophylaxis.

cell biology↗

bioRxiv2024

Involvement of Hepatitis B Core-Related Antigen in Viral Genome Integration in Patients With Prior Hepatitis B Virus Infection

BackgroundThe incidence of non-B non-C hepatocellular carcinoma (NBNC-HCC), which is negative for hepatitis B surface antigen and hepatitis C virus antibodies, is on the rise. Relatively high numbers of NBNC-HCC patients are hepatitis B core antibody (HBcAb) positive, suggesting that previous HBV infection may play a role in NBNC-HCC development, though the exact mechanisms are unclear. This study aimed to investigate whether HBV genomes are integrated into the host genome of HBcAb-positive NBNC-HCC cases and how these integrations may contribute to cancer development and progression. MethodsHBV detection PCR using HBV-specific primers on DNA extracted from HBcAb-positive NBNC-HCC tissue samples was performed. Positive samples were further examined for HBV integration sites using viral DNA-capture sequencing. Additionally, hepatitis B core-related antigen (HBcrAg) serum levels were measured to assess whether they could be predictive for HBV detection PCR results. ResultsAmong 90 HBcAb-positive NBNC-HCC samples, HBV genome amplification was detected in 18 samples, and elevated HBcrAg levels were associated with the HBV detection PCR results. Seventeen of these samples exhibited HBV integration. The HBV genome was integrated near the TERT gene in 7 samples, resulting in significantly increased TERT mRNA levels; in the KMT2B gene (2 samples); and downstream of LOC441666 (2 samples). ConclusionThe integration sites we identified in our samples have been previously reported in HBV-related HCC, suggesting that HBV integration may also contribute to hepatocarcinogenesis in HBcAb-positive NBNC-HCC. Furthermore, HBcrAg could serve as a potential, noninvasive marker for detecting HBV integration in these cases.

microbiology↗

bioRxiv2024

First versatile reverse genetics system for feline coronavirus

Feline infectious peritonitis (FIP) is a fatal disease caused by feline coronavirus (FCoV). Although multiple gene mutations in FCoV likely account for FIP pathogenesis, molecular studies for FCoV have been limited due to the lack of a suitable reverse genetics system. In the present study, we established a rapid PCR-based system to generate recombinant FCoV using the circular polymerase extension reaction (CPER) method for both serotype 1 and 2 viruses. Recombinant FCoV was successfully rescued at sufficient titers to propagate the progeny viruses with high sequence accuracy. The growth kinetics of recombinant FCoV were comparable to those of the parental viruses. We successfully generated recombinants harboring spike gene from a different FCoV strain or a reporter HiBiT-tag using the CPER method. The chimeric virus demonstrated similar characteristics with the parental virus of S gene. The reporter tag stably expressed after five serial passages in the susceptible cells, and the reporter virus could be applied to evaluate the sensitivity of antiviral inhibitors using the luciferase assay system to detect HiBiT tag. Taken together, our versatile reverse genetics system for FCoV shown herein is a robust tool to characterize viral genes even without virus isolation and to investigate the molecular mechanisms of the proliferation and pathogenicity of FCoV. ImportanceFeline infectious peritonitis is a highly fatal disease in cats caused by feline coronavirus variants that can infect systemically. Because of a lack of versatile toolbox of manipulating the feline coronavirus genome, an efficient method is urgently needed for studying virus proteins responsible for the severe disease. Herein, we established a rapid reverse genetics system for the virus and demonstrated the capability of the recombinant viruses to be introduced desired modifications or reporter genes without any negative impacts on virus characteristics in cell culture. Recombinant viruses are also useful to evaluate antiviral efficacy. Overall, our system can be a promising tool to reveal the molecular mechanisms of viral life cycle of feline coronavirus and disease progression of feline infectious peritonitis.

microbiology↗

bioRxiv2024

A cellular assay system revealed the deamidase-mediated regulation of the Arg-mediated N-end rule protein degradation pathway

Regulated protein production and degradation are essential for maintaining proteostasis in eukaryotic cells. The N-end rule, or N-degron pathway, is a protein degradation machinery in which the N-terminal amino acid is the mark of the protein degradation via the proteasome. The N-end rule is a conserved protein disposal machinery in eukaryotic cells. However, the precise cellular mechanisms and their physiological roles are not fully understood. Herein, we report the development of an Arg-mediated N-end rule assay system using artificial substrates expressed in cultured cell lines. We demonstrated that the N-end rule degradation is significantly influenced by the expression levels of N-terminal amino acid-modifying enzymes, including NTAN1, NTAQ1, and ATE1. In the N-terminal Asn protein pathway, an increase in NTAN1 or ATE1 expression promotes its disposal via the N-end rule degradation pathway. Interestingly, overexpression of NTAQ1 decreased the degradation of the protein bearing Gln at the N-terminus. Computational prediction of NTAQ1 and ATE1 complex formation suggests that the outer loop region of NTAQ1 is involved in its interaction with ATE1 and that the NTAQ1 overexpression may negatively affect this interaction. Our findings suggest that the degradation activity of the Arg/N-end rule pathway is positively or negatively regulated by deamidase expression levels and propose a higher degree of control of protein degradation by the Arg/N-end rule within cells.

cell biology↗

bioRxiv2023

Virological characteristics of the SARS-CoV-2 BA.2.86 variant

In late 2023, a lineage of SARS-CoV-2 emerged and was named the BA.2.86 variant. BA.2.86 is phylogenetically distinct from other Omicron sublineages identified so far, displaying an accumulation of over 30 amino acid mutations in its spike protein. Here, we performed multiscale investigations to reveal the virological characteristics of the BA.2.86 variant. Our epidemic dynamics modeling suggested that the relative reproduction number of BA.2.86 is significantly higher than that of EG.5.1. Experimental studies showed that four clinically-available antivirals were effective against BA.2.86. Although the fusogenicity of BA.2.86 spike is similar to that of the parental BA.2 spike, the intrinsic pathogenicity of BA.2.86 in hamsters was significantly lower than that of BA.2. Since the growth kinetics of BA.2.86 is significantly lower than that of BA.2 in both in vitro cell cultures and in vivo, it is suggested that the attenuated pathogenicity of BA.2.86 is due to its decreased replication capacity.

microbiology↗

bioRxiv2023

Virological characteristics of the SARS-CoV-2 Omicron EG.5.1 variant

In middle-late 2023, a sublineage of SARS-CoV-2 Omicron XBB, EG.5.1 (a progeny of XBB.1.9.2), is spreading rapidly around the world. Here, we performed multiscale investigations to reveal virological features of newly emerging EG.5.1 variant. Our phylogenetic-epidemic dynamics modeling suggested that two hallmark substitutions of EG.5.1, S:F456L and ORF9b:I5T, are critical to the increased viral fitness. Experimental investigations addressing the growth kinetics, sensitivity to clinically available antivirals, fusogenicity and pathogenicity of EG.5.1 suggested that the virological features of EG.5.1 is comparable to that of XBB.1.5. However, the cryo-electron microscopy reveals the structural difference between the spike proteins of EG.5.1 and XBB.1.5. We further assessed the impact of ORF9b:I5T on viral features, but it was almost negligible at least in our experimental setup. Our multiscale investigations provide the knowledge for understanding of the evolution trait of newly emerging pathogenic viruses in the human population.

microbiology↗