Biology subjects

Imai, S.

Publications and source records attributed to Imai, S..

16 recordsLinked to original sources

bioRxiv2026

Biosensor-enabled single-molecule enzyme activity profiling for function-based molecular counting

Single-molecule enzyme activity analysis enables proteoform-resolved quantification of catalytically active enzyme molecules but remains limited by the need for enzyme-specific fluorogenic substrates, particularly for enzymes involved in central metabolism. Here we establish a biosensor-based framework that integrates genetically encoded fluorescent metabolite biosensors with single-molecule enzyme activity profiling (SEAP), enabling native enzymatic reactions to be monitored without enzyme-specific fluorogenic substrates. By employing different biosensors, the framework was applied to distinct metabolic enzymes, including lactate dehydrogenase and pyruvate kinase. Biosensor-based SEAP enables absolute quantification of active enzyme molecules, transforming conventional activity measurements (U/mL) into molecular abundance (molecules/mL). Applying this approach to single-cell functional proteoform analysis revealed functional heterogeneity in lactate dehydrogenase activity during T-cell activation. In addition, analysis of circulating enzymes showed that much of the plasma lactate dehydrogenase pool could be quantitatively accounted for by physiological erythrocyte turnover. These results establish genetically encoded fluorescent protein-based metabolite biosensors as modular optical transducers for single-molecule functional enzyme analysis across cellular and circulating samples.

biochemistry↗

bioRxiv2026

Generation of lachrymatory factor synthase-suppressed onion (Allium cepa L.) by Agrobacterium-mediated gene transfer for CRISPR/Cas9 genome editing

Lachrymatory factor, an irritating volatile with tear-inducing property, is produced when onion bulbs are cut or chopped. We aimed to generate onion plants with reduced lachrymatory factor synthase (LFS) activity via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (CRISPR/Cas9) genome editing. Calli induced from primary roots were transformed with Agrobacterium tumefaciens carrying expression cassettes for CRISPR/Cas9, guide RNA, green fluorescent protein (GFP), and hygromycin resistance; callus lines that showed a high-frequency stable GFP expression were selected as "elite callus lines" that were suitable for transformation. Cleaved amplified polymorphic sequence (CAPS), heteroduplex mobility assay (HMA), and Sanger sequencing confirmed mutations introduced into the LFS gene, and plants were regenerated from the confirmed LFS-edited callus lines. The LFS enzyme activity in the leaves and bulbs of the LFS-edited plants was lower than that in control plants, while the LFS-edited plants exhibited severe growth abnormalities and failed to set seed, possibly due to long-term culture to maintain the elite callus line. The present study first demonstrated that onion genome editing, which modified a specific trait of onion, the reduction of LFS activity, was achieved. The results obtained opened the feasible way toward the final goal: the production of tear-free, higher health-functional onions.

plant biology↗

bioRxiv2026

Modular mRNA platform for in vivo antigen-specific immune tolerance

Immune-mediated diseases such as autoimmunity and allergy arise from dysregulated antigen-specific responses, yet current therapies largely rely on broad immunosuppression and rarely re-establish durable, antigen-selective tolerance. Here we introduce Tol-mRNA, a modular tolerogenic mRNA platform designed to deliver multiple immune-regulatory signals in vivo to program antigen-specific tolerance. Tol-mRNA co-encodes an antigen together with two complementary regulatory cues, PD-L1 and TGF-{beta}, enabling coordinated antigen presentation and tolerogenic signaling that robustly induces antigen-specific Tregs and suppresses pathogenic effector responses. In murine disease models, Tol-mRNA conferred therapeutic benefit across distinct immune pathologies. In experimental autoimmune encephalomyelitis, Tol-mRNA ameliorated clinical disease and reduced inflammatory immune activation. In an ovalbumin-driven food allergy model, Tol-mRNA prevented allergic symptoms and dampened type 2 inflammation. Extending these findings to humans, Tol-mRNA demonstrated potent, antigen-dependent immunosuppressive activity and a strong capacity to promote antigen-specific Treg induction in human immune settings. Collectively, these results establish Tol-mRNA as a scalable and versatile approach to reprogram antigen-specific immunity and support its development as a next-generation therapeutic modality for immune-mediated diseases.

immunology↗

bioRxiv2026

Identification of novel hepaciviruses in rock pigeon (Columba livia )

Recent advances in sequencing technology and transcriptome mining have revealed highly divergent hepaciviruses in birds. However, only a limited number of avian hepaciviruses have been identified to date, leaving their diversity and evolutionary history poorly understood. Moreover, deep phylogenetic gaps among known avian hepaciviruses suggest that additional lineages remain undiscovered. Here, we screened publicly available RNA-seq data and identified three previously undescribed hepaciviruses from rock pigeon (Columba livia), rusty-margined flycatcher (Myiozetetes cayanensis), and Hispaniolan amazon (Amazona ventralis), named rock pigeon hepacivirus (RpHV), rusty-margined flycatcher hepacivirus (RfHV), and Hispaniolan amazon hepacivirus (HaHV). Although these three viruses meet the ICTV species demarcation criteria relative to their closest known relatives, the NS5B-based criterion was not satisfied between RfHV and HaHV. Notably, however, their genome sequence identity is low at 43.2%, and their hosts differ at the order level, suggesting that their classification warrants further consideration. Our phylogenetic analysis showed that avian hepaciviruses, including those found in this study, are monophyletic, but phylogenetic incongruence was observed between avian hepaciviruses and their hosts, suggesting past cross-species transmission among avian hepaciviruses. Overall, this study provides novel insights into the diversity and evolution of hepaciviruses.

microbiology↗

bioRxiv2025

mRNA-Based Designer APCs Elicit Robust CD8⁺ and CD4⁺ T Cell Responses

mRNA-based therapeutics have demonstrated notable success in SARS-CoV-2 vaccines and are emerging in cancer immunotherapy. However, conventional mRNA cancer vaccines are limited by the low immunogenicity of tumor-associated and neoantigens. We addressed this limitation by formulating a modular, liposome-based mRNA cocktail comprising three distinct mRNAs encoding tumor antigen, the co-stimulatory molecule CD80, and membrane-tethered IL-2. Administration of this mRNA mixture transforms somatic cells into designer antigen-presenting cells (APCs) in vivo, which simultaneously express the antigen, a co-stimulatory molecule, and a cytokine. These designer APCs more effectively activated tumor antigen-specific CD8 T cells than mRNA encoding the antigen alone and elicited robust anti-tumor immune responses. In addition, substituting IL-2 in the mRNA mixtures with membrane-tethered IL-12 led to the expansion and differentiation of endogenous antigen-specific Th1 helper T cells in vivo. Importantly, this platform activated NY-ESO-1-specific CD8 T cells both in human PBMCs in vitro and in HLA-A*02:01-transgenic mice, highlighting its translational potential. This modular mRNA strategy reprograms somatic cells in situ into designer APCs, providing a flexible and translatable platform for precision immunotherapy.

immunology↗

bioRxiv2025

Detection and genetic characterization of pigeon gammacoronavirus

Most members of the genus Gammacoronavirus infect avian hosts, but far fewer viruses have been characterized than in the other coronavirus genera, leaving their diversity largely unclear. Pigeon gammacoronaviruses were previously detected by consensus PCR, but only partial sequences were determined. In this study, we comprehensively analyzed public RNA-seq datasets and reconstructed two nearly complete genomes of pigeon gammacoronaviruses. Molecular evolutionary analyses showed pigeon gammacoronaviruses belong to the subgenus Igacovirus, and their pairwise distances to the members of this subgenus meet the International Committee on Taxonomy of Viruses (ICTV) species demarcation criteria, supporting their designation as a novel species in this subgenus. Notably, although nucleotide sequences are highly conserved gammacoronaviruses, their 3'-genomic region exhibited differences in gene organization among pigeon coronavirus variants. These findings expand our knowledge of the diversity of gammacoronaviruses.

microbiology↗

bioRxiv2025

High-performance genetically-encoded green and red fluorescent biosensors for pyruvate

Pyruvate is the end-product of glycolysis and a central metabolite involved in many biochemical pathways. However, a lack of high-performance (i.e., {Delta}F/F0 > 10) single fluorescent protein (FP)-based biosensors has hindered efforts to investigate the physiological role of pyruvate. Here, we present the GreenPy1 and ApplePy1 series, which are green FP (GFP)-based and red FP (RFP)-based pyruvate biosensors, respectively. Both series exhibit large fluorescence intensity change ({Delta}F/F0 [~] 20 to >40) and a range of affinities (10s of M to several mM). We demonstrate the utility of these pyruvate biosensors for multicolor imaging of metabolite concentration changes in mammalian cells.

molecular biology↗

bioRxiv2025

Identification of an ambisense kolmiovirus from birds

Circular RNA replicons share compact, highly self-complementary genomes that impose severe constraints on genome organization and coding capacity. Recent studies have uncovered a broad diversity of such replicons, but their transcriptional and coding strategies remain inferred largely from sequence analyses. Here we show experimentally that some kolmioviruses (family Kolmioviridae), which have been described as negative-sense circular RNA satellite viruses, use ambisense transcription to expand coding capacity. We identify avian bornaviruses as helper viruses for Serinus canaria kolmiovirus (scKoV) and show that ambisense transcription drives expression of a previously unrecognized gene, open reading frame 2 (ORF2), that promotes helper-dependent transmission. Comparative analyses reveal ambisense gene expression in additional kolmiovirus lineages, suggesting independent acquisition during evolution. These findings provide experimental evidence that circular RNA replicons with highly constrained genomes can acquire additional protein-coding genes through ambisense transcription.

microbiology↗

bioRxiv2024

PinkyCaMP a mScarlet-based calcium sensor with exceptional brightness, photostability, and multiplexing capabilities

Genetically encoded calcium (Ca2+) indicators (GECIs) are widely used for imaging neuronal activity, yet current limitations of existing red fluorescent GECIs have constrained their applicability. The inherently dim fluorescence and low signal-to-noise ratio of red-shifted GECIs have posed significant challenges. More critically, several red-fluorescent GECIs exhibit photoswitching when exposed to blue light, thereby limiting their applicability in all-optical experimental approaches. Here, we present the development of PinkyCaMP, the first mScarlet-based Ca2+ sensor that outperforms current red fluorescent sensors in brightness, photostability, signal-to-noise ratio, and compatibility with optogenetics and neurotransmitter imaging. PinkyCaMP is well-tolerated by neurons, showing no toxicity or aggregation, both in vitro and in vivo. All imaging approaches, including single-photon excitation methods such as fiber photometry, widefield imaging, miniscope imaging, as well as two-photon imaging in awake mice, are fully compatible with PinkyCaMP.

neuroscience↗

bioRxiv2024

Detection of a novel parahenipavirus from northern short-tailed shrews (Blarina brevicauda )

Parahenipaviruses (genus Parahenipavirus) are non-segmented negative strand RNA viruses belonging to the family Paramyxoviridae of the order Mononegavirales. Parahenipaviruses have been detected in shrews and rodents, some of which have been reported to be associated with human diseases. Although many parahenipaviruses have been identified thus far, there still exist large phylogenetic gaps in parahenipaviruses, suggesting the existence of yet-to-be-identified parahenipaviruses. In this study, we analyzed public RNA-seq data and identified a novel parahenipavirus from northern short-tailed shrews (Blarina brevicauda [Say, 1823]), tentatively named Blarina brevicauda virus (BbV). Based on sequence comparisons between BbV and its most closely related viruses, BbV meets the International Committee on Taxonomy of Viruses species demarcation criteria, and therefore we propose that BbV is a novel species of virus in the genus Parahenipavirus. Furthermore, mapping analysis using RNA-seq data derived from multiple tissues of northern short-tailed shrews suggested that BbV is kidney-tropic. This study provides novel insights into the diversity of parahenipaviruses, which would contribute to a deeper understanding of their evolution and control of infectious diseases.

microbiology↗

bioRxiv2024

Soil nutrition-dependent dynamics of the root-associated microbiome in paddy rice

O_LIPlants accommodate diverse microbial communities (microbiomes), which can change dynamically during plant adaptation to varying environmental conditions. However, the direction of these changes and the underlying mechanisms driving them, particularly in crops adapting to the field conditions, remain poorly understood. C_LIO_LIWe investigate the root-associated microbiome of rice (Oryza sativa L.) using 16S rRNA gene amplicon and metagenome sequencing, across four consecutive cultivation seasons in a high-yield, non-fertilized, and pesticide-free paddy field, compared to a neighboring fertilized and pesticide-treated field. C_LIO_LIOur findings reveal that root microbial community shifts and diverges based on soil fertilization status and plant developmental stages. Notably, nitrogen-fixing bacteria such as Telmatospirillum, Bradyrhizobium and Rhizomicrobium were over-represented in rice grown in the non-fertilized field, implying that the assembly of these microbes supports rice adaptation to nutrient-deficient environments. C_LIO_LIA machine learning model trained on the microbiome data successfully predicted soil fertilization status, highlighting the potential of root microbiome analysis in forecasting soil nutrition levels. Additionally, we observed significant changes in the root microbiome of ccamk mutants, which lack a master regulator of mycorrhizal symbiosis, under laboratory conditions but not in the field, suggesting a condition-dependent role for CCaMK in establishing microbiomes in paddy rice. C_LI

plant biology↗

bioRxiv2023

Surface-engineered extracellular vesicles to modulate antigen-specific T cell expansion for cancer immunotherapy

Extracellular vesicles (EVs), including exosomes, are emerging as novel mediators of cell-cell communications, involved in various processes such as immune activation and immunosuppression. Despite the recent development of several EVs-based cancer immunotherapies, their clinical efficacy remained limited. Here, using fusion with tetraspanin as one of the EV engineering techniques, we created antigen-presenting extracellular vesicles (AP-EVs) to reproduce the functional characteristics of professional antigen-presenting cells (APCs). AP-EVs were also equipped with surface-bound IL-2, a feature not inherent to APCs, which facilitated selective delivery of IL-2 to antigen-specific CD8+ T cells. AP-EVs were engineered to express a peptide-major histocompatibility class I (pMHCI) complex, a costimulatory CD80 molecule, and IL-2, allowing the simultaneous presentation of multiple immune modulators to antigen-specific CD8+ T cells. This promoted the clonal expansion and differentiation of antigen-specific cytotoxic T lymphocytes, leading to potent anticancer immune responses. Combination therapy with AP-EVs and anti-PD-1 demonstrated enhanced anticancer immunity against established tumors compared with anti-PD-1 monotherapy. Our engineered EVs represent a novel effective strategy for cancer immunotherapy.

bioengineering↗

bioRxiv2023

New body typology method based on the combination of coupling motion of pelvis, thorax, and cranium.

The purpose of this study is to deepen the understanding of unconscious intrinsic individuality by proposing and verifying the hypothesis of "Integrated Spine Coupling" which is defined by the combination of the way of the coupling motion of 3 major units of our body: pelvis, thorax, and cranium. This paper, as the first step of the verification, examines how each of the three units -- pelvis, thorax, and cranium as the representative units of cervical, thoracic, and lumber spines -- produces a coupling motion during body rotational movement, and how the types of combinations are related to other movements such as breathing. The measurement results suggest that there exist only four types of coupling motion patterns and it significantly affects the way they breath and move their ankle and toe.

physiology↗

bioRxiv2020

Mechanism of hERG inhibition by gating-modifier toxin, APETx1, deduced by functional characterization

Human ether-a-go-go-related gene potassium channel 1 (hERG) is a voltage-gated potassium channel, the voltage-sensing domain (VSD) of which is targeted by a gating-modifier toxin, APETx1. Although it is known that APETx1 inhibits hERG by stabilizing the resting state, it remains unclear where and how APETx1 interacts with the VSD in the resting state. Here, we prepared a recombinant APETx1, which is structurally and functionally equivalent to the natural product. Electrophysiological analyses using wild type and mutants of APETx1 and hERG revealed that their hydrophobic residues, in addition to a previously reported acidic hERG residue, play key roles in the inhibition of hERG by APETx1. Docking models of the APETx1-VSD complex that satisfy the results of mutational analysis suggest a molecular recognition mode between APETx1 and the resting state of hERG; this would provide a structural basis for designing ligands that control hERG function by binding to the VSD.

molecular biology↗

bioRxiv2018

Re-Evaluating One-step Generation of Mice Carrying Conditional Alleles by CRISPR-Cas9-Mediated Genome Editing Technology

CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA-2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA-2ssODN approach, we performed a generalized linear regression model. We show that factors such as the concentration of the sgRNA, Cas9 protein or the distance between the placement of LoxP insertions were not predictive for the success of this technique. The major predictor affecting the methods success was the probability of simultaneously inserting intact proximal and distal LoxP sequences, without the loss of the DNA segment between the two sgRNA cleavage sites. Our analysis of a large data set indicates that the 2sgRNA-2ssODN method generates a large number of undesired alleles (>99%), and a very small number of desired alleles (<1%) requiring, on average 1,192 zygotes.

genetics↗

bioRxiv2017

Structure Of Allium Lachrymatory Factor Synthase Elucidates Catalysis On Sulfenic Acid Substrate

Natural lachrymatory effects are invoked by small volatile S-oxide compounds. They are produced through alkene sulfenic acids by the action of lachrymatory factor synthase (LFS). Here we present the crystal structures of onion LFS (AcLFS) revealed in solute-free and two solute-stabilized forms. Each structure adopts a single seven-stranded helix-grip fold possessing an internal pocket. Mutagenesis analysis localized the active site to a layer near the bottom of the pocket, which is adjacent to the deduced key residues Arg71, Glu88, and Tyr114. Solute molecules visible on the active site have suggested that AcLFS accepts various small alcohol compounds as well as its natural substrate, and they inhibit this substrate according to their chemistry. Structural homologs have been found in the SRPBCC superfamily, and comparison of the active sites has demonstrated that the electrostatic potential unique to AcLFS could work in capturing the substrate in its specific state. Finally, we propose a rational catalytic mechanism based on intramolecular proton shuttling in which the microenvironment of AcLFS can bypass the canonical [1,4]-sigmatropic rearrangement principle established by microwave studies. Beyond revealing how AcLFS generates the lachrymatory compound, this study provides insights into the molecular machinery dealing with highly labile organosulfur species.\n\nSignificance statementCrushing of onion liberates a volatile compound, syn-propanethial S-oxide (PTSO), which causes lachrymatory effect on humans. We present the crystal structures of onion LFS (AcLFS), the enzyme responsible for natural production of PTSO. AcLFS features a barrel-like fold, and mutagenic and inhibitory analyses revealed that the key residues are present in the central pocket, harboring highly concentrated aromatic residues plus a dyad motif. The architecture of AcLFS is widespread among proteins with various biological functions, such as abscisic acid receptors and polyketide cyclases, and comparisons with these homologs indicate that unique steric and electronic properties maintain the pocket as a reaction compartment. We propose the molecular mechanism behind PTSO generation and shed light on biological decomposition of short-lived sulfur species.

biochemistry↗