Biology subjects

Watanabe, N.

Publications and source records attributed to Watanabe, N..

At least 19 recordsLinked to original sources

bioRxiv2026

Machine-learning-guided enzyme discovery and redox-system engineering for efficient production of the nylon monomer methyl 12-aminododecanoate in Escherichia coli

Methyl 12-aminododecanoate (ADAME) is a key precursor for Nylon 12 synthesis and an attractive target for sustainable microbial production. However, efficient biosynthesis of ADAME requires the coordinated oxidation and transamination of methyl dodecanoate (DAME), and identifying compatible enzymes for this multistep pathway remains challenging. In this study, we applied a machine learning-based bioinformatic screening approach to identify alternative enzymes for DAME-to-ADAME bioconversion. Among 18 selected distantly related AlkB homologs, AlkBGp01 from Alcanivorax sp. P2S70 showed activity toward DAME, producing 12-hydroxydodecanoic acid methyl ester (HDAME) and 12-oxododecanoic acid methyl ester (ODAME) despite low sequence identity to Pseudomonas putida AlkB. Further screening identified compatible redox partners, AlkG60 from the same species as AlkBGp01 and AlkTp04, whose combination with AlkBGp01 resulted in almost sixfold higher ODAME production than that achieved with P. putida AlkBFGJLT. ODAME production was further increased by more than threefold by conjugating these three newly identified proteins. The optimized single-plasmid system comprising conjugated AlkBGp01, AlkG60, and AlkTp04, together with AlaD and newly identified omega-transaminases EAV41574 produced 0.28 mM gDCW-1 ADAME, representing a 5.6-fold improvement over the previously reported P. putida AlkBFGJLT-based system with AlaD-CV2025. These results demonstrate that machine learning-guided enzyme discovery can identify functional distantly related homologs and compatible enzyme combinations for constructing efficient synthetic pathways. This study also highlights the importance of optimizing pathway architecture, including enzyme conjugation and plasmid configuration, for improving microbial production of bio-based monomer precursors.

bioengineering↗

bioRxiv2026

Differential associations of insulin resistance with anterior hippocampal volume in mild cognitive impairment and Alzheimer's disease

Insulin resistance is increasingly recognized as a metabolic factor associated with Alzheimers disease (AD); however, its relevance to hippocampal structural changes--a key pathological feature of AD--across disease stages is not fully understood. To address this issue, we investigated the relationship between insulin resistance, hippocampal gray matter volume, and cognitive performance using data from the Alzheimers Disease Neuroimaging Initiative (ADNI), a large-scale neuroimaging dataset. Insulin resistance was assessed using the Homeostatic Model Assessment of Insulin Resistance (HOMA-IR), and its relationship with brain structure and cognitive performance was evaluated across diagnostic groups. In the mild cognitive impairment (MCI) group, higher insulin resistance was associated with larger anterior hippocampal gray matter volume, whereas in the AD group this association was reversed in direction. Furthermore, in the MCI group, anterior hippocampal gray matter volume was also positively associated with higher Mini-Mental State Examination (MMSE) scores, and an exploratory mediation analysis suggested a significant indirect association linking HOMA-IR, anterior hippocampal volume, and cognitive performance through anterior hippocampal volume. These findings suggest that the relationship between insulin resistance and AD-related brain changes differs across diagnostic groups, highlighting the importance of considering metabolic alterations in relation to disease status.

neuroscience↗

bioRxiv2026

Integrating activation-induced costimulation and cytokine signals enhance TCR-based cell therapies

TCR-based cell therapies offer broad targeting of tumor antigens with high sensitivity but often low durability due to insufficient costimulation (signal 2) and cytokine (signal 3) support. To address this, we developed a dual stimulatory receptor (DSR) consisting of the 4-1BBL ectodomain fused to the thrombopoietin receptor (cMPL) endodomain. DSR engages 4-1BB that is transiently upregulated upon TCR stimulation eliciting signal 2 and simultaneously activates cMPL-driven STAT3/5 phosphorylation providing signal 3. DSR increases the expansion of T cells, preserving their effector function and an effector-associated transcriptional profile upon repeated antigen stimulation. DSR arming significantly improves in vivo antitumor activity of T cells redirected to cancer through engineered TCRs or soluble T-cell engagers in diverse xenograft tumor models by enhancing T cell expansion and persistence post-infusion. These results demonstrate broad utility and establish DSR as a modular receptor for the effective and synchronized delivery of signals 2 and 3 to support TCR-based immunotherapies.

immunology↗

bioRxiv2026

Simultaneous in vivo imaging of Ca2+ signals in periarteriolar cholinergic axonal varicosities and arteriole diameter changes in the mouse cerebral cortex

Basal forebrain cholinergic neurons project widely to the cerebral cortex and participate in cerebrovascular regulation. Although cholinergic axons are distributed around the cerebrovasculature, their functional relationship with arteriolar dynamics remains unclear. In this study, we established an in vivo two-photon imaging approach to simultaneously measure Ca2+ signals in cholinergic axonal varicosities and arteriolar diameters in urethane-anesthetized mice. An adeno-associated virus (AAV) vector (rAAV-ChAT-jGCaMP8s) was injected into the nucleus basalis of Meynert. In vivo imaging of the frontal cortex revealed bead-shaped GCaMP signals around the arterioles. Pinch stimulation transiently increased Ca2+ signals in periarteriolar varicosities, followed by arteriolar dilation, with an approximately 2-s delay between their peaks. Linear regression analysis disclosed a significant relationship between the magnitudes of these changes. This approach enabled simultaneous evaluation of cholinergic axonal activity and arteriolar dynamics in vivo, providing a tool to investigate the cholinergic regulation of cerebrovasculature. HighlightsO_LIAAV-ChAT-GCaMP enables selective imaging of cholinergic projections C_LIO_LITwo-photon imaging reveals bead-shaped Ca2+ signals around arterioles C_LIO_LISensory stimulation increases periarteriolar cholinergic axonal Ca2+ signals C_LIO_LIAxonal Ca2+ signals are associated with arteriole dilation C_LI

physiology↗

bioRxiv2026

Endogenous cell competition kills pathogenic de novo mutant cells but declines under pH stress

Although endogenous cell competition safeguards embryonic development by eliminating unfit cells, the intrinsic origins of unfitness remain unclear. Here, zebrafish whole-genome sequencing and imaging reveal that endogenous cell competition removes cells with spontaneous de novo mutations in key developmental signalling pathways and in human disease-associated genes, such as those associated with Alzheimers disease, epilepsy, autism, and premature aging. These mutations arise frequently during normal development but are efficiently eliminated under physiological conditions, thereby preventing the clonal expansion of harmful cells. However, environmental pH stress disrupts this surveillance by dysregulating Ca2+ signals, allowing mutant cells to persist and resulting in tissue mispatterning and morphological defects. Our findings establish endogenous cell competition as an intercellular communication-dependent genome quality-control mechanism that removes pathogenic mutant cells before clonal expansion. This link between developmental robustness, environmental stress, and mosaic genetic disorders highlights how intrinsic mutations and extrinsic stressors together shape disease susceptibility across the lifespan.

cell biology↗

bioRxiv2026

Engineering CAR-Vδ2 T cells to boost persistence and anti-tumor function

Chimeric antigen receptor (CAR)-modified V{delta}2 T cells are an attractive therapeutic cell platform for cancer immunotherapy. However, their clinical efficacy is limited by short in vivo persistence due to insufficient cytokine support and high susceptibility to activation-induced cell death (AICD). Through comparison of membrane-bound (mb) cytokines, we identified mbIL-18 to support superior anti-tumor activity of CAR-V{delta}2 T cells in vitro and in vivo. To reduce constitutive surface exposure of IL-18 and enable antigen-driven signal 3, we fused MyD88 - the key IL-18R signaling mediator - to an extracellular domain of Fas (Fas88). Antigen stimulation-induced FasL engagement of Fas88 triggered IL-18 signaling while simultaneously protecting V{delta}2 T cells from AICD. Fas88-armed human CAR-V{delta}2 T cells produced superior yet stimulation-dependent in vivo expansion and functional persistence in xenograft models of hematologic and solid malignancies. Together, these findings highlight the importance of IL-18 signaling and AICD resistance for CAR-V{delta}2 T cell activity, enabling a single-transgene modification to limit inflammatory risk and facilitate clinical translation.

synthetic biology↗

bioRxiv2025

The complete mitochondrial genome of Hanabira yukibana Lau, Stokvis, Imahara & Reimer, 2019 (Cnidaria: Anthozoa: Octocorallia)

In this study, we sequenced the complete mitochondrial genome (mitogenome) of Hanabira yukibana (family Clavulariidae), collected in Okinawa, Japan. The complete mitochondrial genome of this specimen was 18,690 bp in length and contained 17 genes (14 protein-coding genes, two rRNA genes, and one tRNA gene). It exhibited gene order pattern A, which is the most common arrangement among octocorals. Molecular phylogenetic analysis indicated that H. yukibana is clustered with the genus Clavularia, including Clavularia inflata (the family Clavulariidae), consistent with the previous research using partial mitochondrial genes. Our study provides an additional mitochondrial genomic resource that contributes to comparative studies of octocoral mitogenomes.

genomics↗

bioRxiv2025

Shear Stress Induces Concentration Gradient Distributions of Membrane Proteins in Live Cells

Cells sense and respond to fluid shear stress. Cell surfaces are exposed to flow, yet the influence of shear stress on the behavior of plasma membrane proteins remains unclear. Here we show that extracellular flow induces the gradient distribution of cell membrane proteins with increasing concentration toward the downstream direction of the flow. Shear stress at 10-30 dynes/cm2 caused formation of concentration gradients of both GPI-anchored proteins and transmembrane proteins, including integrin{beta}1, E-cadherin and the insulin receptor in Xenopus XTC cells. Using single-molecule live-cell imaging, we found that GPI-anchored T-cadherin molecules are dragged along the direction of flow under shear stress. In addition, shear stress induced concentration gradients of membrane proteins in COS-7 cells and human umbilical vein endothelial cells (HUVECs). Our findings suggest that external flow directly transports membrane proteins, establishing concentration gradients that may contribute to the cellular flow-sensing mechanism.

biophysics↗

bioRxiv2024

Genome-scale prediction of gene ontology from mass fingerprints reveals new metabolic gene functions

Mass-based fingerprinting can characterize unknown strains, however expansion of these methods to predict specific gene functions is lacking. Therefore, rapid mass fingerprinting was developed to functionally profile a comprehensive yeast knockout library. Matrix assisted laser desorption ionization (MALDI)-time of flight (TOF) mass fingerprints of 3,238 Saccharomyces cerevisiae knockouts were digitized for correlation with gene ontology (GO) annotations. Random forests and support vector machine (SVM) algorithms precisely assigned GO accessions with AUC scores all above 0.83. SVM was the best predictor with average true positive and true negative rates of 0.975 and 0.991, respectively. The SVM model suggested new functions for 28 uncharacterized yeast genes. Metabolomics analysis of two knockouts (YDR215C and YLR122C) of uncharacterized genes predicted to be involved in methylation-related metabolism, showed altered intracellular contents of methionine-related metabolites. Increased S-adenosylmethionine in YDR215C highlights potential for enhancement of methylation pathways. These results demonstrate that MALDI-TOF fingerprints can be rapidly digitized, resulting in datasets that enable prediction of microbial genotypes and even the function of specific genes. This fingerprinting method can inform optimal bioproduction chassis selection.

systems biology↗

bioRxiv2024

Frame-shift mutation of InCO might cause early flowering of Japanese morning glory and might have contributed to northward expansion

Japanese morning glory (Ipomoea nil), a short day plant, has been used for studying flowering times. Here, quantitative trait loci (QTL) analysis for days from sowing to flowering (DTF) of F2 between I. nil var. Tokyo Kokei Standard (TKS) and I. hederacea line var. Q65, an early flowering variety, revealed four QTLs: QTL Ipomoea Flowering 1-4 (qIF1-4). The position of qIF3, which had the most significant effect among the four QTLs, corresponds with that of I. nil (or I. hederacea) CONSTANS (InCO/IhCO) in the linkage map. There is a single-base In/Del in the coding sequence of InCO/IhCO. The single-base deletion (SBD) causes a frame-shift mutation and loss of function in TKS allele (inco-1). I. nil accessions bearing inco-1 tend to flower early, similarly to rice varieties bearing the loss of function allele of CO ortholog, hd1. Consequently, inco-1 was inferred to reduce DTF. This inferred effect of inco-1 corresponds with the effect of the TKS allele of qIF3. Because inco-1 is found exclusively in Asian accessions, the SBD in inco-1 might have played an important role in the expansion of Japanese morning glories, originally native to the tropical regions of the Americas, into temperate Asia.

plant biology↗

bioRxiv2024

Laminar endocytic machinery revealed by quantitative multiplexed super-resolution imaging

Super-resolution microscopy achieves a few nanometers resolution, but protein quantification and colocalization is limited by its labeling density. Here we present a method to map molecular complexes using the multiplexed data of IRIS super- resolution imaging. We developed antiserum-derived Fab IRIS probes for high- density labeling of endogenous targets, and image analysis, Protein Cluster coloring (PC-coloring), which employs pixel-based principal component analysis and clustering. PC-coloring was first used to map regions of distinct ratios of targets on the image. The molecular complex formation was then evaluated by correlation between all combinations of two proteins in each PC-colored region. We elucidate receptor recruitment and the complex formation in a clathrin-coated structure (CCS) which is pre-assembled prior to endocytosis. Upon EGF stimulation, EGFR-, EGFR-Grb2-complex- and Grb2-dominant regions lined up from the CCS rim. Along the interior of Grb2-dominant regions, Eps15, FCHo1/2 and intersectin-1 formed a complex with Grb2. The interior of Eps15-FCHo1/2 complexes was lined with Eps15-intersectin-1 complexes. The results reveal a laminar complex formation of EGFR, Grb2 and CCS components at the recruitment sites.

cell biology↗

bioRxiv2024

Lipogenic Lung Fibroblast-derived Extracellular Vesicles Mitigate Cigarette Smoke-Induced Chronic Obstructive Pulmonary Disease Pathologies through LAT1-mediated Alveolar Type II Cell Restoration

Emerging research has revealed specific cellular aberrations in Chronic Obstructive Pulmonary Disease (COPD), with a particular focus on alveolar type 2 (AT2) cells, which play a pivotal role in the restoration of damaged lung tissue and promotion of normal cellular differentiation. Lipofibroblasts (LipoFBs), which are stromal fibroblasts that house lipid droplets, have been identified in close proximity to AT2 cells and have been demonstrated to support AT2 function. In this study, we present a comprehensive investigation into the therapeutic potential of extracellular vesicles (EVs) derived from LipoFBs (LipoFB-EVs) in COPD treatment. They effectively mitigate key COPD pathologies such as cellular senescence and inflammatory responses in lung epithelial cells. This is achieved by reducing reactive oxygen species (ROS) levels and modulating DNA damage response pathways. Moreover, LipoFB-EVs demonstrate antifibrotic properties by inhibiting TGF-{beta}-induced myofibroblast differentiation, surpassing conventional antifibrotic drugs. They also aid in restoring impaired AT2 stem cells, which are crucial for lung homeostasis, by enhancing their viability, colony-forming ability, and proliferation. Furthermore, we identify the presence of L-type amino acid transporter 1 (LAT1) within LipoFB-EVs, which mediates amino acid uptake, particularly leucine transport, and contributes to the restoration of AT2 cell dysfunction. Importantly, the administration of LipoFB-EVs in murine models of COPD resulted in significant improvements in airway inflammation, remodeling, obstruction, cellular senescence, and alveolar emphysema induced by both short- and long-term CS exposure. Overall, our findings highlight the therapeutic potential of LipoFB-EVs as a novel regenerative therapy for COPD, offering promising avenues for future clinical interventions.

molecular biology↗

bioRxiv2024

Neural signatures of human psychological resilience driven by acute stress

Neurophysiological mechanisms underlying psychological resilience, the ability to overcome adversity1,2, have been extensively studied in animals. However, in comparison with animals, human resilience is unique in that it is underpinned by higher cognitive functions, such as self-confidence and a positive attitude to challenges3,4. Given these discrepancies, the neurophysiological mechanisms underlying human resilience remain unclear. To address this issue, we recorded multimodal responses after acute stress exposure over 1.5 hours using functional brain imaging and peripheral physiological measurements. Here, we showed that the degree of individual resilience is indexed by multiple changes in neural dynamics 1 hour after acute stress. Both functional magnetic resonance imaging and electroencephalography show that activity in the cortical salience network and power in high-beta and gamma oscillations increase in less resilient individuals. Contrastingly, activity in the cortical default mode network and spontaneous activity in the posterior hippocampus increase in more resilient individuals. Machine learning analysis confirmed that, 1 hour after stress exposure, the functional connectivity in the salience network was the most influential, followed by that in the default mode network, gamma power, high-beta power, and hippocampal activity. The neurophysiological dynamics for resilience do not occur as previously thought, but rather in a time-lagged manner against stress exposure. Our findings Shed light on a new approach to recovery from stress-induced deficits such as delayed neuromodulation after a stressful event.

neuroscience↗

bioRxiv2024

Mar1, an HMG-box protein, regulates n-alkane adsorption and cell morphology of the dimorphic yeast Yarrowia lipolytica

The dimorphic yeast Yarrowia lipolytica possesses an excellent ability to utilize n-alkane as a sole carbon and energy source. Although there are detailed studies on the enzymes that catalyze the reactions in the metabolic processes of n-alkane in Y. lipolytica, the molecular mechanism underlying the incorporation of n-alkane into the cells remains to be elucidated. Because Y. lipolytica adsorbs n-alkane, we postulated that Y. lipolytica incorporates n-alkane through direct interaction with it. We isolated and characterized mutants defective in adsorption to n-hexadecane. One of the mutants harbored a nonsense mutation in MAR1 (Morphology and n-alkane Adsorption Regulator) encoding a protein containing a high mobility group box. The deletion mutant of MAR1 exhibited defects in adsorption to n-hexadecane and filamentous growth on solid media, whereas the strain that overexpressed MAR1 exhibited hyperfilamentous growth. Fluorescence microscopic observations suggested that Mar1 localizes in the nucleus. RNA-seq analysis revealed the alteration of the transcript levels of several genes, including those encoding transcription factors and cell surface proteins, by the deletion of MAR1. These findings suggest that MAR1 is involved in the transcriptional regulation of the genes required for n-alkane adsorption and cell morphology transition. IMPORTANCEY. lipolytica, a dimorphic yeast capable of assimilating n-alkane as a carbon and energy source, has been extensively studied as a promising host for bioconversion of n-alkane into useful chemicals and bioremediation of soil and water contaminated by petroleum. While the metabolic pathway of n-alkane in this yeast and the enzymes involved in this pathway have been well-characterized, the molecular mechanism to incorporate n-alkane into the cells is yet to be fully understood. Due to the ability of Y. lipolytica to adsorbs to n-alkane, it has been hypothesized that Y. lipolytica incorporates n-alkane through direct interaction with it. In this study, we identified a gene, MAR1, which plays a crucial role in the transcriptional regulation of the genes necessary for the adsorption to n-alkane and the transition of the cell morphology in Y. lipolytica. Our findings provide valuable insights that could lead to advanced applications of Y. lipolytica in n-alkane bioconversion and bioremediation.

microbiology↗

bioRxiv2023

Artificial Intelligence Enables the Label-Free Identification of Chronic Myeloid Leukemia Cells with Mitochondrial Morphological Alterations

Long-term tyrosine kinase inhibitor (TKI) treatment for patients with chronic myeloid leukemia (CML) causes various adverse events. Achieving a deep molecular response (DMR) is necessary for discontinuing TKIs and attaining treatment-free remission. Thus, early diagnosis is crucial as a lower DMR achievement rate has been reported in high-risk patients. Therefore, we attempted to identify CML cells using a novel technology that combines artificial intelligence (AI) with flow cytometry and investigated the basis for AI- mediated identification. Our findings indicate that BCR-ABL1-transduced cells and leukocytes from patients with CML showed significantly fragmented mitochondria and decreased mitochondrial membrane potential. Additionally, BCR-ABL1 enhanced the phosphorylation of Drp1 via the mitogen-activated protein kinase pathway, inducing mitochondrial fragmentation. Finally, the AI identified cell line models and patient leukocytes that showed mitochondrial morphological changes. Our study suggested that this AI- based technology enables the highly sensitive detection of BCR-ABL1-positive cells and early diagnosis of CML.

cancer biology↗

bioRxiv2023

Exon 44 skipping in Duchenne muscular dystrophy: NS-089/NCNP-02, a dual-targeting antisense oligonucleotide

Exon-skipping therapy mediated by antisense oligonucleotides (ASOs) is expected to provide a therapeutic option for Duchenne muscular dystrophy (DMD). ASOs for exon skipping reported so far target a single continuous sequence in or around the target exon. In the present study, we investigated ASOs for exon 44 skipping (applicable to approximately 6% of all DMD patients) to improve activity by using a novel ASO design incorporating two connected sequences. Phosphorodiamidate morpholino oligomers targeting two separate sequences in exon 44 were created to simultaneously target two splicing regulators in exon 44, and their exon 44 skipping was measured. NS-089/NCNP-02 showed the highest skipping activity among the oligomers. NS-089/NCNP-02 also induced exon 44 skipping and dystrophin protein expression in cells from a DMD patient to whom exon 44 skipping is applicable. We also assessed the in vivo activity of NS-089/NCNP-02 by intravenous administration to cynomolgus monkeys. NS-089/NCNP-02 induced exon 44 skipping in skeletal and cardiac muscle of cynomolgus monkeys. In conclusion, NS-089/NCNP-02, an ASO with a novel connected-sequence design, showed both in vitro and in vivo exon-skipping activity.

cell biology↗

bioRxiv2023

Transition to the structurally vulnerable nuclear state is an integral part of mouse embryonic development

Upon fertilization, germ cells are reprogrammed to acquire the ability to develop into an entire organism. Whereas extensive studies have focused on epigenetic reprogramming of chromatin states during development, changes of the nucleus that surrounds chromatin are ill-defined. Here, we show that nuclei become structurally and mechanically vulnerable at the 2-cell stage during mouse embryonic development. The 2-cell stage nuclei are extraordinarily plastic and deformable in contrast to those of 1-cell and 4-cell stages. The mechanically vulnerable nuclear state is attained by autophagy-mediated loss of lamin B1 from the nuclear membrane. This developmentally programmed lamin B1 dynamics is required for chromatin organization and major zygotic genome activation. We thus demonstrate that structural reprogramming of nuclei is a major determinant of embryonic gene expression and acquisition of totipotency.

cell biology↗

bioRxiv2022

Identification of a novel family of benzimidazole species-selective Complex I inhibitors as potential anthelmintics

Soil-transmitted helminths (STHs) including Ascaris, hookworm, and whipworm are major human pathogens infecting over a billion people worldwide1,2. There are few existing classes of anthelmintics and resistance is increasing3-5 -- there is thus an urgent need for new classes of these drugs. Here we focus on identifying compounds that interfere with the unusual anaerobic metabolism that STHs use to survive the highly hypoxic conditions of the host gut6-9. This requires rhodoquinone (RQ), a quinone electron carrier that is not made or used by the STH hosts10. We previously showed that C. elegans also uses this rhodoquinone-dependent metabolism (RQDM)11 and established a high throughput assay for RQDM11. We screened a collection of 480 natural products for compounds that kill worms specifically when they rely on RQDM -- these 480 are representatives of a full library of ~25,000 natural products and derivatives12,13. We identify several classes of compound including a novel family of species selective inhibitors of Complex I. These Complex I inhibitors are based on a benzimidazole core but unlike commercial benzimidazole anthelmintics they do not target microtubules14-17. We screened over 1,200 benzimidazoles and identify the key structural requirements for species selective Complex I inhibition. We suggest that these novel benzimidazole species-selective Complex I inhibitors may be potential anthelmintics.

molecular biology↗