Biology subjects

Watanabe, T.

Publications and source records attributed to Watanabe, T..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

A cell-matrix interaction regulates the undifferentiated state and self-renewal capacity of avian primordial germ cells

Primordial germ cells (PGCs) are lineage restricted precursor cells of sperm and eggs. Whilst avian PGCs from chicken can be cultured and modified to produce genome-edited chickens, the long-term culture of PGCs from other bird species has not been achieved. Here, we explored the effects of a cell-matrix interaction on the in vitro propagation of chicken PGCs. Blocking integrin signaling severely reduced the self-renewal of the PGC, indicating that a PGC-matrix interaction is an essential process for self-renewal. We investigated the properties of somatic cell differentiated PGCs and expression analyses suggested that the PGCs undergo a partial epithelial-to-mesenchymal transition caused by excess PGC-matrix interactions. Finally, we conducted a long-term culture of chicken PGCs with matrix components, resulting in significant induction of their somatic conversion. These results suggested that a high level of PGC-matrix interaction can cause somatic conversion, whilst a moderate level is essential for their proliferation. Overall, we identified a molecular aspect of self-renewal and maintaining undifferentiated states of avian PGCs.

developmental biology↗

bioRxiv2024

Induction of cell death in U2OS human cancer cells by Ta3N5

Photocatalysts are widely used in modern society, with their applications extending into the field of medicine. Ultraviolet irradiation is used for photocatalysis to generate reactive oxygen species (ROS). Ta3N5 has attracted attention as a next-generation photocatalyst because it produces ROS upon exposure to visible light. Although ROS generation using visible light irradiation is useful in the field of life sciences, the toxicity of Ta3N5 remains unknown. In the present study, we evaluated the cytotoxicity of Ta3N5 using U2OS cells in terms of the intensity, irradiation time, and wavelength of light and Ta3N5 dose. We also investigated the mode and mechanism of cell death induced by Ta3N5. Cytotoxicity increased with increasing light intensity, irradiation time, and Ta3N5 dose. In contrast, high-intensity blue light irradiation induced cytotoxicity with or without Ta3N5, indicating that irradiation to green and red lights, which can selectively toxify Ta3N5-treated cells, is more useful than irradiation to blue light. Moreover, Ta3N5 induced secondary necrotic death of U2OS cells. These results suggest that Ta3N5 can be used as a photocatalyst in medical and life science fields.

cell biology↗

bioRxiv2024

Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

Lipogenesis-driven metabolic flux is crucial for differentiation of pathogenic Th17 cells. Although our previous CRISPR-based screening identified PLA2G12A as a key player in this process, it has remained obscure how this secreted phospholipase A2 isoform controls Th17 differentiation. Here we show that global, T cell-specific, or fibroblast-specific deletion of PLA2G12A prevents Th17 differentiation and associated diseases including psoriasis and arthritis. PLA2G12A acts on Th17-derived extracellular vesicles (EVs) to produce lysophospholipids including the ROR{gamma}t activator 1-oleoyl-lysophosphatidylethanolamine. These lysophospholipids are further converted by autotaxin to lysophosphatidic acid (LPA), which assists Th17 differentiation mainly via LPA2 receptor. Moreover, PLA2G12A promotes the secretion and uptake of EVs by Th17 cells and alters their cargo contents. Defective Th17 differentiation by PLA2G12A deficiency is rescued by supplementation with PLA2G12A-modified EVs. Importantly, a PLA2G12A-blocking antibody prevents Th17 differentiation and ameliorates psoriasis and arthritis models. Thus, targeting the PLA2G12A-EV-lysophospholipid axis may be useful for treatment of Th17-related diseases.

immunology↗

bioRxiv2024

Viral infection to the raphidophycean alga Heterosigma akashiwo affects both intracellular organic matter composition and dynamics of a coastal prokaryotic community

Marine microalgae play a crucial role in marine ecosystem by supplying dissolved organic matter to heterotrophic prokaryotes, which mediate the microbial loop. Microalgae are often infected by viruses, and in infected cells (virocells), the viruses modulate and often change the host metabolism for propagation and thereby endo-metabolites. However, the impact of algal virocells on prokaryotic communities is not fully been understood. In this study, we investigated whether lysates from virocell of Heterosigma akashiwo, a globally prevalent bloom-forming raphidophycean alga, causes shifts inn prokaryotic community structure, and which metabolic compounds in the viral lysate might affect the surrounding prokaryotic populations. Using microcosm experiments, we cultured prokaryotic communities with a dissolved fraction derived from the viral lysate (VDF) of H. akashiwo. Results revealed that certain prokaryotic populations assigned as the Vibrio spp. pathogenic against fish and crustaceans grew specifically responding to the VDF. These Vibrio species possessed a gene module for branched-chain amino acids transporters, which were revealed to be enriched in VDF by gas chromatography-mass spectrometry analysis. Altogether, our findings suggest that viral infection-induced changes in biochemical properties of H. akashiwo cells can promote the growth of taxonomically and metabolically different prokaryotic populations, potentially impacting higher trophic-level consumers in marine ecosystems. HighlightsO_LIThe effect of viral infection to microalgae on marine prokaryotes was investigated. C_LIO_LIViral lysate of Heterosigma akashiwo promoted growth of certain Vibrio population. C_LIO_LIThose Vibrio likely have ability to utilize the compounds enriched in the lysate. C_LIO_LIThose Vibrio have been known as pathogen of fish or crustacean. C_LIO_LIViral infection to H. akashiwo can indirectly affect higher trophic-level consumers. C_LI

microbiology↗

bioRxiv2024

Evolution of the neural sex-determination system in insects: The doublesex homolog in crickets functions as an X-linked masculinizing factor to regulate morphological but not behavioral sexual dimorphism

In animals, sex-specific social behaviors, such as courtship and aggression, are controlled by sex-specific neural circuits. To understand the neural mechanisms of sex-specific social behaviors from a comparative perspective, it is necessary to understand the sex determination mechanisms of neural circuits and their evolution across animal lineages. To address this, I have focused on the evolution of the neural sex determination system in insects. In the fruit fly Drosophila melanogaster, a model insect with powerful genetics, the molecular mechanisms underlying neuronal sex determination are well understood, and two transcription factor genes, fruitless and doublesex (dsx), have been identified as terminal differentiators. In contrast, those in direct-developing insects, which diverged from the holometabolous lineage, including Drosophila, about 400 million years ago, remain unclear. My previous studies found that the fruitless homolog is unlikely to be integrated into the neuronal sex determination system in direct-developing insects. In this study, I investigated whether the dsx homolog in direct-developing insects contributes to neuronal sex determination to form neural circuits that regulate adult social behaviors. I identified the dsx homolog in the two-spotted cricket Gryllus bimaculatus (Gryllus dsx gene) and revealed that it has a unique isoform configuration, and its encoded proteins contain a cryptic DNA-binding DM domain with accumulated amino acid substitutions. A knockout study revealed that the Gryllus dsx gene functions as an X-linked morphological masculinizing factor but does not determine the sexual orientation or behavioral patterns of adult social behavior in crickets. These results suggest that, at least in crickets, an unknown sex determination factor(s) other than fruitless and dsx plays a central role in neuronal sex determination.

evolutionary biology↗

bioRxiv2024

Transplantation of bioengineered lung using decellularized mouse lungs and primary human endothelial cells.

Lung transplantation is a critical treatment for patients with end-stage lung diseases like idiopathic pulmonary fibrosis, but challenges such as donor shortages and post-transplant complications persist. Bioengineered lungs, integrating patient-specific cells into decellularized animal scaffolds, present a promising alternative. Despite progress in using bioengineered lungs in animal models, functionality and structure remain immature. This protocol addresses a critical barrier in organ bioengineering: the need for a cost-effective experimental platform. By using mouse models instead of larger animals like rats or swine, researchers can significantly reduce the resources required for each experiment, accelerating research progress. The protocol outlines a detailed procedure for lung bioengineering using mouse heart-lung blocks and human primary cells, focusing on isolation strategy for the mouse heart-lung block, decellularization, bioreactor setup, perfusion-based organ culture, and orthotopic transplantation of bioengineered lungs. This mouse-scale platform not only reduces experimental costs but also provides a viable framework for optimizing cell types and numbers for recellularization, testing different cell types using histological and molecular methods, and ensuring blood flow post-transplantation. The method holds potential for broad applications, including studying cell interactions in three-dimensional culture conditions, cell-matrix interactions, and ex vivo cancer modeling, thereby advancing the field of organ bioengineering. SUMMARYThis paper describes how to create bioengineered mouse lungs using the decellularization and recellularization methods. It also describes subsequent orthotopic lung transplantation in detail.

bioengineering↗

bioRxiv2024

Unsupervised learning as a computational principle works in visual learning of natural scenes, but not of artificial stimuli

Unsupervised learning--learning through repeated exposure without instruction or reward--is central to both machine learning and human cognition, including language acquisition and statistical learning. However, its role in visual perceptual learning (VPL) remains debated, as previous studies have not shown VPL for task-irrelevant but visible features, particularly in artificial stimuli. Here, we show that task-irrelevant exposure to natural scene images induces robust VPL, while artificial images that lack complex structure characteristics of natural scene images, known as higher-order statistics, do not. Behavioral and fMRI results suggest that although unsupervised learning underlies VPL, it can be suppressed by top-down attention. Higher-order statistics may evade this suppression, possibly because their slower processing reaches visual areas beyond V1 outside the optimal temporal window for attentional suppression. These findings suggest that unsupervised learning underlies VPL, but its occurrence depends on both higher-order stimulus structure and the brains attentional gating mechanisms.

neuroscience↗

bioRxiv2024

5-Deazaflavin (TND1128) and its hybrid analogs are cytoprotective against hydrogen peroxide (H2O2)-induced oxidative stress.

Increased production of reactive oxygen species (ROS) and oxidative stress are implicated in mitochondrial dysfunction, contributing to the pathogenesis of many neurodegenerative diseases. Research is ongoing into a new treatment approach for neurodegeneration, focusing on reactivating dysfunctional mitochondria. Some 5-deazaflavins, such as 10-ethyl-3-methylpyrimido[4,5-b]quinoline-2,4(3H,10H)-dione (TND1128), and four analogs of 5-deazaflavin, including {beta}-nicotinamide mononucleotide ({beta}-NMN), demonstrate efficient self-redox abilities similar to {beta}-NMN, making them potential activators of mitochondrial energy synthesis. This study examines whether TND1128 and its analogs have protective effects against cellular impairment induced by oxidative stress. These compounds exhibit proliferative potential against normal cells. Moreover, TND1128 and its analogs significantly improved cell viability against hydrogen peroxide (H2O2)-induced oxidative stress injury. Our study confirms the cytoprotective effect of these 5-deazaflavins through mitochondrial activation. We anticipate TND1128 and its analogs will serve as mitochondria-stimulating drugs capable of rescuing deteriorating neurons in aging or diseases.

pharmacology and toxicology↗

bioRxiv2024

Development of a new genotype-phenotype linked antibody screening system

Antibodies are powerful tools for the therapy and diagnosis of various diseases. In addition to conventional hybridoma-based screening, recombinant antibody-based screening has become a common choice; however, its application is hampered by two factors: 1) screening starts after Ig gene cloning and recombinant antibody production only, and 2) the antibody is composed of paired chains, heavy and light, commonly expressed by two independent expression vectors. Here, we introduce a method for the rapid screening of recombinant monoclonal antibodies by establishing a Golden Gate-based dual-expression vector and in-vivo expression of membrane-bound antibodies. Using this system, we demonstrate the rapid isolation of influenza cross-reactive antibodies with high affinity from immunized mice within 7 days. This system is particularly useful for isolating therapeutic or diagnostic antibodies, e.g., during foreseen pandemics. Impact StatementA Golden Gate-based dual-expression vector enables rapid screening, which facilitates efficient isolation of high-affinity cross-reactive antibodies for therapeutic or diagnostic use and provides a crucial advance for pandemic preparedness.

bioengineering↗

bioRxiv2024

Lignin isolated by microwave-assisted acid-catalyzed solvolysis induced cell death on mammalian tumor cells by modulating apoptotic pathways

Lignin, the most abundant renewable aromatic polymer, has been shown to suppress the growth of mammalian tumor cells. Despite being a textbook example, there is little information on the biological activity of lignin in relation to its molecular structure or the molecular mechanisms by which lignin suppresses tumor cells in mammalian species. Here, we prepared Microwave-assisted Acid-catalyzed Solvolysis Lignin (MASL) and assessed its effects on human and mouse tumor cells. Our data showed MASL significantly reduced viability of tumor cells by modulating apoptotic pathways. MASL treatment upregulated TNF-, Fas, and FasL expression levels, while suppressing anti-apoptotic NF-{kappa}B and mTOR pathways in tumor cells. In-vivo experiments were also performed using tumor-bearing mice to confirm the anti-tumor effects of MASL. An administration of MASL significantly suppressed tumor growth in mice in association with elevation of caspase 3 expression. These findings strongly suggest the potential usefulness of low molecular weight lignin as an effective therapeutic against malignancies.

cancer biology↗

bioRxiv2024

First-night effect reduces the beneficial effects of sleep on visual plasticity and modifies the underlying neurochemical processes

Individuals experience difficulty falling asleep in a new environment, termed the first night effect (FNE). However, the impact of the FNE on sleep-induced brain plasticity remains unclear. Here, using a within-subject design, we found that the FNE significantly reduces visual plasticity during sleep in young adults. Sleep-onset latency (SOL), an indicator of the FNE, was significantly longer during the first sleep session than the second session, confirming the FNE. We assessed performance gains in visual perceptual learning after sleep and increases in the excitatory-to-inhibitory neurotransmitter (E/I) ratio in early visual areas during sleep using magnetic resonance spectroscopy and polysomnography. These parameters were significantly smaller in sleep with the FNE than in sleep without the FNE; however, these parameters were not correlated with SOL. These results suggest that while the neural mechanisms of the FNE and brain plasticity are independent, sleep disturbances temporarily block the neurochemical process fundamental for brain plasticity.

neuroscience↗

bioRxiv2023

The FHA domain is essential for the autoinhibition of KIF1A/UNC-104

KIF1A/UNC-104, a member of the kinesin super family motor proteins, plays a pivotal role in the axonal transport of synaptic vesicles and their precursors. Drosophila melanogaster UNC-104 (DmUNC-104) is a relatively recently discovered Drosophila kinesin. Although some point mutations that disrupt synapse formation have been identified, the biochemical properties of DmUNC-104 protein have not been investigated. Here, we prepared recombinant full-length DmUNC-104 protein and determined the biochemical features. We analyzed the effect of a previously identified missense mutation in the FHA domain, called bristly (bris), and found that wild-type DmUNC-104 is a mixture of monomer and dimer and the bris mutation strongly promote the dimerization. Furthermore, we found that disease-associated mutations also promote the dimerization of DmUNC-104. These findings suggest that the FHA domain is essential for the autoinhibition of KIF1A/UNC-104, and that abnormal dimerization of KIF1A is linked to human diseases.

biochemistry↗

bioRxiv2023

Learning of the same task subserved by substantially different mechanisms between patients with Body Dysmorphic Disorder and healthy individuals

It is generally believed that learning of a perceptual task involving low-level neuronal mechanisms is similar between individuals. However, it is unclear whether this assumption also applies to individuals with psychiatric disorders that are known to have altered brain activation during visual processing. We investigated this question in patients with body dysmorphic disorder (BDD), a psychiatric disorder characterized by distressing or impairing preoccupation with nonexistent or slight defects in ones physical appearance, and in healthy controls. Participants completed six training sessions on separate days on a visual detection task for human faces with low spatial frequency (LSF) components. Brain activation during task performance was measured with functional magnetic resonance imaging (fMRI) on separate days prior to and after training. The behavioral results showed that both groups of participants improved on the visual detection task to a similar extent through training. Despite this similarity in behavioral improvement, neuronal changes in the Fusiform Face Area (FFA), a core cortical region involved in face processing, with training were substantially different between groups. First, activation in the right FFA for LSF faces relative to High Spatial Frequency (HSF) faces that were used as an untrained control increased after training in BDD patients but decreased in controls. Second, resting state functional connectivity between left and right FFAs decreased after training in BDD patients but increased in controls. Contrary to the assumption that learning of a perceptual task is subserved by the same neuronal mechanisms across individuals, our results indicate that the neuronal mechanisms involved in learning of a face detection task differ fundamentally between patients with BDD and healthy individuals. The involvement of different neuronal mechanisms for learning of even simple perceptual tasks in patients with BDD might reflect the brains adaptations to altered functions imposed by the psychiatric disorder.

neuroscience↗

bioRxiv2023

Emergence of a senescent and inflammatory pulmonary CD4+ T cell population prior to lung allograft failure

Lung transplantation is a life-saving therapy for end-stage pulmonary disease, but its long-term outlook is poor due to a high incidence of chronic lung allograft dysfunction (CLAD). CLAD results from alloimmune-mediated progressive fibrosis and culminates in death or the need for re-transplantation after a median of 6 years. Existing immunosuppression fails to prevent CLAD, suggesting the existence of alloimmune pathways resistant to these drugs. Here, we used mass cytometry to identify cell populations enriched in the bronchoalveolar lavage (BAL) of patients with subsequent allograft dysfunction. We show that CD4+CD57+PD1+ T cells emerge in stable lung transplant recipients in the first year post-transplant, conferring heightened risks for CLAD and death or re-transplantation. CD4+CD57+PD1+ T cells display features of senescence and secrete inflammatory cytokines. Cellular indexing by transcriptomes and epitopes (CITE-Seq) on BAL CD4+ T cells revealed the existence of 2 oligoclonal CD57+ subsets with putative cytotoxic and follicular helper functions. Finally, we observed that CD4+CD57+PD1+ T cells are associated with lung allograft fibrosis in a mouse model and in human explanted CLAD lungs, where they localize near airway epithelium and B cells. Together, our findings reveal the existence of an inflammatory T cell population that predicts future lung allograft dysfunction and may represent a rational therapeutic target in lung transplant recipients.

immunology↗

bioRxiv2023

Location analysis of presynaptically active and silent synapses in single-cultured hippocampal neurons

A morphologically present but non-functioning synapse is termed a silent synapse. Silent synapses are categorized into "postsynaptically silent synapses," where AMPA receptors are either absent or non-functional, and "presynaptically silent synapses," where neurotransmitters cannot be released from nerve terminals. The presence of presynaptically silent synapses remains enigmatic, and their physiological significance is highly intriguing. In this study, we examined the distribution and developmental changes of presynaptically active and silent synapses in individual neurons. Our findings show a gradual increase in the number of excitatory synapses, along with a corresponding decrease in the percentage of presynaptically silent synapses during neuronal development. To pinpoint the distribution of presynaptically active and silent synapses, i.e., their positional information, we enhanced the traditional Sholl analysis and introduced a novel method termed "donut analysis." Our results indicate that the distribution of presynaptically silent synapses within a single neuron does not exhibit a distinct pattern during synapse development in different areas. However, irrespective of neuronal development, the proportion of presynaptically silent synapses tends to rise as the projection site moves farther from the cell body, suggesting that synapses near the cell body may exhibit higher synaptic transmission efficiency. This study represents the first observation of changes in the distribution of presynaptically active and silent synapses within a single neuron. Additionally, we propose that donut analysis can serve as a valuable analytical tool for evaluating synaptic positional information. Scope statementA morphologically present but non-functioning synapse is termed a silent synapse. The presence of presynaptically silent synapses remains enigmatic, and their physiological significance is highly intriguing. This study focused on the distribution and developmental changes of presynaptically active and silent synapses in individual neurons. To pinpoint the distribution of presynaptically active and silent synapses, we enhanced the traditional Sholl analysis and introduced a novel method termed "donut analysis." We found that the distribution of presynaptically silent synapses within a single neuron does not exhibit a distinct pattern during synapse development in different areas. However, irrespective of neuronal development, the proportion of presynaptically silent synapses tends to rise as the projection site moves farther from the cell body. This is a new paper that applies "Sholl analysis," a method invented 70 years ago that is now the gold standard of morphological analysis of the single neuron. This study represents the first observation of changes in the distribution of presynaptically active and silent synapses within a single neuron. Additionally, we propose that donut analysis can serve as a valuable analytical tool for evaluating synaptic positional information for the design of "synaptic maps" in neural circuits.

neuroscience↗

bioRxiv2023

Geometric-relationship specific transfer in visual perceptual learning

Visual perceptual learning (VPL) is defined as long-term improvement on a visual task as a result of visual experience. In many cases, the improvement is highly specific to the location where the target is presented, which refers to location specificity. In the current study, we investigated the effect of a geometrical relationship between the trained location and an untrained location on transfer of VPL. We found that significant transfer occurs either diagonally or along a line passing the fixation point. This indicates that whether location specificity or location transfer occurs at least partially depends on the geometrical relationship between trained location and an untrained location.

neuroscience↗

bioRxiv2023

Noise reduction as a unified mechanism of perceptual learning in humans, macaques, and convolutional neural networks

Visual perceptual learning (VPL), defined as long-term improvement in a visual task, is considered a crucial tool for elucidating underlying visual and brain plasticity. However, the identification of a unified theory of VPL has long been controversial. Multiple existing models have proposed diverse mechanisms, including improved signal-to-noise ratio, changes in tuning curves, and reduction of noise correlations, as major contributors to improved neural representations associated with VPL. However, each model only accounts for specific aspects of the empirical findings, and there exists no theory that can comprehensively explain all empirical results. Here, we argue that all neural changes at single units can be conceptualized as geometric transformations of population response manifolds in a high-dimensional neural space. This approach enables conflicting major models of VPL to be quantitatively tested and compared within a unified computational theory. Following this approach, we found that changes in tuning curves and noise correlations, as emphasized by previous models, make no significant contributions to improved population representations by visual training. Instead, we identified neural manifold shrinkage due to reduced trial-by-trial neural response variability, a previously unexplored factor, as the primary mechanism underlying improved population representations. Furthermore, we showed that manifold shrinkage successfully accounts for learning effects across various domains, including artificial neural responses in deep neural networks trained on typical VPL tasks, multivariate BOLD signals in humans, and multi-unit activities in monkeys. These converging results suggest that our neural geometry theory offers a quantitative and comprehensive approach to explain a wide range of empirical results and to reconcile previously conflicting models of VPL.

neuroscience↗

bioRxiv2023

Only cortical prediction error signals are involved in visual learning, despite availability of subcortical prediction error signals

Both the midbrain systems, encompassing the ventral striatum (VS), and the cortical systems, including the dorsal anterior cingulate cortex (dACC), play roles in reinforcing and enhancing learning. However, the specific contributions of signals from these regions in learning remains unclear. To investigate this, we examined how VS and dACC are involved in visual perceptual learning (VPL) through an orientation discrimination task. In the primary experiment, subjects fasted for 5 hours before each of 14 days of training sessions and 3 days of test sessions. Subjects were rewarded with water for accurate trial responses. During the test sessions, BOLD signals were recorded from regions including VS and dACC. Although BOLD signals in both areas were associated with positive and negative RPEs, only those in dACC associated with negative RPE showed a significant correlation with performance improvement. Additionally, no significant correlation was observed between BOLD signals associated with RPEs in VS and dACC. These results suggest that although signals associated with positive and negative RPEs from both midbrain and cortical systems are readily accessible, only RPE signals in the prefrontal system, generated without linking to RPE signals in VS, are utilized for the enhancement of VPL.

neuroscience↗