Biology subjects

Watanabe, K.

Publications and source records attributed to Watanabe, K..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Trehalose promotes wound healing in vitro by enhancing the migration of human keratinocytes via the VEGF/JNK/PI3K pathway

BackgroundTrehalose is a naturally occurring disaccharide found in invertebrates but cannot be synthesized by vertebrates. We previously reported that high-concentration trehalose induces a transient senescent-like state in fibroblasts, leading to cell cycle arrest and growth factor secretion via CDKN1A/p21, and this process promoted keratinocyte proliferation, enhancing capillary formation and wound closure in vivo. ObjectiveThis study aimed to investigate the effect of trehalose on human keratinocytes. MethodsPreviously published RNA-seq data of cytokine-untreated samples from our group of trehalose-treated human keratinocytes were re-analyzed, and an in vitro scratch assay was performed using cells treated with mitomycin C. ResultsThe trehalose-treated group exhibited increased wound closure. A significantly increased secretion of vascular endothelial growth factor (VEGF) was observed in keratinocytes treated with high-concentration trehalose, which is one of the most crucial molecules inducing angiogenesis in the skin. Significant upregulation of mRNA level and protein secretion of VEGF was confirmed using qPCR and ELISA, respectively. Furthermore, treatment with axitinib, a VEGF receptor inhibitor, significantly suppressed trehalose-induced activation of keratinocyte migration. Additionally, the increase in trehalose-induced migration activity was significantly inhibited by the Jun N-terminal kinase (JNK) inhibitor SP600125 and the PI3K inhibitor LY294002. ConclusionTrehalose promotes wound healing via VEGF secretion from keratinocytes and the PI3K and JNK pathways. The findings of this study may lead to the development of novel therapeutic agents that can alter the wound healing process. IMPORTANTO_LIManuscripts submitted to Review Commons are peer reviewed in a journal-agnostic way. C_LIO_LIUpon transfer of the peer reviewed preprint to a journal, the referee reports will be available in full to the handling editor. C_LIO_LIThe identity of the referees will NOT be communicated to the authors unless the reviewers choose to sign their report. C_LIO_LIThe identity of the referee will be confidentially disclosed to any affiliate journals to which the manuscript is transferred. C_LI GUIDELINESO_LIFor reviewers: https://www.reviewcommons.org/reviewers C_LIO_LIFor authors: https://www.reviewcommons.org/authors C_LI CONTACTThe Review Commons office can be contacted directly at: office@reviewcommons.org

cell biology↗

bioRxiv2025

Reversed functional gradient in primate prefrontal cortex: posterior dominance and frontopolar deactivation

The frontopolar cortex (FPC) is thought to coordinate the more posterior lateral prefrontal cortex (LPFC) during complex, non-routine behaviors through high-level functions such as management of multiple goals, exploration, and self-generated decision-making. However, direct neurophysiological comparisons with other prefrontal regions are lacking, leaving the FPCs putative dominance untested. Contrary to this view, our comparison of neuronal activity across the full anteroposterior LPFC in macaques during six distinct tasks probing these functions revealed a posterior-to-mid LPFC dominance, with resource-allocation, novelty-detection (including reward prediction error), and modality invariant decision-monitoring signals all showing a common posterior bias. In contrast, regardless of task demands, the FPCs strongest encoding was about the most recently executed action, and it displayed minimal object selectivity, even when objects were task-critical. We identified a turning point in this graded posterior-to-anterior transition from task-positive to task-negative regions around the border between the anterior and middle thirds of the LPFC. These findings challenge the prevailing notion that the LPFC is anterior-dominant across primate species, and provide evolutionary constraints on theories of human prefrontal organization.

neuroscience↗

bioRxiv2025

Nested mobile genetic elements mediating antimicrobial resistance genes mobility within and between cells in clinical and environmental Acinetobacter isolates

Mobile genetic elements (MGEs) play a central role in the acquisition and dissemination of antibiotic resistance genes (ARGs). This study analyzed the distribution of MGEs using the whole-genome sequences of 38 Acinetobacter isolates from patient, environmental, and pig waste samples. Insertion sequence (IS) elements were identified as the most common MGE type, followed by plasmids and prophages, with significant variations depending on isolation sources. Pig waste isolates exhibited the highest mean number of plasmids, while prophages were more prevalent in environment-associated isolates. Interestingly, we observed a significant positive correlation between the number of plasmids and number of defense systems. In contrast, a significant negative correlation was identified between the number of prophages and number of defense systems. Plasmids were the primary vectors of ARGs. Co-localization of multiple ARGs, such as msr(E) and mph(E); blaOXA-58, tet(Y), mph(B), and sul2 within a single plasmid was observed, suggesting potential for co-resistance and multidrug resistance. ARGs were enriched in pdif modules, with up to 10 distinct ARGs observed within a single module. Moreover, IS elements were more densely concentrated in conjugative elements. The study further highlights the role of nested MGEs in enabling hierarchical ARG transfer processes both within and between bacterial cells. Additionally, putative novel genomic resistance islands (GRIs) were identified in non-baumannii Acinetobacter species, representing the first documentation of GRIs outside the Acinetobacter calcoaceticus-baumannii (Acb) complex. This study provides new insights into the mechanisms of ARG dissemination in Acinetobacter, particularly the role of MGEs in facilitating hierarchical gene transfer processes. IMPORTANCEUnderstanding the mechanisms behind antimicrobial resistance gene (ARG) dissemination in various environments is crucial for combating the growing global problem of antimicrobial resistance. This study investigated the distribution patterns of mobile genetic elements (MGEs) across distinct ecological contexts, analyzing samples from patients, pig wastewater, and environmental surface water. By investigating the genomes of Acinetobacter species from various isolation sources, we revealed that pig waste-associated isolates possessed more plasmid-borne ARGs compared to patient- and environment-associated isolates. Further, we demonstrated that ARG-bearing plasmids possessed higher density of IS elements than ARG-free plasmids. Putative novel genomic islands were also observed in non-baumannii species isolated from pig wastewater. These findings elucidate the hierarchical structure of ARG transfer mechanisms mediated by nested MGEs, providing valuable insights into the emergence of resistance across both clinical and environmental ecosystems.

microbiology↗

bioRxiv2025

Formation of a μ-oxo nucleophile enables efficient hydrolysis by a trinuclear metal center in Family II inorganic pyrophosphatase

Efficient catalysis by metalloproteins relies on precise spatial arrangement of metal ions and active-site residues. Family II inorganic pyrophosphatase (PPase) from Shewanella species features a trinuclear metal center and displays higher catalytic activity than binuclear counterparts. Here we elucidate its hydrolytic mechanism using X-ray crystal structure-based extended X-ray absorption fine structure (XCS-EXAFS), site-directed mutagenesis, and density functional theory (DFT) calculations. We identify a catalytic -oxo nucleophile, formed via proton transfer from a bridging -hydroxide to Asp14 and subsequent hydrogen-bond rearrangement to Asp72, as the key species in SN2-type hydrolysis. This conversion defines the rate-limiting step with an activation barrier of 15.5 kcal/mol. Molecular orbital analysis reveals that the trinuclear cluster promotes -oxo formation, aligns the nucleophile for attack, and stabilizes the transition state. The side-chain rotation of the conserved Asp14 is crucial for catalysis. Our results highlight how metalloenzymes exploit geometric and electronic tuning to achieve high reactivity through evolutionarily optimized architectures.

biophysics↗

bioRxiv2025

Microfluidic platforms for probing spontaneous functional recovery in hierarchically modular neuronal networks

Inherent capacity to flexibly reorganize after injury is a hallmark of brain networks, and recent studies suggest that functional consequences of focal damage are strongly influenced by the networks non-random connectivity. Although many of these insights have been derived from animal models and computational simulations, experimental platforms that enable bottom-up investigations of the structure-function relationships underlying damage and recovery processes remain limited. In this study, we used polydimethylsiloxane microfluidic devices to construct hierarchically modular neuronal networks that mimic the architectural features of the mammalian cortex. Laser microdissection was employed to selectively sever intermodular connections, enabling controlled damage to either hub or peripheral connections. Damage to hub connections led to delayed recovery, requiring more than three days for correlations to re-emerge. In contrast, peripheral damage resulted in faster recovery. Experiments that induced repeated injury to neuronal networks further demonstrated that recovery primarily occurred through the formation of alternative pathways rather than restoration of the original connections. These findings highlight how topological features of neuronal networks shape their response to injury and subsequent reorganization, providing mechanistic insights into the intrinsic self-repair capacity of biological systems.

neuroscience↗

bioRxiv2025

An ATP-binding cassette subfamily C is crucial for flavonoid sequestration in the domestic silkworm, Bombyx mori

Some herbivorous insects have evolved sequestration mechanisms, the ability to take up and accumulate plant secondary metabolites for their own benefit. The domestic silkworm, Bombyx mori, and its wild ancestor, B. mandarina, take up flavonoids from mulberry leaves and accumulate the molecules as their glucosides in their tissues and cocoon shell. This sequestration enhances the cocoons protective property against ultraviolet or bacterial proliferation. Here, we show that an ATP-binding cassette transporter subfamily C (ABCC) gene, BmABCC4, plays a crucial role in the flavonoid sequestration in the silkworms. BmABCC4 is located at Green c, a cocoon color-associated locus predicted in 1941 and whose detailed position we previously identified. This transporter is expressed in the midgut and silk glands, and the expression is upregulated in the midgut in late period of final instar larva. Knockout of BmABCC4 significantly reduced the total flavonoid content in the tissues and cocoon shell. Our results suggest that BmABCC4 transports flavonoid glucosides from the midgut cells to hemolymph and from the silk gland cells to the silk gland lumen.

genetics↗

bioRxiv2025

Theanine boosts frontal theta and hippocampal beta and gamma oscillations for familiarity in object recognition

Theanine, a non-proteinogenic amino acid derivative from green tea leaves, enhances object recognition memory in rats through facilitated neurogenesis. In this sense, the cellular mechanism for the theanine-enhanced object recognition memory has been elucidated to some extent, but physiological evidence still remains unclear. To tackle this issue, we chronically fed mice with theanine (or tap water) for three weeks, implanted electrodes into the hippocampus and frontal cortex, both of which are responsible for object recognition memory. We then recorded the local field potentials from the two regions during the novel object recognition task, evaluated the task performance, and broke down the neural signals in the hippocampus and frontal cortex into delta, theta, beta, low gamma, and high gamma frequency bands. The discrimination ratio of theanine-treated mice was higher than that of vehicle-treated mice. We also found that theta oscillations in the frontal cortex and beta and low gamma oscillations in the hippocampus in theanine-treated mice were simultaneously enhanced for familiar objects. These results shed light on the new physiological underpinnings of object recognition memory enhanced by exogenous substances.

pharmacology and toxicology↗

bioRxiv2025

Discrimination of Normal from Slow-Aging Mice by Plasma Metabolomic and Proteomic Features

Tests that can predict whether a drug is likely to extend mouse lifespan could speed up the search for anti-aging drugs. We have applied a machine learning algorithm, XGBoost regression, to seek sets of plasma metabolites that can discriminate control mice from mice treated with an anti-aging diet (caloric restriction) or any of four anti-aging drugs. When the model is trained on any four of these five interventions, it predicts significantly higher lifespan extension in mice exposed to the intervention which was not included in the training set. Plasma peptide data sets also succeed at this task. Models trained on drug-treated normal mice also discriminate long-lived mutant mice from their respective controls, and models trained on males can discriminate drug-treated from control females. Triglycerides are over-represented among the most influential features in the regression models. Triglycerides with longer fatty acid chains tend to be higher in the slow-aging mice, while triglycerides with shorter fatty acid chains tend to decrease. Plasma metabolite patterns may help to select the most promising anti-aging drugs in mice or in humans, and may give new leads into physiological and enzymatic targets relevant to discovery of new anti-aging drugs.

bioinformatics↗

bioRxiv2025

MARK4 enhances stress granule formation and increases tau accumulation

Stress granules (SGs) are membrane-less organelles that are formed in response to cellular stress. SG formation protects cells against stress; however, their dysregulation and longer persistence may disrupt proteostasis. Abnormal activity of Microtubule-affinity regulating kinase 4 (MARK4) has been linked to cancer and Alzheimers disease. Here, we report that MARK4 is a component of SGs and enhances SG formation. MARK4 is found in SGs in mammalian cultured cells and primary neurons with T-cell intracellular antigen 1 (TIA1). MARK4 expression enhances SG formation induced by hydrogen peroxide. The spacer domain of MARK4 mediates its localization to SGs, and its kinase activity is required for the enhancement of SG formation. MARK4 suppresses TIA1 dimerization induced by hydrogen peroxide. Furthermore, we found that MARK4 and TIA1 synergistically promoted tau accumulation in cultured cells, and knockdown of a fly TIA1 homolog suppressed tau toxicity in a Drosophila model. These results indicate that MARK4 regulates TIA1 and SG formation and suggest that MARK4 and TIA1 synergistically contribute to tau toxicity.

cell biology↗

bioRxiv2025

ANI-netID: a genome similarity-network based biological identification system

Fungal identification is based on sequence similarity comparisons, with multiple genes often utilized in conjunction, similar to most taxonomic studies. The identification process involves the individual comparison of the similarities of each gene. However, owing to insufficient information, incomplete lineage sorting, gene transfer, hybridization, gene duplication, and loss, different species may match, depending on the DNA region used for comparison. Additionally, identification methods exist that set a uniform similarity threshold value, but they hit multiple species above the threshold. In this study, we introduced ANI-netID, which was developed to address these challenges. ANI-netID is an identification method based on the phylogenetic species concept, where the threshold value is determined by the combination of individuals with the lowest nucleotide similarity within groups (clique groups), such as species and genera. This checks whether the queried individual fall into the same clique group as the reference individual with the highest similarity. ANI-netID solves not only the above-mentioned problems but also indicates that the individual being identified may represent a new lineage, if the individual does not belong to any clique group.

microbiology↗

bioRxiv2025

Human TIMELESS, a potential circadian clock regulator, plays an essential role in survival and reawakening of metastasis-initiating cells in bone

Bone metastasis is becoming increasingly common globally. In such cases, cancer cells disseminate from the primary site to bone, subsequently entering a dormant state. Following a certain incubation period, the cells reawaken and grow, forming a metastatic mass. These dormant cells are called metastasis-initiating cells (MICs), but their survival and reawakening are poorly understood. Here, we established an in vivo MIC-reawakening mouse model in bone. Microarray analysis demonstrated enrichment for mitochondrial oxidative phosphorylation (OXPHOS) and fatty acid synthesis in isolated MICs. In addition, transcriptional regulator TIMELESS was identified as an independent poor prognostic factor by using clinical transcriptomic datasets, which was validated by immunostaining on 209 breast cancer cases. TIMELESS-deficient breast, prostate, and bladder cancer cell lines exhibited reduced viability and MIC reawakening in bone. Single-cell analysis on the epigenetic landscape of MICs revealed that motif activities of CLOCK and BMAL1/ARNTL were enhanced in a cluster with increased TIMELESS accessibility scores. As TIMELESS regulates stemness through the OXPHOS metabolic state, an inhibitor targeting the metabolic pathway, MP-A08, was identified to suppress MIC reawakening and growth in bone more efficiently than cisplatin. These results suggest that TIMELESS regulates survival and reawakening in MICs and MP-A08 could contribute to adjuvant therapeutic strategies.

cancer biology↗

bioRxiv2025

Epithelial-mesenchymal transition couples with cell cycle arrest at various stages

Numerous computational approaches have been developed to infer cell state transition trajectories from snapshot single-cell data. Most approaches first require projecting high-dimensional data onto a low-dimensional representation; however, this can distort the dynamics of the system. Using epithelial-to-mesenchymal transition (EMT) as a test system, we show that both biology-guided low-dimensional representations and trajectory simulations in high-dimensional state space, not representations obtained with brute force dimensionality-reduction methods, reveal two broad paths of TGF-{beta} induced EMT. The paths arise from the coupling between cell cycle and EMT at either the G1 or G2/M phase, contributing to cell-cycle related EMT heterogeneity. Subsequent multi-plex immunostaining studies confirmed the multiple predicted paths at the protein level. The present study highlights the heterogeneity of EMT paths, emphasizes that caution should be taken when inferring transition dynamics from snapshot single-cell data in two- or three-dimensional representations, and shows that incorporating dynamical information can improve prediction accuracy.

systems biology↗

bioRxiv2025

The pivotal role of a novel free fatty acid receptor GPR164 in the intestinal barrier function

GPR164 is a novel free fatty acid receptor, activated by both short-chain fatty acids and medium-chain fatty acids, and expressed throughout the gastrointestinal tract. Although GPR164 is reported to be involved in the release of gut hormones, the physiological functions of this receptor in the maintenance of intestinal homeostasis remain unclear. In this study, we explored the role of GPR164 in regulating intestinal barrier function using mice lacking Gpr164 gene (Gpr164-/-). A loss-of-function mutation in GPR164 promoted cell proliferation and disrupted the intestinal barrier function in both Caco-2 cell line and mice. Genome-wide RNA-seq analysis revealed that GPR164 deletion caused aberrant wnt/{beta}-catenin signaling, and the intraperitoneal injection of wnt/{beta}-catenin inhibitor ameliorated a series of abnormalities of Gpr164-/- mice. Gpr164-/- mice also exhibited gut microbial dysbiosis and severe inflammation, indicating that deletion of Gpr164 causes similar pathologies observed in patients with inflammation bowel disease (IBD). Thus, our findings uncover the pivotal role of GPR164 in the maintenance of intestinal barrier function, providing an attractive clinical target for IBD.

physiology↗

bioRxiv2025

Crystal structure of a thermophilic family II inorganic pyrophosphatase enabling high-temperature adaptation in Thermodesulfobacterium commune

Inorganic pyrophosphatases (PPases) are crucial for energy metabolism and classified into families with distinct metal ion requirements and structural features. This study is the first to successfully express, purify, and structurally characterize a thermophilic family II PPase isolated from Thermodesulfobacterium commune (TcPPase). TcPPase, which is optimally activated by Co2+ and Mn2+, required both the N- and C-terminal domains for complete catalytic function. Comparative structural analyses of psychrophilic and mesophilic homologs revealed that the enhanced thermal stability of TcPPase was due to its strong hydrophobic interactions, high proline content, dense hydrogen bond network, and additional salt bridges. These findings reveal the molecular basis for the thermal adaptation of family II PPases, providing valuable insights for thermostable enzyme engineering for biotechnological applications.

biochemistry↗

bioRxiv2025

Predator-Induced Tail Coloration Toward Diverse Dragonfly Nymphs in Tadpoles of the Japanese Tree Frog (Dryophytes japonicus)

Anuran larvae can change their coloration and morphology to avoid predation. Particularly, some tadpoles in the genus Dryophytes in the Americas develop deep tail fins and bright orange tail coloration in response to predators such as dragonfly nymphs. These conspicuous tails are hypothesized to attract predator attacks, protecting vital body parts from fatal injuries. However, it remains unclear whether the extent of the induced phenotypic changes differs between various predators with distinct feeding patterns and habitats. To explore this, we reared tadpoles of the East Japan tree frog, Dryophytes leopardus, with four dragonfly species, water bug, water scorpion, or newt, and measured phenotypic changes, especially focusing on tail coloration. We found that the aeshnid Anax nigrofasciatus, a sit- and-wait predator that climbs on aquatic vegetation, induced bright orange and broad tails of tadpoles. Other odonate species that forage in and on the substrate also elicited tail coloration or deeper larval tails, but it took more time to induce the tadpoles response compared to A. nigrofasciatus. In contrast, non-odonate predators did not induce color and morphological changes. This study provides the first evidence of predator-induced tail coloration in an Asian species of Dryophytes, which appears to be a defensive response specifically to dragonfly nymphs, as visually adept predators. Moreover, the differences in the extent and timing of the phenotypic changes among odonate species suggest that anti-predator phenotypes are modulated based on relative predation risks, considering the predators foraging patterns and visual capabilities, thereby allowing tadpoles to minimize physiological costs and unintended risks associated with the induced traits.

ecology↗

bioRxiv2025

APMAT analysis reveals the association between CD8 T cell receptors, cognate antigen, and T cell phenotype and persistence

Elucidating the relationships between a class I peptide antigen, a CD8 T cell receptor (TCR) specific to that antigen, and the T cell phenotype that emerges following antigen stimulation, remains a mostly unsolved problem, largely due to the lack of large data sets that can be mined to resolve such relationships. Here, we describe Antigen-TCR Pairing and Multiomic Analysis of T-cells (APMAT), an integrated experimental-computational framework designed for the high-throughput capture and analysis of CD8 T cells, with paired antigen, TCR sequence, and single-cell transcriptome. Starting with 951 putative antigens representing a comprehensive survey of the SARS-CoV-2 viral proteome, we utilize APMAT for the capture and single cell analysis of CD8 T cells from 62 HLA A*02:01 COVID-19 participants. We leverage this unique, comprehensive dataset to integrate with peptide antigen properties, TCR CDR3 sequences, and T cell phenotypes to show that distinct physicochemical features of the antigen-TCR pairs strongly associate with both T cell phenotype and T cell persistence. This analysis suggests that CD8+ T cell phenotype following antigen stimulation is at least partially deterministic, rather than the result of stochastic biological properties.

immunology↗

bioRxiv2024

Cross-regulation of amino acid synthesis and catabolic electron transfer in bacteria

Amino acids are essential for life, serving primarily as the building blocks of proteins. Although the intracellular metabolic fate of amino acids is known, their roles in the global regulation of metabolic pathways are poorly characterized. Here, we investigated the function of amino acids as signaling molecules that modulate the catabolic activity of bacterial cells. Using the model bacterium Shewanella oneidensis MR-1, we showed that methionine activates catabolic processes, including extracellular electron transfer, at submillimolar concentrations. This regulation is mediated by MetR, a transcription factor that regulates methionine biosynthesis. In addition, the MetR-mediated regulation of catabolic electron transfer was observed in Aeromonas hydrophila. These findings reveal methionine-induced signaling cascades that cross-regulate amino acid biosynthesis and catabolism in bacteria. Our study highlights a novel physiological role for amino acids, suggesting their involvement in the coordinated regulation of anabolism and catabolism, particularly in bacteria that thrive in nutrient-limited habitats.

microbiology↗

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↗