Biology subjects

Okuda, S.

Publications and source records attributed to Okuda, S..

At least 19 recordsLinked to original sources

bioRxiv2026

MassBank Human: The annotated human metabolome

Mass spectrometry-based metabolomics is essential for deciphering human metabolic functions and metabolite dark matter. However, the dark matter of the human metabolome is intricately hidden. Therefore, we developed MassBank Human, a curated library that distills 36,512,540 MS2 spectra from 76 public human datasets into 203,886 high-quality spectra via spectral averaging, recalibration, and filtering to achieve high mass accuracy. We further used three metabolite annotation tools and developed a Zipf-Bayesian rank aggregation framework to aggregate their rankings with false discovery rate control. This framework assigned molecular formulas, chemical classes, and planar structures to 55,564, 22,619, and 14,198 spectra, respectively. Approximately half of the annotated structures potentially originated from food, pollutants, or gut microbiota. In a metabolome analysis of colorectal cancer cells, MassBank Human doubled structural annotation coverage (10.6% to 21.2%), identifying acetylated polyamines as markers of metabolic rewiring. This community resource provides an annotated foundation for exploring human metabolomes.

bioinformatics↗

bioRxiv2026

Chromosome-scale genome assembly of Cycas revoluta provides insights into cycad diversification and species diversity

Cycads are an ancient group of seed plants. Despite their ancient origin, many extant cycad genera exhibit high species diversity. The specialized reproductive traits in cycads, dioecy governed by the XY sex-determination system and the elaborate co-evolutionary synergy with insect pollinators, may facilitate lineage diversification. Here, we present a chromosome-scale genome sequence of Cycas revoluta, the species in which plant spermatozoids were discovered in 1896. The genome sequence spanned 11.6 Gb, 98.4% of which were anchored onto the 11 cycad chromosomes. Repetitive sequences occupied 9.8 Gb, and 31,481 genes were predicted. Based on this genome assembly, the X- and Y-associated genomic regions were characterized, and candidate genes for sex determination were identified. In addition, the genomic positions of genes for sex-related traits were determined. Subsequently, we analyzed transcriptomes for thermogenesis responsible for attracting insect pollinators. Through these comprehensive analyses, we provide new insights into the genomic basis of cycad diversification and species diversity.

genomics↗

bioRxiv2026

Calcium Dynamics During Pollen Tube Reception in Arabidopsis Ovules

In flowering plants, pollen tubes communicate with ovular cells to achieve precise one-to-one pollen tube reception. The final step of this communication between the pollen tube and synergid cells has been extensively investigated and visualized by calcium imaging. Synergid cells exhibit characteristic cytoplasmic calcium concentration oscillations, which are thought to play a critical role in pollen tube reception. However, their significance and relationship with calcium dynamics in the entire ovule remain unclear. Here, we show, using the calcium sensor GCaMP6s, that proteins involved in asparagine-linked glycosylation (N-linked glycosylation) are required for normal calcium oscillations in synergid cells but are not essential for pollen tube reception. Using a semi-in vivo assay in Arabidopsis thaliana, we found that the amplitude of these oscillations prior to rapid pollen tube growth across the filiform apparatus was reduced in mutants lacking the oligosaccharyltransferase (OST) 3/6 subunit or alpha1,2-glucosyltransferase (ALG) 10, both of which are involved in N-linked glycosylation. Notably, these mutants did not exhibit reduced fertility attributable to defects in the female gametophyte but instead showed a polytubey phenotype due to a sporophytic defect. These findings suggest that N-linked glycans mediate communication between synergid cells and the pollen tube and indicate that the typical pattern of calcium oscillations in synergid cells is not essential for triggering pollen tube rupture. Furthermore, we show that sporophytic tissues of the ovule exhibit calcium waves that propagate toward the funiculus in correlation with pollen tube contact and rupture, implying that ovular tissues can potentially transmit these signals distantly beyond the ovule. Together, these findings reveal previously unrecognized intercellular calcium signaling and its significance in pollen tube reception by the ovule.

plant biology↗

bioRxiv2026

Computational Design for Engineering Layered Tissue Architectures via Cell-Cell Interfacial Tension Modulation

The spatial arrangement of cells is fundamental to the mechanical and functional integrity of three-dimensional (3D) tissues, yet engineering spatially well-controlled tissue architectures remains challenging. Here, we computationally investigated how layered tissue architectures can be designed by modulating cell-cell interfacial tension. We performed simulations using a 3D vertex model and systematically varied interfacial tension magnitudes. The simulations generated a range of layered tissue architectures, including planar monolayers, bilayers, and structurally stratified states. In homogeneous cell populations, increasing interfacial tension drove transitions from monolayer to structurally stratified configurations. In heterogeneous populations, differential interfacial tensions induced out-of-plane cell sorting and the formation of compositionally sorted multilayers. Moreover, a recursive tension design strategy enabled hierarchical organization of multiple cell types into separate layers. Notably, this recursive scheme uses only two tension levels (high vs. low) assigned across interfaces and can, in principle, be extended to specify layered architectures with an arbitrary number of layers. Together, these results identify cell-cell interfacial tension as a tunable mechanical parameter for regulating layered tissue architecture and provide design principles for layered tissue engineering and regenerative medicine.

bioengineering↗

bioRxiv2026

Divergent mechanics of inner ear morphogenesis are coupled to developmental tempo over vertebrate evolution

Vertebrate embryos vary in size and developmental tempo based on reproductive strategies; however, organs of ancient origin, such as the eye and inner ear, remain highly conserved in structure and function. How both diverse embryonic traits and conserved organ development occur remains unclear. Here, we identify divergent morphogenetic mechanics of inner ear development, particularly in vesicular primordium expansion, in 12 species representing the major vertebrate lineages. Differences in cell volume regulation determine tissue growth mechanics, which diversify the expansion process but yield a conserved morphological outcome. Heterochronic shifts in organogenesis allow this regulation to evolve in coordination with a prolonged developmental period. These findings suggest that alternative morphogenetic mechanics mitigate the effects of diversified embryonic traits, thereby ensuring consistent morphological outcomes for highly conserved organs.

evolutionary biology↗

bioRxiv2026

A live-imaging system for Arabidopsis leaf primordia at early stages

Live imaging is one of the most powerful methods to reveal the morphogenesis of plant organs. However, the highly three-dimensional structure of plant organs always poses technical challenges. For example, the basal region of leaf primordia is rarely observed because of the shape of leaf primordia and the sudden shift in geometry at the point where the leaf primordium connects to the hypocotyl. In this work, we developed a new live-imaging system that is suitable for observing the developmental process of the basal region of Arabidopsis leaf primordia at early stages. Using this system, we achieved continuous observation of the basal region of early Arabidopsis leaf primordia for more than 50 hours.

plant biology↗

bioRxiv2025

Generative AI-based design of hybrid transcriptional activator proteins with new DNA-binding specificity

Transcriptional control arises from the specific recognition of promoter DNA by transcription factors (TFs), forming the basis of cellular information processing and gene regulation. In synthetic biology, TF-promoter interactions are assembled into genetic circuits to program cellular behaviors. To ensure expected circuit performance, most synthetic genetic circuits rely on combinations of well-characterized and orthogonal transcriptional regulations. This reliance minimizes crosstalk but constrains circuit complexity and information integration. Creating hybrid TFs that combine or interpolate promoter specificities could therefore expand the design space of synthetic regulatory systems. However, it remains unclear whether hybrid functions can be created by mixing amino acid sequences, and how such functional integration could be achieved in a principled manner. Here we show that a variational autoencoder (VAE) trained on LuxR-family DNA-binding domains can generate transcription factors with hybrid and partially novel promoter recognition properties. By sampling intermediate regions of the VAE-learned latent space, we designed hybrid TFs that activate both the lux and las promoters. High-throughput sort-seq assays together with individual in vivo assays revealed that a subset of functional variants exhibited dual-responsive behavior while maintaining sequence-selective DNA recognition. Together, these results provide a data-driven strategy for exploring functional intermediate sequences between closely related proteins.

synthetic biology↗

bioRxiv2025

Axial Rotation Comprises Concurrent Twisting and Bending, Each Differentially Regulated by TGF-β Signaling

Axial rotation (AR), a morphogenetic movement that reshapes the body axis, is widely observed in chordates, including mice and rats. AR involves complex three-dimensional deformations; however, its geometric characteristics and regulatory mechanisms remain poorly understood. Here, using the chordate Ciona robusta (Ciona intestinalis type A), we demonstrate that AR consists of two components--leftward bending and clockwise twisting along the anterior-posterior axis--differentially regulated by TGF-{beta} signaling. A comparison between chorionated and dechorionated embryos revealed that dechorionation randomized the bending direction, while twisting remained consistently clockwise. Inhibition of TGF-{beta} signaling with SB431542 randomized both deformations. Quantitative analysis of twisting angles indicated uniform clockwise twisting along the axis, peaking during the tailbud stage and proceeding in the tail region, independent of the tip, trunk, or myofibril patterning. Although overall twisting was reduced under TGF-{beta} inhibition, the tail exhibited disorganized twisting. The sum of absolute twisting-angle differences in every 10 m remained comparable to the wild type (WT). This suggests that twisting is intrinsically generated, while TGF-{beta} signaling aligns local twisting into a coordinated global direction. Our findings dissected the mechanisms of AR in Ciona and highlight the multilayered regulation underlying the morphogenesis of the chordate body plan and providing a foundation for understanding its biomechanical and molecular bases.

developmental biology↗

bioRxiv2025

An antiparallel cell circulation driven by self-alignment induces phase separation

Active tissues exhibit diverse collective dynamics, yet the cell-cell interactions that generate ordered microscopic flows remain poorly understood. Here, we show that antiparallel cell circulation can emerge from self-aligned, polarity-dependent tension gradients. Using a minimal vertex model of confluent tissues, we studied polar cells that align their polarity with their own velocity and impose polarity-dependent tension gradients along cell-cell contacts, without relying on substrate traction. This behavior can be generalized as a minimal interaction in which forces transmitted between cells act with opposite signs, reminiscent of action-reaction forces, organizing cells into stable interlocking antiparallel lanes. In mixtures of motile and nonmotile cells, this circulation drives phase separation, in which motile cells spontaneously form persistent domains. Accordingly, we identified similar antiparallel circulation patterns in two-dimensional aggregates of Dictyostelium discoideum, supporting the biological relevance of the mechanism. Together, these results demonstrate that self-aligned tension gradients provide a robust and underappreciated route to dynamic microscopic pattern formation in multicellular systems.

biophysics↗

bioRxiv2025

Sequestration-Based Neural Networks That Operate Out of Equilibrium

Classification of high-dimensional information is a ubiquitous computing paradigm across diverse biological systems, including organs such as the brain, down to signaling between individual cells. Inspired by the success of artificial neural networks in machine learning, the idea of engineering genetic circuits that operate as neural networks emerges as a strategy to expand the classification capabilities of living systems. In this work, we design these biomolecular neural networks (BNNs) based on the molecular sequestration reaction, and experimentally characterize their behavior as linear classifiers for increasing levels of complexity. Initially, we demonstrate that a static, DNA-based system can effectively prototype a linear classifier, though we also identify its limitations to easily tune the slope of the decision boundary it generates. We then propose and experimentally validate a BNN at the protein level using a cell-free transcription-translation (TXTL) system, which overcomes the DNA-based systems limitation and behaves as a linear classifier even before it reaches its steady state (or, equivalently, out-of-equilibrium). Ultimately, we test a CRISPR-based design and its out-of-equilibrium behavior in a biological context by successfully constructing a linear classifier within mammalian cells. Overall, by leveraging mathematical modeling and experimental automation, we establish molecular sequestration as a universal scheme for implementing neural networks within living systems, paving the way for transformative advances in synthetic biology and programmable biocomputing systems.

synthetic biology↗

bioRxiv2025

Intermediate Evolutionary State of Motile Sperm and Pollen Tubes in the Extant Gymnosperm Cycas revoluta

The transformation of male reproductive cells is a significant event in the evolution of land plant fertilization systems from zooidogamy to siphonogamy. Basal plants such as bryophytes and pteridophytes have motile sperm, whereas most seed plants have non-motile sperm, delivered by a pollen tube. Despite being seed plants, gymnosperm cycads and ginkgo uniquely form highly multi-flagellated and large motile sperm within pollen tubes. However, the evolutionary state of these male reproductive cells remains unknown. We clarified the gene expression profiles of Cycas revoluta pollen tubes and motile sperm that swam toward female reproductive cells. Male cycad cells had fewer genes associated with transcription, translation, and related processes, which is consistent across land plants. Similar to other male gametes, a sperm-specific histone variant may contribute to transcriptional regulation via chromatin condensation. Plasma membrane-localized proteins probably involving male-female interactions revealed that cycad sperm express both factors found in motile plant sperm and seed plant sperm cells, suggesting a transitional state from motile to non-motile male gametes. In contrast, cycad pollen tubes lacked plasma membrane-localized receptor genes previously reported in angiosperms and cell wall-associated factors involved in pollen tube tip growth. These results support the hypothesis that the function of the cycad pollen tube is confined to the haustoria as previously reported. These results indicate a molecular intermediate state of the cycad fertilization mechanism with motile sperm and pollen tubes, providing important insights into the evolution of land plant fertilization. Significant StatementCycads and ginkgo, which possess primitive motile sperm and derived pollen tubes, are important for understanding the evolution of land plant fertilization from zooidogamy to siphonogamy. We explored gene expression in living, capacitated motile sperm and pollen tubes of Cycas revoluta in a tissue-specific transcriptome analysis. Similar to other land plants, the transcriptional repression mechanism has been suggested in these male cells. Plasma membrane-localized genes revealed the characteristics of motile and non-motile sperm, while molecular evidence of pollen tube guidance and tip growth, as seen in angiosperms, was lacking in cycads. A molecular intermediate state of the cycad fertilization mechanism was clarified, providing important insights into the evolutionary process of land plant fertilization.

plant biology↗

bioRxiv2024

Epithelium Stratifies via Nucleation and Growth Induced by Foam-Geometric Instability

The epithelium undergoes stratification, transitioning from a monolayer to a multilayer structure, across broad phenomena. Recent studies have identified several cell behaviors as triggers, including junctional tension, cell density and geometry, and topological defects. However, how these factors drive stratification throughout the entire epithelium remains poorly understood. Here, we report a mechanism underlying epithelial stratification that mirrors the physics of phase transition. Combining cell culture with three-dimensional vertex modeling, we demonstrate that epithelial stratification is analogous to a structural phase transition driven by the nucleation-growth process, i.e., multilayer origins dispersedly appear and expand across the epithelium via unordered intermediate states. This transition is induced by a mechanical instability inherent in the foam-like geometry of the epithelium. Moreover, the nucleation-growth concept applies to embryonic skin development and intestinal cancer transformation. These findings conceptualize epithelial stratification as a form of a phase transition governed by foam mechanics, offering a physical perspective on various epithelial developments.

biophysics↗

bioRxiv2024

Circumferential actomyosin bundles drive endothelial cell deformations to constrict blood vessels

Following the formation of new blood vessels, vascular remodelling ensues to generate a hierarchical network of vascular tubes with optimal connections and diameters for efficient blood perfusion of tissues. How transitions in endothelial cell (EC) number and shape are coordinated to define vessel diameter during development remains an open question. In this study, we discovered EC deformations, rearrangements and transient formation of self-seam junctions as key mechanisms that explain a negative relationship between cell number and vessel diameter. High-resolution analysis of actin cytoskeleton organization disclosed the generation of tension-bearing, circumferential actomyosin bundles in the endothelial cortex that drive EC deformation and vessel constriction. Importantly, the loss of circumferential actin bundles in krit1/ccm1-deficient ECs causes cell enlargement and impaired vessel constriction that culminate in dilated vessels, characteristic of cerebral cavernous malformation. Our multiscale study therefore underpins circumferential actomyosin-driven EC deformations in controlling vessel size and in the prevention of vascular malformations.

cell biology↗

bioRxiv2024

Strain softening and hysteresis arising from 3D multicellular dynamics during long-term large deformation

Living tissues exhibit complex mechanical properties, including viscoelastic and elastoplastic responses, that are crucial for regulating cell behaviors and tissue deformations. Despite their significance, the intricate properties of three-dimensional (3D) multicellular tissues are not well understood and are inadequately implemented in biomaterial engineering. To address this gap, we developed a numerical method to analyze the dynamic properties of multicellular tissues using a 3D vertex model framework. By focusing on 3D tissues composed of confluent homogeneous cells, we characterized their properties in response to various deformation magnitudes and time scales. Stress relaxation tests revealed that large deformations initially induced relaxation in the shapes of individual cells. This process is amplified by subsequent transient cell rearrangements, homogenizing cell shapes and leading to tissue fluidization. Additionally, dynamic viscoelastic analyses showed that tissues exhibited strain softening and hysteresis during large deformations. Interestingly, this strain softening originates from multicellular structures independent of cell rearrangement, while hysteresis arises from cell rearrangement. Moreover, tissues exhibit elastoplastic responses over the long term, which are well represented by the Ramberg-Osgood model. These findings highlight the characteristic properties of multicellular tissues emerging from their structures and rearrangements, especially during long-term large deformations. The developed method offers a new approach to uncover the dynamic nature of 3D tissue mechanics and could serve as a technical foundation for exploring tissue mechanics and advancing biomaterial engineering.

bioengineering↗

bioRxiv2024

Isotonic and minimally invasive optical clearing media for live cell imaging ex vivo and in vivo

Tissue clearing has been widely used for fluorescence imaging of fixed tissues, but not for live tissues due to its toxicity. Here we develop minimally invasive optical clearing media for fluorescence imaging of live mammalian tissues. Light scattering is minimized by adding spherical polymers with low osmolarity to the extracellular medium. A clearing medium containing bovine serum albumin (SeeDB-Live) is minimally invasive to live cells, allowing for structural and functional imaging of live tissues, such as spheroids, organoids, acute brain slices, and the mouse brain in vivo. SeeDB-Live minimally affects the electrophysiological properties and sensory responses of neurons. We demonstrate its utility for widefield imaging of subcellular voltage dynamics, such as backpropagating action potentials, in acute brain slices. We also utilize SeeDB-Live for widefield voltage imaging of dozens of dendrites in vivo, demonstrating population dynamics. Thus, SeeDB-Live expands the scale and modalities of fluorescence imaging of live mammalian tissues.

neuroscience↗

bioRxiv2024

Ex vivo SIM-AFM measurements reveal the spatial correlation of stiffness and molecular distributions in 3D living tissue

Living tissues each exhibit a distinct stiffness, which provides cells with key environmental cues that regulate their behaviors. Despite this significance, our understanding of the spatiotemporal dynamics and the biological roles of stiffness in three-dimensional tissues is currently limited due to a lack of appropriate measurement techniques. To address this issue, we propose a new method combining upright structured illumination microscopy (USIM) and atomic force microscopy (AFM) to obtain precisely coordinated stiffness maps and biomolecular fluorescence images of thick living tissue slices. Using mouse embryonic skin as a representative tissue with mechanically heterogeneous structures inside, we validate the measurement principle of USIM-AFM. Live measurement of tissue stiffness distributions revealed the highly heterogeneous mechanical nature of embryonic skin as well as the role of collagens in maintaining its integrity. Furthermore, quantitative comparisons of stiffness distributions of preserved tissue samples unveiled the distinct impacts of conventional tissue preservation techniques on the tissue stiffness pattern. This series of experiments highlights the importance of live mechanical testing of tissue-scale samples. Our USIM-AFM technique provides a new methodology to reveal the dynamic nature of tissue stiffness and its correlation with biomolecular distributions in live tissues and thus could serve as a technical basis for exploring tissue-scale mechanobiology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/595975v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@8a32f5org.highwire.dtl.DTLVardef@117a350org.highwire.dtl.DTLVardef@e2fb99org.highwire.dtl.DTLVardef@1f41d24_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Vesicle-Mediated Regulation of Pollen tube Growth in Torenia fournieri

In flowering plants, the growth of pollen tubes carrying sperm cells to the female gametophyte is necessary for double fertilization and seed development. The rate of growth of the pollen tube tip determines the fertility of many artificially cultivated plants and depends on the vesicle-mediated transport of cell membrane and cell wall components. However, the investigation of vesicle transport is hampered by the lack of transgenic methods for economically important plants. Here, we developed a method to transiently inhibit vesicle activity using brefeldin A (BFA) and antisense oligodeoxynucleotides (AS-ODNs) targeting key genes in the wishbone flower (Torenia fournieri), which has been used in studies of sexual reproduction in plants. BFA disrupted vesicle gradient homeostasis, thereby altering cell wall deposition and pollen tube morphology. The AS-ODN targeting Torenia fournieri ANXUR (TfANX), which is implicated in maintaining pollen tube growth and is used as a marker to assess inhibition by AS-ODNs, penetrated cell membranes and inhibited the expression of target genes. The treatment of pollen tubes with AS-ODN against TfRABA4D, which is specifically expressed in pollen and involved in regulating vesicle targeting, resulted in pollen tubes with a bulging phenotype and disrupted pectin deposition, effects similar to those of BFA. In summary, vesicle-mediated mechanisms regulate the patterning of the pollen tube cell wall in T. fournieri and our findings could facilitate the genetic manipulation of horticultural crops.

plant biology↗

bioRxiv2023

Elasto-Plastic Transition in Epithelial Folding

During morphogenesis, epithelial sheets undergo sequential folding to form three-dimensional organ structures. The resulting folds are irreversible, ensuring that morphogenesis progresses in one direction. However, the mechanism establishing the irreversibility of folding remains unclear. Here, we report a novel mechanical property of epithelia that is responsible for folding irreversibility. Using a newly developed mechanical indentation assay, we demonstrate that short-term or low-curvature folding induces an elastic, shape-restoring response. In contrast, combined long-term, high-curvature folding results in plastic, irreversible deformation. This elastic-to-plastic transition occurs in a switch-like manner, with critical thresholds for the folding curvature and duration. Specific cells at the fold initiate this transition, sensing the curvature and duration of folding on their apical side via mechanosensitive signaling pathways, including transient receptor potential canonical (TRPC) 3/6-mediated calcium influx and ligand-independent epidermal growth factor receptor activation. These pathways induce F-actin accumulation into a bracket-like structure across the fold, establishing the transition. The duration threshold is determined and tunable by the actin polymerization rate. These results demonstrate that cells control the irreversibility of epithelial folding by detecting folding characteristics and adaptively switching between elastic and plastic responses. This finding resolves a long-standing question about the directionality of morphogenesis.

developmental biology↗