Biology subjects

Okada, Y.

Publications and source records attributed to Okada, Y..

At least 19 recordsLinked to original sources

bioRxiv2026

Beyond point estimates: quantifying predictive uncertainty reveals hidden dimensions of biological age acceleration and improves risk interpretation

Biological age estimates are increasingly used to study aging, disease risk, and mortality, yet their predictive uncertainty is rarely quantified. Consequently, conventional age-gap measures can treat deviations as equally informative even when the underlying biological age predictions differ substantially in reliability. We developed a framework for uncertainty-aware biological aging that generates calibrated prediction intervals and individualized probabilities of accelerated or decelerated aging alongside point estimates. We applied this framework to the UK Biobank Pharma Proteomics Project, evaluating three composite and eleven organ-specific biological age clocks. Predictive uncertainty varied substantially both within and across clocks, revealing that apparently extreme age gaps can differ markedly in the strength of evidence supporting accelerated or decelerated aging. In particular, low-accuracy clocks, including many organ-specific clocks, provided little evidence for confidently accelerated or decelerated aging. Beyond biological age gaps, prediction-interval width was independently associated with disease risk and mortality, particularly for composite, brain, and immune clocks, suggesting that predictive uncertainty captures an additional dimension of biological aging that may reflect increased molecular heterogeneity and dysregulation associated with aging and disease. We replicated these findings in Biobank Japan and an independent clinical cohort from Stanford. By incorporating individual-specific predictive uncertainty, our framework provides a more informative characterization of biological aging and enables improved individual-level risk stratification for disease prevention and longitudinal monitoring.

bioinformatics↗

bioRxiv2026

Dysregulated flow responses drive pathogenesis in a cellular model of Sturge-Weber syndrome

Vascular diseases comprise a broad spectrum of conditions associated with organ dysfunction, yet how endothelial responses to mechanical forces drive their pathogenesis remains unclear. Sturge-Weber syndrome (SWS), a flow-associated vascular malformation caused by somatic GNAQ mutations, provides a model to investigate this question. Here, using isogenic induced pluripotent stem cell-derived endothelial cells (ECs), perfusable vascular models, and simplified systems under defined flow conditions, we show that SWS ECs exhibited maladaptive responses to laminar flow, including impaired flow alignment, reduced CLDN5 expression, and decreased transendothelial electrical resistance. Importantly, three-dimensional acoustic microscopy uncovered disturbed surface changes in SWS ECs, potentially contributing to changes in mechanosensing signaling. Transcriptomic and chromatin accessibility analyses revealed dysregulated flow-responsive gene programs, some of which were validated in human SWS samples. These findings provide insights into flow-dependent endothelial dysfunction and a framework for therapeutic modulation in SWS and other vascular disorders.

cell biology↗

bioRxiv2026

Cellular Mechanisms of Transcranial Magnetic Stimulation in Climbing Fibers and Purkinje Neurons in the Cerebellum

Although transcranial magnetic stimulation (TMS) is widely used for brain stimulation, fundamental issues about its underlying mechanisms remain unresolved. We investigated some of these issues experimentally using an intact isolated turtle cerebellum in vitro, employing a novel chamber designed to deliver precisely calibrated induced electric fields along cortical depth. Our results show that single-pulse TMS can directly activate Purkinje cells and climbing fibers, and synaptically activate Purkinje cells via climbing fibers - all within the first 1.2 ms. Specifically, current source density analysis showed that TMS directly (non-synaptically) activated (1) climbing fibers near the bend with intracellular current directed toward the axonal terminals and (2) Purkinje cells directly near the axon initial segment with intracellular current directed toward the distal dendrites. The thresholds for direct activation of climbing fibers and Purkinje cells were found to be very similar, 25 {+/-} 1 V/m. The climbing fibers synaptically activated Purkinje cells, as expected, with intracellular current originating in the proximal dendritic trunk and directed toward the distal dendrites. At higher electric fields (> 58 {+/-} 17 V/m), TMS synaptically activated dendritic currents in Purkinje cells. These results provide new insight into how TMS may activate afferent fibers and cell bodies of cortical neurons.

biophysics↗

bioRxiv2026

Kinetic design of reversible probe exchange enables continuous single-molecule tracking beyond the photobleaching limit

Single-molecule fluorescence imaging enables direct tracking of molecular dynamics. Prolonged observation, however, is limited by photobleaching, restricting access to rare events and slow transitions. Exchangeable fluorogenic labeling has been developed to overcome this limitation, yet continuous single-molecule tracking has not been achieved. We define two quantitative kinetic conditions for continuous tracking: pre-bleach dissociation and within-frame rebinding. Mapping reported systems onto a kinetic landscape delineates the regime required for continuous tracking. To reach this regime, we turned to odorant-binding proteins, whose biological role favors rapid ligand capture and release. We paired this scaffold with high-contrast fluorogenic probes to maximize rebinding capacity while preserving rapid dissociation. The resulting system, EverGreen, sustains uninterrupted single-molecule observation for over 24 hours and establishes a general kinetic design framework beyond the photobleaching limit.

biophysics↗

bioRxiv2026

Genome compartments guide protamine replacement and genome stability during spermiogenesis

Compartment-scale genome organisation persists in mammalian sperm, yet how histone-protamine replacement is orchestrated in space and time during spermiogenesis remains unclear. Here we combine stage-resolved purification of mouse spermatids with spike-in-normalised ATAC-seq and PRM1 CUT&Tag to map chromatin accessibility and protamine incorporation across spermiogenesis. We uncover a transient, genome-wide hyper-accessible phase coincident with replacement that is uncoupled from transcription and suppressed in catalytic PHF7-mutant spermatids. PRM1 loading initiates within accessible A-compartment chromatin and later spreads across both A- and B-compartments, whereas protamine-null mice demonstrated that PRM1/ PRM2 deficiency selectively destabilises A-compartment closure. Sequencing of short DNA fragments from Prm1/Prm2 dosage-reduced epididymal sperm reveals early enrichment of breakage at protamine-targeted A-compartment regions, which dissipates as fragmentation becomes genome-wide during epididymal transit. Together, our data place histone-protamine replacement within a compartment-centred framework that links 3D genome domains to the timing, targeting and integrity of the sperm genome.

genomics↗

bioRxiv2026

Federated single-cell QTL meta-analysis reveals novel disease mechanisms

Genetic effects on gene expression are often cell type-specific and obscured in bulk analyses. To resolve this context-dependent regulation, we performed a federated cis-eQTL meta-analysis across 12 PBMC datasets (2,032 individuals, 2.5 million cells). Across six immune cell types, we identified cis-eQTLs for 6,592 genes and fine-mapped 14,985 independent loci. Notably, the 42% of eQTLs that were undetected in a bulk eQTL study on 43,301 whole blood samples also showed stronger enrichment for disease GWAS loci. We further identified three genome-wide significant and 65 suggestive loci affecting the abundance of (rare) immune cell types and validated these using previously reported hematological GWAS and bulk-derived trans-eQTLs. Integrating single-cell cis-eQTLs with bulk trans-eQTLs enabled us to anchor 6,382 trans-eGenes (37.2% novel) to upstream regulators and reconstruct directed gene regulatory relationships. For example, a hemorrhoidal disease-associated variant showed a CD4+ T cell-specific cis-eQTL on BACH1 that colocalized with 45 immune and metabolic trans-eGenes. These results demonstrate the power of single-cell QTL meta-analysis in interpreting complex trait genetics.

genetics↗

bioRxiv2025

Early pathogenesis of spinal and bulbar muscular atrophy uncovered by human iPSC-derived motor neurons highlights pathogenic neuropeptides as therapeutic targets

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disorder caused by the expansion of the polyglutamine tract in the androgen receptor (AR). Motor neurons (MNs) derived from patient-specific induced pluripotent stem cells (iPSCs) robustl y recapitulated early SBMA phenotypes driven by endogenous mutant AR in the absence of testosterone (dihydrotestosterone) and detectable mutant AR aggregation. Notably, endoplasmic reticulum stress markedly exacerbated SBMA pathology. Cross-species integrative analyses of patient-derived neurons and spinal cords of transgenic mouse models revealed high expression of multiple disease-associated neuropeptides, including urotensin II (UTS2), in patient spinal MNs that was correlated with disease onset and progression in iPSC-derived MNs. Downstream signaling analyses of these neuropeptides revealed convergent molecular pathways whose pharmacological inhibition rescued cellular phenotypes. Together, these results establish a human disease model harboring endogenous mutant AR that closely reproduces early SBMA pathology and provides molecular leads for elucidating disease mechanisms and biomarkers and developing therapeutic targets.

neuroscience↗

bioRxiv2025

Revisiting the in vivo Functions of Coilin in Mice

Cajal bodies (CBs), first identified in the early 20th century, are classic nuclear structures organized primarily by the protein coilin. Although CBs have long been implicated in snRNA modification and maturation, their precise roles in mice remain unclear. Here, we revisit the functions of CBs by using genome editing to generate mice that lack nearly the entire coilin protein, retaining only the first seven amino acids. Mass-spectrometry analyses showed reduced 2'-O-methylation of U2 snRNA in coilin mutant liver; however, these reductions had minimal impact on overall gene expression and pre-mRNA splicing patterns. Additionally, detailed analysis recapitulated previously reported testicular size reductions and newly revealed decreased proportions of high-quality sperm. We also identified novel coilin-containing condensates distinct from genuine CBs in the nuclear pocket of elongating spermatids. Our findings implicate that coilin orchestrates membraneless organelles in various compartments, playing multifunctional roles beyond classical CB formation.

molecular biology↗

bioRxiv2025

Rare k-mers reveal centromere haplogroups underlying human diversity and cancer translocations

Centromeres are among the most diverse and dynamically evolving regions of the human genome and are commonly affected in various human cancers. However, organized into highly repetitive -satellite higher-order repeats (HORs), human centromere sequences have long resisted detailed genomic analysis. Although the development of long-read sequencing platforms has enabled the analysis of complete centromere sequences, their application to a large set of samples is still largely limited, preventing our understanding of centromere variation and haplotype structures across large human populations and the structural basis of centromere-involving translocations in cancer. Here we show that rare k-mers present in centromeric regions can serve as effective markers for dissecting the complexity of centromere structure, particularly that of active -satellite HOR arrays (aHOR arrays), across human populations and for understanding centromere-involving abnormalities in cancer. Based on rare k-mer-based clustering, centromere aHOR arrays are clustered into discrete haplogroups (aHOR-HGs) with distinct structural features. These k-mers were also used to develop a framework that enables the inference of haplogroups in a given sample based on short-read whole genome sequencing (WGS) data (ascairn). By applying ascairn to large-scale human population datasets (n > 3,300), we revealed the diversity of aHOR-HGs and their geographic histories across populations. The rare k-mer-based approach was also applied to investigate the structure of 1p/19q co-deletion, a highly recurrent centromere-involving translocation in IDH-mutated oligodendrogliomas. Analyzing short-read WGS data from 142 cases with 1p/19q co-deletion using rare k-mers, we showed that breakpoints of 1p/19q co-deletion were mapped to aHOR arrays in chromosomes 1 (D1Z7) and 19 (D19Z3), which was validated by long-read sequencing of two 1p/19q co-deletion-positive cases. Notably, the translocation preferentially involved haplogroups composed of haplotypes containing larger regions susceptible to rearrangement. These results highlight the role of rare k-mers in dissecting the complexity of centromere sequences and their evolutionary history as well as understanding centromere-involving abnormalities associated with human diseases.

genomics↗

bioRxiv2025

Temporary parasitic ants modify nestmate discrimination patterns of host workers

Social insects rely on nestmate discrimination systems to maintain highly organized societies. Some social parasites can evade these systems to exploit their hosts. In this study, we report that the temporarily socially parasitic ant Lasius umbratus may modify the nestmate discrimination patterns of its host, L. japonicus. Aggression assays revealed that unparasitized hosts exhibited high aggression toward non-nestmate conspecifics and parasites. In contrast, parasitized hosts showed significantly lower aggression toward non-nestmate conspecifics and parasites, while maintaining considerable aggression toward a non-parasitic congener as a positive control. This suggests that parasites modify the nestmate discrimination patterns of the hosts rather than merely suppressing aggression. Furthermore, chemical analysis of cuticular hydrocarbons revealed that hosts from mixed-species colonies of hosts and parasites had profiles resembling those of unparasitized hosts, whereas parasites from mixed-species and parasite-only colonies showed distinct profiles. Notably, parasites from mixed-species colonies had hydrocarbon profiles resembling those of their hosts, indicating that L. umbratus employs chemical mimicry to alter host behaviour. Our findings shed light on the intricate strategies of social parasites and the behavioural and chemical mechanisms sustaining highly organized ant societies.

animal behavior and cognition↗

bioRxiv2025

Enhanced axonal transport in large vertebrates: KIF5A adaptations in giraffes and pythons

Efficient axonal transport is essential for neuronal function, particularly in species with exceptionally long axons. The Kinesin-1 motor protein KIF5A plays a key role in this process, but whether and how it adapts to the transport demands of large vertebrates remains unclear. Here, we show that KIF5A from giraffes (GcKIF5A) and pythons (PbKIF5A), moves 25% faster than its mouse counterpart (MmKIF5A) on neuronal microtubules in vitro and within axons of cultured mouse hippocampal neurons. This enhanced velocity, driven by three unique amino acid substitutions (R114Q, S155A and Y309F), facilitates long-distance transport in these species and represents a case of convergent evolution. Our structural analysis reveals that accelerated ADP release underlies the increased speed of GcKIF5A. Despite exhibiting a reduced force generation, GcKIF5A maintains efficient cargo transport under load. Furthermore, GcKIF5A exerts less drag in mixed-motor environments, an adaptation particularly beneficial for multiple motor-driven long-distance axonal transport. These findings reveal that KIF5A has evolved specific adaptations to facilitate efficient axonal transport in large vertebrates, highlighting the evolutionary plasticity of kinesin motors.

biophysics↗

bioRxiv2025

Biplanar nulling coil system for OPM-MEG using printed circuit boards

Optically pumped magnetometers (OPMs) are a promising sensor technology for non-invasive measurement of human electrophysiological signals, in particular the magnetoencephalogram (MEG). OPMs do not need cryogenic cooling and can be placed conformal to the subjects scalp, thus greatly reducing the sensor-to-source distance and improving signal sensitivity. OPMs, however, require near-zero background magnetic field to achieve linearity and minimize signal distortion. Prior work has proposed the use of biplanar field nulling coils to remove the uniform and gradient components of the background magnetic field. Biplanar coils have been expensive to construct, involving tedious error-prone manual winding of over 1000 m of copper wire. In this work, we designed and fabricated background field nulling coils (three uniform and three gradient components) on two-layer Printed Circuit Boards (PCBs). We used an open-source software (bfieldtools) to determine the current loops needed to produce the target magnetic field in a 50-cm-diameter spherical volume. We developed a software-based approach to connect the discrete current loops into a continuous conducting path traversing the two layers of the PCB. For ease of manufacture, the designed (1.5 x 1.5 m2) coils were cut along the symmetry axis and printed as pairs of 1.5 x 0.75 m2 PCBs (2 oz Cu), soldered together and mounted on a sliding aluminum frame. The efficiency of the coils (1.3 - 7.1 nT/mA) was similar or higher than previously reported in the literature. We mapped the field inside the target region after field nulling inside our single-layer shielded room and were able to reduce the largest component of the background field from 21 to 2 nT. Using our nulling coil system, we were able to operate OPMs in a lightly shielded room (background field varying from 6.5 to 108 nT in the floor-to-ceiling direction) to record somatosensory evoked fields (SEFs) comparable to those measured using SQUID-based MEG in a 3-layer shielded room. We disseminate the software and hardware as an open-source package opmcoils. This work will facilitate access to more affordable field nulling coils for OPM-MEG and help to realize the potential of OPM-MEG as an accessible sensor technology for use in human neuroscience.

neuroscience↗

bioRxiv2025

Single-shot label-free nanoscopy for quantitative organelle visualization on standard commercial microscopes

Label-free microscopy techniques that overcome the limitations of fluorescence microscopy are increasingly needed in biological research. Quantitative phase imaging has emerged as a powerful label-free method for live cell imaging, particularly at multicellular scales. However, a critical gap exists in achieving easy-to-implement, high-speed, high-resolution microscopy at subcellular scales. Here, we report upgraded polarization-resolved differential phase contrast nanoscopy (UpDPC); this single-shot computational phase microscopy (CPM) enhances resolution and sensitivity to subcellular levels. UpDPC can be readily integrated into commercial fluorescence microscopes, enabling 100 nm gap separation, over 100 Hz imaging via single-shot phase retrieval, and 100 ms computation of phase image during observation. Our results demonstrate that high-performance CPM provides reproducible and comprehensive visualization of various organelles, offering a practical and scalable implementation for basic research and clinical applications.

cell biology↗

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

Mitochondrial direct repeat reduction as a strategy for enhancing human longevity: the case of the common repeat

Aging, characterized by a series of functional declines correlated with advancing chronological age, has a significant mitochondrial DNA (mtDNA) component, with somatic mtDNA deletions playing a central role. In post-mitotic or slow-dividing cells like neurons and skeletal muscles, selfish mtDNA deletions clonally expand within a cell, ultimately leading to the deterioration and death of host cells and appearence of age-related phenotypes. Thus reducing the burden of somatic deletions could have far- reaching systemic benefits for the entire human body. Given the crucial role of direct nucleotide repeats in the formation of mitochondrial deletions, we hypothesize that minimizing these repeats in the human mitochondrial genome could enhance healthspan by decreasing somatic deletions. To investigate this hypothesis, we focus on the "common repeat", a 13-base pair perfect direct repeat sequence (ACCTCCCTCACCA) located at positions 8470-8482 and 13447-13459, respectively. This perfect repeat: (i) is highly prevalent, with its potential deleterious consequences affecting the majority of humans; (ii) represents one of the most fragile sites, highly prone to forming deletions; (iii) when disrupted, is associated with a decreased somatic deletion load and enhanced human healthspan; (iv) is likely to experience positive selection in the present or near future due to indirect fitness effects, such as the "grandmother effect", and direct fitness effects, such as (v) a decreased mutation rate. These observations support the argument that reducing the mtDNA somatic deletion load through targeted disruption of these repeats, or by using naturally occurring polymorphisms with disrupted repeats in mitochondrial medicine, could be an effective approach to increasing human longevity.

genetics↗

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

A versatile platform for single fluorescent protein-based fluorescence lifetime biosensors

Single fluorescent protein (FP)-based FLIM (fluorescence lifetime imaging) biosensors are potent tools for quantitatively imaging intracellular processes with high spatial and temporal resolution. They only require a single wavelength for detection, which facilitates multi-color imaging. However, the development of single FP-based FLIM biosensors has been limited by the absence of a general design framework and the complexity of the screening process. In this study, we engineered FLIM biosensors capable of detecting ATP (adenosine triphosphate), cAMP (cyclic adenosine monophosphate), citrate, and glucose by inserting each sensing domain into the mTurquoise2 fluorescent protein between Tyr-145 and Phe-146 using peptide linkers. Through efficient linker screening, we successfully developed FLIM biosensors exhibiting an effective dynamic range from 0.5 to 1.0 ns upon analyte binding. This demonstrates that the qmTQ2-ATP-0 backbone is a universal platform for developing mTQ2-based biosensors. As a proof-of-concept, we demonstrated the capabilities of these FLIM biosensors in monitoring the intracellular dynamics of ATP and cAMP alongside dual-color imaging. Therefore, our work presents an accessible methodology for establishing a single FP-based FLIM biosensor platform for quantitative imaging.

molecular biology↗

bioRxiv2024

BMP4 regulates asymmetric Pkd2 distribution in mouse nodal immotile cilia and ciliary mechanosensing required for left-right determination

BackgroundMouse nodal immotile cilia mechanically sense the bending direction for left-right (L-R) determination and activate the left-side-specific signaling cascade, leading to increased Nodal activity. Asymmetric distribution of Pkd2, a crucial channel for L-R determination, on immotile cilia has been reported recently. However, the causal relationship between the asymmetric Pkd2 distribution and direction-dependent flow sensing is not well understood. Furthermore, the underlying molecular mechanism directing this asymmetric Pkd2 distribution remains unclear. ResultsThe effects of several recombinant proteins and inhibitors on the Pkd2 distribution were analyzed using super-resolution microscopy. Notably, bone morphogenetic protein 4 (BMP4) affected the Pkd2 distribution. Additionally, three-dimensional manipulation of nodal immotile cilia using optical tweezers revealed that excess BMP4 caused defects in the mechanosensing ability of the cilia. ConclusionsExperimental data together with model calculations suggest that BMP4 regulates the asymmetric distribution of Pkd2 in nodal immotile cilia, thereby affecting the ability of these cilia to sense the bending direction for L-R determination. This study, for the first time, provides insight into the relationship between the asymmetric protein distribution in cilia and their function.

developmental biology↗