Search bioRxiv⌕ Search

Biology subjects

Takahashi, M.

Publications and source records attributed to Takahashi, M..

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

Mutation-agnostic gene insertion therapy for RHO-associated autosomal dominant retinitis pigmentosa using zinc finger nucleases

PurposeAutosomal dominant retinitis pigmentosa caused by mutations in the rhodopsin gene (RHO-adRP) is among the most prevalent inherited retinal dystrophies. With nearly 100 distinct pathogenic variants identified to date, the mutational heterogeneity of RHO-adRP severely limits the clinical utility of mutation-specific therapeutic strategies. We aimed to develop a mutation-agnostic gene insertion therapy using homology-independent targeted integration (HITI) mediated by zinc finger nuclease ZF-ND1 targeting the human RHO 5'-UTR, and to validate its preclinical efficacy, safety, proof-of-concept, and proof-of-mechanism. MethodsWe developed two AAV serotype 5 (AAV5) vectors: one encoding the ZF-ND1 pair (AAV5-ZFN) and one carrying the therapeutic donor cassette (AAV5-RHO), delivered by subretinal co-injection. ZF pairs targeting the RHO 5'-UTR were arranged and refined by in vitro validation; AAV vector optimization and mechanistic verification were performed in human induced pluripotent stem cell (hiPSC)-derived retinal organoids-derived retinal organoids; longitudinal proof-of-concept efficacy and safety were assessed in a humanized RHO-T17M rat disease model by 6-month optical coherence tomography (OCT); and proof-of-mechanism was evaluated in non-human primate retina. ResultsWe identified a ZF-ND1 pair achieving cleavage efficiency comparable to the SpCas9 RNP previously validated for HITI-mediated editing in mouse retina, and optimized the ZF array composition and NLS configuration for efficient editing especially in post-mitotic photoreceptors. HITI-mediated donor integration was confirmed across multiple cell types and ZFN:donor ratios. In the humanized rat disease model, the therapeutic vector provided outer nuclear layer (ONL) preservation by 6 months, with AAV5-ZFN:AAV5-RHO ratios of 1:1 and 1:2 maintaining ONL thickness above the preservation threshold. In the non-human primate retina, the fraction of HITI-edited rod photoreceptors exceeded the 20% therapeutic correction threshold in the successfully treated individual. ConclusionsThese findings support the advancement of this therapeutic vector to first-in-human trials as a mutation-agnostic insertion therapy applicable to all patients with RHO-adRP, irrespective of the specific causative variant.

genetics↗
bioRxiv2026

OxyBLI: A Genetics-Based Approach for In Vivo Real-Time Visualization of Tissue Oxygenation Dynamics

Accurate measurement of cellular oxygen levels is essential for understanding the balance between oxygen demand and supply in tissues. However, conventional methods only yield compromised results. We harnessed the oxygen dependence of bioluminescence to develop OxyBLI--a noninvasive optical method that directly monitors oxygen levels in specific cell populations of intact experimental animals. We characterized OxyBLI signals across various critical situations associated with common interventions. Hypoxic breathing and subsequent systemic tissue hypoxia caused blood to be redistributed in a way that prioritized brain oxygenation. In contrast, hyperoxic breathing sharply increased tissue oxygenation, which promptly returned to the target level owing to a vasoconstrictor response. These findings are expected to help resolve the long-standing clinical issue regarding the risks and benefits of administering supplemental oxygen to acutely ill patients. Our multifaceted approach, which presents multiple challenges to individual animals over time, will advance our understanding of the delicate interaction between hypoxia and hyperoxia.

physiology↗
bioRxiv2026

In vivo brain temperature measurement using quantum dot imaging temperature sensors

Temperature regulation in the brain is essential for maintaining neuronal function and preventing thermally induced damage. Here, we report the development and in vivo application of quantum dots (QDs) to high-resolution thermometry in the mouse brain using two-photon excitation microscopy. These QDs, via the red-to-green photoluminescence (PL) intensity ratios, enabled stable temperature measurements in both normal and chronically hypo-perfused cerebral tissue. Our findings show that localized neuronal activity leads to transient heat generation, which is rapidly dissipated by cerebrovascular responses. In a chronic hypoperfusion model, impaired vascular function resulted in exaggerated and prolonged brain temperature elevations. This thermometry system provides unprecedented insight into the mechanisms of cerebral thermoregulation and highlights the importance of vascular cooling in protecting the brain from heat-induced stress, particularly in pathological conditions such as stroke.

bioengineering↗
bioRxiv2026

Inflammasome activation drives gasdermin-independent plasma membrane rupture by clustering ninjurin-1 in macrophages

Inflammasome assembly rapidly triggers caspase-1 activation to initiate pyroptosis, an inflammatory cell death characterized by the release of cytosolic contents, including interleukin (IL)-1{beta}/. Here, we report that inflammasome activation drives necrotic cell death independent of gasdermin D (GSDMD) and GSDME, which are essential executors of pyroptosis by forming a pore on the plasma membrane and increasing membrane permeability. NLRP3 inflammasome activation induced necrotic cell death, coupled with IL-1{beta}/ release in Gsdmd-/-Gsdme-/- macrophages. Mechanistically, the oligomerization of ninjurin-1 (NINJ1) was caused by inflammasome activation even in the absence of GSDMD and GSDME. Concordantly, glycine, an inhibitor of NINJ1, blocked plasma membrane permeabilization triggered by inflammasome activation in Gsdmd-/-Gsdme-/- macrophages, but not in WT macrophages. The dimerizer-mediated ASC oligomerization promoted NINJ1-mNeonGreen cluster formation in the absence of GSDMD and GSDME. Moreover, NINJ1 deficiency prevented membrane permeabilization initiated by ASC oligomerization in Gsdmd-/-Gsdme-/- immortalized bone marrow-derived macrophages (iBMDM). Blocking of phosphatidylserine (PtdSer) exposure, a feature of inflammasome-driven necrotic cell death, by Xkr8 deficiency inhibited plasma membrane permeabilization in Gsdmd-/-Gsdme-/- iBMDM. These results suggest that inflammasome-triggered activation of caspase-1 itself drives inflammatory necrotic cell death independent of gasdermins.

cell biology↗
bioRxiv2026

SLFN-generated 3'-truncated tRNALeu or tRNASer together with tRNase ZL works as a sequence-specific RNA cutter

A complex of the 3'-truncated tRNAArg that lacks 9 nt and tRNase ZL works as a GCCC-recognizing RNA cutter. It recognizes an RNA substrate via four Watson-Crick-Franklin base-pairings with the 3'-truncated tRNAArg. Human SLFN11 and SLFN13 can generate 3'-truncated tRNALeu that lacks 10 nt and 3'-truncated tRNASer that lacks 11 nt, respectively, from their corresponding mature tRNAs. Here, we investigated if these 3'-truncated tRNAs together with tRNase ZL work as sequence-specific RNA cutters. We examined five RNA targets for cleavage by recombinant human tRNase ZL in the presence of the 3'-truncated tRNALeu or tRNASer. We demonstrated that the 3'-truncated tRNALeu and tRNASer together with tRNase ZL indeed work as [~]6-base-recognizing and 7-base-recognizing RNA cutters, respectively.

biochemistry↗
bioRxiv2026

Comparative genome analysis of Corynebacterium ulcerans Japanese isolates revealed domestically and globally diverse geographical distribution of the organism of different types.

Corynebacterium ulcerans is a zoonotic pathogen causing infections in humans that are clinically indistinguishable from those of Corynebacterium diphtheriae. In many developed countries, companion animals such as dogs and cats, are the predominant sources of infection by C. ulcerans, as is the case in Japan. To date, we have collected 106 Japanese clinical and domestic animal isolates of C. ulcerans and a recently branched species, C. ramonii, most of which were isolated from humans and cats. In the present study, we performed a comparative analysis of whole-genome sequences from Japanese isolates and 597 published isolates from Japan and outside Japan. The Japanese isolates were divided into two distinct lineages: C. ulcerans and C. ramonii. The MLST types of C. ulcerans Japanese isolates exhibited a unique distribution pattern, with one major type (ST337) accounting for 69.0%, which is very rare in Europe. In addition to Japan, distinct MLST pattern compositions were observed across geographically distinct regions. The sequence types were associated with lysogenized prophage types that encode the diphtheria toxin (tox) gene and were partially associated with toxin production under certain conditions. Through SNV analysis, transmission from animals to humans has been suggested in some clinical cases. Significance StatementBy analyzing more than 700 genomes, we demonstrate striking geographic differences in multilocus sequence types and toxigenic prophage compositions between Japanese and European isolates. Notably, we identify the predominance of sequence type ST337 in Japan, a lineage that is rare in Europe, and show that prophage types encoding the diphtheria toxin are closely associated with specific sequence types and toxin production phenotypes. Our single-nucleotide variant analyses further provide genomic evidence supporting zoonotic transmission between companion animals and humans in several clinical cases.

microbiology↗
bioRxiv2025

Structure-Function Coupling Aligns with a Unimodal-to-Transmodal Gradient in the Mouse Cortex

Understanding how anatomical connectivity shapes brain activity is essential for clarifying brain function and disorders. In the human brain, the regional heterogeneity in structure-function (S-F) coupling is well characterized by measuring correlations between structural and functional connectivity. However, it remains unclear whether the same principles apply to the mouse cortex, where biological mechanisms can be studied directly. Here, we mapped S-F coupling across the mouse cortex by combining high-resolution structural connectivity derived from axonal tracing with resting-state functional connectivity measured by wide-field calcium imaging. Our findings revealed that structural connectivity imposes a robust yet regionally variable constraint on functional connectivity. As in humans, the S-F coupling was strong in primary sensorimotor areas and weaker in association areas, demonstrating a gradient from unimodal-to-transmodal cortical organization. This spatial variation covaried with intrinsic cellular properties, including myelination, excitation-inhibition balance, synaptic density, and gene expression profiles, but did not align with the anatomically defined cortical hierarchy. Our findings highlight graded S-F coupling as a common organizational principle in both the mouse and human cortex, providing a framework for future mechanistic studies using mouse models.

neuroscience↗
bioRxiv2025

Spinal interneuronal populations encode static hindlimb posture in the cat

Proprioceptive signals from primary afferents reflect changes in single-joint angles, whereas neuronal population in the cerebral cortex represent whole-limb postures. Where and how this transformation emerges along the somatosensory axis from peripheral proprioceptive receptors remains unclear. We simultaneously recorded many lumbosacral spinal neurons in two decerebrate, immobilized cats while a robotic device held the hindlimb at 16 static endpoint positions spanning hip-knee configurations. Using high-density multielectrode recordings, we asked how spinal populations encode static limb state. At the single-neuronal level, activities in a majority of neurons covaried with a single joints angle (hip or knee), a smaller subset showed combined modulation by both joints, and a distinct subset ( single-endpoint neurons) fired selectively at one unique hip-knee configuration near the sampled joint-range limits and was quiescent in adjacent postures. Population analyses revealed a low-dimensional structure: the first two principal components tracked knee and hip angles, respectively, whereas a third component isolated a boundary-aligned, posture-specific pattern, with loadings peaking at the same extreme configurations preferred by single-endpoint neurons. Decoders trained on ensemble activity reconstructed both joint angles and the limbs endpoint position in body-centered coordinates, indicating that the recorded spinal-interneuron populations contain sufficient information to reconstruct whole-limb kinematics. Together, these findings are consistent with a hierarchical organization whereby joint-based representations within spinal-interneuron populations could contribute to the emergence of limb-centered representations in the ascending proprioceptive pathways. The boundary preference of single-endpoint neurons supports a possible categorical coding scheme at workspace limits that may provide spinal "landmarks" for switching control modes, enhancing stability near kinematic extremes, and supporting recalibration of proprioceptive population codes. Key PointsO_LIWe simultaneously recorded many lumbar spinal neurons in two decerebrate, immobilized cats while a robot held the hindlimb at 16 static positions to test how spinal populations encode posture. C_LIO_LIMany neurons varied with a single joint angle (hip or knee), a smaller subset showed combined hip-knee modulation, and a distinct subset was active only at one specific endpoint posture. C_LIO_LIPopulation analyses revealed a low-dimensional structure: the first two principal components tracked knee and hip angles, while a third captured a posture-specific pattern aligned with single-endpoint neurons. C_LIO_LIDecoders trained on ensemble activity reconstructed joint angles and the limbs endpoint, indicating that spinal interneuronal populations contain sufficient information for whole-limb kinematics. C_LIO_LIThese findings are consistent with a hierarchical organization whereby joint-based representations within spinal-interneuron populations could contribute to the emergence of limb-centered representations in the ascending proprioceptive pathways. C_LI

neuroscience↗
bioRxiv2025

Arabidopsis epigenetic factor AS2 attenuates nucleolar stress by camptothecin and establishes leaf polarity by repressing a CDK inhibitor

The Arabidopsis thaliana leaf, exhibiting a symmetrically extended flat morphology, consists of two distinct cellular domains: adaxial and abaxial layers. The ASYMMETRIC LEAVES2 (AS2) gene is essential for specifying the adaxial domain, and its protein forms nucleolar structures, termed AS2 bodies, at ribosomal-DNA loci. Numerous nucleolus-related genes have been reported to be cooperatively involved in leaf adaxialization together with AS2. However, the molecular relationships between AS2 and these genes remain unclear. To identify chemical modulators of AS2 function, we screened a chemical library containing natural products and identified eight molecules, including camptothecin, that induced filamentous leaves in the as2 mutant. Camptothecin is an inhibitor of topoisomerase I, which is required for transcription of ribosomal-RNA genes, and induces nucleolar stress. Treatment with 10-hydroxy-camptothecin increased KIP-RELATED-PROTEIN5/INHIBITOR-OF-CYCLIN-DEPENDENT-KINASE3 (KRP5/ICK3) transcript levels, encoding a CDK inhibitor, and caused notable changes in the number and morphology of AS2 bodies. Given its role in leaf morphogenesis, our findings suggest that AS2 is a key factor in establishing leaf adaxial-abaxial polarity by regulating cell proliferation and protecting against nucleolar stress through coordinated interactions with nucleolar proteins.

developmental biology↗
bioRxiv2025

A Novel MiRNA Detection System Combining TWJ-SDA,Multistep L-TEAM, and CRISPR-Cas3

MicroRNAs (miRNAs) serve critical regulatory roles in gene expression and are valuable biomarkers for early disease detection. However, their inherent low concentration in biological fluids poses significant detection challenges. Although traditional methods like real-time quantitative PCR (RT-qPCR) are highly sensitive, they require thermal cycling, limiting their application in point-of-care testing (POCT). Here, we present an isothermal amplification-based miRNA detection system integrating Three-Way Junction (TWJ) formation, Multistep Low-Temperature Amplification (L-TEAM), and CRISPR-Cas3-mediated signal amplification. The integration of the Multistep L-TEAM with the TWJ method achieves high sensitivity, detecting miRNA at concentrations as low as 10 femtomolar within 50 minutes, and effectively distinguishes single-nucleotide mismatches. When CRISPR-Cas3-mediated reaction was integrated, it still proved effective for confirming the presence of the target, but its quantitative reliability requires further optimization. We developed a predictive model using machine learning to facilitate rational optimization of experimental conditions through contribution analysis and to establish a methodology for designing more favorable sequences. The modular nature of our method permits adaptation to diverse miRNA targets without modifications to the fundamental amplification mechanism.

molecular biology↗
bioRxiv2025

The interferon-stimulated gene product HERC5 inhibits human LINE-1 retrotransposition with an ISGylation-independent mechanism

Text AbstractMobilization of long interspersed element-1 (LINE-1 or L1) compromises genome stability and can cause sporadic genetic disease. Accordingly, cells have evolved multiple mechanisms to restrict L1 retrotransposition. Several interferon-stimulated genes (ISGs) that interact with cytoplasmic L1 ribonucleoproteins (RNPs), which contain the L1-encoded proteins ORF1p and ORF2p, have been identified as suppressors of L1 retrotransposition. We previously reported that the ISG protein HECT and RLD domain containing E3 ubiquitin-protein ligase 5 (HERC5) efficiently inhibits L1 retrotransposition. While HERC5 is known to restrict numerous viruses through ISGylation, how HERC5 inhibits L1 remains to be elucidated. Here, we show that HERC5 inhibits L1 retrotransposition through an ISGylation-independent mechanism. HERC5 interacts with L1 RNA and reduces ORF1p levels, a function that requires the ORF1 coding sequence. We further demonstrate that HERC5 decreases L1 translation efficiency and prevents the assembly of L1 RNPs. Our comparative analysis suggests that HERC5 may have acquired its L1-inhibitory function during the evolution of the small HERC family. These findings uncover a previously unidentified mechanism by which an ISG protein recognizes and inhibits L1 and suggest a role for HERC5 as an evolutionarily adapted restriction factor that expands the repertoire of cellular defenses against retrotransposons.

molecular biology↗
bioRxiv2025

Retrospective metabolomics via dual-dimensional deconvolution using ZT Scan DIA 2.0

We present a scanning data-independent acquisition (DIA) strategy, ZT Scan DIA, combined with dual-dimensional tandem mass spectrometry spectral filtering and deconvolution along both the quadrupole and retention time axes to reconstruct compound-specific MS2 spectra from complex mixtures. This approach is particularly effective for hydrophilic metabolomics data, where spectral similarity-based annotation is widely used, increasing annotation rates by 119-193% compared with conventional data-dependent acquisition (DDA) and window-based DIA methods. In lipidomics, deconvolution improved annotation precision by removing contaminant product ions and enabled separate quantification of co-eluting isomers using MS2 chromatograms, although common diagnostic ions could also be erroneously removed. Nevertheless, optimization of analysis parameters minimized this negative effect. Furthermore, we developed a practical data processing pipeline in which raw ZT Scan DIA-MS2 chromatograms are directly used for isomer separation and MS2-based quantification, covering 1,393 and 3,020 molecules for human plasma and mouse liver tissues, respectively. All data processing steps, including direct import of vendor raw data, are supported in MS-DIAL.

bioengineering↗
bioRxiv2025

A probiotic bacterium modulates antitumor γδ T-cell responses in lung cancer

The link between the intestinal microflora and cancer outcomes has been recognized for over a decade. Several recent studies have demonstrated that the gut microbiome is associated with the efficiency of T-cell checkpoint blockade therapy for cancer raising interest in strategies to harness this effect via consumption of live microorganisms (probiotics). The probiotic Clostridium butyricum strain MIYAIRI 588 (CBM588) enhances both response rates and overall survival in patients receiving immune checkpoint blockade therapy for non-small cell lung cancer and metastatic renal cell carcinoma but the mechanism underpinning this benefit remains unclear. Here, we show that CBM588 spores induce a population of V{gamma}9V{delta}2 T-cells from the peripheral blood of healthy donors and lung cancer patients. A subset of these T-cells responded to, and directly lysed, cancer cell lines via a butyrophilin 3A-dependant mechanism. In patients taking CBM588 alongside checkpoint blockade, peripheral V{delta}2 T-cells expressed the activation marker CD69 more frequently than those receiving checkpoint blockade alone and the frequency of V{delta}2CD69 cells increased following initiation of CBM588 treatment (p = 0.0041). Our findings provide a potential mechanism by which the manipulation of the intestinal microflora might directly contribute to cancer prognosis by activating immune effector cells with intrinsic anticancer properties.

immunology↗
bioRxiv2025

Hidden Markov Models Reveal Behavioral State Dynamics in Depth-Related Locomotion in Mice

Understanding how mice process and respond to visual depth cues is crucial for studying visual perception, yet traditional behavioral analyses often miss key aspects of this process, such as the dynamic transitions between behavioral states, the influence of environmental context, and the integration of multiple spatial cues that shape depth-related behaviors. Here we demonstrate that mouse responses to visual depth cues are more sophisticated than previously recognized, involving both direct avoidance behaviors and complex modulations of exploratory patterns. By combining a modified circular apparatus with Hidden Markov Model analysis, we reveal that mice transition between three distinct behavioral states--resting, exploring, and navigating--in response to visual depth cues. Using this framework, we uncover several fundamental aspects of mouse visual processing: depth perception has an optimal range of spatial frequencies, with strongest responses to patterns between 6-8 cm; visual processing integrates multiple spatial cues rather than triggering simple avoidance; and initial strong cliff-avoidance responses evolve into more nuanced behavioral adaptations over time. Comparisons between wild-type C57BL/6J mice (Mus musculus), retinal degeneration models (rd1-2J, C57BL/6J background, Mus musculus), and control conditions confirm that these behavioral patterns specifically reflect visual processing rather than general exploratory behavior. These findings reveal that mouse depth perception involves sophisticated neural processing that modulates overall exploratory behavior rather than simply triggering avoidance responses. Our approach establishes a new framework for analyzing complex behavioral sequences in neuroscience research, demonstrating how refined behavioral analysis can reveal previously undetectable aspects of sensory processing.

animal behavior and cognition↗
bioRxiv2025

Genomic characterization of sub-populations in human pluripotent stem cell-derived retinal progenitor cells that drive retinal layer structure

The mechanism underlying retinal spheroid layer formation was investigated using Rax::GFP-positive retinal progenitor cells from human embryonic stem cell-derived retinal organoids. Single-cell RNA sequencing revealed that well-layered spheroids transiently activated canonical WNT2B-FZD7 signaling followed by temporary expression of non-canonical WNT5A. Despite structural differences in vitro, non- layered and well-layered retinal spheroids on differentiation Day 60 successfully developed into cone and rod subtypes of photoreceptors and established synaptic connections with host bipolar cells after transplantation in a retinal degeneration rat model, resulting in light-evoked functional recovery. Additionally, part of the presumable Rax::GFP-positive retinal progenitor cells differentiated into non- retinal lineages, including ciliary marginal zone-like, retinal pigment epithelium, and spinal cord-like tissues in vitro, reflecting the developmental plasticity of RAX-positive cells. These findings suggest that canonical and non-canonical WNT signaling pathways sequentially orchestrate early retinal morphogenesis, whereas environmental factors within the host retina strongly drive the alignment and functional integration of graft photoreceptors.

cell biology↗
bioRxiv2025

Local, but not circulating, complement C3 governs immune checkpoint blockade efficacy

The roles of circulating complement C3, produced in hepatocytes, in immune defense are well defined in the literature. Interestingly, the C3 gene evolved before the circulatory system, suggesting an essential but yet unknown role of locally produced C3. Here, we investigated the effect of C3 on cancer immunotherapy and found that cancer-associated fibroblast (CAF)-derived C3, not hepatocyte-derived C3, determines the efficacy of immune checkpoint blockade (ICB). Tumors developed in CAF-specific C3 knockout mice exhibited resistance to anti-PD-1 therapy, with increased immunosuppressive M2-like macrophage infiltration. Mechanistically, the C3 degradation product iC3b suppressed myeloid cell infiltration via complement receptor 3 signaling, and the therapeutic targeting of this pathway restored ICB sensitivity of immunotherapy-resistant tumors. In human cancers, stromal C3 expression correlates with reduced M2-like macrophage infiltration and improved immunotherapeutic outcomes. These data demonstrate the role of locally produced C3 in the innate immune response, which may have been conserved across many organisms.

cancer biology↗