Biology subjects

Sakamoto, S.

Publications and source records attributed to Sakamoto, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Oxidized EGCG redirects α-synuclein aggregation through shear-selected interactions

-Synuclein (Syn) aggregation is central to Parkinson's disease pathology, and the green-tea polyphenol epigallocatechin gallate (EGCG) has been studied as an inhibit fibril formation. However, EGCG autoxidation and analyses under static, non-fibrillating conditions have obscured whether activity arises from EGCG itself or oxidized species and which interactions persist during Syn assembly. Here we combine HPLC, LC-MS, TEM and solution NMR to compare unoxidized and oxidized EGCG (oxEGCG), while Rheo-NMR monitors Syn during shear-promoted assembly. Unoxidized EGCG showed no detectable binding under static, reducing conditions, whereas oxEGCG bound Syn, inhibited fibrillation and remodeled fibrils. Under shear flow, many oxEGCG-dependent perturbations partially reverted toward free Syn, whereas interactions near the NAC-C-terminal boundary were preferentially retained, coinciding with formation of oligomers and short, thick fibrils. These findings identify oxidation state and flow as determinants of EGCG action on Syn and show that Rheo-NMR distinguishes interactions that persist during shear-promoted assembly.

biochemistry↗

bioRxiv2026

Early detection of pancreatic ductal adenocarcinoma by single-molecule profiling of pancreatic enzyme activities

Pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer-related mortality, largely due to diagnosis at advanced stages. Early detection through minimally invasive liquid biopsy holds promise for improving patient outcomes. Here, we report a blood-based liquid biopsy platform based on single-molecule enzyme activity profiling (SEAP), which detects proteoform-level alterations in circulating pancreatic enzymes at single-molecule resolution. Using a tissue-centric biomarker discovery strategy, we identified activity signatures of pancreas-specific digestive enzymes associated with PDAC. A combinatorial classifier detected PDAC (stage I-IV) with 95.5% specificity and 75.0% sensitivity (74.2% for stage I-II) across 690 blood samples collected from multiple hospitals and biobanks. Performance was further validated in an independent cohort enriched for early-stage disease, where 54.2% (13/24) of stage IA, 64.3% (9/14) of stage IB, and 21.4% (3/14) of stage 0 lesions were classified as positive. These findings support the clinical potential of SEAP for early detection of PDAC.

biochemistry↗

bioRxiv2026

Synthesis based on covalent capture and release (SCCR): a programmable strategy for automated preparation of protease-activatable molecules

Enzyme-activatable chemical tools, including fluorogenic probes and prodrugs, are essential in chemical biology and targeted therapeutics but remain challenging to access in structurally diverse forms because their synthesis is often bespoke and difficult to standardize. Here, we introduce synthesis based on covalent capture and release (SCCR) as a programmable chemical strategy that enables the modular assembly of protease-activatable molecules through specifically designed protecting-group logic. The SCCR framework establishes a standardized capture-elongation- release workflow that decouples molecular diversification from individual synthetic optimization, thereby enabling automated preparation of complex libraries. Using this chemistry, we generated a diverse set of fluorogenic probes and applied them to single-molecule enzyme activity analyses to identify candidate activity-based biomarkers of liver diseases. The generality of the SCCR strategy was further demonstrated by extending the same chemical logic to the preparation of antibody-drug conjugate (ADC) linkers, allowing systematic evaluation of plasma stability and cytotoxic potential. By establishing a programmable capture-release chemistry for the synthesis of enzyme-activatable molecules, this work provides a generalizable chemical foundation for the scalable and automated construction of functional small-molecule tools across biological and translational research.

biochemistry↗

bioRxiv2026

Mechanism of ATXN8OS CTA/CTG repeat-associated non-AUG translation revealed by approaches ranging from cell-free translation to live-cell imaging.

Microsatellite repeat expansions contribute to the pathogenesis of many neurodegenerative disorders. In spinocerebellar ataxia type 8 (SCA8), abnormal expansion of CTA/CTG repeats in the 3' untranslated region of the ATXN8OS (ATXN8 Opposite Strand) gene has been implicated in disease pathology. Although the occurrence of repeat-associated non-AUG (RAN) translation from the ATXN8 transcript has been reported, whether and how RAN translation occurs from the ATXN8OS transcript has remained unexplored. Here, using a cell-free translation system and cultured cells, we showed that ATXN8OS undergoes robust AUG-independent translation in a repeat length-dependent manner. Mechanistic analyses revealed that translation of the poly L (0) frame initiates at a non-AUG codon located upstream of the repeats. Moreover, using live-cell imaging at a single mRNA level, we directly visualized ribosomal -1 frameshifting from the poly L (0) frame to the poly T-poly A (+2) frame during translation elongation. We further showed that ATXN8OS translation was enhanced upon activation of the integrated stress response. Together, these findings establish both the occurrence and the molecular mechanisms of ATXN8OS translation from expanded CTA/CTG repeats and provide insights into the pathogenic processes underlying SCA8. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/700793v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1f836fborg.highwire.dtl.DTLVardef@e1b77forg.highwire.dtl.DTLVardef@d605c3org.highwire.dtl.DTLVardef@11475d9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Single-Cell Profiling of the Developing Organ of Corti Identifies Etv4/5/1 as Key Regulators of Pillar Cell Identity

The mammalian auditory sensory epithelium, the organ of Corti, contains a number of unique cell types, including the inner and outer pillar cells that form the walls of the tunnel of Corti. The limited number of pillar cells and their close physical proximity to other cells within the organ of Corti has limited efforts to transcriptionally characterize their development. To identify potential regulators of pillar cell formation, we isolated cochlear duct epithelial cells at time points between embryonic day 11 and 16 for single cell RNA-sequencing. The resulting data were used to build a developmental trajectory from undifferentiated precursor cells to each of the major cell types in the organ of Corti including inner pillar cells. Bioinformatic analyses including SCENIC, TradeSeq and CellOracle identified the Etv4/5/1 transcription factors as likely regulators of inner pillar cell development. To specifically examine the role of Etv4/5/1 in pillar cell development, conditional triple mutants were generated. Results indicate defects in the formation of inner pillar cells as well as changes in other cochlear cells likely as a result of secondary interactions. Finally, we demonstrate that expression of Etv4/5/1 in pillar cells is dependent on Fgfr3 and identify downstream targets of Fgfr3/Etv signaling in inner pillar cells. These results provide significant insights regarding the specification and early development of inner pillar cells, which will have implications for understanding congenital deficits and potential applications in the development of regenerative strategies.

developmental biology↗

bioRxiv2026

Transmembrane PhoxID: Photo-proximity labelling across the plasma membrane

Transmembrane proteins perform essential roles in cellular transport, signalling, and communication. The function and dynamics of these proteins are precisely regulated by interactions on both sides of the plasma membrane; thus, mapping the composition of these interactomes is a fundamental challenge in molecular biology. Proximity labelling methods are powerful tools for this purpose; however, existing approaches that rely on membrane-impermeable reactive species are limited by the ability to detect only one side, either the extracellular or the intracellular region, of transmembrane proteins. Here, we capitalized on the cell permeability of singlet oxygen to carry out proximity labelling of the cytoplasmic side of transmembrane proteins using an extracellularly anchored photosensitizer. We applied this method, termed transmembrane PhoxID (tmPhoxID), to several receptors (GRID2, GABAAR, and GRM1) in the living mouse brain and successfully determined their specific intracellular interactomes. Notably, network analysis of the identified proteins revealed that this method can characterize the native components of transsynaptic nanocolumns formed at parallel fibre-Purkinje cell synapses. Furthermore, our study revealed a previously uncharacterized GABAAR-CAMKV interaction in mice and human brains. Our results provide a proof of concept for transmembrane and transcellular proximity labelling, providing a powerful platform for analysing the interactome of transmembrane proteins.

neuroscience↗

bioRxiv2025

Quantitative analysis of synaptic zinc in the brain by covalent chemistry-based semisynthetic biosensors

Labile zinc ions (Zn{superscript 2}) are stored in glutamatergic vesicles at specific excitatory synapses in the central nervous system and released into synaptic clefts in an activity-dependent manner. Although Zn{superscript 2} is suggested to modulate various neuroreceptor functions, its precise roles remain unclear due to a lack of tools capable of quantitatively analyzing Zn{superscript 2} with synapse-level spatial resolution. Here, we developed neuroreceptor-based semisynthetic sensors that record synaptic Zn{superscript 2} dynamics by covalent chemistry in the living mouse brain. Using a chemical knock-in strategy, we introduced activity-based Zn2+-probes with distinct affinities into endogenous -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid or {gamma}-aminobutyric acid type A receptors. These in-brain-constructed Zn{superscript 2} sensors enabled, to our knowledge, for the first time, quantitative, region-specific mapping of Zn{superscript 2} released into synaptic clefts. Imaging-based analyses revealed differences in Zn{superscript 2} concentrations at excitatory and inhibitory synapses across hippocampal regions during kainate-induced seizures, providing new insights into the physiological and pathological functions of synaptic Zn{superscript 2}.

neuroscience↗

bioRxiv2025

Platform of single-molecule enzyme activity-based liquid biopsy for detection of pancreatic adenocarcinoma at early stages

Liquid biopsy analyzes biomolecules in biofluids to provide critical information for the early detection of diseases and the development of more effective treatment strategies. Among these biomolecules, the functional states of proteins offer particularly valuable insights due to their direct correlation with phenotypic changes. However, current approaches for assessing protein function in liquid biopsies are often limited by low detection sensitivity. Here, we present a liquid biopsy platform that analyzes single-molecule protease/peptidase activity to detect pathological alterations in enzyme function in blood samples. The platform demonstrates potential for identifying patients with early-stage (stage I-II) pancreatic ductal adenocarcinoma (PDAC).

biochemistry↗

bioRxiv2025

Canonical translation factors eIF1A and eIF5B modulate the initiation step of repeat-associated non-AUG translation.

Nucleotide repeat expansions, such as the GGGGCC repeats in C9orf72, associated with C9-ALS, are linked to neurodegenerative diseases. These repeat sequences undergo a non-canonical translation known as repeat-associated non-AUG (RAN) translation. Unlike canonical translation, RAN translation initiates from non-AUG codons and occurs in all reading frames. To identify potential regulators of RAN translation, we employed a bottom-up approach using a human factor-based reconstituted cell-free translation system to recapitulate RAN translation. This approach revealed that omission of either eIF1A or eIF5B enhanced the translation in all reading frames of C9orf72-mediated RAN translation (C9-RAN), suggesting that eIF1A and eIF5B act as repressors of RAN translation. eIF1A and eIF5B are known to contribute to the fidelity of translation initiation. In HEK293T cells, double knockdown of eIF1A and eIF5B further promoted C9-RAN compared to single knockdowns, indicating that these factors regulate C9-RAN through distinct initiation steps. Furthermore, under eIF1A knockdown conditions, the enhancement of RAN translation via the integrated stress response (ISR) was not observed in HEK293T cells, indicating that eIF1A is involved in the ISR-mediated non-AUG translation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/654993v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@160e2adorg.highwire.dtl.DTLVardef@1c1cc8forg.highwire.dtl.DTLVardef@5d01d0org.highwire.dtl.DTLVardef@220507_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Ethylene modulates cell wall mechanics for root responses to compaction

Soil stresses impact crop yields, presenting global agricultural challenges. Soil compaction triggers root length reduction and radial expansion driven by the plant hormone ethylene. We report how ethylene controls cell wall properties to promote root radial expansion. We demonstrate how soil compaction stress, via ethylene, upregulates Auxin Response Factor1 in the root cortex, which represses Cellulose Synthase (CESA) genes. CESA repression drives radial expansion of root cortical cells by modifying the thickness and mechanics of their cell walls, which result in a "stiff epidermis-soft cortex" contrast. Our research thus connects ethylene signaling with root mechanics via cell wall strength, and reveals how dynamic regulation of cellulose synthesis crucially controls root growth in compacted soil.

plant biology↗

bioRxiv2025

Spectral acoustic contributions to musical pleasure are dynamically shaped by autonomic neural inputs

Most people enjoy music and often use music to regulate their emotions. Although pleasure derived from music-listening has been shown to be mediated by dopaminergic signals in the mesolimbic reward network, its relationship with physiology is still poorly understood. Here, we introduced time-warped representational similarity analysis (twRSA) to directly map dynamic representations of multiple modalities across variable-duration stimuli. Our method revealed that although time-varying spectral and tonal acoustic features predicted changes in autonomic neural responses (measured via cardiac, pupil, and respiratory activity) during music-listening, only a small subset was in fact relevant to listeners on-line pleasure ratings. Despite that, we identified a weak mediation effect of physiology on shaping musical pleasure. Our results thus indicate that whilst musical pleasure may be embodied in bodily responses, the mapping between subjective experience and physiology is likely one-to-many--in line with psychological construction theories of emotion--and not one-to-one as is commonly assumed in classical basic emotion theories.

neuroscience↗

bioRxiv2024

In-brain construction of receptor-based protease sensors by coupling ligand-directed chemistry and click chemistry

The chemical modification of natural proteins in living systems is highly desirable toward the cutting-edge research in chemistry-biology interface. Recent advances in bioorthogonal protein modification have enabled the production of chemically functional proteins in cultured cell systems. However, few methods are applicable in vivo because of the complexity of the three-dimensional constructs of living systems with diverse, heterogeneous cell populations and flow systems filled with tissue fluids. Here, we report a genetic engineering-free method to modify receptor proteins with various probes in the living mouse brain by combining in-brain ligand-directed chemistry with bioorthogonal click chemistry, and propose a chemical guideline for the reaction design. The rapid and selective tethering of a set of fluorescent peptides to AMPA-type glutamate receptors (AMPARs) allowed the construction of receptor-based fluorescent sensors. These probes enabled mapping of the activity of matrix metalloproteinase-9 proximal to AMPARs in the living brain to be realized with high spatial resolution. Our strategy provides new opportunities for the precise analysis of particular in vivo microenvironments that has not been able to be addressed by conventional methods. Such analysis should contribute to the understanding of the molecular basis for complicated in vivo events, such as the regulation of neuroplasticity, the most important challenge in neuroscience.

biochemistry↗

bioRxiv2023

Enhanced muscle regeneration in mdx mice, Duchenne muscular dystrophy animal model, proven by CD44 & MYH3 expression, on oral feeding of N-163 strain of Aureobasidium Pullulans produced B-Glucan

IntroductionDuchenne muscular dystrophy (DMD) is a rare genetic disease, causing muscle degeneration due to lack of dystrophin with inadequate muscle regeneration culminating in muscle dysfunction. The N-163 strain of Aureobasidium Pullulans produced Beta-1,3-1,6-glucan (Neu REFIX) reported to be safe with anti-inflammatory and anti-fibrotic efficacy earlier, herein we evaluated its effects on muscle regeneration in mdx mice. MethodsForty-five mice in three groups (n=15 each): Group 1 (normal), Group 2 (mdx control), and Group 3 (mdx fed Neu REFIX) were evaluated for 45 days. IGF-1, Dystrophin, CD44 and MYH3 in diaphragm, plasma and skeletal muscle were evaluated by ELISA and immunohistochemistry. ResultsMean IGF-1 expression was 20.32% and 16.27% higher in plasma (p = 0.03) and diaphragm respectively in Neu-REFIX group. Mean dystrophin was higher in Neu-REFIX group by 70.3% and 4.7% in diaphragm and plasma respectively than control. H-score intensity of CD44+ was >2.0 with an MYH3-positivity 20% higher in Neu-REFIX than control. ConclusionOral administration of Neu REFIX was safe. Significantly enhanced plasma IGF-1 beside increased Dystrophin, MYH3 and CD44, proving a restoration of muscle regeneration and differentiation, especially in diaphragm, makes us recommend it as a disease modifying adjuvant in both early and advanced stages of DMD.

genetics↗

bioRxiv2023

Phototriggered profiling of receptor-proximal proteins in vivo in minutes

Neurotransmitter receptors are regulated by an extensive and dynamic network of protein-protein interactions. Understanding how these networks control synaptic transmission and give rise to higher-order brain functions necessitates their investigation in the live mammalian brain. However, tools available for this purpose lack the temporal resolution necessary to capture rapid changes in the interactome in live animals and require potentially disruptive genetic modifications to the animal under study. Here, we describe a method for light-activated proximity labelling in the live mouse brain that relies solely on small-molecule reagents and achieves a minute-order temporal resolution. Named PhoxID (photooxidation-driven proximity labelling for protein identification), this method involves selectively tethering a chemical photosensitiser to neurotransmitter receptors of interest and enabled us to identify characteristic as well as less studied interactors of the endogenous -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and the {psi}-aminobutyric acid type A receptor (GABAAR) with just minutes of in-brain green light irradiation. Furthermore, PhoxIDs temporal precision allowed us to capture molecular snapshots of the AMPAR-proximal proteome in the postnatal developing cerebellum, leading to the discovery of age-dependent shifts. Overall, this work establishes a highly flexible and generalisable platform to study receptor interactomes and proximal microenvironments in genetically intact specimens with an unprecedented temporal resolution.

neuroscience↗

bioRxiv2023

Bioorthogonal chemical labelling of endogenous neurotransmitter receptors in living mouse brains

Neurotransmitter receptors are essential components of synapses for communication between neurons in the brain. Because the spatiotemporal expression profiles and dynamics of neurotransmitter receptors involved in many functions are delicately governed in the brain, in vivo research tools with high spatiotemporal resolution for receptors in intact brains are highly desirable. Covalent chemical labelling of proteins without genetic manipulation is now a powerful method for analyzing receptors in vitro. However, selective target receptor labelling in the brain has not yet been achieved. This study shows that ligand-directed alkoxyacylimidazole (LDAI) chemistry can be used to selectively tether synthetic probes to target endogenous receptors in living mouse brains. The reactive LDAI reagents with negative charges were found to diffuse well over the whole brain and could selectively label target endogenous receptors, including AMPAR, NMDAR, mGlu1, and GABAAR. This simple and robust labelling protocol was then used for various applications: three-dimensional spatial mapping of endogenous receptors in the brains of healthy and disease-model mice; multi-colour receptor imaging; and pulse-chase analysis of the receptor dynamics in postnatal mouse brains. Here, results demonstrated that bioorthogonal receptor modification in living animal brains may provide innovative molecular tools that contribute to the in-depth understanding of complicated brain functions.

biochemistry↗

bioRxiv2022

Pulvinar Slits: Cellulose-deficient and De-esterified Homogalacturonan-rich Structures in a Legume Motor Cell

The cortical motor cells (CMCs) in a legume pulvinus execute the reversible deformation in leaf movement that is driven by changes in turgor pressure. In contrast to the underlying osmotic regulation proper, the cell wall structure of CMCs that contributes to the movement has yet to be characterized in detail. Here, we report that the cell wall of CMCs has circumferential slits with low levels of cellulose deposition. This structure is unique and distinct from that of any other primary cell walls reported so far; thus, we named them "pulvinar slits." Notably, we predominantly detected de-methyl-esterified homogalacturonan inside pulvinar slits, with a low deposition of highly methyl-esterified homogalacturonan, as with cellulose. In addition, Fourier-transform infrared spectroscopy analysis indicated that the cell wall composition of pulvini is different from that of other axial organs, such as petioles or stems. Moreover, monosaccharide analysis showed that pulvini are pectin-rich organs like developing stems and that the amount of galacturonic acid in pulvini is greater than in developing stems. Computer modeling suggested that pulvinar slits facilitate anisotropic extension in the direction perpendicular to the slits in the presence of turgor pressure. When tissue slices of CMCs were transferred to different extracellular osmotic conditions, pulvinar slits altered their opening width, indicating their deformability. In this study, we thus characterized a distinctive cell wall structure of CMCs, adding new knowledge to repetitive and reversible organ deformation as well as the structural diversity and function of the plant cell wall.

plant biology↗

bioRxiv2021

Revisiting PFA-mediated tissue fixation chemistry: FixEL enables trapping of small molecules in the brain to visualize their distribution dynamics

Various small molecules have been used as functional probes for tissue imaging in medical diagnosis and pharmaceutical drugs for disease treatment. The spatial distribution, target selectivity, and diffusion/extrusion kinetics of small molecules in structurally complicated specimens are critical for function. However, robust methods for precisely evaluating these parameters in the brain have been limited. Herein we report a new method termed "Fixation-driven chemical crosslinking of exogenous ligands (FixEL)" which traps and images exogenously administered molecules-of-interest (MOI) in complex tissues. This method relies on proteins-MOI interactions, and chemical crosslinking of amine-tethered MOI with paraformaldehyde used for perfusion fixation. FixEL is used to obtain images of the distribution of the small molecules and their dynamics, which addresses selective/nonselective binding to proteins, time-dependent localization changes, and diffusion/retention kinetics of MOI such as PET tracer derivatives or drug-like small molecules. Clear imaging of a nanobody distributed in the whole brain was also achieved with high spatial resolution using 2D/3D mode.

neuroscience↗

bioRxiv2021

Morphological variation and phylogeny of Karenia selliformis (Gymnodiniales, Dinophyceae) in an intensive cold-water algal bloom in eastern Hokkaido, Japan in September-November 2021

Harmful algal blooms responsible for mass mortalities of marine organisms have so far been rare in Hokkaido, northern Japan, although fish killing blooms have been frequently reported from western Japanese coasts. In September-November 2021, a huge and prolonged cold-water bloom occurred along the Pacific coast of eastern Hokkaido, Japan, and was associated with intensive mortalities of sea urchin, fish, octopus, shellfish, etc. In this study, morphology and phylogeny of the dominant and co-occurred unarmored dinoflagellates of the Kareniaceae in the bloom were examined by using light microscopy, scanning electron microscopy and molecular phylogeny inferred from ITS and LSU rDNA (D1-D3) sequences. Morphological observation and molecular phylogeny showed that the dominant species was Karenia selliformis, with co-occurrences of other kareniacean dinoflagellates, Kr. longicanalis, Kr. mikimotoi, Karlodinium sp., Takayama cf. acrotrocha, Takayama tuberculata and Takayama sp. The typical cell forms of K. selliformis in the bloom were discoid, dorsoventrally flattened, and larger than the cell sizes in previous reports, 35.3-43.6 (39.4{+/-}2.1) m in length. Transparent cells of Kr. selliformis lacking or having several shrunken chloroplasts and oil droplets were also found. Cells of Kr. selliformis had morphological variation, but the species could be distinguished from other co-occurred Karenia species by its numerous (46-105) and small granular (2.9-4.6 m in diameter) chloroplasts and the nucleus positioned in the hypocone. Cell density of Kr. selliformis exceeding 100 cells/mL was recorded in the range of temperature 9.8-17.6{degrees}C. The rDNA sequences determined from Kr. selliformis in the blooms of Hokkaido, Japan in 2021 were identical to those from another bloom in Kamchatka, Russia in 2020. HighlightsO_LIA marine fauna-destructive harmful algal bloom in the Pacific coast of eastern Hokkaido, Japan in September-November 2021 was dominated by Karenia selliformis. C_LIO_LICells of Karenia selliformis typical in the bloom were discoid and possessing numerous small chloroplasts, approximately 70 in number. C_LIO_LICells of Karenia selliformis showed morphological variation in size and shape, and transparent motile cells lacking or having degraded chloroplasts were also present. C_LIO_LICo-occurred kareniaceans in the bloom were Karenia longicanalis, Karenia mikimotoi, Karlodinium sp. and Takayama spp. C_LIO_LIrDNA sequences of Karenia selliformis in the blooms of Hokkaido in 2021 and Kamchatka in 2020 were identical, which belong to the group I of Kr. selliformis. C_LI

ecology↗