Biology subjects

Uchida, T.

Publications and source records attributed to Uchida, T..

13 recordsLinked to original sources

bioRxiv2026

Single-Molecule Activity Profiling of Glycosidase Proteoforms Using Water-Soluble Fluorogenic Probes

In this study, we report a series of highly water-soluble fluorogenic probes for glycosidases useful for microdevice-based single-molecule enzyme activity profiling (SEAP). The assay was able to detect trace glycosidase activities in blood with high sensitivity and with proteoform resolution. This platform revealed the potential of -mannosidase as sensitive blood-based liquid-biopsy biomarkers for liver injury, highlighting the potential of this approach for activity-based diagnostics.

biochemistry↗

bioRxiv2026

Solid-Phase Synthesis of ProTide Fluorogenic Probes Enables Systematic Profiling of Carboxypeptidase Activity

Carboxypeptidases play diverse roles in physiological and pathological processes, yet comprehensive analysis of their activities in complex biological samples remains challenging. Here we report a solid-phase synthesis strategy for fluorogenic ProTide-based probes that enables systematic profiling of carboxypeptidase activities based on defined C-terminal amino acid motifs. By modular synthesis of dipeptide-fluorophore conjugates, we generated a focused probe set that revealed distinct substrate preferences among carboxypeptidases, including carboxypeptidase A and B family enzymes. Integration of these probes with a single-molecule enzyme activity assay allowed ultrasensitive detection of circulating carboxypeptidase activities in human blood samples. Application of this platform to clinical specimens demonstrated that specific carboxypeptidase activities are elevated in patients with pancreatic cancer compared with healthy controls, whereas closely related enzymes showed limited diagnostic value. These results establish a scalable chemical strategy for activity-based profiling of exopeptidases and highlight circulating carboxypeptidase activity as a functional enzymatic signature associated with pancreatic cancer.

biochemistry↗

bioRxiv2026

Possible Novel Sulfolipid Utilization Pathway in Giant Clams and Other Aquatic Invertebrates: Implications for Photosymbiosis and Sulfur Cycling

Photosymbiosis with dinoflagellates of the family Symbiodiniaceae enables giant clams to thrive in oligotrophic coral reef environments. However, mechanisms by which clams utilize algal-derived biomolecules remain largely unexplored. Using newly available genome resources for photosymbiotic bivalves (Tridacna and Fragum), we conducted a comparative genomic analysis to identify positively selected genes from these photosymbiotic bivalve lineages, that are potentially involved in symbiotic adaptations. Among candidate genes, we focused on sulfoquinovosidase (SQase), an enzyme that hydrolyzes sulfoquinovose (SQ) from sulfoquinovosyl diacylglycerol (SQDG), a sulfur-containing sulfolipid abundant in photosynthetic membranes. Although SQDG degradation has been characterized in bacteria, the distribution and role of SQase in animals have not been systematically examined. We found that possibly functional SQase homologs are widely distributed among aquatic invertebrates, but are largely absent in terrestrial taxa. In silico predictions showed that most animal SQases possess signal peptides and enter the secretory pathway, with lineage-specific gains and losses of membrane association, suggesting functional diversification. Transcriptomic analyses further demonstrated that SQase is predominantly expressed in digestive organs in diverse taxa, whereas in giant clams, it is also highly expressed in the outer mantle, the tissue primarily involved in harboring symbionts enriched in SQDG. Together, these results suggest that in aquatic invertebrates, SQase functions as a digestive enzyme for algal-derived sulfolipids and that photosymbiotic bivalves have expanded its deployment to tissues specialized for symbiosis. Our findings uncover a previously unrecognized metabolic interface between photosymbiosis and sulfur utilization in aquatic invertebrates, with broader implications for host-symbiont interactions and marine sulfur cycling.

zoology↗

bioRxiv2025

Genomic insights into photosymbiosis in giant clams: Comparisons with coral strategies

Giant clams are representative bivalves in coral reef ecosystems that host photosynthetic dinoflagellates extracellularly and rely on their photosynthates, functioning as "solar-powered animals." Unlike corals, which harbor intracellular dinoflagellates, molecular mechanisms and evolutionary history underlying this symbiosis remain largely unknown. Here, we integrated chromosome-scale genome assembly, transcriptome profiling, and bleaching experiments involving Tridacna crocea to explore the genetic basis of extracellular symbiosis. Signals associated with sterol transport by Niemann-Pick disease type C2 (NPC2) transporters and carbon-concentrating mechanisms suggest that giant clams share some nutrient exchange strategies with corals. Strikingly, the nitrate transporter NRT2, a "plant-like" gene previously thought to be absent in animals, represents an unexpected evolutionary retention that enables nitrate-based nutrient supply, highlighting a fundamental divergence from coral symbiosis. Together, our findings reveal both conserved and distinct molecular strategies of photosymbiosis in reef-dwelling marine invertebrates and provide insights into evolution and ecological resilience of coral reef ecosystems.

zoology↗

bioRxiv2025

Oligo DNA-based quantum dot (QD) single-particle tracking for multicolor single-molecule imaging

Quantum dot single-particle tracking (QD-SPT), a powerful tool for analyzing membrane domains that are critical to various cellular processes, is widely used in membrane molecular dynamics research. QDs, which possess both a broad excitation range and a narrow emission bandwidth, are inherently suited for multicolor imaging at various wavelengths. However, applying multicolor QD-SPT with multiple biomolecular targets has been challenging due to the limited methods for specifically conjugating QDs to biomolecules. Here, we propose a DNA hybridization-based QD labeling method that generates several specific interactions based on sequence. QD fused with 20-mer oligo DNA specifically labeled membrane lipid 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (DPPE) covalently bound to complementary oligo DNA, forming a stable label that is suitable for SPT. The combination of polyA-polyT sequence as the linker oligo caused more QDs to fuse to DPPE compared with a random sequence linker. Oligo DNA-based QD-SPT accurately reflected the diffusion dynamics of DPPE measured using the single-fluorescence tracking technique and was compatible with conventional QD-SPT utilizing secondary antibody Fab. Using different pairs of oligo DNA sequences, we successfully achieved multicolor QD-SPT that distinguishes the lateral diffusion of DPPE and a membrane protein, GABAA receptor (GABAAR), within the same cell. The oligo DNA-based QD labeling method developed in this study is anticipated to substantially advance simultaneous multicolor QD-SPT of different living cell membrane molecules specific to DNA sequences.

biophysics↗

bioRxiv2025

Modeling of hepatitis B virus infection spread in primary human hepatocytes

Chronic hepatitis B virus (HBV) infection poses a significant global health threat, causing severe liver diseases including cirrhosis and hepatocellular carcinoma. We characterized HBV DNA kinetics in primary human hepatocytes (PHH) over 32 days post-inoculation (pi) and used agent-based modeling (ABM) to gain insights into HBV lifecycle and spread. Parallel PHH cultures were mock-treated or HBV entry inhibitor Myr-preS1 (6.25 g/mL) was initiated 24h pi. In untreated PHH, 3 viral DNA kinetic patterns were identified: (1) an initial decline, followed by (2) rapid amplification, and (3) slower amplification/accumulation. In the presence of Myr-preS1, viral DNA and infected cell numbers in phase 3 were effectively blocked, with minimal to no increase. This suggests that phase 2 represents viral amplification in initially infected cells, while phase 3 corresponds to viral spread to naive cells. The ABM reproduced well the HBV kinetic patterns observed and predicted that the viral eclipse phase lasts between 18 and 38 hours. After the eclipse phase, the viral production rate increases over time, starting with a slow production cycle of 1 virion per day, which gradually accelerates to 1 virion per hour after 3 days. Approximately 4 days later, virion production reaches a steady state production rate of 4 virions/hour. The estimated median efficacy of Myr-preS1 in blocking HBV spread was 91% (range: 90-92%). The HBV kinetics and the predicted estimates of the HBV eclipse phase duration and HBV production cycles in PHH are similar of those predicted in uPA/SCID mice with human livers. ImportanceWhile primary human hepatocytes (PHH) are the most physiologically relevant culture system for studying HBV infection in vitro, comprehensive understanding of HBV infection kinetics and spread in PHH is lacking. In this study, we characterize HBV viral kinetics and employ agent-based modeling (ABM) to provide quantitative insights into the HBV production cycle and viral spread in PHH. The ABM provides an estimate of HBV eclipse phase duration, HBV production cycles and Myr-preS1 efficacy in blocking HBV spread in PHH. The results resemble those predicted in uPA/SCID mice with human livers, demonstrating that estimated HBV infection kinetic parameters in PHH in vitro mirrors that observed in in vivo HBV infection chimeric mouse model.

systems biology↗

bioRxiv2024

Induction of the aggresome and insoluble tau aggregation using an optogenetic tool

Tauopathy is a spectrum of diseases characterized by fibrillary tau aggregate formation in neurons and glial cells in the brain. Tau aggregation originates in the brainstem and entorhinal cortex and then spreads throughout the brain in Alzheimers disease (AD), which is the most prevalent type of tauopathy. Understanding the mechanism by which locally developed tau pathology propagates throughout the brain is crucial for comprehending AD pathogenesis. Therefore, a novel model of tau pathology that artificially induces tau aggregation in targeted cells at specific times is essential. This study describes a novel optogenetic module, OptoTau, which is a human tau with the P301L mutation fused with a photosensitive protein CRY2olig, inducing various forms of tau according to the temporal pattern of blue light illumination pattern. Continuous blue light illumination for 12 h to Neuro2a cells that stably express OptoTau (OptoTauKI cells) formed clusters along microtubules, many of which eventually accumulated in aggresomes. Conversely, methanol-resistant tau aggregation was formed when alternating light exposure and darkness in 30-min cycles for 8 sets per day were repeated over 8 days. Methanol-resistant tau was induced more rapidly by repeating 5-min illumination followed by 25-min darkness over 24 h. These results indicate that OptoTau induced various tau aggregation stages based on the temporal pattern of blue light exposure. Thus, this technique exhibits potential as a novel approach to developing specific tau aggregation in targeted cells at desired time points. SignificanceThis study developed an approach to manipulate tau aggregation in a blue light-dependent manner using cells that stably express OptoTau, which is an optogenetic tool based on the CRY2olig module. Tau accumulation in aggresomes or stable tau aggregation were selectively induced by blue light illumination conditions. These results are crucial as they provide a new technological basis for establishing a singular point of tau aggregation in specific targeted cells at a particular time.

biophysics↗

bioRxiv2023

A genome and tissue-specific transcriptomes of the large-polyp coral, Fimbriaphyllia (Euphyllia) ancora: Recipe for a coral polyp

Coral "polyps" are composed of several tissues; however, their characteristics are largely unexplored. Here we report biological characteristics of the four tissues that comprise polyps: tentacle (Te), mesenterial filament (Me), body wall (Bo), and mouth with pharynx (MP), using comparative genomic, morpho-histological, and transcriptomic analyses of the large-polyp coral, Fimbriaphyllia ancora. A draft F. ancora genome assembly of 434 Mbp was created. Morpho-histological and transcriptomic characterization of the four tissues showed that they have distinct differences in structure, primary cellular composition, and transcriptional profiles. Tissue-specific, highly expressed genes (HEGs) of Te are related to biological defense, predation, and coral-algal symbiosis. Me expresses multiple digestive enzymes, whereas Bo expresses innate immunity and biomineralization-related molecules. Many receptors for neuropeptides and neurotransmitters are expressed in MP. The established dataset and new insights into tissue functions will facilitate a deeper understanding of coral biology.

genomics↗

bioRxiv2023

CD4+ T-cell immunity of SARS-CoV-2 patients determine pneumonia development

Most humans infected with SARS-CoV-2 will recover without developing pneumonia. A few SARS-CoV-2 infected patients, however, develop pneumonia, and occasionally develop cytokine storms. In such cases, it is assumed that there is an inadequate immune response to eliminate viral infected cells and an excessive inappropriate immune response causing organ damage, but little is known about this mechanism. In this study, we used single cell RNA sequencing and mass cytometry to analyze peripheral blood T cells from patients hospitalized with proven COVID-19 infection in order to clarify the differences in host immune status among COVID-19 pneumonia cases, non-pneumonia cases, and healthy controls. The results showed that a specific CD4+ T cell cluster with chemokine receptor expression patterns, CXCR3+CCR4-CCR6+ (Th1/17), was less abundant in COVID-19 pneumonia patients. Interestingly, these CD4+ T-cell clusters were identical to those we have reported to correlate with antitumor immunity and predict programmed cell death (PD)-1 blockade treatment response in lung cancer. The Th1/17 cell percentages had biomarker performance in diagnosing pneumonia cases. In addition, CTLA-4 expression of type17 helper T cells (Th17) and regulatory T cells (Treg) was found to be significantly lower. This indicates that functional suppression of Th17 was less effective and Treg function was impaired in pneumonia cases. These results suggest that imbalance of CD4+ T-cell immunity generates excessive immunity that does not lead to viral eradication. This might be a potential therapeutic target mechanism to prevent severe viral infections. ImportanceIn this observational study, 49 consecutive patients with SARS-CoV-2 infection confirmed by PCR testing and admitted to Saitama Medical University Hospital and Saitama Medical University International Medical Centre between December 4, 2020 and January 17, 2022 were included. Of these 49 patients, 29 were diagnosed with COVID-19 pneumonia by computed tomography (CT) scan (Table 1). The unique CD4+ T-cell immunity with less abundant Th1/17 CD4+ T-cell cluster and low expression of CTLA-4 in Th17 and Treg was consistently found in SARS-CoV-2 pneumonia patients on admission and 1-week of admission. The imbalance of CD4+ T-cell immunity may contribute to develop pneumonia in SARS-CoV-2 virus infected patients by delaying viral clearance and resulting in an excessive immune response. O_TBL View this table: org.highwire.dtl.DTLVardef@1aac015org.highwire.dtl.DTLVardef@128c14dorg.highwire.dtl.DTLVardef@aacabaorg.highwire.dtl.DTLVardef@e39c8eorg.highwire.dtl.DTLVardef@13b439a_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 1C_FLOATNO O_TABLECAPTIONPatient demographics C_TABLECAPTION C_TBL

immunology↗

bioRxiv2021

All-trans retinoic acid induces GADD34 gene expression via transcriptional regulation by Six1-TLE3 and post-transcriptional regulation by p38-TTP in skeletal muscle

All-trans retinoic acid (ATRA) increases the sensitivity to unfolded protein response (UPR) in differentiating leukemic blasts. The downstream transcriptional factors of PERK, a major arm of UPR, regulates muscle differentiation. However, the role of growth arrest and DNA damage-inducible protein 34 (GADD34), one of the downstream factors of PERK, and the effects of ATRA on GADD34 expression in muscle remain unclear. In this study, we identified ATRA increased the GADD34 expression independent of the PERK signal in the gastrocnemius muscle of mice. ATRA up-regulated GADD34 expression through the transcriptional activation of it via inhibiting the interaction of homeobox Six1 and transcription co-repressor TLE3 with the MEF3-binding site on the GADD34 gene promoter in myoblasts. ATRA also inhibited the interaction of TTP, which induces mRNA degradation, with AU-rich element on GADD34 mRNA via p38 MAPK, resulting in the instability of GADD34 mRNA. Overexpressed GADD34 in myoblasts changes the type of myosin heavy chain in myotubes. These results suggest ATRA increases GADD34 expression via transcriptional and post-transcriptional regulation in myoblasts, which changes muscle fiber type in myotubes.

biochemistry↗

bioRxiv2021

Validity and sensitivity of an inertial measurement unit-driven biomechanical model of motor variability for gait

Motor variability in gait is frequently linked to fall risk, yet field-based biomechanical joint evaluations are scarce. We evaluated the validity and sensitivity of an inertial measurement unit (IMU)-driven biomechanical model of joint angle variability for gait. Fourteen healthy young adults completed seven-minute trials of treadmill gait at several speeds and arm swing amplitudes. Joint kinematics were estimated by IMU- and optoelectronic-based models using OpenSim. We calculated range of motion (ROM), magnitude of variability (meanSD), local dynamic stability ({lambda}max), persistence of ROM fluctuations (DFA), and regularity (SaEn) of each angle over 200 continuous strides, and evaluated model accuracy (e.g., RMSD: root mean square difference), consistency (ICC2,1: intraclass correlation), biases, limits of agreement, and sensitivity to within-participant gait responses (effects of Speed and Swing). RMSDs of joint angles were 1.7-7.5{degrees} (pooled mean of 4.8{degrees}), excluding ankle inversion. ICCs were mostly good- excellent in the primary plane of motion for ROM and in all planes for meanSD and {lambda}max, but were poor-moderate for DFA and SaEn. Modeled Speed and Swing responses for ROM, meanSD, and {lambda}max were similar. Results suggest that the IMU-driven model is valid and sensitive for field-based assessments of joint angles and several motor variability features.

bioengineering↗

bioRxiv2020

Transcriptome analysis of sevoflurane exposure effects at the different brain regions

BackgroundsSevoflurane is a most frequently used volatile anaesthetics, but its molecular mechanisms of action remain unclear. We hypothesized that specific genes play regulatory roles in whole brain exposed to sevoflurane. Thus, we aimed to evaluate the effects of sevoflurane inhalation and identify potential regulatory genes by RNA-seq analysis. MethodsEight-week old mice were exposed to sevoflurane. RNA from four medial prefrontal cortex, striatum, hypothalamus, and hippocampus were analysed using RNA-seq. Differently expressed genes were extracted. Their gene ontology terms and the transcriptome array data of the cerebral cortex of sleeping mice were analysed using Metascape, and the gene expression patterns were compared. Finally, the activities of transcription factors were evaluated using a weighted parametric gene set analysis (wPGSA). JASPAR was used to confirm the existence of binding motifs in the upstream sequences of the differently expressed genes. ResultsThe gene ontology term enrichment analysis result suggests that sevoflurane inhalation upregulated angiogenesis and downregulated neural differentiation in the whole brain. The comparison with the brains of sleeping mice showed that the gene expression changes were specific to anaesthetized mice. Sevoflurane induced Klf4 upregulation in the whole brain. The transcriptional analysis result suggests that KLF4 is a potential transcriptional regulator of angiogenesis and neural development. ConclusionsKlf4 was upregulated by sevoflurane inhalation in whole brain. KLF4 might promote angiogenesis and cause the appearance of undifferentiated neural cells by transcriptional regulation. The roles of KLF4 might be key to elucidating the mechanisms of sevoflurane induced functional modification in the brain.

genetics↗

bioRxiv2020

Cystal structure of inhibitor-bound human MSPL/TMPRSS13 that can activate high pathogenic avian influenza

Infection of certain influenza viruses is triggered when its hemagglutinin (HA) is cleaved by host cell proteases such as proprotein convertases and type II transmembrane serine proteases (TTSP). HA with a monobasic motif is cleaved by trypsin-like proteases, including TMPRSS2 and HAT, while the multibasic motif found in high pathogenicity avian influenza HA is cleaved by furin, PC5/6, or MSPL. MSPL belongs to the TMPRSS family and preferentially cleaves [R/K]-K-K-R{downarrow} sequences. Here, we solved the crystal structure of the extracellular region of human MSPL in complex with an irreversible substrate-analog inhibitor. The structure revealed three domains clustered around the C-terminal -helix of the SPD. The inhibitor structure and its putative model show that the P1-Arg inserts into the S1 pocket, whereas the P2-Lys and P4-Arg interacts with the Asp/Glu-rich 99-loop that is unique to MSPL. Based on the structure of MSPL, we also constructed a homology model of TMPRSS2, which is essential for the activation of the SARS-CoV-2 spike protein and infection. The model may provide the structural insight for the drug development for COVID-19.

biophysics↗