Biology subjects

Arakawa, H.

Publications and source records attributed to Arakawa, H..

10 recordsLinked to original sources

bioRxiv2026

Reverse genetics system for emerging tick-borne orthonairovirus highlights dispensable N-terminal region of Gn associated with replication in tick vectors

A growing number of emerging and re-emerging tick-borne orthonairoviruses belonging to the Sulina and Tamdy genogroups have recently been identified in association with febrile diseases in East Asia. Yezo virus (YEZV) is one such viruses and is genetically distinct from well-characterized Crimean-Congo hemorrhagic fever virus. Orthonairovirus glycoproteins (Gn and Gc) expressed from a single glycoprotein precursor (GPC) gene mediate interaction with host cells, yet the organization and functions of the YEZV glycoproteins remain largely undefined. To characterize YEZV glycoproteins in the context of infection, we developed a reverse genetics system that enables recovery of recombinant YEZV entirely from cloned cDNAs. The recombinant wild-type virus exhibited growth properties comparable to those of the parental isolate in vitro and maintained pathogenicity in vivo. Proteomic analysis of purified virus particles produced in mammalian cells showed that peptide coverage of GPC-derived products began at residue 69. We therefore used the reverse genetics system to examine the functional importance of the GPC subregion upstream of residue 69. A mutant with a deletion of GPC residues 28 to 68 (rYEZV GPC{Delta}28-68), which retained the predicted signal peptide, was successfully recovered, suggesting that this subregion is dispensable for producing infectious virus. rYEZV GPC{Delta}28-68 propagated at levels comparable to those of the wild-type virus in mammalian cells and exhibited similar pathogenicity in a mouse model. Interestingly, the mutant reached lower viral titers in tick-derived ISE6 cells and in ticks in vivo, suggesting that this N-terminal subregion may contribute to virus replication in ticks. Together, these findings demonstrate the utility of the newly established reverse genetics system for dissecting the functions of the YEZV glycoproteins in mammalian and tick systems.

microbiology↗

bioRxiv2026

Real-time mass defect-driven prediction of glycopeptide precursors enables enrichment-free serum glycoproteomics

Glycopeptide enrichment remains a cornerstone in glycoproteomics, but bias and reproducibility issues continue to hinder biological insight and clinical translation. Employing curated glycoproteomics datasets and machine learning, we trained a glycopeptide classifier to recognize N-glycopeptide precursors through mass defect signatures. Integration of the classifier into a data-dependent acquisition framework facilitated real-time prediction of N-glycopeptides from human serum and revealed sex differences in murine plasma fucosylation opening avenues for enrichment-free glycoproteomics.

bioinformatics↗

bioRxiv2026

Spatiotemporal spinal integration of descending and spinal volleys in spinal motor circuits revealed by compound motor evoked potentials

Descending corticospinal and afferent pathways underlying spinally evoked motor potential both contribute to motor output, yet how their interaction at the spinal and peripheral levels is organized spatially within a muscle remains unclear. This study investigated the spatiotemporal characteristics of descending modulation of spinally evoked motor potentials by combining subthreshold transcranial magnetic stimulation (TMS) with transcutaneous spinal cord stimulation (tSCS). In Experiment 1, spinally evoked motor potentials were recorded from multiple lower-limb muscles at various interstimulus intervals (ISIs) defined relative to central conduction time (CCT). Subthreshold TMS facilitated spinally evoked motor potentials from CCT onward across all recorded muscles, with additional bilateral facilitation observed at longer ISIs. In Experiment 2, high-density surface electromyography (HDsEMG) revealed distinct intramuscular activation patterns in the tibialis anterior. The center of gravity (CoG) of TMS-induced motor evoked potentials was located more proximally than that of spinally evoked motor potentials. Notably, the CoG of facilitation maps was shifted further proximally than that of both single-stimulus responses. These findings suggest that descending and afferent inputs preferentially recruit partially distinct motoneuron pools within the same muscle. The proximal bias of facilitation indicates recruitment of additional motoneurons rather than uniform amplification of existing activity. Together, these results demonstrate that the interaction between descending and afferent inputs is both timing-dependent and spatially non-uniform, providing new insight into sensorimotor integration in the human lower limb.

neuroscience↗

bioRxiv2026

Distinct neural input strategies to motor units in the soleus and medial gastrocnemius during quiet standing

Standing posture control critically depends on the activation of the soleus (SOL) and medial gastrocnemius (MG), which serve distinct functional roles. Identifying underlying neural mechanisms has been challenging, as conventional invasive techniques sample only limited motor units (MUs). Recent advances in high-density surface electromyography (HDsEMG) have enabled analysis of MU activity and estimation of common synaptic inputs to spinal motoneurons. Therefore, we aimed to elucidate the common synaptic inputs underlying the distinct MU behaviors of SOL and MG during standing. We recorded HDsEMG from the SOL and MG, alongside electroencephalography, from 20 male participants during standing and isometric voluntary contractions. EMG signals were decomposed into individual MU activity, with common synaptic inputs estimated through intramuscular and corticomuscular coherence analyses (IMC and CMC). Compared to SOL, the MG exhibited significantly higher delta-, alpha-, and beta-band IMC during standing. In task comparisons, alpha-band IMC increased during standing specifically in the MG. Furthermore, although beta-band CMC decreased in both muscles while standing, IMC was preserved in the MG but markedly reduced in SOL. This dissociation suggests that the common neural drive to the MG during standing is likely derived from subcortical rather than cortical pathways. These results demonstrate that the SOL and MG are governed by distinct neural control strategies, which likely underlie their functional roles. Given the low CMC, the MG relies on strong common synaptic input from subcortical pathways (e.g., vestibulospinal and reticulospinal) to produce rapid corrective torque, whereas the SOL functions with lower neural synchrony to ensure steady ankle stiffness. Key pointsO_LIThe soleus and medial gastrocnemius play distinct roles in standing control, however, due to technical limitations, it has been difficult to identify the underlying neural mechanisms responsible for these differences. C_LIO_LIUsing high-density surface electromyography, we examined motor unit activity and neural inputs to these muscles during standing. C_LIO_LIThe medial gastrocnemius shows greater common synaptic input, potentially facilitating rapid ankle plantarflexion torque generation to correct postural sway. C_LIO_LIThe soleus exhibits lower motor unit synchrony, enabling stable and continuous ankle plantarflexion torque generation for body weight support. C_LIO_LIThis study demonstrates that the soleus and medial gastrocnemius are governed by distinct neural control strategies, which likely underlie their distinct functional roles. C_LI Abstract figure legend O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/698550v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@be53aorg.highwire.dtl.DTLVardef@f6793aorg.highwire.dtl.DTLVardef@190e8f9org.highwire.dtl.DTLVardef@af5e4b_HPS_FORMAT_FIGEXP M_FIG C_FIG Motor unit spike trains were decomposed from high-density surface electromyograms recorded from the medial gastrocnemius (MG; left; red) muscle and soleus (SOL; right; blue). To compare the neural input to the spinal motor neurons between them, we quantified the intramuscular coherence (IMC) of motor unit spike trains within the delta, alpha, and beta bands. MG exhibited greater IMC than SOL during standing, indicating stronger common synaptic input, likely mediated by vestibulospinal and reticulospinal pathways. In contrast, SOL showed lower IMC, suggesting a greater contribution of independent synaptic input. As a consequence, high motor-unit synchrony in the MG supports rapid, phasic torque generation for postural sway attenuation, whereas low synchrony in the SOL enables smooth, steady torque production for weight bearing during standing.

physiology↗

bioRxiv2025

Neutron crystallography of the covalent intermediate of β-glucosidase reveals remodeling of the catalytic center

Anomer-retaining glycoside hydrolases (GHs) generally catalyze a double displacement reaction via a covalent intermediate; however, neutron crystallography of glycoside ligand-bound states has not been performed. In this study, we investigated {beta}-glucosidase Td2F2 from GH family 1 as a model enzyme for anomer-retaining GHs. We determined joint X-ray/neutron structures of Td2F2 in ligand-free form, covalent intermediate with a 2-deoxy-2-fluoro glucoside (2F-Glc) inhibitor, and glucose product complex using hydrogen/deuterium-exchanged crystals at room temperature, with neutron diffraction resolutions of 1.80-1.70 [A]. Extensive hydrogen bonds recognizing the hydroxy groups of 2F-Glc were identified, along with the positions of deuterium atoms. The acid/base catalyst residue Glu166 was anchored by a hydrogen bond network pivoted by Asn293. Tyr295 forms a hydrogen bond with the catalytic nucleophile residue Glu352 in the ligand-free and glucose complex forms, while the active center undergoes significant reorganization, including side chain displacements of Glu352 and Tyr295, as well as the incorporation of a water molecule. An alternative conformation of Tyr295 was observed in the 2F-Glc structure at room temperature, suggesting its role in positioning the nucleophilic water during the deglycosylation step. The tyrosine hydrogen bonded to the nucleophile is also conserved in many other anomer-retaining GH families, underscoring its importance in catalysis. Based on the deuterium/hydrogen positions determined from neutron structures, we proposed a detailed reaction mechanism for Td2F2. Significance StatementGlycoside hydrolases perform diverse functions in organisms, with over 180 known enzyme families. Although hydrogen bonds and proton transfer play important roles in enzymatic reactions, hydrogen atoms are generally invisible in macromolecular X-ray crystallography. In anomer-retaining glycoside hydrolases, general acid/base catalysis and the formation and hydrolysis of a covalent glycosyl-enzyme intermediate have been postulated. Here, we report neutron crystal structures of a {beta}-glucosidase, where hydrogen and deuterium atoms were visualized at high resolution. An intricate hydrogen-bonding network and remarkable remodeling at the catalytic center were observed during covalent intermediate formation, revealing a detailed catalytic mechanism. The enzyme belongs to glycoside hydrolase family 1 and represents numerous enzymes employing the anomer-retaining mechanism.

biochemistry↗

bioRxiv2023

Testis organ culture system capable of evaluating testicular toxicity

Successful treatment of pediatric cancers often results in long-term health complications, including potential effects on fertility. Therefore, assessing the male reproductive toxicity of anti-cancer drug treatments and the potential for recovery is of paramount importance. However, in vivo evaluations are time-intensive and require large numbers of animals. To overcome these constraints, we utilized an innovative organ culture system that supports long-term spermatogenesis by placing the testis tissue between a base agarose gel and a polydimethylsiloxane ceiling, effectively mirroring the in vivo testicular environment. The present study aimed to determine the efficacy of this organ culture system for accurately assessing testicular toxicity induced by cisplatin, using acrosin-green fluorescent protein (GFP) transgenic neonatal mouse testes. The testis fragments were treated with different concentrations of cisplatin-containing medium for 24 hours and incubated in fresh medium for up to 70 days. The changes in tissue volume and GFP fluorescence over time were evaluated to monitor the progression of spermatogenesis, in addition to the corresponding histopathology. Cisplatin treatment caused tissue volume shrinkage and reduced GFP fluorescence in a concentration-dependent manner. Recovery from testicular toxicity was also dependent on the concentration of cisplatin received. The results demonstrated that this novel in vitro system can be a faithful replacement for animal experiments to assess the testicular toxicity of anti-cancer drugs and their reversibility, providing a useful method for drug development. Short AbstractAssessing the male reproductive toxicity of anti-cancer drugs and the potential for recovery is of paramount importance, however, in vivo evaluations are time-intensive and require large numbers of animals. We utilized an innovative organ culture system that mirrors the in vivo testicular environment to determine its efficacy in accurately assessing testicular toxicity induced by cisplatin. The results demonstrate that this system can be a faithful replacement for animal experiments to assess the testicular toxicity of anti-cancer drugs and their reversibility.

developmental biology↗

bioRxiv2023

CD38 assigns physiological function to soluble uric acid

Excessive elevation or reduction of soluble uric acid (sUA) levels has been linked to some of pathological states, raising another subject that sUA at physiological levels may be essential for the maintenance of health. Yet, the fundamental physiological functions and molecular targets of sUA remain largely unknown. Using enzyme assays and in vitro and in vivo metabolic assays, we demonstrate that sUA directly inhibits the hydrolase and cyclase activities of CD38 via a reversible non-competitive mechanism, thereby limiting nicotinamide adenine dinucleotide (NAD+) degradation. CD38 inhibition is restricted to sUA in purine metabolism, and a structural comparison using methyl analogs of sUA such as caffeine metabolites shows that 1,3-dihydroimidazol-2-one is the main functional group. Moreover, sUA at physiological levels prevents crude lipopolysaccharide (cLPS)-induced systemic inflammation and monosodium urate (MSU) crystal-induced peritonitis in mice by interacting with CD38. Together, this study unveils an unexpected physiological role for sUA in controlling NAD+ availability and innate immunity through CD38 inhibition, providing a new perspective on sUA homeostasis and purine metabolism.

physiology↗

bioRxiv2023

Identification of a mitochondrial targeting sequence in cathepsin D and its localization in mitochondria

Cathepsin D (CTSD) is a major lysosomal protease harboring an N-terminal signal peptide (amino acids 1-20) to enable vesicular transport from endoplasmic reticulum to lysosomes. Here, we report the possibility of a mitochondrial targeting sequence and mitochondrial localization of CTSD in cells. Live-cell imaging analysis with enhanced green fluorescent protein (EGFP)-CTSD indicated that CTSD localizes to mitochondria. CTSD amino acids 21-35 are responsible for its mitochondrial localization, which exhibit typical features of mitochondrial targeting sequences, and are evolutionarily conserved. A proteinase K protection assay and sucrose gradient analysis showed that a small population of endogenous CTSD molecules exists in mitochondria. These results suggest that CTSD is a dual-targeted protein that may localize in both lysosomes and mitochondria.

cell biology↗

bioRxiv2020

Mieap forms membraneless organelles to compartmentalize and facilitate cardiolipin metabolism

Biomolecular condensates (BCs) are formed by proteins with intrinsically disordered regions (IDRs) via liquid-liquid phase separation. Mieap/Spata18, a p53-inducible protein, participates in suppression of colorectal tumors by promoting mitochondrial quality control. However, the regulatory mechanism involved remains unclear. Here, we report that Mieap is an IDR-containing protein that drives formation of BCs involved in cardiolipin metabolism. Mieap BCs specifically phase separate the mitochondrial phospholipid, cardiolipin. Mieap directly binds to cardiolipin in vitro. Lipidomic analysis of cardiolipin suggests that Mieap promotes enzymatic reactions in cardiolipin biosynthesis and remodeling. Accordingly, four cardiolipin biosynthetic enzymes, TAMM41, PGS1, PTPMT1, and CRLS1, and two remodeling enzymes, PLA2G6 and TAZ, are phase-separated by Mieap BCs. Mieap-deficient cells exhibit altered crista structure, leading to decreased respiration activity and ATP production in mitochondria. These results suggest that Mieap may form membrane-less organelles to compartmentalize and facilitate cardiolipin metabolism, thus potentially contributing to mitochondrial quality control.

cell biology↗

bioRxiv2020

Gastrointestinal absorption of pimozide is enhanced by inhibition of P-glycoprotein

Following the death due to cardiac arrest of a patient taking pimozide, sertraline and aripiprazole antipsychotic/antidepressant combination therapy, a role of drug-drug interaction was suggested. Here, we investigated P-glycoprotein (P-gp)-mediated interaction among the three drugs using in vitro methods. Sertraline or aripiprazole significantly increased the permeability of pimozide in Caco-2 cell monolayers. ATPase assay indicated that pimozide is a P-gp substrate, and might act as a P-gp inhibitor at higher concentrations. The values of the kinetic parameters of carrier-mediated efflux, calculated from the concentration dependence of pimozide efflux from LLC-GA5-COL150 cells expressing human P-gp, were as follows: maximum transport rate (Jmax) = 84.9 {+/-} 8.9 pmol/min/mg protein, half-saturation concentration (Kt) = 10.6 {+/-} 4.7 M, first-order rate constant (kd) = 0.67 {+/-} 0.14 pmol/min/mg protein. Further, the efflux ratio of pimozide in LLC-GA5-COL150 cells was significantly decreased in the presence of sertraline or aripiprazole. These results indicate that pimozide is a substrate of P-gp, and its efflux is inhibited by sertraline and aripiprazole. Thus, P-gp inhibition by sertraline and/or aripiprazole may alter the gastrointestinal permeability of co-administered pimozide, resulting in an increased blood concentration of pimozide, which may increase the likelihood of pimozides known life-threatening side effect of QT prolongation.

pharmacology and toxicology↗