Biology subjects

Ito, D.

Publications and source records attributed to Ito, D..

15 recordsLinked to original sources

bioRxiv2026

Microbiota-derived pantothenic acid rapidly restores colonic mucus barrier after antibiotic-induced dysbiosis

The intestinal mucus layer forms the primary physical barrier separating the host epithelium from the gut microbiota. Antibiotic-induced dysbiosis compromises this barrier, yet the microbial metabolites and host mechanisms responsible for its rapid restoration remain poorly understood. Here, we identify pantothenic acid (vitamin B5) as a microbial metabolite that rapidly restores colonic mucus integrity following antibiotic administration. Integrated 16S rRNA gene sequencing and metabolomic profiling of murine feces revealed a positive association between Muribaculaceae abundance, fecal pantothenic acid level, and colonic mucus thickness. Among four isolated Muribaculaceae species, only Sangeribacter muris produced pantothenic acid. An exogenous pantothenic acid supplement quickly restored the thin mucus layer. This rapid response occurred without detectable transcriptional activation. This response did not recur in isolated colonic epithelium or colonic organoids, indicating that pantothenic acid acts through a non-epithelial, non-autonomous mechanism. Together, these findings identify a previously unrecognized microbe-metabolite-host signaling pathway that rapidly regulates colonic mucus barrier homeostasis and reveal a local barrier-protective function of microbiota-derived pantothenic acid.

microbiology↗

bioRxiv2026

Cytotoxicity-based High-throughput Screening System for CAR T Cell

Some chimeric antigen receptor (CAR) T cell therapies have shown strong clinical efficacy, yet systematic screening of new CAR designs remains constrained by labor-intensive, low-throughput evaluation methods. To address this limitation, we developed a cytotoxicity-centered, high-throughput screening platform that integrates single-cell pooled screening with fully automated arrayed screening enabling both large-scale library handling and quantitative functional resolution for systematic CAR design exploration. Using a mutation-based CAR design approach guided by protein fitness prediction, we generated a 4-1BB-based CAR library with approximately 10 theoretical variants while minimizing the prevalence of low-activity designs. In pooled screening, CAR T cells were evaluated at the single-cell level based on cytotoxicity and proliferation, enabling rapid enrichment of high-performing variants from a highly diverse library. Subsequent automated arrayed screening quantitatively measured cytotoxicity with high reproducibility, providing high-resolution functional data suitable for comparative ranking. Selected CAR variants demonstrated superior antitumor efficacy in a leukemia xenograft model compared with a template CAR. Furthermore, systematic analysis of mutation sites from an enhanced CAR variant identified essential mutation combinations underlying functional enhancement. Together, this study establishes a cytotoxicity-focused screening framework that provides a robust approach for optimizing CAR architectures and accelerating the development of CAR T-cell therapies.

synthetic biology↗

bioRxiv2025

NADPH phosphatase activity of Mesh1 controls sleep in Drosophila

Recent findings have shown that metazoans accumulate the bacterial second messenger guanosine tetraphosphate (ppGpp) and possess Mesh1, a gene encoding a ppGpp hydrolase domain. Mesh1 deficiency affects sleep behavior and eclosion under starvation in Drosopila, and ferroptosis in human cells. However, human Mesh1 also exhibits NADPH/NADP+ phosphatase activity in vitro, making it unclear whether these phenotypes result from loss of ppGpp hydrolysis, NADPH/NADP+ dephosphorylation, or both. To address this, we performed biochemical and genetic analysis of Drosophila Mesh1. We first found that Drosophila Mesh1 dephosphorylates NADPH, but not NADP+, in vitro. We subsequently sought to generate Drosophila Mesh1 point mutants specifically impaired in NADPH phosphatase activity. Based on structural data, we mutated W138 and R142, residues that are predicted to interact with NADPH but not ppGpp. W138 was replaced with phenylalanine results in complete loss of NADPH phosphatase activity with retained ppGpp hydrolase activity. Flies carrying the W138F mutation, introduced via genome editing, exhibited shortened total sleep and increased sleep fragmentation in behavioral assays, without changes in intracellular ppGpp levels. These results indicate that the NADPH phosphatase activity of Mesh1, and specifically the W138 residue, is essential for normal sleep regulation in Drosophila.

molecular biology↗

bioRxiv2025

PEG10-ORF1 programs trophoblast progenitor development for placental labyrinth formation

The emergence of the placenta was a significant event in mammalian evolution, enabling therian mammals to give birth to live young through viviparity. A key genomic change associated with this process was likely the acquisition of the therian-specific paternally expressed 10 (PEG10) from a metavirus, as Peg10-null mutant mice exhibit early embryonic lethality due to the absence of both the spongiotrophoblast and labyrinth layers in the placenta. PEG10 encodes a GAG-like open reading frame 1 (ORF1) and a POL-like ORF2, and produces two proteins, PEG10-ORF1 and PEG10-ORF1/2 fusion proteins, via the -1 frameshift mechanism conserved in retroviruses and metaviruses. Here, we demonstrate PEG10-ORF1 plays an essential and distinct role in labyrinth development. Mice lacking ORF1 but retaining the ORF1/2 protein initially developed normally in both embryos and placentas; however, later exhibited underdevelopment of the placental labyrinths complex microarchitecture, accompanied by mid-to late gestational growth retardation and lethality. These severe defects are most likely attributable to dysregulated development of labyrinth trophoblast precursor (LaTP) cells. Beyond its essential role in early placental formation, we previously demonstrated that the DSG protease domain within PEG10-ORF1/2--a metaviruse-derived feature--is critical for maintaining fetal capillaries during late gestation. Thus, PEG10 fulfills multiple indispensable functions in placental formation and maintenance by exploiting a metavirus-derived multi-protein and -peptide production system, despite being a single gene. This study provides additional evidence underscoring the profound role of PEG10 acquisition in therian placental evolution. Significance StatementThe placenta is an essential organ for viviparous reproduction in therian mammals; therefore, its evolutionary emergence is of significant interest. One key event may have been the acquisition of PEG10, a metavirus (retrovirus-like LTR retrotransposon)- derived gene, by the common therian ancestor. We previously showed that Peg10 is required for early placental development and that its protease domain is essential for late-gestational maintenance of fetal capillaries within the placental labyrinth. Here, we provide additional genetic evidence that Peg10 is also indispensable for mid-gestational development of the placental labyrinthine microvasculature. Together, these findings illustrate how multiple, stage-specific functions of a single metavirus-derived gene, PEG10, coordinate placental formation and maintenance, highlighting the evolutionary importance of PEG10 acquisition in therian placentation.

developmental biology↗

bioRxiv2025

Enhancing CAR-T cell activity prediction via fine-tuning protein language models with generated CAR sequences

Chimeric antigen receptor (CAR)-T cell therapy has shown remarkable success in treating hematological malignancies; however, several challenges remain, including limited efficacy against solid tumors, T cell exhaustion, and lack of T cell persistence, which have restricted its clinical efficacy across various indications. Sequence optimization of CAR constructs offers a promising strategy to enhance therapeutic efficacy of CAR-T cells. Recent advances in machine learning, especially protein language models (PLMs), enable prediction of mutational effects based on sequence representations. Nevertheless, applying PLMs to CARs is challenging due to the artificial nature of CARs and the absence of comprehensive CAR sequence databases. In this study, we developed a computational framework to predict CAR-T cell activity by fine-tuning ESM-2 with the CAR sequences generated using sequence augmentation. These CAR sequences were constructed by recombining homologous domains of CARs, enabling task-specific adaptation of the model. To evaluate prediction performance, we experimentally assessed the cytotoxicity of CAR-T cells expressing mutated CAR variants and compared these results with model predictions. Our results demonstrated that fine-tuned ESM-2 significantly improves prediction performance of CAR-T cell activity. Furthermore, we showed that training parameters--such as sequence diversity, number of training steps, and model size--substantially influence prediction performance. This work highlights the potential of combining sequence augmentation with fine-tuning PLMs to advance data-driven CAR-T cell design.

bioinformatics↗

bioRxiv2024

Dysregulated expanded endocannabinoid system as therapeutic targets of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the selective loss of upper and lower motor neurons. ALS patients often manifest systemic metabolic abnormalities such as glucose intolerance. Herein, to elucidate the systemic metabolic changes related to ALS progression, we performed metabolomics analysis on the serum of ALS patients and identified several metabolites associated with the disease progression, including metabolites involved in the expanded endocannabinoid system (ECS). In particular, the levels of N-acyl taurines (NAT) were correlated with the longitudinal change in the revised ALS functional rating scale (ALSFRS-R) rating. In vitro experiments with ALS cell models and in vivo studies with SOD1G93A transgenic mice revealed that PF-04457845, a fatty amide acid hydrolase (FAAH) inhibitor, up-regulated the expanded ECS, particularly the levels of NATs and N-acyl ethanolamine and ameliorates motor neuron degeneration through the regulation of microglial polarization, synapse plasticity, and neuronal development. Our study indicates that dysregulation of the expanded ECS is associated with ALS progression and a target for novel disease-modifying therapies.

neuroscience↗

bioRxiv2023

Recruitment status and host-fish-limitation threats to endangered freshwater pearl mussel (Margaritifera laevis) in eastern Hokkaido, northern Japan

Recruitment failure is a major threat to freshwater mussel (Order Unionoida) populations worldwide. Assessments of the recruitment status and determining the bottleneck factors of mussel recruitment are crucial for preventing future declines in mussel populations. In this study, we investigated the recruitment status (i.e., size structure and the proportion of juveniles within a population) of the endangered freshwater pearl mussel Margaritifera laevis in 22 rivers in eastern Hokkaido, northern Japan. We also quantified the density of the host fish Oncorhynchus masou masou and examined the relationship between the proportion of juveniles and host-fish density to assess host-limitation threats to M. laevis recruitment. Our assessments showed that 13 (59.1%) rivers had no signs of recent recruitment within 10 years, with a low mean proportion of juveniles (mean:0.02 [range:0.00-0.09] fraction), indicating that these populations are threatened by sustained recruitment failure. The proportion of juveniles was positively associated with host-fish density, suggesting that host-fish limitation could be a bottleneck factor for M. laevis recruitment. These results highlight the urgent need for prompt conservation measures, including the enhancement of host-fish availability, to sustain M. laevis populations in the study region.

ecology↗

bioRxiv2021

Multiple stressors and recruitment failure of long-lived endangered freshwater mussels with a complex life cycle

O_LIMultiple stressors can interactively affect the population of organisms; however, the process by which they affect recruitment efficiency remains unclear for empirical populations. Recruitment efficiency can be regulated at multiple stages of life, particularly in organisms with complex life cycles. Understanding the interactive effects of multiple stressors on recruitment efficiency and determining the bottleneck life stages is imperative for species conservation. C_LIO_LIThe proportion of <20-year-old juveniles of the endangered freshwater pearl mussel Margaritifera togakushiensis, which has an obligate parasitic larval stage, was investigated in 24 rivers from eastern Hokkaido, northern Japan to reveal the influence of nutrients, fine sediment, and their combined effects on juvenile recruitment efficiency. The following indices for recruitment at adult, parasitic, and post-parasitic juvenile stages were obtained from 11 of these rivers: gravid female density, glochidia density (the number of glochidia infections per stream area), and juvenile survival rate. This study explored the bottleneck stages of recruitment efficiency and the interactive effects of the two stressors on these stages. C_LIO_LITwenty-four population status assessments determined that the proportion of juveniles ranged from 0.00 to 0.53, and juveniles were absent from four rivers. The results showed that the parasitic and post-parasitic juvenile stages were bottlenecks for recruitment efficiency. Juvenile survival rates had a more significant positive effect on recruitment efficiency in rivers with a high glochidia density. Juvenile survival rate was decreased by the synergistic interaction of nutrients and fine sediment, although factors limiting glochidia density were not found. C_LIO_LIThe nutrient concentration of rivers in the study region was well explained by the proportion of agricultural land cover and urban areas in the watersheds, but no relationship was detected between fine sediment abundance and land use. C_LIO_LIThis study suggests that nutrient management at a catchment scale can be effective for re-establishing the recruitment of M. togakushiensis, particularly in rivers with a high content of fine sediments. The results also emphasise the importance of considering both parasitic and post-parasitic juvenile stages of mussels to maximise the positive effects of stressor mitigation. C_LI

ecology↗

bioRxiv2021

Exploring ensemble structures of Alzheimer's amyloid β (1-42) monomer using linear regression for the MD simulation and NMR chemical shift

Aggregation of intrinsically disordered amyloid {beta} (A{beta}) is a hallmark of Alzheimers disease. Although complex aggregation mechanisms have been increasingly revealed, structural ensembles of A{beta} monomers with heterogeneous and transient properties still hamper detailed experimental accesses to early events of amyloidogenesis. We herein developed a new mathematical tool based on multiple linear regression to obtain the reasonable ensemble structures of A{beta} monomer by using the solution nuclear magnetic resonance (NMR) and molecular dynamics simulation data. Our approach provided the best-fit ensemble to two-dimensional NMR chemical shifts, also consistent with circular dichroism and dynamic light scattering analyses. The major monomeric structures of A{beta} including {beta}-sheets in both terminal and central hydrophobic core regions and the minor partially-helical structures suggested initial structure-based explanation on possible mechanisms of early molecular association and nucleation for amyloid generation. A wide-spectrum application of the current approach was also indicated by showing a successful utilization for ensemble structures of folded proteins. We propose that multiple linear regression in combination to experimental results will be highly promising for studies on protein misfolding diseases and functions by providing a convincing template structure. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/457317v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1b4e6eeorg.highwire.dtl.DTLVardef@1a533eeorg.highwire.dtl.DTLVardef@f48eb3org.highwire.dtl.DTLVardef@1c4bf9a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Dual effects of presynaptic membrane mimetics on α-synuclein amyloid aggregation

Aggregation of intrinsically disordered -synuclein (SN) under various conditions is closely related to synucleinopathies. Although various biological membranes have shown to alter the structure and aggregation propensity of SN, a thorough understanding of the molecular and mechanical mechanism of amyloidogenesis in membranes remains unanswered. Herein, we examined the structural changes, binding properties, and amyloidogenicity of three variations of SN mutants under two types of liposomes, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and presynaptic vesicle mimetic (Mimic) membranes. While neutrally charged DOPC membranes elicited marginal changes in the structure and amyloid fibrillation of SNs, negatively charged Mimic membranes induced dramatic helical folding and biphasic amyloid generation. At low concentration of Mimic membranes, the amyloid fibrillation of SNs was promoted in a dose-dependent manner. However, further increases in the concentration constrained the fibrillation process. These results suggest the dual effect of Mimic membranes on regulating the amyloidogenesis of SN, which is rationalized by the amyloidogenic structure of SN and condensation-dilution of local SN concentration. Finally, we propose physicochemical properties of SN and membrane surfaces, and their propensity to drive electrostatic interactions as decisive factors of amyloidogenesis.

biophysics↗

bioRxiv2020

A unique adhesive motif of protein disulfide isomerase P5 supports its function via dimerization

P5, also known as PDIA6, is a PDI-family member that plays an important role in the ER quality control. Herein, we revealed that P5 dimerizes via a unique adhesive motif contained in the N-terminal thioredoxin-like domain. This motif is apparently similar to, but radically different from conventional leucine-zipper motifs, in that the former includes a periodic repeat of leucine or valine residues at the third or fourth position spanning five helical turns on 15-residue anti-parallel -helices, unlike the latter of which the leucine residues appear every two helical turns on [~]30-residue parallel -helices at dimer interfaces. A monomeric P5 mutant with the impaired adhesive motif showed structural instability and local unfolding, and behaved as an aberrant protein that induces the ER stress response. Disassembly of P5 to monomers compromised its ability to inactivate IRE1 via reduction of intermolecular disulfide bonds and its Ca2+-dependent regulation of chaperone function in vitro. Thus, the leucine-valine adhesive motif supports structure and physiological function of P5.

biochemistry↗

bioRxiv2019

ppGpp functions as an alarmone in metazoa

Guanosine 3,5-bis(pyrophosphate) (ppGpp) functions as a second messenger in bacteria to adjust their physiology in response to environmental changes. In recent years, the ppGpp-specific hydrolase, metazoan SpoT homolog-1 (Mesh1), was shown to have important roles for growth under nutrient deficiency in Drosophila melanogaster. Curiously, however, ppGpp has never been detected in animal cells, and therefore the physiological relevance of this molecule, if any, in metazoans has not been established. Here, we report the detection of ppGpp in Drosophila and human cells and demonstrate that ppGpp accumulation induces metabolic changes, cell death, and eventually lethality in Drosophila. Our results provide the first evidence of the existence and function of the ppGpp-dependent stringent response in animals.

molecular biology↗

bioRxiv2019

Plastidial (p)ppGpp synthesis by the Ca2+-dependent RelA-SpoT homolog regulates the adaptation of chloroplast gene expression to darkness in Arabidopsis

In bacteria, the hyper-phosphorylated nucleotides, guanosine 5-diphosphate 3-diphosphate (ppGpp) and guanosine 5-triphosphate 3-diphosphate (pppGpp), function as secondary messengers in the regulation of various metabolic processes of the cell, including transcription, translation, and enzymatic activities, especially under nutrient deficiency. The activity carried out by these nucleotide messengers is known as the stringent response. (p)ppGpp levels are controlled by two distinct enzymes, namely, RelA and SpoT, in Escherichia coli. RelA-SpoT homologs (RSHs) are also conserved in plants and algae where they function in the plastids. The model plant Arabidopsis thaliana contains four RSHs: RSH1, RSH2, RSH3, and Ca2+-dependent RSH (CRSH). Genetic characterizations of RSH1, RSH2, and RSH3 were undertaken, which showed that the (p)ppGpp-dependent plastidial stringent response significantly influences plant growth and stress acclimation. However, the physiological significance of CRSH-dependent (p)ppGpp synthesis remains unclear, as no crsh-null mutant has been available. Here to investigate the function of CRSH, a crsh-knockout mutant of Arabidopsis was constructed using a site-specific gene-editing technique, and its phenotype was characterized. A transient increase of ppGpp was observed for 30 min in the wild type (WT) after light-to-dark transition, but this increase was not observed in the crsh mutant. Similar analyzes were performed with the rsh2rsh3 double and rsh1rsh2rsh3 triple mutants of Arabidopsis and showed that the transient increments of ppGpp in the mutants were higher than those in the WT. The increase of (p)ppGpp in the WT and rsh2rsh3 accompanied decrements in the mRNA levels of psbD transcribed by the plastid-encoded plastid RNA polymerase. These results indicated that the transient increase of intracellular ppGpp at night is due to CRSH-dependent ppGpp synthesis and the (p)ppGpp level is maintained by the hydrolytic activities of RSH1, RSH2, and RSH3 to accustom plastidial gene expression to darkness.

plant biology↗

bioRxiv2018

Electric field dependent effects of motor cortical TDCS

Transcranial direct current stimulation (TDCS) can modulate motor cortical excitability. However, its after-effects are highly variable between individuals. Individual cranial and brain anatomy may contribute to this variability by producing varying electric fields in each subjects brain. Here we show that these fields are related to excitability changes following anodal TDCS of the primary motor cortex (M1). We found in two experiments (N=28 and N=9) that the after-effects of TDCS were proportional to the individual electric field in M1, calculated using MRI-based models. Individuals with the lowest and highest local electric fields in M1 tended to produce opposite changes in excitability. Furthermore, the effect was field-direction dependent and non-linear with stimulation duration or other experimental parameters. The electric field component pointing into the brain was negatively proportional to the excitability changes following 1 mA 20 min TDCS of right M1 (N=28); the effect was opposite after 1 mA 10 min TDCS of left M1 (N=9). Our results demonstrate that a large part of variability in the after-effects of motor cortical TDCS is due to inter-individual differences in the electric fields. We anticipate that individualized electric field dosimetry could be used to control the neuroplastic effects of TDCS, which is increasingly being explored as a treatment for various neuropsychiatric diseases.

neuroscience↗

bioRxiv2018

An ancestral role of pericentrin in centriole formation through SAS-6 recruitment

The centrosome is composed of two centrioles surrounded by a microtubule-nucleating pericentriolar matrix (PCM). Centrioles regulate matrix assembly. Here we ask whether the matrix also regulates centriole assembly. To define the interaction between the matrix and individual centriole components, we take advantage of a heterologous expression system using fission yeast. Importantly, its centrosome, the spindle pole body (SPB), has matrix but no centrioles. Surprisingly, we observed that the SPB can recruit several animal centriole components. Pcp1/pericentrin, a conserved matrix component that is often upregulated in cancer, recruits a critical centriole constituent, SAS-6. We further show that this novel interaction is conserved and important for centriole biogenesis and elongation in animals. We speculate that the Pcp1/pericentrin-SAS-6 interaction surface was conserved for one billion years of evolution after centriole loss in yeasts, due to its conserved binding to calmodulin. This study reveals an ancestral relationship between pericentrin and the centriole, where both regulate each other assembly, ensuring mutual localisation.\n\nShort summaryThe pericentriolar matrix (PCM) is not only important for microtubule-nucleation but also can regulate centriole biogenesis. Ito et al. reveal an ancestral interaction between the centriole protein SAS-6 and the PCM component pericentrin, which regulates centriole biogenesis and elongation.

cell biology↗