Biology subjects

Ohno, H.

Publications and source records attributed to Ohno, H..

At least 19 recordsLinked to original sources

bioRxiv2026

Membrane progesterone receptors PAQR6 and PAQR8 in the ruminant uterus and ovary

Progesterone governs the establishment and maintenance of pregnancy in ruminants, acting through the nuclear progesterone receptor (PGR) and, potentially, through membrane progesterone receptors (mPRs) of the PAQR family. The cellular distribution of mPRs in ruminant reproductive tissues, however, remains undefined. We generated and validated rabbit polyclonal antibodies against the intracellular C-terminal peptides of bovine PAQR6 (mPR{delta}) and PAQR8 (mPR{beta}), and used them, with quantitative PCR of the uterus and conceptus, to map these receptors in the ovine uterus and conceptus and in the bovine ovary. Both antisera showed high ELISA titer against their immunizing peptides, negligible preimmune reactivity, and complete loss of tissue signal after peptide pre-absorption. By qPCR, PAQR6, PAQR8 and PGR transcript levels did not change significantly across the peri-implantation period in either the conceptus or the endometrium; in contrast, the progesterone-responsive gene ZBTB16 increased markedly in the conceptus (up to ~45-fold by day 21, p < 0.05 at days 17, 19 and 21), whereas FKBP5 showed a numerical increase without reaching statistical significance. Both genes remained relatively stable in the endometrium, indicating conceptus-specific changes in progesterone-responsive gene expression. At the protein level, PAQR6 and PAQR8 localized to the luminal and glandular epithelium of the endometrium across the cycle and early pregnancy, and to follicular (granulosa) and luteal cells of the ovary. Dual immunofluorescence in the ovine endometrium showed that both PAQR6 and PAQR8 are enriched at the apical surface of the luminal epithelium and are spatially distinct from the basolateral adherens-junction marker E-cadherin. These results provide a protein-level map of PAQR6 and PAQR8 in the ruminant reproductive tract, identify the apical epithelial surface as a site of receptor enrichment, and show that the endometrial epithelium retains membrane progesterone receptors while progesterone-responsive gene expression changes dynamically in the conceptus during the peri-implantation period. The validated ruminant-reactive antibodies provide a resource for future functional studies.

developmental biology↗

bioRxiv2026

Loss of the fatty acid β-oxidation gene acaa-2 impairs timely hatching under poor maternal diet in C. elegans

Developmental programs must remain robust despite fluctuations in maternal nutrition, yet the genetic basis of this robustness is incompletely understood. Using an unbiased, diet-dependent forward genetic screen in the nematode Caenorhabditis elegans, we searched for mutants with normal embryogenesis on standard food but defective development when mothers experienced poor nutrition. We isolated a mutation in acaa-2, which encodes an enzyme catalyzing the final step of mitochondrial fatty acid {beta}-oxidation, and confirmed its role using an independent deletion allele. acaa-2 mutant embryos exhibit a marked hatching delay specifically when mothers consume a low-quality diet. Despite remaining motile within the eggshell, mutant embryos are associated with increased unoccupied intra-eggshell space, consistent with impaired late embryonic growth. Our findings reveal a previously unrecognized role for acaa-2 and suggest that mitochondrial fatty acid {beta}-oxidation acts as a metabolic buffer that promotes developmental robustness under maternal nutrient limitation.

developmental biology↗

bioRxiv2026

Size-dependent effect of site-specific polyethylene glycol modification of trastuzumab on the antibody functions

Polymer modification of antibodies has attracted attention for enhancing antibody functionality. Modification with polyethylene glycol (PEG) or biopolymers such as antibody-oligonucleotide conjugates has been investigated. Although direct effects at the modification site have been understood, distal effects of modifications on intrinsic antibody functions such as antigen-binding and Fc receptor-binding activities remain poorly characterized. In this study, we prepared site-specifically PEGylated trastuzumab to evaluate the effects of the modification site and the size of PEG chains on antibody functions. Reporter gene assays revealed a significant reduction in antibody-dependent cell cytotoxicity (ADCC) activity in a site- and size-dependent manner. Both the reduction in interaction with the antigen and Fc{gamma}RIIIa were observed in a size-dependent manner. Interestingly, the effect of the modification site was only observed for ADCC activity, which suggests that the higher-order structure of the modified antibody plays a critical role in effector function. These results provide molecular design guidelines for the polymer modification of antibodies.

biochemistry↗

bioRxiv2026

Cell-type-specific expression of classical and membrane progesterone receptors at the human maternal-fetal interface

Progesterone is essential for the establishment and maintenance of pregnancy, primarily through the classical nuclear progesterone receptor (PGR). However, progesterone also exerts rapid, non-genomic effects through membrane progesterone receptors, including members of the progestin and adipoQ receptor (PAQR) family, whose cellular distribution and potential roles at the human maternal-fetal interface remain poorly understood. We therefore sought to define the cell-type-specific expression landscape of classical and membrane progesterone receptors during early human pregnancy. We analyzed publicly available single-cell RNA-sequencing datasets of the first-trimester human maternal-fetal interface to characterize the expression of PGR and PAQR family members across trophoblast, immune, stromal, and vascular cell populations. Major expression patterns were examined across independent datasets and further assessed using spatial transcriptomic data. PGR expression was predominantly localized to decidual stromal and perivascular populations, whereas individual PAQR family members showed distinct cellular distributions. PAQR6 was enriched in decidual natural killer cells, PAQR7 was broadly expressed in trophoblast populations, PAQR8 was prominently expressed in fetal stromal and macrophage populations, including Hofbauer cells, and PAQR9 showed prominent enrichment in syncytiotrophoblasts. Selected cell-type-specific patterns were further supported by spatial transcriptomic analysis. These findings provide a cell-type-resolved framework for investigating how distinct progesterone receptor systems mediate progesterone signaling across maternal and fetal cellular compartments during early human placentation.

developmental biology↗

bioRxiv2026

Maternal progesterone signaling establishes lifelong oral homeostasis

Pregnancy is accompanied by profound endocrine remodeling, yet the mechanisms by which maternal hormonal signals establish long-term tissue homeostasis remain largely unknown. Here we identify maternal progesterone signaling as a developmental cue that establishes lifelong oral homeostasis through a hormone-lipid-microbiome axis. We show that the membrane progesterone receptor mPR{delta} is selectively expressed in the developing and maternal submandibular glands, where it mediates non-genomic progesterone signaling to promote epithelial differentiation by driving the selective mobilization of docosahexaenoic acid (DHA). Loss of this pathway disrupts salivary gland maturation, reshapes the oral microbial ecosystem through the selective expansion of Pasteurellaceae, and causes local inflammation as well as systemic metabolic dysfunction. Mechanistically, antibiotic treatment abolishes these phenotypes, whereas transfer of the oral microbiota recapitulates disease, demonstrating that developmental defects in the host are translated into long-term pathology through the oral microbiome. Remarkably, maternal--but not adult--DHA supplementation restores salivary gland development and microbial homeostasis and prevents adult disease phenotypes, identifying a critical developmental window during which oral homeostasis is durably established. Collectively, these findings reveal a previously unrecognized maternal endocrine mechanism that establishes lifelong host-microbiome homeostasis and identify developmental programming as a fundamental principle linking maternal physiology to adult health. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/743932v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@178cc90org.highwire.dtl.DTLVardef@1be04beorg.highwire.dtl.DTLVardef@29759dorg.highwire.dtl.DTLVardef@1e89994_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2026

OddCAPS: a simple, low-cost, universal technique for detecting single nucleotide variants

Derived Cleaved Amplified Polymorphic Sequences (dCAPS) assays have been widely performed historically to detect known base substitutions in many model organisms--notably Caenorhabditis elegans, Arabidopsis thaliana, and yeast--where chemical mutagens that induce point mutations are frequently used. With the rise of whole-genome sequencing and genome editing technologies, dCAPS is increasingly applied to detect diverse nucleotide changes across a broader range of species. However, a key limitation of dCAPS is that genomic target sites amenable to primer designs that both preserve PCR amplification and create recognition sites for inexpensive, high-performance restriction enzymes are scarce. Here we report One-step dual-primer dCAPS (OddCAPS), a modification that uses three primers in one reaction to overcome this constraint. Two of these primers, an intermediate primer and a dCAPS primer, sequentially introduce 1-2 base substitutions each into the amplicon, enabling up to four engineered base changes near the nucleotide of interest. By using the intermediate primer at 1/10-1/100 the concentration of the other primers, the desired product is generated directly in a single-tube, one-step PCR. Increasing the number of engineered substitutions improves the chance of using a researchers preferred restriction enzyme. In principle, having eight common restriction enzymes (BamHI, EcoRI, NheI, SalI, BglII, ClaI, HindIII, and MluI) suffices to detect any single-nucleotide variant in any biological or synthetic DNA sequence with this approach.

molecular biology↗

bioRxiv2026

LRP1 is an entry receptor for the botulinum toxin complex in the gut

Botulinum neurotoxin (BoNT) is an etiologic agent of food poisoning caused by Clostridium botulinum. The large progenitor toxin complex (L-PTC) crosses the intestinal epithelial barrier to deliver BoNT to target neurons; however, it is not clearly understood how BoNT enters the host. Here, we identified low-density lipoprotein receptor-related protein 1 (LRP1) as a major enterocyte transcytosis receptor for the hyper-oral-toxic L-PTC serotype B-Okra (L-PTC/BOkra). We found that hemagglutinin (HA), a neurotoxin-associated protein within the L-PTC/BOkra complex, binds to LRP1 via N-glycans. HA/BOkra co-localized with LRP1 within the internalized vesicles in cultured cells and enterocytes. LRP1 deletion inhibited the apical-to-basal transcytosis of L-PTC/BOkra in an intestinal epithelial cell line, and this effect was rescued by LRP1 re-expression. Finally, intestinal epithelial cell-specific LRP1-deficient mice displayed reduced susceptibility to toxicity caused by oral administration of L-PTC/BOkra. Taken together, these results indicate that N-glycosylated LRP1 mediates L-PTC/BOkra transcytosis via enterocytes, enabling BoNT to traverse the intestinal epithelial barrier.

microbiology↗

bioRxiv2026

Changes in dysbiosis and gene expression in the gut of wharf roach (Ligia Spp.) fed with expanded polystyrene

The East Asian region, known for its high levels of human and fishery activities, experiences serious plastic pollution in the marine environment, especially in seawater and along coastlines. Wharf roaches (Ligia spp.) collected from the coast of western Japan frequently ingest expanded polystyrene (EPS), which is then excreted as microplastic through their feces. However, the impact of EPS exposure and ingestion on the gut microbiome of wharf roaches remains unclear. Thus, this study aimed to investigate the effects of EPS ingestion on the gut microbiota of wharf roaches by examining their gut microbiota and gene expression. The expression levels of more than 400 genes, including those associated with xenobiotic metabolism, and the abundance of gut microbial community were altered. Microbial analysis revealed that at least five archaeal types, two to four bacterial types, three to seven eukaryotic types, and three viral types were involved in a correlation network composed of strong associations. Among them, Haloquadratum, Halalkalicoccus, and Methanospirillum (archaea); Volvox (eukaryote); and Varicellovirus and T4-like viruses showed significantly increased abundance. Furthermore, covariance structure analysis indicated that the viruses and methanogens played key causal roles as characteristic factors related to EPS administration. In conclusion, EPS disrupts the intestinal environment of wharf roaches and serves as a potential material for viral activation and methane production. Building on our previous field study that identified wharf roaches as potential indicators of coastal EPS pollution, this study provides novel insights into the ecological impacts of EPS ingestion and consequences of plastic pollution.

pharmacology and toxicology↗

bioRxiv2025

Epigenomic and transcriptomic germ-free ageing atlas reveals sterile inflammation as an intrinsic ageing feature

Inflammageing is a hallmark of ageing. Commensal microbiota plays crucial roles in maintaining tissue homeostasis, yet its impact on cellular ageing and inflammageing remains poorly understood. Here we present a comprehensive single-cell epigenomic and transcriptomic atlas of tissues from mice aged under specific pathogen-free (SPF) or germ-free (GF) conditions. Microbiota conferred beneficial effects in young mice but accelerated various ageing features in old, such as age-related AP-1 pathway upregulation, senescence and transcriptomic alterations, likely due to age-associated dysbiosis. Strikingly, inflammatory signatures persisted across cell types in aged GF mouse tissues, establishing sterile inflammation as an intrinsic feature of ageing. Age-associated B cells expanded equally under GF and SPF conditions, raising the possibility that they function as intrinsic, microbiota-independent drivers of inflammageing and potential therapeutic targets. The atlas provides a resource for distinguishing intrinsic ageing features from those modulated by the microbiota, illuminating mechanisms of cellular ageing and potential anti-ageing interventions. HighlightsSterile inflammation and age-associated B cell expansion are prominent intrinsic ageing features The upregulation of age-associated AP-1 pathways and senescence of alveolar macrophages are attenuated in germ-free condition Germ-free condition induces premature ageing-like features in young mouse tissues but delays ageing features in old mice across multiple cell types

genomics↗

bioRxiv2025

Conversion of an agonistic anti-TNFR2 biparatopic antibody into an antagonist by insertion of peptide linkers into the hinge region

Biparatopic antibodies (BpAbs) bind two different antigen epitopes to form characteristic immunocomplexes. Many BpAbs have been developed for enhanced cross-linking to induce signal transduction or cell internalization, whereas few were reported with smaller immunocomplexes to suppress unwanted signaling. Here, we developed a strategy to induce 1:1 immunocomplex formation to maximize antagonistic function. Various peptide linkers were introduced into the hinge regions of IgG-like agonist BpAbs against tumor necrosis factor receptor 2. Loss of crosslinking activity was observed for one BpAb, allowing the conversion of its function from an agonist to an antagonist. However, cross-linking activity was retained for another agonist BpAb, which binds to a different epitope pair. In a combined analysis of cryo-electron microscopy and coarse-grained molecular dynamics simulations, effect of epitope combination on the stability of 1:1 complexes was observed. These results lead to an understanding of the mechanism and design of BpAbs to adopt a 1:1-binding mode.

biochemistry↗

bioRxiv2024

Split RNA switch: Programmable and precise control of gene expression by ensemble of pre- and post-translational regulation

Regulating gene expression in response to biomolecules is a powerful strategy for monitoring intracellular environments and controlling cellular programs. RNA switch is a synthetic mRNA-based technology that controls gene expression at the translational level in response to cellular RNA and protein molecules, thus enabling cell type-specific gene regulation and showing promise for gene therapy, regenerative medicine, and cell therapy. However, single RNA switches often lack the specificity required for practical applications due to low ON/OFF ratios and difficulty in finding distinct and single biomolecule targets. To address these issues, we developed "split RNA switches" that integrate outputs from multiple RNA switches by exploiting protein splicing, a post-translational modification mechanism. We demonstrated that split RNA switches significantly improve the ON/OFF ratio of microRNA (miRNA)-responsive ON switch systems by canceling undesirable leaky OFF level. We achieved efficient and robust target cell purification based on endogenous miRNA profiles, which was impossible with an ON switch alone. Additionally, we constructed multi-output and multi-input RNA-based synthetic circuits using split RNA switches to enable the detection of multiple miRNAs for precise gene control with logical operations. Split RNA switches illustrate a novel application of protein splicing and demonstrate the potential of post-translational processing as a comprehensive solution for advancing translational control technologies toward widespread mRNA therapies.

synthetic biology↗

bioRxiv2024

Identification of M-Sec as a unique cellular regulator of CSF-1 receptor activation

Fms, the CSF-1 receptor encoding tyrosine kinase, is essential for tissue macrophage development, and the therapeutic target for many tumors. However, it is not completely understood how Fms activation is regulated. Here, we identify the cellular protein M-Sec as a unique regulator of Fms. In macrophages, Fms forms large aggregates via unknown mechanisms. We found that the inhibition or knockdown of reduced Fms aggregate formation and functional response of macrophages to CSF-1, which was consistent with reduced Fms activation after CSF-1 stimulation. When expressed in 293 cells, M-Sec augmented Fms aggregate formation and CSF-1-induced Fms activation. CSF-1 and M-Sec bind the cellular phosphatidylinositol 4,5-biphosphate (PIP2). The removal of PIP2-binding motif of Fms or M-Sec, or the depletion of cellular PIP2 reduced Fms aggregate formation. Moreover, M-Sec altered cellular distribution of PIP2. Since CSF-1-induced dimerization of Fms is critical for its activation, our findings suggest that M-Sec augments large Fms aggregate formation via PIP2, which brings Fms monomers close to each other and enables the efficient dimerization and activation of Fms in response to CSF-1.

molecular biology↗

bioRxiv2024

Maternal progesterone and adipose mPRε in pregnancy regulate the embryonic nutritional state

Sex steroid hormones such as progesterone play a pivotal role in reproductive functions and maintaining pregnancy; however, the impact of progesterone on the interaction between mother and embryo is unclear. Here, we demonstrate that the relationship between maternal progesterone and membrane progesterone receptor epsilon (mPR{varepsilon}) in adipose tissue regulates embryonic nutritional environment and growth after birth in mice. The activation of adipose mPR{varepsilon} by increased progesterone during pregnancy enhanced maternal insulin resistance through the production of prostaglandins, thereby efficiently providing glucose to embryos. The offspring of mPR{varepsilon}-deficient mothers exhibited metabolic dysfunction, whereas mPR{varepsilon}-deficient mothers with high-fat-diet-induced obesity exhibited improved insulin sensitivity. These findings establish the importance of progesterone as a nutritional regulator between mother and embryo, and suggest that mPR{varepsilon} modulators could be developed to treat pregnant glycemic control disorders such as gestational diabetes mellitus, as well as metabolic syndrome in offspring. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/609823v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1ecd968org.highwire.dtl.DTLVardef@b5492aorg.highwire.dtl.DTLVardef@1d692ecorg.highwire.dtl.DTLVardef@47de8a_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2024

Generation of antagonistic biparatopic anti-CD30 antibody from an agonistic antibody by precise epitope determination and utilization of structural characteristics of CD30 molecule

CD30 is a type I membrane protein that has been successfully targeted for lymphoma therapy using Brentuximab vedotin, an antibody-drug conjugate. Recently, the potential of blocking CD30-dependent NF-{kappa}B intracellular signaling has gained attention for treating inflammatory disorders. Development of antibody-based CD30 antagonists would broaden therapeutic strategies. A challenge in developing antagonistic antibodies is that the bivalent form of natural antibody format inevitably cross-links trace amounts of CD30 molecules, leading to signal transduction. In this study, we developed a series of biparatopic antibodies with each pair of antibody variable domains (Fvs) binding to distinct epitopes on CD30, and evaluated their biological activities and binding modes. Initially, we precisely identified epitope sites of the nine antibodies precisely by assessing binding to multiple orthologous CD30 proteins and mutants. We then produced 36 biparatopic antibodies covering all possible combinations of the nine Fvs, and analyzed their biological activities. Among these, we identified both potent agonists and antagonists. Notably, a significant proportion of the biparatopic antibodies displayed reduced agonistic activities, including 1:1-binding antagonists derived from a Fv of a strong agonist previously developed for lymphoma therapy, AC10. The mechanism of signaling activity induction is discussed using epitope information, which leads to the strategies of the development of biparatopic antibodies.

biochemistry↗

bioRxiv2024

Label-free imaging of cellular organization in living mammalian cells via external apodization phase-contrast microscopy

Developing techniques to visualize intracellular structures, which influence the spatiotemporal functionality of biomolecules, is essential for elucidating mechanisms governing cellular behavior. In this study, we demonstrate that label-free external apodization phase-contrast (ExAPC) microscopy serves as a valuable tool for the simultaneous observation of various intracellular structures with high spatiotemporal resolution, while successfully mitigating halo artifacts. Additionally, through quantitative analysis of images obtained by combining ExAPC microscopy with fluorescence microscopy, we identified distinct heterogeneities in biomolecular condensates, lipid droplets, and mitochondria. Our findings highlight the potential of ExAPC microscopy to provide detailed insights into alterations in intracellular structures associated with diverse cellular processes, corroborating the existing knowledge and potentially contributing to the discovery of novel cellular mechanisms.

cell biology↗

bioRxiv2023

Gut microbiome-based prediction of autoimmune neuroinflammation

Gut commensals are linked to neurodegenerative diseases, yet little is known about causal and functional roles of microbial risk factors in the gut-brain axis. Here, we employed a pre-clinical model of multiple sclerosis in mice harboring distinct complex microbiotas and six defined strain combinations of a functionally-characterized synthetic human microbiota. Discrete microbiota compositions resulted in different probabilities for development of severe autoimmune neuroinflammation. Nevertheless, assessing presence or the relative abundances of a suspected microbial risk factor failed to predict disease courses across different microbiota compositions. Importantly, we found considerable inter-individual disease course variations between mice harboring the same microbiota. Evaluation of multiple microbiome-associated functional characteristics and host immune responses demonstrated that the immunoglobulin A-coating index of Bacteroides ovatus before disease onset is a robust individual predictor for disease development. Our study highlights that the "microbial risk factor" concept needs to be seen in the context of a given microbial community network, and host-specific responses to that community must be considered when aiming for predicting disease risk based on microbiota characteristics.

microbiology↗

bioRxiv2023

Salmonella cancer therapy metabolically disrupts tumours at the collateral cost of T cell immunity

Bacterial cancer therapy (BCT) is a promising therapeutic for solid tumours. Salmonella enterica Typhimurium (STm) is well-studied amongst bacterial vectors due to advantages in genetic modification and metabolic adaptation. A longstanding paradox is the redundancy of T cells for treatment efficacy; instead, STm BCT depends on innate phagocytes for tumour control. Here, we used distal T cell receptor (TCR) reporter mice (Nr4a3-Tocky-Ifng-YFP) and a colorectal cancer (CRC) model to interrogate T cell activity during BCT with attenuated STm. We found that colonic TILs exhibited a variety of activation defects, including IFN-{gamma} production decoupled from TCR signalling, decreased polyfunctionality and reduced TCM formation. Modelling of T-cell-tumour interactions with a tumour organoid platform revealed an intact TCR signalosome, but paralysed metabolic reprogramming due to inhibition of the master metabolic controller, c-Myc. Restoration of c-Myc by deletion of the bacterial asparaginase ansB reinvigorated T cell activation, but at the cost of decreased metabolic control of the tumour by STm. This work shows for the first time that T cells are metabolically defective during BCT, but also that this same phenomenon is inexorably tied to intrinsic tumour suppression by the bacterial vector.

immunology↗

bioRxiv2022

Development of a 1:1-binding biparatopic anti-TNFR2 antagonist by epitope selection

Conventional bivalent antibodies against cell surface receptors often initiate unwanted signal transduction by crosslinking two antigen molecules. Biparatopic antibodies (BpAbs) bind to two different epitopes on the same antigen, thus altering crosslinking ability. In this study, we developed BpAbs against tumor necrosis factor receptor 2 (TNFR2), which is an attractive immune checkpoint target. Using different pairs of variable regions specific to topographically distinct TNFR2 epitopes, we successfully regulated the size of BpAb-TNFR2 immunocomplexes to result in controlled agonistic activities. One particular antagonist BpAb bound TNFR2 in 1:1 ratio without unwanted signal transduction, with its structural basis revealed by cryo-electron microscopy. This antagonist suppressed the proliferation of regulatory T cells expressing TNFR2. Therefore, the BpAb format would be useful in designing specific and distinct antibody functions.

biochemistry↗