Biology subjects

Sato, Y.

Publications and source records attributed to Sato, Y..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Local, but not circulating, complement C3 governs immune checkpoint blockade efficacy

The roles of circulating complement C3, produced in hepatocytes, in immune defense are well defined in the literature. Interestingly, the C3 gene evolved before the circulatory system, suggesting an essential but yet unknown role of locally produced C3. Here, we investigated the effect of C3 on cancer immunotherapy and found that cancer-associated fibroblast (CAF)-derived C3, not hepatocyte-derived C3, determines the efficacy of immune checkpoint blockade (ICB). Tumors developed in CAF-specific C3 knockout mice exhibited resistance to anti-PD-1 therapy, with increased immunosuppressive M2-like macrophage infiltration. Mechanistically, the C3 degradation product iC3b suppressed myeloid cell infiltration via complement receptor 3 signaling, and the therapeutic targeting of this pathway restored ICB sensitivity of immunotherapy-resistant tumors. In human cancers, stromal C3 expression correlates with reduced M2-like macrophage infiltration and improved immunotherapeutic outcomes. These data demonstrate the role of locally produced C3 in the innate immune response, which may have been conserved across many organisms.

cancer biology↗

bioRxiv2025

Development of a digital analysis system for a novel 3D culture-based colony formation to detect malignantly transformed cells in human cell-based therapeutic products

The presence of malignantly transformed cells in human cell-based therapeutic products (hCTPs) is a significant safety concern. Although such cellular impurities in hCTPs can be assessed by detecting anchorage-independent growth using conventional soft agar colony formation (SACF) assays, the sensitivity of these assays is often insufficient. To overcome this limitation, we previously developed a novel tumorigenicity-associated testing method, the digital SACF (D-SACF) assay, which combines a partitioned culture of test cells to concentrate target cells with colony detection via image analysis. However, conventional soft agar culture involves complicated operations, such as preparing multilayered culture media and temperature control, and further technical optimization is required for the widespread adoption of the D-SACF assay. In this study, we focused on a new culture system incorporating a three-dimensional (3D) culture method using a liquid medium containing the low-molecular-weight agar polymer LA717 in low-adhesion culture vessels. We initially confirmed conditions for the efficient high-density 3D culture of normal cells using LA717-supplemented medium in low-adhesion 96-well plates. Using human mesenchymal stem/stromal cells (MSCs) as a normal cell model and HeLa cells as a transformed cell model, we demonstrated that the new 3D culture system effectively maintained the dispersion of MSCs and prevented their aggregation, while transformed HeLa cells exhibited robust anchorage independence, thereby establishing the new liquid/low-molecular-weight agar colony formation (LACF) method as an alternative to SACF. Finally, by systematizing the digital analysis system for the LACF assay (D-LACF assay), which streamlines the overall workflow from the performance evaluation of the test method to product testing and result interpretation, the limitations of the conventional soft agar-based D-SACF assay were addressed and its practicality and utility were enhanced. This in vitro evaluation system is expected to provide a promising approach for improving the quality and safety of hCTPs.

bioengineering↗

bioRxiv2025

Class I myosins direct circumferential F-actin flows to define cell chirality

Eukaryotic cells possess intrinsic chirality in their structure, motility, and intracellular dynamics, which are designated cell chirality. Cell chirality participates in the left-right asymmetric morphogenesis and tissue integrity. However, the mechanisms of cell chirality formation remain elusive. In Drosophila, two evolutionarily conserved myosin I genes, Myosin 1D (Myo1D) and Myosin 1C (Myo1C), respectively, dictate the dextral and sinistral chirality of the cells and body. Here, we reported that Myo1D and Myo1C respectively directed the clockwise and counterclockwise circumferential flow of F-actin in Drosophila macrophages. Both induced the corresponding circular cytoplasm flows and depended on Myosin2 (Myo2). In a modified in vitro motility assay using near-physiological actin concentrations, Myo1D triggered the self-organization of the F-actin ring (chiral F-actin ring) that rotated clockwise; conversely, Myo1C induced the random flow of F-actin. The chiral F-actin ring implied that the F-actin bundle was parallelly and annularly polarized concerning its barbed pointed end. Considering that Myo1D and Myo1C are localized to the dorsal plasma membrane of macrophages, Myo1D and Myo1C might organize the parallelly polarized F-actin in macrophages. Our results suggest that Myo2 might drive the clockwise circumferential flow of F-actin along its parallel and annular polarity induced by Myo1D, which may be a molecular basis of cell and organ chirality.

cell biology↗

bioRxiv2025

Spatial heterogeneity of disease infection attributable to neighbor genotypic identity in barley cultivars

Pest damage exhibits considerable spatial heterogeneity among individual plants in the field. While such spatial heterogeneity has often been treated as a nuisance in crop breeding trials, underlying biotic factors and loci remain poorly understood. To quantify the spatial variation in disease infection and associate it with neighboring genotypes, we applied two methods, Spatial Analysis of Field Trials with Splines (SpATS) and Neighbor Genome-Wide Association Study (Neighbor GWAS), to barley cultivars. Having compiled the CIMMYT Australia ICARDA Germplasm Evaluation (CAIGE) data, we first applied SpATS to three disease phenotypes such as the net form net blotch, spot form net blotch, and scald damage. This SpATS analysis showed extraneous phenotypic variation unexplained by smooth spatial trends, thereby leading us to focus on neighboring genotypes as an extraneous biological factor. We then applied the Neighbor GWAS model and found that neighbor genotypic identity explained 0.1-0.3 fractional variation in the three disease phenotypes. The Neighbor GWAS method also detected two significant or marginally significant variants on the barley 7H chromosome, which were associated with neighbor genotypic influence on the net form net blotch and scald damage. These variants were estimated to have beneficial effects that reduce disease damage by their allelic mixtures. Our findings suggest that neighbor genotypic identity can account for spatial variation in disease infection, providing a key to reduce pest damage by variety mixtures in field crops.

plant biology↗

bioRxiv2025

Ab-trapping - a peripheral staining artifact in antibody-based microscopy and genomics

Antibodies (Ab) are essential for detecting specific epitopes in microscopy and genomics, but can produce artifacts leading to erroneous interpretations. Here, we characterize a novel artifact, Ab-trapping, in which antibodies bind at the periphery of a cellular structure and do not diffuse further into its interior. This causes anomalous peripheral staining for multiple critical targets, including endogenous or ectopically expressed nuclear proteins like nucleolar proteins, histone variants and their modifications like H3K9me2. Ab-trapping can affect any assay relying on Ab diffusion, including immunofluorescence microscopy and recent genomics approaches like CUT&Tag. Critically, computational modeling and experimental validation reveal that Ab-trapping is caused by high epitope abundance, high Ab affinity, and low diffusion rates. Consequently, its effects can be mitigated by using alternative Abs and optimizing incubation conditions. Ab-trapping is therefore a considerable artifact that should be considered when designing experiments and interpreting results.

cell biology↗

bioRxiv2025

Longitudinal analysis of antibody titers after primary and booster mRNA COVID-19 vaccination can identify individuals at risk for breakthrough infection

A key issue in the post-COVID-19 era is the ongoing administration of COVID-19 vaccines. Repeated vaccination is essential for preparing against currently circulating and newly emerging SARS-CoV-2 variants while enabling people to continue with daily life. Optimizing vaccination strategies is crucial to efficiently manage medical resources and establish an effective vaccination framework. Therefore, it is important to quantitatively understand vaccine-induced immunity dynamics and to be able to identify poor responders with lower sustained antibody titers as potential priorities for revaccination. We investigated longitudinal antibody titer data in a cohort of 2,526 people in Fukushima, Japan, from April 2021 to November 2022 for whom basic demographic and health information was available. Using mathematical modeling and machine learning, we stratified the time-course patterns of antibody titers after 2 primary doses and 1 booster dose of mRNA COVID-19 vaccines. We identified 3 notable populations, which we refer to as the durable, the vulnerable, and the rapid-decliner populations, approximately half of which remained in the same population after the booster dose. Notably, the rapid-decliner population experienced earlier infections than the others. Furthermore, when comparing IgG(S) titers, IgA(S) titers, and T-spot counts between participants who experienced breakthrough infections after booster vaccination and those who did not, we found that IgA(S) titers were significantly lower in breakthrough infected participants during the early stage after booster vaccination. Our computational approach is adaptable to various types of vaccinations. This flexibility can inform policy decisions on vaccine distribution to enhance immunity both in future pandemics and in the post-COVID-19 era.

immunology↗

bioRxiv2025

Machine Learning-Assisted Decoding of Temporal Transcriptional Dynamics via Fluorescent Timer

Investigating the temporal dynamics of gene expression is crucial for understanding gene regulation across various biological processes. Using the Fluorescent Timer protein (Timer), the Timer-of-cell-kinetics-and-activity (Tocky) system enables analysis of transcriptional dynamics at the single-cell level. However, the complexity of Timer data has limited its broader application. Here, we introduce an integrative approach combining molecular biology and machine learning to elucidate Foxp3 transcriptional dynamics through flow cytometric Timer analysis. We have developed a Convolutional Neural Networks (ConvNet) approach that incorporates image conversion and Gradient-weighted Class Activation Mapping (Grad-CAM) for class-specific feature identification at the single-cell level. Biologically, we developed a novel CRISPR mutant of Foxp3-Tocky lacking the Conserved Non-coding Sequence 2 (CNS2), which has successfully elucidated CNS2-dependent Foxp3 transcription dynamics, revealing novel roles of CNS2 in regulating Foxp3 transcription frequency under specific conditions. Furthermore, generating new data from WT Foxp3 Tocky mice at various ages, the Grad-CAM methods successfully revealed distinct dynamics of Foxp3 expression from neonatal to aged mice, highlighting prominent thymus-like features of neonatal splenic Foxp3+ T cells. In conclusion, our study uncovers previously unrecognised Foxp3 transcriptional dynamics, establishing a proof-of-concept for integrating CRISPR, Tocky, and machine learning methods as advanced techniques to understand transcriptional dynamics in vivo.

immunology↗

bioRxiv2025

AI-assisted protein design to rapidly convert antibody sequences to intrabodies targeting diverse peptides and histone modifications

Intrabodies are engineered antibodies that function inside living cells, enabling therapeutic, diagnostic, and imaging applications. While powerful, their development has been hindered by challenges associated with their folding, solubility, and stability in the reduced intracellular environment. Here, we present an AI-driven pipeline integrating AlphaFold2, ProteinMPNN, and live-cell screening to optimize antibody framework regions while preserving epitope-binding complementarity-determining regions. Using this approach, we successfully converted 19 out of 26 antibody sequences into functional single-chain variable fragment (scFv) intrabodies, including a panel targeting diverse histone modifications for real-time imaging of chromatin dynamics and gene regulation. Notably, 18 of these 19 sequences had failed to convert using the standard approach, demonstrating the unique effectiveness of our method. As antibody sequence databases expand, our method will accelerate intrabody design, making their development easier, more cost-effective, and broadly accessible for biological research.

cell biology↗

bioRxiv2025

Nectriatides with no antifungal activity bind to ergosterol and potentiate the antifungal activity of amphotericin B against Candida albicans

Fungal cyclotetrapeptide nectriatide and its synthetic linear derivatives (nectriatide-based compounds, NCTs), having no antifungal activity, potentiated antifungal activity of amphotericin B (AmB) against Candida albicans. The mechanism of action was investigated using fluorescein-conjugated and biotin-tagged probes. Microscopy revealed fluorescent-probe localization at the C. albicans cell membrane. The biotinyl probe binding assay towards membrane lipids showed the highest affinity for ergosterol but no affinity for cholesterol. Ergosterol binding was confirmed by a liposome disruption assay. LC-MS quantification of AmB in C. albicans revealed that NCTs increased AmB binding to C. albicans. These results indicate that NCTs bind to ergosterol on the fungal plasma membrane, subsequently localizing AmB, explaining the observed potentiation of AmB fungicidal activity.

microbiology↗

bioRxiv2025

Starship giant transposons dominate plastic genomic regions in a fungal plant pathogen and drive virulence evolution

Starships form a recently discovered superfamily of giant transposons in Pezizomycotina fungi, implicated in mediating horizontal transfer of diverse cargo genes between fungal genomes. Their elusive nature has long obscured their significance, and their impact on genome evolution remains poorly understood. Here, we reveal a surprising abundance and diversity of Starships in the phytopathogenic fungus Verticillium dahliae. Remarkably, Starships dominate the plastic genomic compartments involved in host colonization, are enriched in virulence-associated genes, and exhibit genetic and epigenetic characteristics associated with adaptive genome evolution. We further uncover extensive horizontal transfer of Starships between Verticillium species and, strikingly, from distantly related Fusarium fungi. Finally, we demonstrate how Starship activity facilitated the de novo formation of a novel virulence gene. Our findings illuminate the profound influence of Starship dynamics on fungal genome evolution and the development of virulence.

microbiology↗

bioRxiv2024

Integrated transcriptomic, metabolomic, and lipidomic analyses reveal a unique lipid profile of regulatory T cells upon activation

Regulatory T cells (Tregs) exhibit stable FOXP3 expression and regulate the immune response through suppressive activity. Their unique metabolic properties include reduced glycolysis and increased oxidative phosphorylation. We combined transcriptomic, metabolomic, and lipidomic analyses to dissect the metabolic dynamics of Tregs upon activation. Combined metabolomic and lipidomic analyses showed that freshly isolated and activated Tregs had distinct metabolomic and lipidomic properties, respectively. Activated Tregs contained omega-3 long-chain polyunsaturated fatty acid (PUFA)-rich diglycerides and triglycerides. These were supported by transcriptomics data, showing upregulation of PPAR-alpha and PPAR-gamma. Activated Tregs exhibited increased ceramide production, consistent with the upregulation of ceramide synthase and sphingomyelin synthase. Confocal microscopy revealed that Tregs were enriched in lysosomes and peroxisomes compared with effector T cells upon activation. Our data confirm the unique metabolic properties of Tregs, especially those characterized by omega-3 long-chain PUFA-rich triglycerides and ceramides, together with enriched lysosomes and peroxisomes, which correspond to metabolic alterations.

immunology↗

bioRxiv2024

Direct Intercellular Vesicle Exchange between Adjacent Cells

Intercellular communication plays a central role in the development and integrity of multicellular organisms. Vesicle transfer, especially through extracellular space, has recently been highlighted as a critical intercellular communication modality, carrying nucleic acids, proteins, and others to distant cells. Previously, we demonstrated that extracellular vesicles induce "phenotypic synchronization of cells (PSyC)" during stem cell differentiation. While examining the mechanism underlying PSyC, we discovered a novel form of cellular communication mediated by direct intercellular vesicle exchange (DIVE) between adjacent cells across the plasma membrane (PM). By achieving cell-wide and high-spatiotemporal resolution imaging of vesicles labeled with fusion proteins of CD63 or CD81 to StayGold, a photostable fluorescent marker, we observed small vesicles (50-500 nm in diameter) directly transferred between adjacent cells. These vesicles moved at approximately 1 {micro}m/s and crossed PM in approximately 10-20 seconds. Furthermore, multiple vesicles traversed nearly identical sites of PM, suggesting the presence of specific routes or structures, potentially including a pore, mediating the vesicle transfer. Three-dimensional electron microscopy provided supportive observations for traversing vesicles with single membrane. These vesicles, named InterCellular Vesicles (InterCVs), were observed to colocalize with nucleic acids, including mRNA, microRNA, and DNA, suggesting the exchange of nucleic acid-mediated information, potentially inducing PSyC, between adjacent cells. Our discovery, DIVE, reveals a previously unknown modality of cell-cell communication, with the potential to reshape our understanding of cellular biology.

cell biology↗

bioRxiv2024

Structural, genetic, and adaptive basis of superhydrophobicity in rice leaves

In most plants, leaf surfaces exhibit water-repellent properties, which protect against pathogens and weather. Plants such as lotus and rice have evolved outstanding water-repellent properties, so-called superhydrophobicity. However, the structural and genetic basis of these properties in crop plants remains unclear. In this study, we identified the genes and microstructures responsible for superhydrophobicity by analyzing water repellence in rice mutants and cultivars, as well as wild Oryza species. Analysis of the surface structure of mutant leaves revealed that the degree of water repellency in rice largely depends on the accumulation of cuticular wax. Meanwhile, although papillae differentiation contributes minorly to water repellency, it is suggested to play a crucial role in achieving superhydrophobicity. These morphological characteristics are regulated by at least five previously characterized genes. We also found that superhydrophobicity is present in ancestral species, is retained by most cultivated rice, and appears to have evolved independently of rice domestication. Understanding the mechanisms of water repellence and their applications should provide important insight into the natural design of water repellence, as well as a basis for future innovation.

plant biology↗

bioRxiv2024

Universal base editing for hemophilia B

The repair of pathological gene variants is an ultimate aim for treating genetic diseases; however, it is not practical to develop different therapeutic reagents for each of the many variants that can occur in a gene. Here, we investigated whether base editing to induce a gain-of-function variant in blood coagulation factor IX (FIX) can increase FIX activity as a treatment strategy for hemophilia B. We engineered a G:C to A:T substitution at c.1151 of F9 by cytosine base editing to generate R338Q, known as the Shanghai F9 variant, which markedly potentiates coagulation factor activity. An adeno-associated virus vector harboring the base editor converted more than 60% of the target G:C to A:T and increased FIX activity in HEK293 cells harboring patient-derived F9 variants, as well as in knock-in mice harboring a human F9 cDNA. Furthermore, administration of lipid nanoparticles embedded with the base editor mRNA and gRNA increased FIX activity in mice. These data indicate that cytosine base editing to generate R338Q in FIX can become a universal genome editing strategy for hemophilia B.

bioengineering↗

bioRxiv2024

Type 2 diabetes remodels collateral circulation and promotes leukocyte adhesion following ischemic stroke

AbstractType 2 diabetes mellitus (T2DM) is associated with impaired leptomeningeal collateral compensation and poor stroke outcome. Neutrophils tethering and rolling on endothelium after stroke can also independently reduce flow velocity. However, the chronology and topological changes in collateral circulation in T2DM is not yet defined. Here, we describe the spatial and temporal blood flow dynamics and vessel remodeling in pial arteries and veins and leukocyte- endothelial adhesion following middle cerebral artery (MCA) stroke using two-photon microscopy in awake control and T2DM mice. Relative to control mice prior to stroke, T2DM mice already exhibited smaller pial vessels with reduced flow velocity. Following stroke, T2DM mice displayed persistently reduced blood flow in pial arteries and veins, resulting in a poor recovery of downstream penetrating arterial flow and a sustained deficit in microvascular flow. There was also persistent increase of leukocyte adhesion to the endothelium of veins, coincided with elevated neutrophils infiltration into brain parenchyma in T2DM mice compared to control mice after stroke. Our data suggest that T2DM-induced increase in chronic inflammation may contribute to the remodeling of leptomeningeal collateral circulation and the observed hemodynamics deficiency that potentiates poor stroke outcome. HighlightsO_LIBlood flow and leukocyte imaging in awake mice by two-photon microscopy before and after stroke under physiological conditions C_LIO_LIT2DM induces collateral remodeling prior to stroke C_LIO_LIT2DM reduces blood flow and impedes recovery in pial arteries and veins after ischemic stroke C_LIO_LIPoor recovery of penetrating arterial flow and sustained deficit in microvascular flow after ischemic stroke in T2DM mice C_LIO_LIT2DM increases persistent leukocyte adhesion to endothelium of veins and elevates neutrophils infiltration into the brain parenchyma after ischemic stroke. C_LI

neuroscience↗

bioRxiv2024

ENDOTHELIAL PROX1 INDUCES BLOOD-BRAIN BARRIER DISRUPTION IN THE CENTRAL NERVOUS SYSTEM

The central nervous system (CNS) parenchyma has conventionally been believed to lack lymphatic vasculature, likely due to a non-permissive microenvironment that hinders the formation and growth of lymphatic endothelial cells (LECs). Recent findings of ectopic expression of LEC markers including Prospero Homeobox 1 (PROX1), a master regulator of lymphatic differentiation, and the vascular permeability marker Plasmalemma Vesicle Associated Protein (PLVAP), in certain glioblastoma and brain arteriovenous malformations (AVMs), has prompted investigation into their roles in cerebrovascular malformations, tumor environments, and blood-brain barrier (BBB) abnormalities. To explore the relationship between ectopic LEC properties and BBB disruption, we utilized endothelial cell-specific Prox1 overexpression mutants. When induced during embryonic stages of BBB formation, endothelial Prox1 expression induces hybrid blood-lymphatic phenotypes in the developing CNS vasculature. This effect is not observed when Prox1 is overexpressed during postnatal BBB maturation. Ectopic Prox1 expression leads to significant vascular malformations and enhanced vascular leakage, resulting in BBB disruption when induced during both embryonic and postnatal stages. Mechanistically, PROX1 downregulates critical BBB-associated genes, including {beta}-catenin and Claudin-5, which are essential for BBB development and maintenance. These findings suggest that PROX1 compromises BBB integrity by negatively regulating BBB-associated gene expression and Wnt/{beta}-catenin signaling.

developmental biology↗

bioRxiv2024

Identification of SPP1-positive macrophages by single-cell spatial analysis in human lung tissues with mycobacterial infection

Tuberculosis and non-tuberculous mycobacterial (NTM) diseases are infections caused by Mycobacterium tuberculosis and non-tuberculous mycobacteria such as the Mycobacterium avium complex, leading to the formation of granulomatous lesions with caseous necrosis in the lungs. Although granulomatous tissues are infiltrated by numerous inflammatory cells, including macrophages, lymphocytes, and neutrophils, the mechanisms underlying granuloma formation caused by mycobacteria remain unclear. In this study, we performed single-cell spatial analysis on lung tissue samples from patients with tuberculosis and NTM diseases to investigate the infiltrating cell populations. We analyzed seven lung lesions and identified individual cell types infiltrating the granulomatous tissue. Based on gene expression profiles, at least four macrophage subtypes were identified. Notably, SPP1-positive macrophages predominantly found infiltrating the granulomatous tissue. Langhans giant cells expressed SPP1, and numerous SPP1-positive macrophages without giant cell morphology were also observed around the granulomas. RNA-seq analysis revealed elevated SPP1 expression in mycobacterium-infected tissues. The SPP1-CD44 signaling pathway was active in SPP1-positive macrophages and their neighboring cells in mycobacterium-infected tissues. SPP1-positive macrophages were also observed around granulomas in other granulomatous diseases, such as granulomatosis with polyangiitis and sarcoidosis. These findings suggest that SPP1-CD44 signaling in SPP1-positive macrophages may play a role in the pathology of granulomatous diseases, including mycobacterial infections. Brief summarySPP1-CD44 signaling in SPP1-positive macrophages may play a role in granuloma formation in mycobacterial and other granulomatous diseases.

pathology↗

bioRxiv2024

Bundle sheath cell-dependent chloroplast movement in mesophyll cells of C4 plants analyzed using live leaf-section imaging

In C4 plants, mesophyll (M) chloroplasts aggregate toward bundle sheath (BS) cells in response to environmental stress, which would contribute to C4 photosynthetic cycle adjustment between M and BS cells. However, it remains unclear whether mesophyll chloroplast movement is an intercellular response mediated by BS cells. One major challenge to resolving this is the difficulty in observing how M chloroplasts aggregate toward adjacent BS cells due to scattering and absorption of observation light in live-leaf tissues. We established a live leaf-section imaging technique that enables the long-term observation of sections of chemically unfixed leaf blades, with which we quantitatively analyzed M chloroplast movements. Another challenge in clarifying the contribution of BS cells to M chloroplast movement is the selective ablation of BS cells without impairing their function of M cells. To investigate the necessity of BS cells for M chloroplast movement, we developed a method to remove BS cells only based on differences in shape and size between M and BS cells. We also found that chloroplasts in M cells without adjacent BS cell contents did not show typical aggregative movement but showed a light-avoidance response. This indicates that the M chloroplast aggregative movement occurs during communication with BS cells. HighlightWe established live leaf-section imaging to observe individual chloroplast movements in multi-layered cells and found that bundle sheath cells are involved in the aggregative movement of mesophyll chloroplasts.

plant biology↗