Biology subjects

Mizuno, Y.

Publications and source records attributed to Mizuno, Y..

At least 19 recordsLinked to original sources

bioRxiv2026

Proximity Labeling-Assisted Flow Cytometry Improves Fluorescence Contrast in Extracellular Vesicle-Enriched Fractions

Extracellular vesicles (EVs) are potential sources of circulating biomarkers, but disease-associated EVs are often scarce and their nanoscale dimensions limit analysis by conventional flow cytometry. To improve the detection of disease-associated EV signals using widely available instruments, we developed a workflow combining preparation of serum EV-enriched fractions by size-exclusion chromatography, CHL1-initiated enzyme-mediated activation of radical sources (EMARS) proximity labeling, and conventional flow cytometry. EMARS deposits fluorescein on molecules proximal to antibody-bound CHL1, thereby increasing target-initiated fluorescence. In a representative comparison, EMARS increased the signal-to-background ratio from 1.21 with conventional antibody labeling to 6.28. Because unlabeled EVs and debris interfered with analysis, we used an AI-assisted procedure to explore FITC-H cutoffs for retained P1 events. ChatGPT compared 65 candidate cutoffs across statistical and event-retention metrics and recommended 1000; the authors reviewed the complete results before adopting this value. At this data-selected cutoff, mean fluorescence intensity was higher in interstitial lung disease (ILD) than in non-ILD samples among 28 evaluable samples (P = 0.002; area under the receiver operating characteristic curve [AUC] = 0.846); neither value was adjusted for cutoff selection. Post hoc nested leave-oneout cross-validation (LOOCV), in which cutoff selection was repeated within each outer fold, yielded an AUC of 0.716 with 29 of 34 samples evaluable. The association with ILD requires validation in independent cohorts, but these exploratory findings suggest that proximity labeling may enhance measurable fluorescence contrast in serum EV-enriched fractions and support further evaluation of disease-associated EV signals using conventional flow cytometers.

biochemistry↗

bioRxiv2026

Fermented Botanical Product Modulates Soil Bacterial Communities and Enhances Plant-Growth-Promoting Activity for Sustainable Agriculture

Although biostimulants have attracted attention for sustainable agricultural systems, their efficacy remains poorly understood. In this study, we evaluated the effects of fermented botanical product (FBP) produced by fermenting and aging 41 types of fruits, grains, seaweed, and root vegetables with brown sugar for more than three years. Three crops, tomato, rice, and komatsuna (Brassica rapa), were cultivated with the application of 5,000- or 10,000-fold diluted FBP in greenhouses or fields. Application of diluted FBP promoted plant growth, as indicated by increased fresh weights of shoots, leaves, and roots, fruit production in tomato, and rice husk yield. As diluted FBP contained low nutrient levels, an indirect mechanism of plant growth promotion was suggested. Bacterial community structure analysis indicated changes in alpha diversity, beta diversity, and the predominant phyla in FBP-applied soils without plants and in soils cultivated with tomato, rice, and komatsuna. In addition, the abundance of plant-growth-promoting bacteria, such as Arthrobacter, Pseudomonas, Paraburkholderia, and Planifilum, increased in soils treated with diluted FBP. Furthermore, ammonium formation activity was observed in komatsuna cultivation soils treated with diluted FBP, whereas phosphate-solubilizing activity was enhanced in soils from all three crop cultivation systems treated with diluted FBP. These results suggest that diluted FBP influences bacterial communities and promotes crop growth through indirect effects, including increases in plant-growth-promoting bacteria, ammonium production, and phosphate solubilization. Alternatively, FBP may directly stimulate plant growth. Therefore, FBP may be a useful biostimulant for sustainable agricultural systems. HighlightsO_LIDiluted FBP promoted the growth of tomato, rice, and komatsuna (Brassica rapa). C_LIO_LIDiluted FBP altered the bacterial community structure in cultivated soils. C_LIO_LIFBP increased the abundance of plant-growth-promoting bacteria in cultivated soils. C_LIO_LIFBP stimulated ammonium formation and phosphate solubilization in cultivated soils. C_LIO_LIFBP may be a useful biostimulant for sustainable agricultural systems. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/728655v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@4772dorg.highwire.dtl.DTLVardef@12c40e8org.highwire.dtl.DTLVardef@594513org.highwire.dtl.DTLVardef@c5e5a6_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Informational and Normative Influence on Conformity in Autism

This preregistered study examined whether adults with autism spectrum disorder (ASD) show reduced social conformity and whether any such reduction depends on the type of social influence. Social conformity, defined as the tendency to adjust judgments to align with those of others, is typically driven by normative (acceptance-seeking) and informational (accuracy-seeking) motives. Thirty adults with ASD and 30 matched neurotypical (NT) adults completed two tasks: a preference rating task indexing normative influence, and a dot-counting task indexing informational influence with monetary rewards. Contrary to our predictions, adults with ASD conformed as much as NT adults in the preference rating task but showed significantly reduced conformity in the dot-counting task. Exploratory analyses indicated that this reduction was driven by a distinct subgroup of nine adults with ASD who never revised their initial estimates despite informative social cues, resulting in poorer accuracy and lower rewards. When this subgroup was excluded, group differences in conformity were no longer evident. These findings suggest that, overall, adults with ASD are as susceptible as NT adults to normative influence but less responsive to informational influence, highlighting the importance of distinguishing between types of social influence and considering individual differences when characterizing social behavior in ASD.

neuroscience↗

bioRxiv2025

Elevated Na+/K+ Ratio in Alzheimers Disease: A Potential Biomarker for Braak Stage

Alzheimers disease (AD) is a neurodegenerative disorder characterized by cognitive decline, synaptic dysfunction, and the accumulation of amyloid plaques and neurofibrillary tangles. While prior research has focused mainly on protein aggregation and neuroinflammation, emerging evidence suggests that ionic imbalances, particularly involving sodium (Na+) and potassium (K+), may contribute to AD progression. Na+ and K+ are critical for maintaining neuronal membrane potential, regulating action potential firing, and supporting neurotransmitter function. Although studies primarily focused on absolute Na+ concentrations, the Na+/K+ ratio may provide a more sensitive marker of ionic dysregulation. Given that the Na+/K+ gradient is actively maintained by the Na/K-ATPase pump, a target known to be vulnerable in AD, we hypothesized that the Na+/K+ ratio is altered in AD. We analyzed postmortem tissue from the prefrontal cortex, thalamus, and cerebrospinal fluid (CSF) of 97 human subjects (67 AD, 30 controls). AD cases exhibited a significant increase in the Na+/K+ ratio in the thalamus and CSF, driven primarily by elevated Na+ levels. The Na+/K+ ratio positively correlated with Braak tangle stage, suggesting an association with AD progression. These findings provide novel insights into ionic dysregulation in AD and suggest that the CSF Na+/K+ ratio may serve as a valuable biomarker of disease severity and progression. Future research should explore the potential of targeting ionic homeostasis as a therapeutic strategy in AD.

biochemistry↗

bioRxiv2025

Pseudovirus-Mediated Proximity Labeling Identifies Candidate Host Cell Membrane Proteins Involved in Viral Attachment

Viral infections represent a significant threat to humanity, as exemplified by the COVID-19 pandemic. To mitigate the associated damage, it is essential to understand the biological characteristics of causative viruses. In this study, we report a novel method for identifying host cell molecules (including virus receptor) required for viral attachment, referred to here as "Host Cell Attachment Factors (HCAFs)". Our approach utilizes proximity labeling (PL) technology: pseudoviruses engineered to express a PL enzyme are applied to host cells, where they bind and initiate proximity labeling. Since HCAFs are expected to be localized near viral attachment sites, candidate HCAFs are specifically tagged by the PL process. Analysis of influenza HCAFs enabled the identification of multiple candidate molecules. Subsequent virus attachment experiments using CHO cells suggested that NRP1 may serve as an HCAF for influenza viruses. Given its simplicity and rapid turnaround, this method is adaptable to a wide range of enveloped viruses, including pandemic viruses that require emergency analysis.

microbiology↗

bioRxiv2024

Light-harvesting by antenna-containing rhodopsins in pelagic Asgard archaea

Aquatic bacterial rhodopsin proton pumps have been recently reported to utilize hydroxylated carotenoids1,2. Here, by combining a marine chromophore extract with purified archaeal rhodopsins identified in marine metagenomes, we report on light energy transfer from diverse hydroxylated carotenoids (lutein, diatoxanthin, and fucoxanthin) to heimdallarchaeial rhodopsins (HeimdallRs)3,4 from uncultured marine planktonic members of the "Ca. Kariarchaeaceae" ("Ca. Asgardarchaeota")5. These light-harvesting antennas absorb in the blue-light range and transfer energy to the green-light absorbing retinal chromophore within HeimdallRs. Furthermore, antenna enhancement of proton pumping by HeimdallRs is also observed under white-light illumination along with a carotenoid-binding-induced structural change in the protein. Our results indicate that the use of light-harvesting antennas in microbial rhodopsins is observed not only in bacteria but also in marine archaea.

microbiology↗

bioRxiv2024

The seminal vesicle is a juvenile hormone-responsive tissue in adult male Drosophila melanogaster

Juvenile hormone (JH) is one of the most essential hormones controlling insect metamorphosis and physiology. While it is well known that JH affects many tissues throughout the insects life cycle, the difference in JH responsiveness and the repertoire of JH-inducible genes among different tissues has not been fully investigated. In this study, we monitored JH responsiveness in vivo using transgenic Drosophila melanogaster flies carrying a JH response element-GFP (JHRE-GFP) construct. Our data highlight the high responsiveness of the epithelial cells within the seminal vesicle, a component of the male reproductive tract, to JH. Specifically, we observe an elevation in the JHRE-GFP signal within the seminal vesicle epithelium upon JH analog administration, while suppression occurs upon knockdown of genes encoding the intracellular JH receptors, Methoprene-tolerant and germ cell-expressed. Starting from published transcriptomic and proteomics datasets, we next identified Lactate dehydrogenase as a JH-response gene expressed in the seminal vesicle epithelium, suggesting insect seminal vesicles undergo metabolic regulation by JH. Together, this study sheds new light on biology of the insect reproductive regulatory system.

physiology↗

bioRxiv2023

Distinct adrenal gland macrophages regulate corticosteroid production

The adrenal glands are hormone secreting glands that sit on top of the kidneys. Adrenal glands produce glucocorticoids, mineralocorticoids, and catecholamines, and are therefore critical regulators of the stress response, the immune response, metabolism, and blood pressure. Despite being identified for more that 30 years, our understanding of adrenal macrophages remains incomplete. In numerous other tissues, macrophages carry out a plethora of physiological and homeostatic roles in addition to their classical immune functions. The aim of this study was to characterise the macrophage compartment of the adrenal gland and assess its contribution to adrenal function. Using an in vivo approach, we herein describe two morphologically and spatially distinct subsets of adrenal macrophages - dendritic-like macrophages that are present throughout the gland in young and old mice, and "foamy" lipid-laden macrophages that accumulate in the murine adrenal cortex in an age and diet-dependent manner. Furthermore, we present data showing that these foamy-like macrophages accumulate cholesterol and thereby regulate adrenal hormonal output, at steady state and in the context of obesity. We hereby provide novel insights into the physiological roles of macrophages in the adrenal gland and the mechanisms by which adrenal hormone production is regulated.

immunology↗

bioRxiv2023

Ablation of Max expression induces meiotic onset in sexually undifferentiated germ cells

Meiosis is a specialized type of cell division that occurs only in germ cells physiologically. We have previously demonstrated that MYC-associated factor X (MAX) is involved in blocking ectopic and precocious meiotic onsets in embryonic and germline stem cells, respectively, as a central component of the PRC1 subtype PRC1.6. In this study, we investigated the role of the Max gene in germ cells in vivo. Our data revealed that mitotically active germ cell-specific disruption of Max was coupled to meiotic onset in male and female germ cells. However, such Max-null germ cells did not undergo meiotic processes progressively, but were stalled at its relatively early stages and eventually eliminated by apoptosis. Our data also revealed that Max, which is generally known as an obligate heterodimerization partner for MYC and MXD transcription factors, showed high expression in sexually undifferentiated male and female germ cells, but female germ cells exhibited an abrupt decline in its expression at the timing of or immediately prior to physiological meiotic onset. Moreover, computational analyses identified the regulatory region that supported high levels of Max expression in sexually undifferentiated germ cells.

developmental biology↗

bioRxiv2022

KARRIKIN INSENSITIVE2 (KAI2)-dependent signaling pathway controls vegetative reproduction in Marchantia polymorpha

In vegetative reproduction of Marchantia polymorpha, propagules, called gemmae, are formed in gemma cups. Despite its significance for survival, control of gemma and gemma cup formation by environmental cues is not well understood. We show here that the number of gemmae formed in a gemma cup is a genetic trait. Gemma formation starts from the central region of the floor of the gemma cup, proceeds to the periphery, and terminates when the appropriate number of gemmae are initiated. The MpKARRIKIN INSENSITIVE2 (MpKAI2)-dependent signaling pathway promotes gemma cup formation and gemma initiation. The number of gemmae in a cup is controlled by modulating the ON/OFF switch of the KAI2-dependent signaling. Termination of the signaling results in the accumulation of MpSMXL, a suppressor protein. In the Mpsmxl mutants, gemma initiation continues, leading to the formation of a highly increased number of gemmae in a cup. Consistent with its function, the MpKAI2-dependent signaling pathway is active in gemma cups where gemmae initiate, as well as in the notch region of the mature gemma and midrib of the ventral side of the thallus. In this work, we also show that GEMMA CUP-ASSOCIATED MYB1 works downstream of this signaling pathway to promote gemma cup formation and gemma initiation. We also found that the availability of potassium affects gemma cup formation independently from the KAI2-dependent signaling pathway in M. polymorpha. We propose that the ancestral function of the KAI2-dependent signaling pathway may be to optimize vegetative reproduction by adapting to the environment.

plant biology↗

bioRxiv2022

Energy transfer in ubiquitous rhodopsin pumps with xanthophyll antennas

Energy transfer from light-harvesting ketocarotenoids to light-driven proton pumps xanthorhodopsins has been previously demonstrated in two unique cases: an extreme halophilic bacterium1 and a terrestrial cyanobacterium2. Attempts to find carotenoids that bind and transfer energy to rhodopsin proton pumps from the abundant marine and freshwater photoheterotrophs have thus far failed3-5. Here, using functional metagenomics combined with chromophore extraction from the environment, we detected light energy transfer from the widespread hydroxylated carotenoids zeaxanthin and lutein to the retinal moiety of xanthorhodopsins and proteorhodopsins. The light-harvesting carotenoids transfer up to 42% of the harvested energy in the violet/blue-light range to the green-light absorbing retinal chromophore. Our data suggest that these antennas have a significant impact on rhodopsin phototrophy in the worlds lakes, seas and oceans.

microbiology↗

bioRxiv2022

Female reproductive dormancy in Drosophila melanogaster is regulated by DH31-producing neurons projecting into the corpus allatum

Female reproductive dormancy in insects is a process that drastically suppresses oogenesis to conserve energy under adverse environments. In many insects, including the fruit fly, Drosophila melanogaster, reproductive dormancy is induced under low-temperature and short-day conditions by the downregulation of juvenile hormone (JH) biosynthesis by the corpus allatum (CA). Previous studies have suggested that brain neurons that project directly to the CA are important for the regulation of reproductive dormancy. However, the role of CA-projecting neurons in JH-mediated reproductive dormancy has not yet been confirmed by molecular genetic studies. In this study, we report that, in adult D. melanogaster, the neuropeptide diuretic hormone 31 (DH31) is produced by brain neurons that project into the CA. DH31-producing-CA-projecting neurons are connected downstream with a subset of circadian clock neurons, such as s-LNvs, which are known to be involved in reproductive dormancy regulation. The CA expresses the gene encoding the DH31 receptor, which is required for DH31-triggered elevation of intracellular cAMP in the CA. Knocking down Dh31 in these CA-projecting neurons or DH31 receptor in the CA leads to a failure in the decrease of the JH titer, normally observed under dormancy-inducing conditions, leading to abnormal yolk accumulation in the ovaries. Our findings provide the first molecular genetic evidence demonstrating that CA-projecting peptidergic neurons play an essential role in regulating reproductive dormancy by suppressing JH biosynthesis. Significance StatementDormancy is an adaptive physiological response to environmental changes that are unsuitable for survival. Adult females of many insect species undergo reproductive dormancy in which oogenesis is drastically arrested; it is induced by a decrease in juvenile hormone (JH) titers. However, we are yet to fully understand the molecular mechanisms underlying the control of JH biosynthesis under dormancy-inducing conditions. In this study using the fruit fly, we demonstrated that brain neurons projecting directly to the JH-producing organ, corpus allatum, play an essential role in regulating reproductive dormancy via the neuropeptide DH31. As the morphologically-similar neurons have previously been suggested to be involved in reproductive dormancy regulation, this study provides a fundamental molecular and neuronal basis for reproductive dormancy in insects.

physiology↗

bioRxiv2021

Cellular adhesion is a controlling factor in neutrophil extracellular trap formation induced by antineutrophil cytoplasmic antibodies

Anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis (AAV) is a life-threatening condition characterized by improper activation of neutrophils and release of neutrophil extracellular traps (NETs) in small vessels. This study aimed to explain the role of NETs in AAV pathogenesis by investigating a link between neutrophil adhesion and NET release. We leveraged an imaging flow cytometry-based assay and three-dimensional culture to demonstrate that neutrophil adhesion is essential for ANCA induced NET formation. We confirmed this requirement for cell adhesion using standard microscopy on ultra-low attachment hydrogel surfaces and demonstrate that this depends on the focal adhesion kinase pathway as determined using inhibitors for multiple targets in this process. ANCA increased expression of {beta}2 integrins on neutrophils, and we confirmed that these integrins were required for NET formation using blocking antibodies. Finally, inhibitors for oxidative burst prevented NET formation, and this oxidative burst was mediated by the focal adhesion pathway. Overall, our findings reveal a central role for neutrophil attachment in NET formation in response to ANCA, helping to explain the restricted localization pattern of vessel damage, and suggesting that targeting neutrophil adhesion factors may be beneficial in preventing pathological damage from NETs during AAV.

immunology↗

bioRxiv2021

An ancestral function of strigolactones as symbiotic rhizosphere signals

In flowering plants, carotenoid-derived strigolactones (SLs) have dual functions as hormones that regulate growth and development, and as rhizosphere signaling molecules that induce symbiosis with arbuscular mycorrhizal (AM) fungi. Here, we report the identification of bryosymbiol (BSB), a previously unidentified SL from the bryophyte Marchantia paleacea. BSB is also found in vascular plants, indicating that it is ancestral in land plants. BSB synthesis is enhanced at AM symbiosis permissive conditions and BSB deficient mutants are impaired in AM symbiosis. In contrast, the absence of BSB synthesis has little effect on the growth and gene expression. We show that the introduction of the SL receptor of Arabidopsis renders M. paleacea cells BSB-responsive. These results suggest that BSB is not perceived by M. paleacea cells due to the lack of cognate SL receptors. We propose that SLs originated as AM symbiosis-inducing rhizosphere signaling molecules and were later recruited as plant hormone.

plant biology↗

bioRxiv2021

A population of neurons that produce hugin and express the diuretic hormone 44 receptor gene projects to the corpora allata in Drosophila melanogaster

The corpora allata (CA) are essential endocrine organs that biosynthesize and secrete the sesquiterpenoid hormone, namely juvenile hormone (JH), to regulate a wide variety of developmental and physiological events in insects. Previous studies had demonstrated that the CA are directly innervated with neurons in many insect species, implying the innervations to be important for regulating JH biosynthesis in response to internal physiology and external environments. While this is also true for the model organism, Drosophila melanogaster, which neurotransmitters are produced in the CA-projecting neurons are yet to be clarified. In this study on D. melanogaster, we aimed to demonstrate that a subset of neurons producing the neuropeptide hugin, the invertebrate counterpart of the vertebrate neuromedin U, directly projects to the adult CA. A synaptic vesicle marker in the hugin neurons was observed at their axon termini located on the CA, which were immunolabeled with a newly-generated antibody to the JH biosynthesis enzyme JH acid O-methyltransferase (JHAMT). We also found the CA-projecting hugin neurons to likely express a gene encoding the specific receptor for diuretic hormone 44 (Dh44). Moreover, our data suggested that the CA-projecting hugin neurons have synaptic connections with the upstream neurons producing Dh44. To the best of our knowledge, this is the first study to identify a specific neurotransmitter of the CA-projecting neurons in D. melanogaster, and to anatomically characterize a neuronal pathway of the CA-projecting neurons and their upstream neurons.

zoology↗

bioRxiv2020

Major components in the KARRIKIN INSENSITIVE2-ligand signaling pathway are conserved in the liverwort, Marchantia polymorpha

KARRIKIN INSENSITIVE2 (KAI2) was first identified in Arabidopsis thaliana as a receptor of karrikin, a smoke-derived germination stimulant. KAI2 is also considered a receptor of an unidentified endogenous molecule called the KAI2-ligand (KL). Upon KAI2 activation, signals are transmitted through degradation of D53/SMXL proteins via ubiquitination by a Skp-Cullin-F-box (SCF) E3 ubiquitin ligase complex. All components in the KL signaling pathway exist in the liverwort Marchantia polymorpha, namely MpKAI2A and MpKAI2B, MpMAX2 encoding the F-box protein, and MpSMXL, indicating that the signaling pathway became functional in the common ancestor of bryophytes and seed plants. Genetic analysis using knock-out mutants of these KL signaling genes, produced using the CRISPR system, indicated that MpKAI2A, MpMAX2 and MpSMXL act in the same genetic pathway and control early gemma growth. Introduction of MpSMXLd53, in which a domain required for degradation is mutated, into wild-type plants caused phenotypes resembling those of the Mpkai2a and Mpmax2 mutants. In addition, Citrine fluorescence was detected in tobacco cells transiently transformed with the 35S:MpSMXL-Citrine gene construct and treated with MG132, a proteasome inhibitor. On the other hand, introduction of 35S:MpSMXLd53-Citrine conferred Citrine fluorescence without MG132 treatment. These findings imply that MpSMXL is subjected to degradation, and that degradation of MpSMXL is crucial for KL signaling in M. polymorpha. We also showed that MpSMXL is negatively regulated by KL signaling. Taken together, this study demonstrates that basic mechanisms in the KL signaling pathway are conserved in M. polymorpha.

plant biology↗

bioRxiv2020

Two DNA binding domains of Mga act in combination to suppress ectopic activation of meiosis-related genes in mouse embryonic stem cells

Mouse embryonic stem cells (ESCs) have high potential for meiotic entry, like germ cells. Although the physiological meaning of this potential is not known, it is certain that a rigid safeguarding system is required to prevent ectopic onset of meiosis. PRC1.6, a non-canonical PRC1, is known for its suppression of precocious and ectopic meiotic onset in germ cells and ESCs, respectively, in which MGA has important roles in DNA binding as well as in constructing the complex as a scaffolding component. As a salient feature, MGA bears two distinct DNA-binding domains termed bHLHZ and T-box. However, how these features contribute to the functions of PRC1.6, particularly in the repression of meiotic genes, remains largely obscure. Here, we demonstrated that both DNA binding domains of Mga repress distinct sets of genes in murine ESCs, and substantial numbers of meiosis-related genes are included in both gene sets. In addition, our data demonstrated that both DNA binding domains of Mga, in particular bHLHZ, are crucially involved in repressing the expression of Meiosin, which plays essential roles in meiotic entry in collaboration with Stra8, revealing at least part of the molecular mechanisms that link negative and positive regulation of meiotic onset.

cell biology↗

bioRxiv2019

Consistency in macroscopic human brain responses to noisy time-varying visual inputs

It is an open question as to whether macroscopic human brain responses to repeatedly presented external inputs show consistent patterns across trials. We here provide experimental evidence that human brain responses to noisy time-varying visual inputs, as measured by scalp electroencephalography (EEG), show a signature of consistency. The results indicate that the EEG-recorded responses are robust against fluctuating ongoing activity, and that they respond to visual stimuli in a repeatable manner. This consistency presumably mediates robust information processing in the brain. Moreover, the EEG response waveforms were discriminable between individuals, and were invariant over a number of days within individuals. We reveal that time-varying noisy visual inputs can harness macroscopic brain dynamics and can manifest hidden individual variations.

neuroscience↗