Biology subjects

Nagao, K.

Publications and source records attributed to Nagao, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Adrenal remodeling encodes stress axis memory to shape fentanyl seeking

The hypothalamic-pituitary-adrenal (HPA) axis is implicated in substance use disorders, yet the features of this circuit that contribute to addiction vulnerability remain unclear. Here, we show that fentanyl engages with different components of the HPA axis, and this engagement varies across timescales. Within minutes, fentanyl activated corticotropin-releasing hormone neurons in the paraventricular nucleus of the hypothalamus (PVNCRH) and transiently increased adrenocorticotropic hormone (ACTH) levels. Hours to days later, fentanyl suppressed PVN activity, reduced adrenal size, and impaired corticosterone release. Conversely, ACTH-induced adrenal hypertrophy, increased corticosterone, suppressed fentanyl-evoked PVNCRH activity, and elevated fentanyl-seeking behavior. These findings identify adrenal remodeling as a physiological record of stress history that shapes neuroendocrine and behavioral responses to fentanyl, mechanistically linking stress and addiction while revealing treatment targets for opioid dependence.

neuroscience↗

bioRxiv2026

Identification of a transient receptor potential channel that is regulated by phospholipid asymmetry

AbstractPhospholipid asymmetry is a hallmark of mammalian cell membranes and reflects the selective distribution of distinct phospholipid species between the two leaflets of the lipid bilayer. Although this asymmetry is tightly maintained, the membrane proteins whose functions depend on it remain largely unknown. To perturb phospholipid asymmetry experimentally, we expressed a constitutively active phospholipid scramblase and thereby identified transient receptor potential melastatin 8 (TRPM8) as an ion channel regulated by this membrane property. Activation of TRPM8 by both l-menthol and innocuous cold was markedly suppressed following disruption of phospholipid asymmetry. Likewise, selective depletion of phosphatidylserine (PS), a phospholipid enriched in the cytoplasmic leaflet, using a cytosolically targeted PS decarboxylase attenuated TRPM8 activation, indicating that cytoplasmic PS is required for proper TRPM8 function. Mechanistically, our findings suggest that cytoplasmic PS supports efficient TRPM8 activation by maintaining the biochemical state of the channel. Together, these findings identify TRPM8 as a phospholipid asymmetry-dependent ion channel and establish an experimental strategy for systematically identifying membrane proteins regulated by phospholipid asymmetry. This work provides a foundation for future studies investigating the biological significance of this fundamental membrane property.

biochemistry↗

bioRxiv2026

CHARIOT-AAV: Conjugation of diverse vectors to adeno-associated viruses for delivery of large genes

Systemic, tissue-specific delivery of large transgenes exceeding the packaging capacity of adeno-associated viruses (AAVs) remains a key translational challenge for molecular therapeutics. Vectors with larger capacities, such as lentiviral vectors (LVVs) and lipid nanoparticles (LNPs), often lack adjustable, tissue-specific tropisms. Here we report CHARIOT-AAV (Crosslinked Hybrid Architectures for Robust, Interchangeable, and Organ-specific Targeting with AAV), a platform where diverse delivery vectors are conjugated to AAVs, thereby achieving tissue-specific tropism of AAVs and expanded cargo capacity. AAV-AAV conjugates packaging split SpCas9 constructs in AAV.CAP-B10 capsids demonstrate a [~]2-fold increase in brain gene editing efficiency over unconjugated AAV cocktails after intravenous injection. In addition to AAV-AAV conjugates, AAV-LVV and AAV-LNP conjugates achieve AAV-guided delivery of genetic payloads to target cells. Furthermore, AAV-LNP conjugates enable systemic delivery of mRNAs to brain endothelial cells. CHARIOT-AAV thus provides a modular platform for systemic, tissue-specific delivery of diverse therapeutics beyond the limits of individual vectors.

bioengineering↗

bioRxiv2026

Wireless Magnetomechanical Stimulation of Targeted Vagal Gut-Brain Circuits

Causal manipulation of gut-brain neural circuits empowers studies of metabolism and interoception. However, the anatomy and cytoarchitecture of peripheral ganglia relaying gut-brain circuits pose challenges to deployment of optical or electrical stimulation probes. To enable implant-free, cell-type specific, and temporally precise control of defined gut-brain pathways, we develop a neuromodulation platform based on magnetic nanodiscs (MNDs) targeted to peripheral neurons via genetically delivered anchoring moieties. The anchored MNDs selectively transduce externally applied weak magnetic fields to mechanical torque, thereby activating endogenous mechanosensitive pathways in specified cell types with sub-second latency. When targeted to nodose ganglia neurons expressing oxytocin or glucagon-like peptide 1 receptors, MND-mediated stimulation enables robust and reversible activation of gut-brain signaling, which engages hindbrain satiety circuits and regulates feeding behavior. These findings establish MND-mediated stimulation as a genetically targetable, implant-free strategy for modulating gut-brain neural circuits and highlight its potential in studies of brain-body physiology and bioelectronic medicines.

bioengineering↗

bioRxiv2026

Longitudinal in vivo human wound healing model defines key role for smooth muscle cells in ECM remodeling

BackgroundEffective skin wound healing is essential for restoring tissue integrity following injury. Repair proceeds through phases of hemostasis, inflammation, proliferation, and remodeling, but molecular mechanisms governing these stages remain poorly defined. Vascular niche cells (VNCs)-including endothelial cells, vascular smooth muscle cells (SMCs), and fibroblasts-are central regulators of healing, but the lack of longitudinal in vivo human data has limited identification of VNC-derived signals that distinguish effective repair from pathological healing such as ulcers. Thus, defining the regulation of VNCs in wound healing addresses a critical knowledge gap. MethodsWe developed a protocol for wound healing using dermal forearm punch biopsies to track longitudinal repair in healthy volunteers. Single-cell and spatial transcriptomics were performed to identify and validate signaling activities within VNCs. ResultsWe spatiotemporally defined the inflammation, proliferation, and remodeling phases of human skin wound healing with a focus on VNCs. Spatial analysis localized this activity for VNCs and immune cells within a heterogenous granulation zone that later led to re-epithelializion. Angiogenesis was dominated by Vegf, Egf and Hif1 signaling. Extracellular matrix (ECM) remodeling occurred through Collagen, Laminin, Thrombospondin, and Fibronectin. SMCs emerged as dominant drivers of injury-induced remodeling including basement membrane and interstitial ECM components compared to fibroblasts. This SMC-led program was further defined by robust induction of TIMP1, an inhibitor of matrix degradation, which localized to granulation tissue and correlated with re-epithelialization and wound resolution. Lastly, we compared remodeling factors between healing and non-healing human diabetic foot ulcers (DFUs). SMCs in non-healing DFUs had deficient expression for core remodeling factors, including TIMP1, indicating SMC activity is needed for effective healing. ConclusionWe identified an SMC-driven model of wound repair in which TIMP1-dependent activity underpins granulation zone formation. Failure of this program defined a mechanistic basis for impaired healing in ulcers, identifying SMCs and TIMP1 as therapeutic targets.

molecular biology↗

bioRxiv2025

Linking Germline Telomere Removal to Global Programmed DNA Elimination in Tetrahymena Genome Differentiation

In the ciliate Tetrahymena, telomeres of the germline micronucleus (MIC) are removed and replaced by de novo telomere addition during somatic macronuclear (MAC) development. In this study, we investigated the kinetics and mechanism of the MIC telomere elimination. Comparison of the MIC and MAC genome sequences indicated that the MIC telomeres are excised from chromosomes as part of larger MIC-limited sequences (MLSs) through chromosomal breakage. We confirmed this using an optimized oligo-FISH protocol and found that their elimination occurs in parallel with other programmed DNA elimination processes. CRISPR-Cas9 disruption of a MLS-associated Chromosome Breakage Sequence (CBS) showed that elimination of the MLS was not blocked but instead led to loss of adjacent MAC-destined sequence (MDS), suggesting abnormal co-elimination. In biparental crosses of the CBS mutant, however, both MLS and MDS were retained, DNA elimination was broadly disrupted, and no viable progeny were produced. These findings indicate that chromosome breakage at MLS-associated CBSs is essential for the proper separation of MLSs and MDSs, ensuring correct DNA elimination and successful sexual progeny development. We propose that the MIC telomere elimination is subsumed within the broader process of programmed DNA elimination.

cell biology↗

bioRxiv2025

Light stimulation system for measuring pupillary light responses based on Maxwellian view optical system using a retina presentation type viewfinder

BackgroundMelanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGC) contribute predominantly to non-image-forming effects of light, such as light-induced circadian phase shift, melatonin suppression, and pupillary light reflex. Post-illumination pupil response (PIPR) has been well-used for the evaluation of ipRGC sensitivity for humans since it is able to detect ipRGC function independently from conventional cones. However, pharmacological mydriasis or a specialized light stimulation system, such as the Maxwellian view (MV) optical system, is required to avoid dynamic pupil constriction in the stimulated eye for effective PIPR measurements, which makes these approaches less practical and difficult to implement in standard experimental settings. In this context, we propose the application of a retinal projection viewfinder based on the MV system as a practical approach for measuring PIPR, and evaluate the feasibility of this approach by comparing its performance with a typical LED-based optical system. MethodsTwenty-two healthy participants underwent pupillometry using both the MV-based viewfinder and a typical LED-based system. Monochromatic red and blue light stimuli were presented for durations of 1 and 10 seconds. Pupil responses, including maximum constriction, PIPR amplitude after 6 seconds from the light offset, area under the curve (AUC) values of PIPR, and sustained slopes, were analyzed using a linear mixed-effects model to assess the differences between the two systems. ResultsThe MV-based viewfinder significantly enhanced net PIPR amplitude (p < 0.05) and sustained slope (p < 0.01) during 10-second light stimulation compared to the LED system, demonstrating its capability to effectively measure ipRGC-driven responses. In contrast, no significant differences were observed in the net AUC values. These results highlight that the MV-based viewfinder enables effective PIPR measurements by delivering constant and controlled light stimulation directly to the retina, minimizing the effects of dynamic pupil constriction during light stimulation. ConclusionsThe MV-based viewfinder showed feasibility as an effective method for measuring PIPR without requiring pharmacological dilation or complex optical instrumentation. This approach has strong potential for advancing the assessment of human ipRGC function using pupillary responses.

neuroscience↗

bioRxiv2025

Adeno-associated viruses escort nanomaterials to specific cells and tissues

The delivery of nanotherapeutics to specific tissues relies on bespoke targeting strategies or invasive surgeries. Conversely, adeno-associated viruses (AAVs) can target specific tissues following intravenous injections. Here we show that cell-targeting properties of AAVs could be broadly conferred to nanomaterials. We develop a strategy to couple AAV capsids to nanoparticles that is invariant of viral serotype or nanomaterial chemistry and permits control over stoichiometry of the AAV-nanoparticle chimeras. The chimeras selectively escort nanoparticles into cell classes governed by AAV serotypes. When applied to magnetic nanoparticles, the AAV-nanoparticle chimeras enable magnetically localized gene delivery. In vivo, we show that leveraging the brain-targeting AAV serotype CAP-B10 achieves nanoparticle delivery to the parenchyma with [~]10% efficiency (% injected dose/g[brain]) while avoiding accumulation in the liver. The enhanced delivery efficiency and tissue specificity highlight the potential of AAV-chimeras as a versatile strategy to escort broad classes of nanotherapeutics to the brain and beyond.

bioengineering↗

bioRxiv2024

Effect of environmental conditions on seed germination and seedling growth in Cuscuta campestris

The dodder, Cuscuta, is an obligate parasitic plant that cannot survive without a host plant and causes major damage to crop yields. To know its growth characteristics before parasitism, we examined the effect of various environmental conditions on seed germination and seedling growth in Cuscuta campestris. As for the effect of light on germination, far-red light was rather preferable to red light and the reversible response of the seeds to red and far-red light was confirmed, implicating a phytochrome-mediated signaling opposite to that in many seed plants. Among amino acids, aspartic acid and alanine had a promotive effect and histidine had an inhibitory effect on germination. We further found that, in addition to gibberellic acid, methyl jasmonate was stimulatory to germination and shoot elongation. While 2,4-D extended the viability of trichomes around the root cap, kinetin induced the formation of scale leaves on the shoot and calli at the base of the shoot and the root tip. RT-PCR experiments confirmed that expression of a putative RbcS gene for photosynthesis showed no response to light but that of a Phytochrome A homolog was increased in the dark. Our results indicate that some of the molecular mechanisms to respond to light and hormone signals are uniquely modified in dodder seedlings, providing a clue for understanding the survival strategy of parasitic plants.

plant biology↗

bioRxiv2024

Fiber-based Probes for Electrophysiology, Photometry, Optical and Electrical Stimulation, Drug Delivery, and Fast-Scan Cyclic Voltammetry In Vivo

Recording and modulation of neuronal activity enables the study of brain function in health and disease. While translational neuroscience relies on electrical recording and modulation techniques, mechanistic studies in rodent models leverage genetic precision of optical methods, such as optogenetics and imaging of fluorescent indicators. In addition to electrical signal transduction, neurons produce and receive diverse chemical signals which motivate tools to probe and modulate neurochemistry. Although the past decade has delivered a wealth of technologies for electrophysiology, optogenetics, chemical sensing, and optical recording, combining these modalities within a single platform remains challenging. This work leverages materials selection and convergence fiber drawing to permit neural recording, electrical stimulation, optogenetics, fiber photometry, drug and gene delivery, and voltammetric recording of neurotransmitters within individual fibers. Composed of polymers and non-magnetic carbon-based conductors, these fibers are compatible with magnetic resonance imaging, enabling concurrent stimulation and whole-brain monitoring. Their utility is demonstrated in studies of the mesolimbic reward pathway by simultaneously interfacing with the ventral tegmental area and nucleus accumbens in mice and characterizing the neurophysiological effects of a stimulant drug. This study highlights the potential of these fibers to probe electrical, optical, and chemical signaling across multiple brain regions in both mechanistic and translational studies.

neuroscience↗

bioRxiv2023

Ether phospholipids modulate somatosensory responses by tuning multiple receptor functions in Drosophila

Transient receptor potential (TRP) and PIEZO channels are known receptors for physical stimuli, such as temperature and mechanical touch, respectively, in sensory nerves. As these receptors are localized in the plasma membrane, the modulation of sensory receptor activity by plasma membrane lipids has recently attracted attention. In this study, we focused on ether phospholipids (ePLs), which are abundant in neurons, and analyzed their role in somatosensation using Drosophila as a model. Reduced mechanosensory behavior was observed with ePL synthesizing gene knockout or knockdown in mechano-sensitive PIEZO-expressing neurons. The activation of PIEZO channels was significantly augmented in the presence of ePLs. Furthermore, we observed that ePLs modulate the thermosensory behavior and reduce thermal threshold of the thermosensitive TRPA1 channels. Finally, we revealed that ePLs affect membrane tension and lipid order of the plasma membrane in culture cells. Our study identified ePLs as a modulator of multiple somatosensation modalities in Drosophila, which underscore the significance of functional interaction between membrane lipid and sensory channel proteins.

molecular biology↗

bioRxiv2023

Bioinformatic and fine-scale chromosomal mapping uncover the essence and evolution of eliminated chromosomes in the Japanese hagfish, Eptatretus burgeri, through repetitive DNA family analysis

In the Japanese hagfish, Eptatretus burgeri, approximately 21% of the genomic DNA in germ cells (2n=52) consists of 16 chromosomes (eliminated [E]-chromosomes) that are eliminated from presumptive somatic cells (2n=36). To uncover the eliminated genome (E-genome), we have identified 16 eliminated repetitive DNA families from eight hagfish species, with 11 of these repeats being selectively amplified in the germline genome of E. burgeri. Furthermore, we have demonstrated that six of these sequences, namely EEEb1-6, are exclusively localized on all 16 E-chromosomes. This has led to the hypothesis that the eight pairs of E-chromosomes are derived from one pair of ancestral chromosomes via multiple duplication events over a prolonged evolutionary period. NGS analysis has recently facilitated the re-assembly of two distinct draft genomes of E. burgeri, derived from the testis and liver. This advancement allows for the prediction of not only nonrepetitive eliminated sequences but also over 100 repetitive and eliminated sequences, accomplished through K-mer-based analysis. In this study, we report four novel eliminated repetitive DNA sequences (designated as EEEb7-10) and confirm the relative chromosomal localization of all eliminated repeats (EEEb1-10) by fluorescence in situ hybridization (FISH). With the exception of EEEb10, all sequences were exclusively detected on EEEb1-positive chromosomes. Surprisingly, EEEb10 was detected as an intense signal on EEEb1-positive chromosomes and as a scattered signal on other chromosomes in germ cells. The study further divided the eight pairs of E-chromosomes into six groups based on the signal distribution of each DNA family, and fiber-FISH experiments showed that the EEEb2-10 family was dispersed in the EEEb1-positive extended chromatin fiber. These findings provide new insights into the mechanisms underlying chromosome elimination and the evolution of E-chromosomes, supporting our previous hypothesis.

molecular biology↗

bioRxiv2022

Organelle-selective click labeling coupled with flow cytometry allows high-throughput CRISPR screening of genes involved in phosphatidylcholine metabolism

Lipids comprise biomembranes and are involved in many crucial cell functions. While cellular lipid synthesis and transport appear to be governed by intricate protein networks, the whole scheme is insufficiently understood. Although functional genome-wide screening should contribute to deciphering the regulatory networks of lipid metabolism, technical challenges remain - especially for high-throughput readouts of lipid phenotypes. Here, we coupled organelle-selective click labeling of phosphatidylcholine (PC) with flow cytometry-based CRISPR screening technologies to convert organellar PC phenotypes into a simple fluorescence readout for genome-wide screening. This technique, named O-ClickFC, was successfully applied in genome-scale CRISPR-knockout screens to identify previously reported genes associated with PC synthesis (PCYT1A, ACACA), vesicular membrane trafficking (SEC23B, RAB5C), and non-vesicular transport (PITPNB, STARD7). Moreover, this work revealed previously uncharacterized roles of FLVCR1 as a new choline transporter; CHEK1 as a post-translational regulator of the PC-synthetic pathway, and TMEM30A as responsible for translocation of PC to the outside of the plasma membrane bilayer. These findings demonstrate the versatility of O-ClickFC as an unprecedented platform for genetic dissection of cellular lipid metabolism.

cell biology↗

bioRxiv2021

SmcHD1 underlies the formation of H3K9me3 blocks on the inactive X chromosome in mice

Stable silencing of the inactive X chromosome (Xi) in female mammals is critical for the development of embryos and their postnatal health. SmcHD1 is essential for stable silencing of the Xi, and its functional deficiency results in derepression of many X-inactivated genes. Although SmcHD1 has been suggested to play an important role in the formation of higher order chromatin structure of the Xi, the underlying mechanism is largely obscure. Here we explore the epigenetic state of the Xi in SmcHD1-deficient epiblast stem cells (EpiSCs) and mouse embryonic fibroblasts (MEFs) in comparison with their wild-type counterparts. The results suggest that SmcHD1 underlies the formation of H3K9me3-enriched blocks on the Xi, which, although the importance of H3K9me3 has been largely overlooked in mice, play a critical role in the establishment of the stably silenced state. We propose that the H3K9me3 blocks formed on the Xi facilitate robust heterochromatin formation in combination with H3K27me3, and the substantial loss of H3K9me3 caused by SmcHD1 deficiency leads to aberrant distribution of H3K27me3 on the Xi and derepression of X-inactivated genes.

genetics↗

bioRxiv2021

The mechanosensitive Ca2+-permeable ion channel PIEZO1 promotes satellite cell function in skeletal muscle regeneration

Muscle satellite cells (MuSCs), myogenic stem cells in skeletal muscle, play an essential role in muscle regeneration. During the regeneration process, cues from the surrounding microenvironment are critical for the proliferation and function of MuSCs. However, the mechanism by which mechanical stimuli from the MuSCs niche is converted into biochemical signals to promote muscle regeneration is yet to be determined. Here, we show that PIEZO1, a calcium ion (Ca2+)-permeable cation channel that is activated by membrane tension, mediates the spontaneous Ca2+ influx to controls the regenerative function of MuSCs. Our genetically engineering approach in mice revealed that PIEZO1 is functionally expressed in MuSCs, and the conditional deletion of Piezo1 in MuSCs delays myofiber regeneration after myofiber injury, which is at least in part due to the growth defect in MuSCs via the reduction in RhoA-mediated actomyosin formation. Thus, we provide the first evidence in MuSCs that PIEZO1, a bona fide mechanosensitive ion channel, promotes the proliferative and regenerative function during skeletal muscle regeneration.

cell biology↗

bioRxiv2020

Single-cell transcriptomics, scRNA-Seq and C1 CAGE discovered distinct phases of pluripotency during naïve-to-primed conversion in mice

BackgroundTwo types of mammalian pluripotent stem cells (PSC), i.e. naive and primed possess distinct cellular characteristics. It is largely unknown how these differences are generated during naive-to-primed transition process. We have established a robust in vitro transition system using a Wnt inhibitor for the first time and analyzed dynamic changes in cellular status via single-cell RNA-sequencing and C1 CAGE analyses. ResultsAnalysis of known marker genes suggested that the cell transition process progresses as expected. However, cluster analyses revealed a sudden increase in expression profile diversities three and four days after induction of the transition. These expression diversities can be reconciled by the presence of two subpopulations with distinct transcription profiles emerging at these time points. One of the subpopulations appears transiently, and surprisingly these cells showed a global downregulation of gene expression. Moreover, initiation of random X chromosome inactivation (XCI) coincides with the appearance of these transient cells. The other subpopulation can be maintained as a stem cell line and possesses expression profiles more similar to those of primed epiblast stem cells (EpiSC) than embryonic stem cells (ESC). However, there are important differences in gene expression related to epithelial-mesenchymal transition (EMT), suggesting that this subpopulation may represent a novel pluripotent state that has an intermediate cellular phenotype between ESC and EpiSC. ConclusionsThese findings should contribute to our understanding of the establishment and maintenance of distinct differentiation statuses of mammalian PSCs and provide new insights into the pluripotency spectrum in general.

genomics↗

bioRxiv2020

Elucidation of the antiviral mechanism of cystine and theanine through transcriptome analysis of mice and comparison with COVID-19 gene set data

We previously showed that oral administration of cystine and theanine (CT) to mice confers resistance to influenza virus infection. In human studies, CT prevented colds in healthy subjects and enhanced antibody production after influenza vaccination in elderly individuals with a poor nutritional status. The mechanism of action of CT is thought to be glutathione (GSH)-mediated regulation of intracellular redox, which might affect innate immune systems such as macrophages to exert physiological effects. The effect of CT on influenza is independent of viral type, and this treatment has a broad range of antiviral activities. To explore the mechanisms of CT in viral infection, we performed transcriptome profiling of spleen tissues isolated from influenza A virus (IAV)-infected mice. We identified unique gene signatures in response to CT in the IAV-infected mice. Genes upregulated by CT included redox-regulated genes such as GCLC/GCLM (subunits of glutamate cysteine ligase, a rate-limiting enzyme of GSH biosynthesis), TXN1, TXN2, TXNRD2, and SOD1, suggesting that the intracellular redox environment is substantially altered by CT. However, genes downregulated in response to CT included chemokine/chemokine receptor genes (CCL5, CCL19, CXCL9, CXCL12, CXCR3, CXCR4, and ACKR3), some of which are related to cytokine storm. A comparison with public COVID-19-related gene set data showed that the upregulated gene signature was highly similar to the downregulated gene sets of SARS-CoV/SARS-CoV-2-infected cells and the upregulated gene set of attenuated SARS-CoV-infected cells. In conclusion, the unique gene signatures observed in response to orally administered CT in IAV-infected mouse spleen tissues suggested that CT may attenuate viral infection, replication and associated symptoms such as cytokine storm.

molecular biology↗

bioRxiv2017

Genome-wide stability of the DNA replication program in single mammalian cells

Here, we report the establishment of a single-cell DNA replication sequencing method, scRepli-seq, which is a simple genome-wide methodology that measures copy number differences between replicated and unreplicated DNA. Using scRepli-seq, we demonstrate that replication domain organization is conserved among individual mouse embryonic stem cells (mESCs). Differentiated mESCs exhibited distinct replication profiles, which were conserved from cell to cell. Haplotype-resolved scRepli-seq revealed similar replication timing profiles of homologous autosomes, while the inactive X chromosome was clearly replicated later than its active counterpart. However, a small degree of cell-to-cell replication timing heterogeneity was present, and we discovered that developmentally regulated domains are a source of such variability, suggesting a link between cell-to-cell heterogeneity and developmental plasticity. Together, our results form a foundation for single-cell-level understanding of DNA replication regulation and provide insights into 3D genome organization.

genomics↗