Biology subjects

Nakano, S.

Publications and source records attributed to Nakano, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Region-specific molecular regulatory programs define epithelial identity, progenitor states, and mucus homeostasis in human distal airways

Small distal airways differ from proximal large airways in structure, airflow dynamics, and epithelial composition, and represent a central site of muco-obstructive lung disease pathogenesis. However, due in part to their inaccessibility, the molecular mechanisms that establish regional epithelial identity and govern mucociliary defense in distal airway epithelia remain poorly defined. Here, we integrate transcriptomic, secretomic, and chromatin accessibility analyses of matched primary human large and small airway epithelial cultures to define region-specific regulatory networks. We identify distal airway-specific transcriptional and chromatin programs required for maintaining epithelial identity and mucus homeostasis. Loss of NKX2-1 impairs distal airway secretory cell (DASC) differentiation and shifts mucus properties toward a disease-associated state. Lineage-resolved organoid assays identify an NKX2-1-high distal airway basal cell population with hybrid basal-secretory features as a selective progenitor for DASCs. Collectively, these findings establish a molecular framework for distal airway epithelial biology and define mechanisms regulating region-specific mucociliary host defense.

Cell Biology↗

bioRxiv2026

A cyanobacterial adenine prenyltransferase enables longer-chain N6 prenylation

Adenine, a ubiquitous nucleobase found in biomolecules such as nucleic acids, cofactors, and signaling molecules, mediates diverse molecular interactions. Here, we identify TvAPT, an adenine prenyltransferase from the cyanobacterium Trichormus variabilis NIES-23. TvAPT efficiently catalyzes the unprecedented N6-prenylation of adenine-containing substrates using extended C10 and C15 prenyl donors, whereas canonical adenine prenyltransferases are generally limited to C5-dimethylallylation. X-ray structural analyses and protein engineering revealed that an enlarged prenyl-binding pocket enables the accommodation of extended prenyl donors, providing a structural basis for rationally tuning prenyl-donor preference. Together, these findings establish TvAPT as an adenine prenyltransferase that accepts a range of adenine-containing substrates, with a preference for nucleoside 5-monophosphates. Beyond expanding the known catalytic scope of adenine prenyltransferases, TvAPT provides access to nucleotide derivatives with enhanced membrane permeability and analogues of plant signaling molecules.

biochemistry↗

bioRxiv2025

Monitoring the poroelastic response of collagen gels with embedded senescent fibroblasts reveals tissue softening associated with collagen network reorganization

The poroelastic properties of tissues regulate molecular transport and mechanical signaling, yet their evolution during aging remains poorly understood. In particular, senescent fibroblasts accumulate in aged tissues, contributing to extracellular matrix (ECM) remodeling, but their impact on tissue mechanics and permeability is unclear. In this study, we developed a microfluidic-based in vitro model to assess the poroelastic properties of collagen gels embedded with senescent fibroblasts over time. Our approach integrates periodic pressure actuation with real-time pressure monitoring in a sealed air cavity, enabling the detection of fluid permeation and solid matrix deformations. We analyze our data using analytical and numerical models based on a porohyperelastic framework. This framework combines compressible Neo-Hookean elasticity with the Kozeny-Carman permeability relationship. We demonstrate that senescent fibroblasts induce a progressive softening of the ECM without altering its permeability. Immunostaining reveals that this softening correlates with structural reorganization of the collagen network, characterized by increased branching and network remodeling. Our findings provide insights into the biomechanical effects of senescent fibroblasts on ECM homeostasis. We further argue that our platform offers a unique solution to investigate ECM remodeling not only in aging but also fibrosis, cancer progression, or regenerative medicine strategies.

biophysics↗

bioRxiv2024

p16High immune cell - controlled disease tolerance as a broad defense and healthspan extending strategy

The ability of an organism to overcome infectious diseases has traditionally been linked to killing invading pathogens. Accumulating evidence, however, indicates that, apart from restricting pathogen loads, organismal survival is coupled to an additional yet poorly understood mechanism called disease tolerance. Here we report that p16High immune cells play a key role in establishing disease tolerance. We found that the FDA-approved BNT162b2 mRNA COVID-19 vaccine is a potent and rapid inducer of p16High immune subsets both in mice and humans. In turn, p16High immune cells were indispensable for counteracting different lethal conditions, including LPS-induced sepsis, acute SARS-CoV-2 infection and ionizing irradiation. Mechanistically, we propose that activation of TLR7 or a low physiological activity of STING is sufficient to induce p16High immune subset that, in turn, establishes a low adenosine environment and disease tolerance. Furthermore, containing these signals within a beneficial range by deleting MDA5 that appeared sufficient to maintain a low activity of STING, induces p16High immune cells and delays organ deterioration upon aging with improved healthspan. Our data highlight the beneficial role of p16High immune subsets in establishing a low adenosine environment and disease tolerance.

cell biology↗

bioRxiv2024

NR4A3 deficiency ameliorates contact hypersensitivity and exacerbates psoriasis by regulating gene expression in dendritic cells

NR4A3 is a transcription factor that belongs to the nuclear receptor superfamily. To reveal the roles of NR4A3 in skin diseases, we constructed contact hypersensitivity (CHS)- and imiquimod-induced psoriasis models in Nr4a3-/- mice. In the CHS induced Nr4a3-/- mice, ear swelling was significantly reduced, accompanied by suppressed migration of dendritic cells (DCs), in which CCR7 expression was reduced. In contrast, ear swelling in psoriasis model Nr4a3-/- mice was enhanced. The expression levels of inflammatory cytokines in psoriasis skin lesions and in TLR7-ligand-stimulated DCs were increased by NR4A3 deficiency. Subcutaneous (s.c.) injection of hapten-treated Nr4a3+/+ DCs induced ear swelling in Nr4a3-/- mice, and s.c. injection of TLR7-ligand-stimulated Nr4a3-/- DCs caused significant ear swelling. Compared with Nr4a3+/+ DCs, Nr4a3-/- DCs expressed lower levels of CCR7 and IRF4, and higher levels of TLR7, IRF7, IFN-{beta}, and PU.1. Taken together, NR4A3 deficiency ameliorates CHS and exacerbates psoriasis by regulating the expression of CCR7, TLR7, and related transcription factors in DCs.

immunology↗

bioRxiv2024

Prolonged airway explant culture enables study of health, disease, and viral pathogenesis

In vitro models play a major role in studying airway physiology and disease. However, the native lungs complex tissue architecture and non-epithelial cell lineages are not preserved in these models. Ex vivo tissue models could overcome in vitro limitations, but methods for long-term maintenance of ex vivo tissue has not been established. We describe methods to culture human large airway explants, small airway explants, and precision-cut lung slices for at least 14 days. Human airway explants recapitulate genotype-specific electrophysiology, characteristic epithelial, endothelial, stromal and immune cell populations, and model viral infection after 14 days in culture. These methods also maintain mouse, rabbit, and pig tracheal explants. Notably, intact airway tissue can be cryopreserved, thawed, and used to generate explants with recovery of function 14 days post-thaw. These studies highlight the broad applications of airway tissue explants and their use as translational intermediates between in vitro and in vivo studies.

bioengineering↗

bioRxiv2023

Maternal sterol 27-hydroxylase is crucial for securing fetal development

The maternal body helps in providing nutrients and degrading toxic metabolites instead of the fetal body; disruptions in these mechanisms affect normal fetal development. Sterol 27-hydroxylase (Cyp27a1) is involved in the alternative pathway of bile acid synthesis, which is enhanced during pregnancy. However, its role in fetal development remains unclear. Here, we demonstrate that maternal Cyp27a1 activity is essential for progression of normal pregnancy and fetal organ formation. Depletion of maternal Cyp27a1 reduced the pregnancy rate and litter size. Newborn mice died of respiratory distress syndrome resulting from the absence of mature alveolar epithelial cells. These phenotypes were caused by 7-hydroxycholesterol (7-HC) accumulating in Cyp27a1-deficient mice. Mechanistically, 7-HC destabilized the Fau protein, mediating ribosome assembly, the downregulation of which caused poor polysome formation, lower protein synthesis, and impaired lung maturation. Overall, this study revealed an essential mechanism of securing fetal development by degrading a toxic metabolite in the maternal body.

developmental biology↗

bioRxiv2023

A hyperpolarizing neuron recruits undocked innexin hemichannels to transmit neural information in Caenorhabditis elegans

While depolarization of neuronal membrane is known to evoke the neurotransmitter release from synaptic vesicles, hyperpolarization is regarded as a resting state of chemical neurotransmission. Here we report that hyperpolarizing neurons can actively signal neural information by employing undocked hemichannels. We show that UNC-7, a member of the innexin family in Caenorhabditis elegans, functions as a hemichannel in thermosensory neurons and transmits temperature information from the thermosensory neurons to their post-synaptic interneurons. By monitoring neural activities in freely behaving animals, we find that hyperpolarizing thermosensory neurons inhibit the activity of the interneurons and that UNC-7 hemichannels regulate this process. UNC-7 is required to control thermotaxis behavior and functions independently of synaptic vesicle exocytosis. Our findings suggest that innexin hemichannels mediate neurotransmission from hyperpolarizing neurons in a manner that is distinct from the synaptic transmission, expanding the way of neural circuitry operations.

neuroscience↗

bioRxiv2022

Horizontal transmission of penicillin binding protein 1A caused a nationwide spread of β-lactam resistance in pneumococci

The emergence and spread of drug-resistant bacteria continue to be a global crisis. The mechanism of the resistance spread via mobile genetic elements such as plasmids is well known, however, the impact of the natural transformation on the spread remains unclear. Streptococcus pneumoniae is well known to be a transformable pathogen by natural competence and they become {beta}-lactam resistance by the acquisition of chromosomal genetic elements including mutated PBPs by natural transformation. To trace the transmission of pneumococcal PBPs among nationwide pediatric population and analyze the impact of transformed PBPs to {beta}-lactam resistance, we collected and analyzed more than 1300 isolates of S. pneumoniae through nationwide surveillance study for pediatric pneumococcal diseases between 2012-2017 in Japan. We discovered a high prevalence of a specific PBP1A type (pbp1a-13) in {beta}-lactam resistant pneumococci that had a 370SSMK substitution in their {beta}-lactam binding SXXK motif, suggesting that this pbp1a-13 transferred horizontally between different clones resulting in emergence and spread of {beta}-lactam resistant pneumococcal clones. Divergence dating analysis suggested that pbp1a-13 was inserted into major resistant lineages in the early 1990s through the 2000s, before introduction of pneumococcal conjugate vaccines in Japan. Our additional analysis for pneumococcal isolates that were recovered in the 90s in Japan suggested that pbp1a-13 in GPSC1 (serotype 19F-CC236) and GPSC14 (serotype 23F-CC242) isolates were the origin of the currently spread pbp1a-13. We provide evidence of pbp1a horizontal transmission at a nationwide scale and highlight the importance of PBP profile monitoring for identifying the emergence and spread of resistant pneumococci lineages.

microbiology↗

bioRxiv2022

Isolation and characterization of bacteriophages against IMP-6-producing Klebsiella pneumoniae isolated from clinical settings in Japan

Carbapenemase-producing Enterobacteriaceae (CPE) are one of the most detrimental species of antibiotic-resistant bacteria worldwide. Phage therapy has emerged as an effective strategy for the treatment of infections caused by CPE pathogens. In west Japan, the increasing occurrence of Klebsiella pneumoniae harboring the pKPI-6 plasmid, which encodes blaIMP-6, is a growing concern. To manage such major antimicrobial-resistant pathogens, we isolated 29 novel phages from sewage in Japan, targeting 31 strains of K. pneumoniae and one strain of Escherichia coli harboring the pKPI-6 plasmid. Electron microscopy analysis indicated that of the 29 isolated phages, 21 (72.4%), 5 (17.2%), and 3 (10.3%) belonged to Myoviridae, Siphoviridae, and Podoviridae, respectively. Host range analysis revealed that 20 Myoviridae members in isolated phages infected 25-26 strains of K. pneumoniae, indicating that most of the isolated phages have a broad host range. The K. pneumoniae Kp21 can only be infected by phage oKp_21, while Kp22 can be infected by more than 20 phages. We applied a phage cocktail, which consists of 10 phages, against Kp21 and Kp22 and found that the phage cocktail delayed the emergence of phage-resistant bacteria for Kp21 strain but not for the Kp22 strain. Furthermore, phage-resistant Kp21 (Kp21r) became prone to be infected from other bacteriophages as a "trade-off" of resistance to phage oKp_21. Our proposed phage set has an adequate number of phages to combat the K. pneumoniae strain isolated in Japan. Notably, our work demonstrates how a suitable phage cocktail diminishes the occurrence of phage-resistant bacteria. ImportanceKlebsiella pneumoniae harboring the plasmid carrying blaIMP-6 is becoming an increasingly hazardous species in Japan. We collected and characterized 29 novel bacteriophages that infect K. pneumoniae carrying the pKPI-6 plasmid, isolated in clinical settings of west Japan. Our phages showed broad host ranges. We applied a phage cocktail treatment constructed from 10 phages against two host strains, Kp21 and Kp22, which show different phage susceptibility patterns each other. Although the phage cocktail delayed phage-resistant Kp21 emergence, the emergence of phage-resistant Kp22 could not be delayed. Moreover, phage-resistant Kp21 became sensitive to other phages, which did not originally infect wild-type Kp21. Our study demonstrates how a suitable phage cocktail can diminish the occurrence of phage-resistant bacteria.

microbiology↗

bioRxiv2022

Genetic screens identified dual roles of MAST kinase and CREB within a single thermosensory neuron in the regulation of C. elegans thermotaxis behavior

Animals integrate sensory stimuli presented at the past and present, assess the changes in their surroundings and navigate themselves toward preferred environment. Identifying the molecular and circuit mechanisms of such sensory integration is pivotal to understand how the nervous system generates perception and behavior. Previous studies on thermotaxis behavior of Caenorhabditis elegans suggested that a single thermosensory neuron AFD plays an essential role in integrating the past and present temperature information and is essential for the neural computation that drives the animal toward the preferred temperature region. However, the molecular mechanisms by which AFD executes this neural function remained elusive. Here we report multiple forward genetic screens to identify genes required for thermotaxis. We reveal that kin-4, which encodes the C. elegans homolog of MAST kinase, plays dual roles in thermotaxis and can promote both cryophilic and thermophilic drives. We also uncover that a thermophilic defect of mutants for mec-2, which encodes a C. elegans homolog of stomatin, can be suppressed by a loss-of-function mutation in the gene crh-1, encoding a C. elegans homolog CREB transcription factor. Calcium imaging analysis from freely-moving animals suggest that mec-2 and crh-1 function in AFD and regulate the neuronal activity of its post-synaptic interneuron AIY. Our results suggest that a stomatin family protein can control the dynamics of neural circuitry through the transcriptional regulation within a sensory neuron.

genetics↗

bioRxiv2022

A model of persistent post SARS-CoV-2 induced lung disease for target identification and testing of therapeutic strategies

COVID-19 survivors develop post-acute sequelae of SARS-CoV-2 (PASC), but the mechanistic basis of PASC-associated lung abnormalities suffers from a lack of longitudinal samples. Mouse-adapted SARS-CoV-2 MA10 produces an acute respiratory distress syndrome (ARDS) in mice similar to humans. To investigate PASC pathogenesis, studies of MA10-infected mice were extended from acute disease through clinical recovery. At 15-120 days post-virus clearance, histologic evaluation identified subpleural lesions containing collagen, proliferative fibroblasts, and chronic inflammation with tertiary lymphoid structures. Longitudinal spatial transcriptional profiling identified global reparative and fibrotic pathways dysregulated in diseased regions, similar to human COVID-19. Populations of alveolar intermediate cells, coupled with focal upregulation of pro-fibrotic markers, were identified in persistently diseased regions. Early intervention with antiviral EIDD-2801 reduced chronic disease, and early anti-fibrotic agent (nintedanib) intervention modified early disease severity. This murine model provides opportunities to identify pathways associated with persistent SARS-CoV-2 pulmonary disease and test countermeasures to ameliorate PASC.

microbiology↗

bioRxiv2021

A method for identifying environmental stimuli and genes responsible for genotype-by-environment interactions from a large-scale multi-environment data set

It has not been fully understood in real fields what environment stimuli cause the genotype-by-environment (G x E) interactions, when they occur, and what genes react to them. Large-scale multi-environment data sets are attractive data sources for these purposes because they potentially experienced various environmental conditions. Here we developed a data-driven approach termed Environmental Covariate Search Affecting Genetic Correlations (ECGC) to identify environmental stimuli and genes responsible for the G x E interactions from large-scale multi-environment data sets. ECGC was applied to a soybean (Glycine max) data set that consisted of 25,158 records collected at 52 environments. ECGC illustrated what meteorological factors shaped the G x E interactions in six traits including yield, flowering time, and protein content and when they were involved. For example, it illustrated the relevance of precipitation around sowing dates and hours of sunshine just before maturity to the interactions observed for yield. Moreover, genome-wide association mapping on the sensitivities to the identified stimuli discovered candidate and known genes responsible for the G x E interactions. Our results demonstrate the capability of data-driven approaches to bring novel insights on the G x E interactions observed in fields. Key messageThe proposed method is able to identify environmental stimuli and genes responsible for the G x E interactions observed in multi-environmental trials. The method is based on similarity search between genetic correlation and environmental stimuli among environments.

plant biology↗

bioRxiv2020

Lactobacilli in a clade ameliorate age-dependent decline of thermotaxis behavior in Caenorhabditis elegans

The causality and mechanism of dietary effects on brain aging are still unclear due to the long time scales of aging. The nematode Caenorhabditis elegans has contributed to aging research because of its short lifespan and easy genetic manipulation. When fed the standard laboratory diet, Escherichia coli, C. elegans experiences an age-dependent decline in temperature-food associative learning, called thermotaxis. To address if diet affects this decline, we screened 35 lactic acid bacteria as alternative diets and found that animals maintained high thermotaxis ability when fed a clade of Lactobacilli enriched with heterofermentative bacteria. Among them, Lactobacillus reuteri maintained the thermotaxis of aged animals without affecting their lifespan and motility. The effect of Lb. reuteri depends on the DAF-16 transcription factor functioning in neurons. Furthermore, RNA sequencing analysis revealed that differentially expressed genes between aged animals fed different bacteria were enriched with DAF-16 targets. Our results demonstrate that diet can impact brain aging in a daf-16 dependent manner without changing the lifespan.

neuroscience↗

bioRxiv2020

Comparison of 12 molecular detection assays for SARS-CoV-2

Molecular testing for SARS-CoV-2 is the mainstay for accurate diagnosis of the infection, but the diagnostic performances of available assays have not been defined. We compared 12 molecular diagnostic assays, including 8 commercial kits using 155 respiratory samples (65 nasopharyngeal swabs, 45 oropharyngeal swabs, and 45 sputum) collected at 2 Japanese hospitals. Sixty-eight samples were positive for more than one assay and one genetic locus and were defined as true positive samples. All the assays showed a specificity of 100% (95% confidence interval, 95.8 to 100). The N2 assay kit of the US Centers for Disease Control and Prevention (CDC) and the N2 assay of the Japanese National Institute of Infectious Disease (NIID) were the most sensitive assays with 100% sensitivity (95% confidence interval, 94.7 to 100), followed by the CDC N1 kit, E assay by Corman, and NIID N2 assay multiplex with internal control reactions. These assays are reliable as first-line molecular assays in laboratories when combined with appropriate internal control reactions.

microbiology↗

bioRxiv2019

cGMP dynamics underlie thermosensation in C. elegans

Animals sense ambient temperature so that they can adjust their behavior to the environment; they avoid noxious heat and coldness and stay within a survivable temperature range. C. elegans can sense temperature, memorize past cultivation temperature and navigate towards preferable temperature, for which a thermosensory neuron, AFD, is essential. AFD responds to temperature increase from the past cultivation temperature by increasing intracellular Ca2+ level. We aimed to reveal how AFD encodes and memorizes the information of temperature. Although cGMP synthesis is crucial for thermosensation by AFD, whether and how cGMP level temporally fluctuates in AFD remained elusive. We therefore monitored cGMP level in AFD and found that cGMP dynamically responded to temperature change in a manner dependent on past cultivation temperature. Given that cGMP dynamics is supposed to be upstream of Ca2+ dynamics, our results suggest that AFDs memory is formed by simpler molecular mechanisms than previously expected from the Ca2+ dynamics. Moreover, we analyzed how guanylyl cyclases and phosphodiesterases, which synthesize and degrade cGMP, respectively, contributed to cGMP and Ca2+ dynamics and thermotaxis behavior.

neuroscience↗

bioRxiv2019

Novel method to determine diagnosis-defining refraction points

Diagnosis of a certain disease generally relies on definitions established by professional medical societies and comprise the patients history, physical examination, and test results. These include physical compositions such as body mass index (BMI), and laboratory tests such as serum creatinine and albumin in urine samples. In general, laboratory tests are based on mathematical methods, e.g. defining critical values from the mean {+/-} k{sigma} of a population, where k is a natural number and the standard deviation is {sigma} (\"mean {+/-} k{sigma}-method\"). In most cases k is defined as 2, leading to reference ranges defining 95% of test results as normal. However, this method mostly depends on a normal distribution of values.\n\nHere we applied a novel method (\"SoFR-method\") based on data sorting to define refraction points, which carry informative value as diagnostic criteria. Applying the SoFR-method, standard measures such as critical BMI-values are categorized by equal robustness as by the mean {+/-} k{sigma}-method. However, the SoFR-method showed higher validity when analyzing non-normalized values such as creatinine and albumin, as well as hepatocyte growth factor (HGF) and hemoglobin in a novel Perioscreen assay in saliva of diabetic and non-diabetic patients.\n\nTaken together, we defined a novel method based on data sorting of test results from patients to effectively define refraction points which might guide more accurately clinical diagnoses and define relevant thresholds.

epidemiology↗

bioRxiv2019

Mammalian NSUN2 introduces 5-methylcytidines into mitochondrial tRNAs

Post-transcriptional modifications in mitochondrial tRNAs (mt-tRNAs) play critical roles in mitochondrial protein synthesis, which produces respiratory chain complexes. In this study, we used mass spectrometric analysis to map 5-methylcytidine (m5C) at positions 48-50 in eight mouse and six human mt-tRNAs. We also confirmed the absence of m5C in mt-tRNAs isolated from Nsun2 knockout (KO) mice, as well as from NSUN2 KO human culture cells. In addition, we successfully reconstituted m5C at positions 48-50 of mt-tRNA in vitro with NSUN2 protein in the presence of S-adenosylmethionine (SAM). Although NSUN2 is predominantly localized to the nucleus and introduces m5C into cytoplasmic tRNAs and mRNAs, structured illumination microscopy (SIM) clearly revealed NSUN2 foci inside mitochondria. These observations provide novel insights into the role of NSUN2 in the physiology and pathology of mitochondrial functions.

molecular biology↗