Biology subjects

Nishi, K.

Publications and source records attributed to Nishi, K..

16 recordsLinked to original sources

bioRxiv2026

Nardilysin fine-tunes the mammalian circadian clock through selective modulation of PER2 function

Circadian rhythms in mammals are generated by negative feedback loops, in which CLOCK and BMAL1 bind to E-box to activate transcription of Period (Per) and Cryptochrome (Cry) and the E-box-dependent transactivation is inhibited by PER and CRY proteins. Although the core transcriptional feedback loop of the circadian clock has been well defined, how this machinery interfaces with broader nuclear regulatory systems remains incompletely understood. Here, we identify nardilysin (NRDC), a metalloendopeptidase previously implicated in nuclear transcriptional regulation and metabolic homeostasis, as an unexpected modulator of the circadian clock. NRDC deficiency led to elevated PER2 protein levels in the liver and enhanced PER2 dynamics in cell-autonomous circadian oscillators, and was accompanied by a significant shortening of behavioral rhythms in mice. Biochemical analyses demonstrated that NRDC selectively associates with PER2 and CRY2 and antagonizes PER2-mediated repression of CLOCK-BMAL1-dependent transcription. Genome-wide chromatin immunoprecipitation analyses reveal that NRDC is enriched at promoter-proximal E-box-containing regions, frequently co-localizing with CLOCK binding sites. Together, these findings uncover a previously unrecognized link between circadian timing and protease-based nuclear regulation, positioning NRDC as a critical modulator of PER2 function and circadian period determination.

molecular biology↗

bioRxiv2026

HDAC3 inhibition stabilizes the IL-37 receptor module to enhance anti-inflammatory signaling in cystic fibrosis airway epithelium

Airway inflammation in cystic fibrosis (CF) persists despite advances in CFTR modulator therapy. IL-37b suppresses innate immune signaling through a receptor complex containing IL-18R and wild-type SIGIRR (WT-SIGIRR; IL-1R8), but this pathway is compromised in CF airway epithelial cells by the dominant-negative exon 8-skipped SIGIRR isoform ({Delta}8-SIGIRR). Here, a natural-product screen identified short-chain fatty acids as preferential enhancers of WT-SIGIRR. Pan-HDAC inhibition with panobinostat increased WT-SIGIRR, reduced {Delta}8-SIGIRR, and restored IL-37b-dependent suppression of the TLR3 ligand poly(I:C)-induced IL-8 production. Isoform-selective inhibitor screening and siRNA knockdown identified HDAC3 as a regulator of the IL-37 receptor module. Low concentrations of RGFP966 and HDAC3 silencing increased WT-SIGIRR and IL-18R protein abundance without inducing their mRNA levels. HDAC3 inhibition delayed proteasome-dependent WT-SIGIRR turnover and stabilized IL-18R, thereby enhancing IL-37b-mediated anti-inflammatory signaling in CF airway epithelial cells.

cell biology↗

bioRxiv2026

Nardilysin regulates Slc2a2 expression through ISLET1 recruitment to an evolutionarily conserved enhancer in pancreatic β-cells

GLUT2 (Slc2a2) is a key glucose transporter in pancreatic {beta}-cells, and its reduced expression is closely linked to defective glucose-stimulated insulin secretion (GSIS) and diabetes. We previously reported that pancreatic {beta}-cell-specific nardilysin (NRDC)-deficient mice (BetaKO) exhibit severe diabetic phenotype with defective GSIS and reduced Slc2a2 expression in islets. However, because BetaKO mice also showed reduced MafA, a key upstream regulator of Slc2a2, along with an increased -cell/{beta}-cell ratio and other secondary changes that could influence GLUT2 levels, the mechanism by which NRDC regulates Slc2a2 transcription remained unclear. Here, we demonstrate that NRDC controls Slc2a2 expression in a {beta}-cell autonomous and MafA-independent manner. By integrating publicly available ATAC-seq and ChIP-seq datasets, we identified four active enhancer regions around the murine Slc2a2 locus, two of which are evolutionarily conserved in human islets. Luciferase assays revealed that NRDC selectively controls the activity of a conserved enhancer located 39k bp downstream of the Slc2a2 transcriptional start site. Chromatin immunoprecipitation (ChIP) and re-ChIP assays further revealed that NRDC binds to this enhancer and is required for efficient recruitment of ISLET1, a transcription factor upstream of Slc2a2. These findings indicate that NRDC directly regulates Slc2a2 in addition to MafA, highlighting multifaceted roles of NRDC in pancreatic {beta}-cell gene regulation.

cell biology↗

bioRxiv2025

Mathematical modeling for primitive form of habituation in an amoeba

Learning abilities, once thought to be unique to higher animals, have been reported to exist in their primitive form in single-celled organisms. This has triggered a growing interest in carefully examining the nature and mechanisms of the primitive versions of learning abilities, which would provide important clues for understanding the evolution of behavioral capabilities in organisms. In this study, we focused on previous experimental studies showing that the slime mold Physarum polycephalum, a model organism for studying protist behavior, exhibits the ability to adapt to chemical environments. We propose a possible dynamic mechanism underlying this habituation, reproducing reported experimental observations with accuracy. By refining a mathematical model that was as simple as possible and based on non-specific biochemical processes within cells, we clarified a plausible mechanistic framework. Based on these results, we examined the similarities and differences between this framework and previously proposed habituation models of single-cell movement and animal neural-circuit regulation. These findings are significant because they open new avenues for research into the generality and evolutionary origins of acclimation learning.

systems biology↗

bioRxiv2025

SarcAsM: AI-based multiscale analysis of sarcomere organization and contractility in cardiomyocytes

Cardiomyocyte function critically depends on sarcomere dynamics and their organization in myofibrils. To uncover how cardiomyocyte function emerges from individual sarcomere dynamics, comprehensive analysis of Z-bands (nanometer scale), sarcomeres ([~]2 {micro}m scale), and myofibrils ([~]10 to 100 {micro}m scale) is required. Tools for such multiscale analyses are presently lacking. Here we introduce the Sarcomere Analysis Multitool (SarcAsM), which combines deep learning and graph-based methods for automated, fast, and unbiased structural assessment of sarcomeric Z-bands, sarcomeres, and myofibrils as well as their organization in larger myofibril domains. SarcAsM features a generalist deep learning model, pre-trained on a broad range of experimental and published images, ensuring its versatile application and immediate usability across diverse datasets. Finally, we demonstrate the versatile utility of SarcAsM in analyzing sarcomere structure and dynamics under acute and chronic drug exposure in hiPSC-derived cardiomyocytes with fluorescently tagged Z-bands. SarcAsM is available as open-source Python package and stand-alone application.

bioinformatics↗

bioRxiv2025

Disentanglement of batch effects and biological signals across conditions in the single-cell transcriptome

Batch effects, which arise due to technical variations across different experimental factors, pose a significant challenge for single-cell transcriptomic analysis. Although various batch correction methods have been developed to mitigate these effects, they often indiscriminately mix data from different batches, leading to the removal of biologically meaningful signals. This limitation hinders comparative analyses across multiple conditions, an essential aspect of scientific research. Recent approaches attempt to address this issue by mapping data to separate spaces, but they prevent direct comparisons between conditions. Here, we propose Kanade, a batch correction method based on a variational autoencoder. Kanade explicitly disentangles batch effects from biological signals by specializing latent variables for different types of information. Using both simulated and real datasets, we demonstrate that Kanade selectively correct batch effects while preserving essential biological features, enabling more accurate comparative analyses in single-cell transcriptomics.

bioinformatics↗

bioRxiv2024

Stochastic tug-of-war among sarcomeres mediates cardiomyocyte response to environmental stiffness

Cardiac contraction is driven by the collective action of cardiomyocytes, that contain parallel bundles of myofibrils consisting of linear chains of sarcomeres, the basic force-generating units. The dynamics of individual sarcomeres within intact cardiomyocytes remain incompletely understood. While most models assume uniform, synchronized contractions, recent studies hint at unexpected heterogeneity whose origins and significance are not yet clear. By combining the culture of fluorescent sarcomere-reporter hiPSC-derived cardiomyocytes on micropatterned soft gels of different stiffness (5 - 85 kPa) with AI-based tracking of sarcomere motion, we found that increasingly stiff substrates inhibited overall cardiomyocyte contraction, but, surprisingly, did not diminish individual sarcomere dynamics. Instead, sarcomeres competed in a tug-of-war causing increasing heterogeneity, including rapid length oscillations and overextensions (popping). Statistical analysis showed that the heterogeneous dynamics were not caused by static structural differences but were largely stochastic. Stochastic heterogeneity is thus an intrinsic property of cardiac sarcomeres and likely mediates the adaptation of cardiomyocyte contractility to mechanical constraints. A mesoscopic model of coupled sarcomeres shows that these phenomena can be explained by a non-monotonic force-velocity relationship and stochastic fluctuations, where dynamic instability at a critical yielding force creates heterogeneity. Stochastic heterogeneity compensates for structural disorder by randomizing yield events beat-to-beat, preventing damage to specific sarcomeres. Our findings recast cardiac sarcomeres as active, dynamically unstable, and stochastic units engaged in a stochastic tug-of-war, where transient, velocity-dependent forces dominate. We propose that pathological disorder in cardiomyopathy drives a transition from protective stochastic fluctuations to more deterministic, persistently overloaded sarcomeres.

biophysics↗

bioRxiv2024

Myosin II actively regulates Drosophila proprioceptors

Auditory receptors can be motile to actively amplify their mechanical input. Here we describe a novel and different type of motility that, residing in supporting cells, shapes physiological responses of mechanoreceptor cells. In Drosophila larvae, supporting cap cells transmit mechanical stimuli to proprioceptive chordotonal neurons. We found that the cap cells are strongly pre-stretched at rest to twice their relaxed length. The tension in these cells is modulated by non-muscle myosin-II motors. Activating the motors optogenetically causes contractions of the cap cells. Cap-cell-specific knockdown of the regulatory light chain of myosin-II alters mechanically evoked receptor neuron responses, converting them from phasic to more tonic, impairing sensory adaptation. Hence, two motile mechanisms seem to operate in concert in insect chordotonal organs, one in the sensory receptor neurons, based on dynein, and the other in supporting cells, based on myosin. One Sentence SummaryMyosin II motors in contractile cells pre-stretch Drosophila stretch receptors.

cell biology↗

bioRxiv2024

Alteration of long and short-term hematopoietic stem cell ratio causes myeloid-biased hematopoiesis

Myeloid-biased hematopoiesis is a well-known age-related alteration. Several possibilities, including myeloid-biased hematopoietic stem cell (HSC) clones, may explain this. However, the precise mechanisms remain controversial. Utilizing the Hoxb5 reporter system to prospectively isolate long-term HSCs (LT-HSCs) and short-term HSCs (ST-HSCs), we found that young and aged LT-HSCs co-transplanted into the same recipients demonstrated nearly equivalent myeloid lineage output, contrary to the theory of myeloid-biased HSC clones. Transcriptomics indicated no significant myeloid gene enrichment in aged LT-HSCs compared to their young counterparts. Instead, transplanting reconstituted young HSCs with the ratio of LT/ST-HSCs seen in aged mice can significantly skew the lineage output to myeloid cells. In addition, while the niche environment in the bone marrow minimally affects myeloid-biased hematopoiesis, aged thymi and spleens substantially hinder lymphoid hematopoiesis, resulting in further myeloid-domination. Thus, we demonstrate that myeloid-biased hematopoiesis in aged organisms originates due to alteration of the ratio between LT-HSCs and ST-HSCs rather than in heterogeneous HSC clones with various cell fates.

developmental biology↗

bioRxiv2023

Nonequilibrium bending fluctuations reveal microtubule mechanics in-vivo and their regulation by glutamylation

Cells can be described as active composite materials. The mechanical properties of cells are controlled by complex polymer networks and are dynamically tuned for diverse cellular processes driven by force-generating motor proteins. Microtubules are the most rigid protein polymers in the cytoskeleton, and their material properties have been measured in vitro by active bending or by analyzing thermal bending fluctuations. Microtubule mechanics in living cells are extremely difficult to probe directly, while fluctuations are difficult to interpret because they are generated by active forces in a surrounding cytoplasm with poorly understood material properties. Here we introduce a method to measure the elastic properties of microtubules in living cells by making use of motor-generated forces that drive bending fluctuations. Bending dynamics are governed by three main factors: microtubule material properties, cytoskeletal active forces, and the response characteristics of the surrounding cytoplasm. We show theoretically that, when one factor can be independently determined, the other two can be derived from observed fluctuations. Using this method we discovered that polyglutamylation, a post-translational modification enriched on microtubule arrays that need to withstand large mechanical forces such as those in axons or cilia, increases microtubule stiffness in living cells. Our work provides a theoretical and experimental framework to study microtubule mechanics and their regulation by tubulin modifications and microtubule effectors in complex cellular environments. The approach can be extended to other large aspect ratio cellular structures such as the endoplasmic reticulum or the mitochondrial network.

biophysics↗

bioRxiv2023

Analysis of chronic host-aspergilloma interactions using a novel mouse model

An aspergilloma is a fungus ball caused by chronic infection of Aspergillus species in a pre- existing cavity, such as a destroyed lung or the sinuses. Patients with pulmonary aspergilloma are at risk of sudden life-threatening hemoptysis. Antifungal therapy is administered to aspergilloma patients who are ineligible for surgery, but its efficacy is limited. Understanding the pathophysiology of aspergilloma is crucial for developing further treatment strategies. The mechanism behind the long-term host response to aspergilloma is poorly understood. We created a novel mouse model to analyze the host response to aspergilloma by implanting a fungus ball of Aspergillus fumigatus into an air-filled subcutaneous cavity. Our findings indicate that a live fungus ball led to tissue invasion, even in immunized mice. When a fungus ball consisting of dead hyphae was implanted, it persisted for over three months and induced pathological findings simulating human aspergilloma, including an inflammatory cell infiltration into the fungus ball and angiogenesis in the cavity wall. Dead fungus ball induced Th1 and Th17 inflammatory cytokines and vascular endothelial growth factor. Neutrophils infiltrated the inside of the fungus ball immediately after implantation, and macrophages surrounded it after a one-week delay. The macrophages around the fungus ball were swollen with phagocytosed fragments of dead hyphae and transformed into foam cells containing fat droplets. We also confirmed in vitro that macrophages were damaged and transformed into foam cells by direct contact with dead hyphae. This model holds promise to provide new insights into the fungal-host interaction during aspergillomas. NOTATION OF PRIOR ABSTRACT PUBLICATION/PRESENTATIONNone IMPORTANCEChronic aspergillosis, which affects over 3 million people worldwide with a 5-year survival rate of 50%, is understudied compared to other forms. Our study focuses on aspergilloma, a key aspect of chronic aspergillosis, using a groundbreaking mouse model that mirrors clinical features over three months. By studying host-fungal interactions within aspergillomas, we discovered Th1 and Th17 inflammatory responses to dead fungal hyphae. Initially, neutrophils dominate, later giving way to macrophages with a lipid-accumulating foamy phenotype. This transition may impede aspergilloma clearance. In addition, even dead hyphae induce vascular endothelial growth factor and promote angiogenesis. Our findings, which are critical for the prevention of fatal hemoptysis, highlight the need for innovative treatments that target fungal clearance and challenge the limited efficacy of antifungal agents against dead fungal bodies. This research represents a significant step forward in the understanding of chronic aspergillosis.

microbiology↗

bioRxiv2023

Relationship Between Domain and Function of the Yeast RNase T2, Rny1p, Which Mediates rRNA Degradation upon Starvation

RNase T2 is ubiquitous across diverse organisms, playing essential roles despite its simple enzymatic activity. In Saccharomyces cerevisiae, RNase T2, known as Rny1p, localizes in vacuoles and mediates rRNA degradation during autophagy of ribosomes. In this study, we elucidated novel aspects of ribosome degradation mechanisms and the function of Rny1p. First, we discovered that most ribosomes are degraded by selective autophagy, where Rsa1p is the specific receptor of ribosomes to be degraded. Complex structure prediction suggested that Rsa1p also interacts with Atg8p. Furthermore, we observed that the accumulation of rRNA in vacuoles, due to the lack of Rny1p, leads to a decrease in bulk autophagic activity. This decrease in autophagic activity may explain the inability of Rny1p-deficient strains to adapt to starvation conditions. Second, our structural prediction and biochemical analyses indicate that a C-terminal extension, characteristic in fungal RNase T2 including Rny1p, is not necessary for rRNA degradation but for anchoring to the cell wall. Together with molecular phylogenetic analysis, a species-specific role of RNase T2 conferred by the C-terminal extension is suggested.

molecular biology↗

bioRxiv2023

DNA metabarcoding workflow utilizing nanopore long-read sequencing and consensus generation for rapid identification of fungal taxa with high phylogenetic resolution

The study of microbiota has been revolutionized by the development of DNA metabarcoding. This sequence-based approach enables the direct detection of microorganisms without the need for culture and isolation, which significantly reduces analysis time and offers more comprehensive taxonomic profiles across broad phylogenetic lineages. While there has been an accumulating number of researches on bacteria, molecular phylogenetic analysis of fungi still remains challenging due to the lack of standardized tools and the incompleteness of reference databases limiting the accurate and precise identification of fungal taxa. Here, we present a DNA metabarcoding workflow for characterizing fungal microbiota with high taxonomic resolution. This method involves amplifying longer stretches of ribosomal RNA operons and sequencing them using nanopore long-read sequencing technology. The resulting reads were error-polished to generate consensus sequences with 99.5-100% accuracy, which were then aligned against reference genome assemblies. The efficacy of this method was explored using a polymicrobial mock community and patient-derived specimens, demonstrating the marked potential of long-read sequencing combined with consensus calling for accurate taxonomic classification. Our approach offers a powerful tool for the rapid identification of pathogenic fungi and has the promise to significantly improve our understanding of the role of fungi in health and disease.

microbiology↗

bioRxiv2020

Reconstruction of the urinary tract at the appropriate time reduces fibrosis of the metanephros in rats as judged by imaging

Chronic kidney disease leads to high morbidity rates among humans. It is a serious disease that requires curative treatments other than kidney transplantation. Recently, we successfully established the iPS-derived generated kidney, which might produce urine. The urine can be directed to the native bladder with a stepwise peristaltic ureter system, followed by anastomosis with the recipient ureter for reconstruction of the urinary tract. However, the growth of the regenerated kidney varies significantly, whereas the time window of the anastomosis is quite narrow. Therefore, this study was conducted to evaluate the growth of transplanted metanephros with bladder periodically and noninvasively using computed tomography and ultrasonography. Ultrasonographic findings showed high correlations with computed tomographic findings and clearly evaluated metanephros with bladder. We found that the degree of growth of the metanephros with bladder after the transplantation differed in each individual. However, most of them reached the appropriate period for urinary tract reconstruction within 3 weeks after transplantation. Optimizing the stepwise peristaltic ureter system anastomosis by ultrasonography reduced long-term tubular dilation of the metanephros, thereby decreasing fibrosis caused by transforming growth factor-{beta}. This may be significantly related to long-term maturation of fetal grafts. These results provide new insights into transplanting regenerated kidneys in higher animals. We are one step closer to the first human trial of kidney generation.

developmental biology↗

bioRxiv2020

Inhibition mechanism of human sterol O-acyltransferase 1 by competitive inhibitor

Sterol O-acyltransferase 1 (SOAT1) is an endoplasmic reticulum (ER) resident, multi-transmembrane enzyme that belongs to the membrane-bound O-acyltransferase (MBOAT) family 1. It catalyzes the esterification of cholesterol to generate cholesteryl esters for cholesterol storage 2. SOAT1 is a target to treat several human diseases 3. However, its structure and mechanism remain elusive since its discovery. Here, we report the structure of human SOAT1 (hSOAT1) determined by cryo-EM. hSOAT1 is a tetramer consisted of a dimer of dimer. The structure of hSOAT1 dimer at 3.5 [A] resolution reveals that the small molecule inhibitor CI-976 binds inside the catalytic chamber and blocks the accessibility of the active site residues H460, N421 and W420. Our results pave the way for future mechanistic study and rational drug design of SOAT1 and other mammalian MBOAT family members.

biochemistry↗

bioRxiv2017

Propofol induces metabolic reprogramming and cell death in a mitochondrial electron transport chain-dependent manner

The intravenous anesthetic propofol (2,6-diisopropylphenol) has been used for the induction and maintenance of anesthesia in operating rooms and for sedation in intensive care units. Although there is no widely accepted definition of propofol infusion syndrome (PRIS), PRIS is defined as the development of metabolic acidosis, rhabdomyolysis, hyperkalemia, hepatomegaly, renal failure, arrhythmia, and progressive cardiac failure. In vitro evidence suggests that PRIS is related to the impaired mitochondrial function. There are indications that preexisting mitochondrial disorders predispose to PRIS. However, the precise molecular mechanisms, including mitochondrial defects and a metabolic conversion by propofol, are largely unknown as yet. To elucidate the underlying cellular and molecular mechanisms of PRIS, we investigated the effects of propofol on the cellular metabolic mode and cell death. We demonstrated that clinically relevant concentrations of propofol, used within a clinically relevant exposure time, suppressed the mitochondrial function, caused the generation of reactive oxygen species, and induced a metabolic switch, from oxidative phosphorylation to glycolysis, by targeting complexes I and III of mitochondria. The data also indicated that a predisposition to mitochondrial dysfunction, caused by a genetic mutation or pharmacological suppression of the electron transport chain by biguanides such as metformin and phenformin, promoted the cell death and caspase activation induced by propofol.

pharmacology and toxicology↗