Biology subjects

Nishida, M.

Publications and source records attributed to Nishida, M..

17 recordsLinked to original sources

bioRxiv2026

Development of a genetically encoded supersulfide-dependent translocation reporter

Supersulfides are emerging sulfur-containing signaling molecules involved in redox regulation, mitochondrial function, and protein S-sulfhydration. However, their dynamic behavior in living mammalian systems remains poorly understood because existing analytical methods require destructive sample preparation or lack sufficient intracellular applicability. Here, we developed a genetically encoded supersulfide-dependent translocation reporter (SuTR) for mammalian cells and in vivo imaging. Although the previously reported probe psGFP failed to respond to supersulfides in mammalian cells, fusion of psGFP with the sulfide-responsive transcriptional repressor (SqrR) generated SuTR, a novel reporter that exhibited supersulfide-dependent translocation from the nucleus to the cytoplasm. Na2S2 and Na2S induced dose-dependent cytosolic translocation of SuTR, whereas Na2S showed no effect. Fluorescence recovery after photobleaching (FRAP) analysis revealed accelerated fluorescence recovery shortly after supersulfide stimulation, and overexpression of the endogenous supersulfide-producing enzyme Cysteinyl-tRNA Synthetase 2 (CARS2) similarly altered reporter dynamics. Mutational analyses demonstrated that reporter responsiveness depends on the DNA-binding activity of SqrR. Furthermore, SuTR successfully detected supersulfide induction in mouse liver in vivo following Na2S administration. These findings establish SuTR as a genetically encoded reporter for monitoring supersulfide dynamics in mammalian cells and tissues. HighlightsO_LIWe developed SuTR, a genetically encoded supersulfide-dependent translocation reporter. C_LIO_LISupersulfides induce nuclear-to-cytoplasmic translocation of SuTR C_LIO_LIFRAP enables rapid detection of endogenous and exogenous supersulfide responses C_LIO_LISuTR activity depends on the DNA-binding function of SqrR C_LIO_LISuTR enables visualization of supersulfide dynamics in mouse liver in vivo C_LI

Molecular Biology↗

bioRxiv2026

Lipid A counteracts doxorubicin-induced systemic dysfunction by boosting mitochondrial activity

Vaccine adjuvants are critical for enhancing immune responses and sustaining antibody production. Although their safety profiles are well established, assessments have largely focused on metabolic and excretory organs such as the liver and kidneys, with limited attention to the heart. Here, we systematically evaluated the cardiac effects of five representative adjuvants in mice: alum, MF59, AS03, Sigma Adjuvant Systems, and lipid A. None of the adjuvants impaired baseline cardiac contractile function. Notably, lipid A uniquely enhanced mitochondrial respiratory capacity in rat and human induced pluripotent stem cell-derived cardiomyocytes and promoted mitochondrial membrane hyperpolarization. We next examined its therapeutic potential in a doxorubicin (Dox)-induced heart failure model characterized by mitochondrial dysfunction. Co-administration of lipid A with influenza hemagglutinin (HA) antigen significantly ameliorated cardiac dysfunction. In parallel, lipid A prevented the Dox-induced decline in anti-HA antibody titers, an effect associated with preservation of splenic B cell populations. Collectively, these findings reveal a previously unappreciated cytoprotective dimension of lipid A, demonstrating that it not only potentiates immune responses but also counteracts chemotherapy-induced functional decline by enhancing mitochondrial activity.

pharmacology and toxicology↗

bioRxiv2025

Senotherapeutic potential against xeroderma pigmentosum

Xeroderma pigmentosum (XP) is an inherited photoaging syndrome caused by mutations in genes involved in the nucleotide excision repair (NER) pathway. XP patients exhibit hypersensitivity to ultraviolet (UV) radiation, leading to accelerated skin aging and requiring lifelong sun avoidance. Here, we demonstrate that UV-induced DNA damage triggers cellular senescence and up-regulates senescence-associated secretory phenotype (SASP) genes in melanocytes derived from an XP patient. To explore the potential therapeutics for XP, we developed a cisplatin-based drug screening system and identified JAK inhibitors and curcuminoids as promising senomorphic agents. In addition, two classes of senolytic agents, BCL-2-like protein inhibitors and HSP90 inhibitors, effectively eliminate senescent melanocytes. Further analysis demonstrates that senomorphic treatment effectively counteracts senescence and reduces SASP gene expression in XP-derived melanocytes. Moreover, genes in senescence-related pathways, including the JAK/STAT, Type I interferon (IFN-I), and PI3K/AKT pathways, which are activated by both UV irradiation and cisplatin treatment, are down-regulated by senomorphic treatment. This study highlights a potential senotherapeutic strategy for XP, which may help alleviate photoaging symptoms in XP patients.

cell biology↗

bioRxiv2025

Impact of Low-Dose Narrowband UV Radiation on Skin Tissue and Mast Cells in a Canine Model

Narrowband ultraviolet radiation (NB-UVR) therapy is widely utilized in dermatology worldwide. While the immunosuppressive effects of ultraviolet radiation are well-documented, its non-inflammatory effects remain poorly understood. This study evaluated the effects of sub-minimal erythema dose (MED) narrowband ultraviolet radiation using a canine model. The effects of narrowband-ultraviolet B (310 nm) and A2 wavelengths (320 and 330 nm) were compared. Initially, we demonstrated the therapeutic potential of NB-UVR in dogs with spontaneous atopic dermatitis (n = 6, p < 0.05). Following 300 mJ/cm{superscript 2} sub-MED ultraviolet radiation exposure, intradermal skin testing, skin mast cell counts, and RNA sequencing analysis were conducted. Additionally, cell viability (WST assay) and apoptosis (DAPI staining) were assessed in vitro using a canine mast cell line. Ultraviolet radiation exposure significantly reduced intradermal skin test scores across all wavelengths and decreased skin mast cell counts at 310 nm (n = 3, p < 0.05). In vitro, cell viability was significantly reduced at 310 and 320 nm (p < 0.05), and 310 nm irradiation specifically increased apoptotic cell numbers (p < 0.05). RNA sequencing identified 71 differentially expressed genes at 320 nm, with enrichment analysis highlighting muscle cell components and functions, potentially associated with arrector pili muscle activity. These findings suggest that 310 nm irradiation induces apoptosis, while 320 nm elicits non-inflammatory modulatory effects, highlighting distinct wavelength-specific therapeutic mechanisms of narrowband ultraviolet radiation.

immunology↗

bioRxiv2024

Physiological Evaluation of Far Infrared-Emitting Garments on Sleep and Thermoregulation

Garments engineered to emit far-infrared (FIR) spectral frequencies have potential conditioning benefits. This study aimed to explore the physiological effects of FIR-emitting clothes on sleep propensities involving thermoregulation and autonomic nervous function. In this double-blind, randomized, placebo-controlled, crossover study, fifteen healthy male participants (age 21.6 {+/-} 1.5 years, body mass index 23.1 {+/-} 1.9, Pittsburgh Sleep Quality Index 5.0 {+/-} 2.0) wore a whole-body FIR garment (wavelength: 5-20 m at 40{degrees}C), while a total body garment with the 100% polyester fiber without ceramic components was used as control for the intervention. To compare the physiological variables between the two electroencephalograms, the tympanic membrane temperature, sweating rate, skin temperature, and humidity were measured during sleep. Heart rate variability during sleep was also calculated. The percentage of rapid eye movement (REM) sleep was significantly higher in the FIR compared than in control (p = 0.027). We found a significant main effect of tympanic membrane temperature in time, equivalent to core body temperature. (2-way ANOVA; (F(26, 234)=4.046, p<0.001). Furthermore, the sweating rate was significantly lower when sleeping with FIR predominantly in the final phase of sleep (garment x time interaction (F(26,572)=55.05, p<0.001). Although there were no significant differences in skin surface temperature between the two garments, FIR provided significantly lower skin humidity during the middle of night sleep compared with the control. Regarding LF, a significant garment type x time interaction (F(1.87, 48.57)=3.36, p=0.046). Post-hoc analysis revealed that the LF value of FIR was higher in the final phase of sleep than that of control (p=0.027). Then present results suggest that sleeping with FIR in comparison to with control, has a beneficial effect to facilitate restorative sleep, presumably involving proper thermoregulation.

neuroscience↗

bioRxiv2024

Characterization of high affinity IgM and IgG monoclonal antibodies against norovirus variants GII.4 and GII.17

Human norovirus, a leading cause of viral gastroenteritis, results in significant global health and economic burden, requiring sensitive and accurate diagnosis and effective therapeutics and vaccines. In this study, we immunized mice with the virus like capsid particles of GII.4, a mainstream strain, and GII.17, a modern strain that began to circulate in 2014, and used hybridoma technology to generate hybridoma cells that produce norovirus-binding antibodies against GII.4 and GII.17, respectively. Selection of these hybridoma cells yielded monoclonal IgG and IgM antibodies against these strains. Characterization of these antibodies revealed that avidity effect by multivalent binding is necessary for IgM to bind to norovirus at high efficiency, while IgG achieve high affinity even by monovalent binding. Surface plasmon resonance and ELISA data suggest that the high density of antigen protrusion domain in the norovirus capsid, containing approximately protomers, facilitates IgM to bind to norovirus capsid with high efficiency.

biochemistry↗

bioRxiv2023

Growth inhibitory factor/metallothionein-3 is a sulfane sulfur-binding protein

Cysteine-bound sulfane sulfur atoms in proteins have received much attention as key factors in cellular redox homeostasis. However, the role of sulfane sulfur in zinc regulation has been underinvestigated. We report here that cysteine-bound sulfane sulfur atoms serve as ligands to hold and release zinc ions in growth inhibitory factor (GIF)/metallothionein-3 (MT-3) with an unexpected C-S-S-Zn structure. Oxidation of such a zinc/persulfide cluster in Zn7GIF/MT-3 results in the release of zinc ions, and intramolecular tetrasulfide bridges in apo-GIF/MT-3 efficiently undergo S-S bond cleavage by thioredoxin to regenerate Zn7GIF/MT-3. Three-dimensional molecular modeling confirmed the critical role of the persulfide group in the thermostability and Zn-binding affinity of GIF/MT-3. The present discovery raises the fascinating possibility that the function of other Zn-binding proteins is controlled by sulfane sulfur.

biochemistry↗

bioRxiv2023

NMR analysis of the homodimeric structure of a D-Ala-D-Ala metallopeptidase, VanX, from vancomycin-resistant bacteria

Bacteria that have acquired resistance to most antibiotics, particularly those causing nosocomial infections, create serious problems. Among these, the emergence of vancomycin-resistant enterococci was a tremendous shock considering that vancomycin is the last resort for controlling methicillin-resistant Staphylococcus aureus. Therefore, there is an urgent need to develop an inhibitor of VanX, a protein involved in vancomycin resistance. Although the crystal structure of VanX has been resolved, its asymmetric unit contains six molecules aligned in a row. We have developed a structural model of VanX as a stable dimer in solution, primarily utilizing nuclear magnetic resonance (NMR) residual dipolar coupling. Despite the 46 kDa molecular mass of the dimer, which is typically considered too large for NMR studies, we successfully assigned the main chain using an amino acid-selective unlabeling method. Because we found that the Zn2+-coordinating active sites in the dimer structure were situated in the opposite direction to the dimer interface, we generated an active monomer by mutating the dimer interface. The monomer consists of only 202 amino acids and is expected to be used in future studies to screen and improve inhibitors using NMR.

biochemistry↗

bioRxiv2023

In Vivo mRNA Hacking with Staple Oligomers Prevents Myocardial Hypertrophy

Summary paragraphThe elucidation of gene-silencing mechanisms by RNA interference (RNAi) and antisense oligomers has drawn increasing attention to nucleic acid medicine. However, several challenges remain to be overcome, such as in vivo stability1, target selectivity 2,3, drug delivery4,5, and induced innate immunity6. Here, we report a new, versatile, and highly-selective method to hack RNA by controlling RNA structure using short oligonucleotides (RNA hacking: RNAh) in living cells. The oligonucleotide, named Staple oligomer, hybridizes specifically to a target mRNA and artificially induces an RNA higher-order structure, RNA G-quadruplex (RGq)7, on the mRNA. As a result, the RGq allows effective suppression of the target protein translation. This technology does not require cooperation with bioprocesses including enzymatic reactions as in RNAi or antisense technologies, permitting the introduction of artificial nucleic acids into Staple oligomers to increase their in vivo stability without compromising their effectiveness. The method was validated by translational regulation of the mRNAs of TPM3, MYD88, and TRPC6, in a cell-free system and in living mammalian cells. In vivo application of the technology to TRPC6 mRNA allowed us to prevent cardiac hypertrophy in transverse aortic constriction (TAC)-treated mice with no detectable off-target effects. This technology provides new insights into gene therapy after RNAi and antisense technologies.

molecular biology↗

bioRxiv2022

Metabolic clogging of mannose triggers genomic instability via dNTP loss in human cancer cells

Mannose has anti-cancer activity that inhibits cell proliferation and enhances the efficacy of chemotherapy. How mannose exerts its anti-cancer activity, however, remains poorly understood. Here, using genetically engineered human cancer cells that permit the precise control of mannose metabolic flux, we demonstrate that the large influx of mannose exceeding its metabolic capacity induced metabolic remodeling, leading to the generation of slow-cycling cells with limited deoxyribonucleoside triphosphates (dNTPs). This metabolic remodeling impaired dormant origin firing required to rescue stalled forks by cisplatin, thus exacerbating replication stress. Importantly, pharmacological inhibition of de novo dNTP biosynthesis was sufficient to retard cell cycle progression, sensitize cells to cisplatin, and inhibit dormant origin firing, suggesting dNTP loss-induced genomic instability as a central mechanism for the anti-cancer activity of mannose.

cancer biology↗

bioRxiv2022

Activation of the urotensin-II receptor by anti-COVID-19 drug remdesivir induces cardiomyocyte dysfunction

Remdesivir is an antiviral drug used for COVID-19 treatment worldwide. Cardiovascular (CV) side effects have been associated with remdesivir; however, the underlying molecular mechanism remains unknown. Here, we performed a large-scale G-protein-coupled receptor (GPCR) screening in combination with structural modeling and found that remdesivir is a selective agonist for urotensin-II receptor (UTS2R). Functionally, remdesivir treatment induced prolonged field potential in human induced pluripotent stem cell (iPS)-derived cardiomyocytes and reduced contractility in neonatal rat cardiomyocytes, both of which mirror the clinical pathology. Importantly, remdesivir-mediated cardiac malfunctions were effectively attenuated by antagonizing UTS2R signaling. Finally, we characterized the effect of 110 single-nucleotide variants (SNVs) in UTS2R gene reported in genome database and found four missense variants that show gain-of-function effects in the receptor sensitivity to remdesivir. Collectively, our study illuminates a previously unknown mechanism underlying remdesivir-related CV events and that genetic variations of UTS2R gene can be a potential risk factor for CV events during remdesivir treatment, which collectively paves the way for a therapeutic opportunity to prevent such events in the future. One Sentence SummaryRemdesivir s activity as a selective agonist of urotensin-II receptor underlies its known cardiotoxicity in anti-viral therapy.

pharmacology and toxicology↗

bioRxiv2021

Glutamine deficiency in solid tumors confers resistance to ribosomal RNA synthesis inhibitors

Ribosome biogenesis involves the processing of precursor ribosomal RNAs (pre-rRNAs) and sequential assembly with ribosomal proteins. Here we report that nutrient deprivation severely impairs pre-rRNA processing and leads to the accumulation of unprocessed rRNAs. Upon nutrient restoration, the accumulated pre-rRNAs are processed into mature rRNAs that are utilized for ribosome biogenesis. Failure to accumulate pre-rRNAs under nutrient deprivation leads to perturbed ribosome assembly during nutrient restoration and subsequent apoptosis via uL5/uL18-mediated activation of p53. Restoration of glutamine alone activates p53 by triggering uL5/uL18 translation. Induction of uL5/uL18 protein synthesis by glutamine was dependent on the translation factor eukaryotic elongation factor 2 (eEF2), which was in turn dependent on Raf/MEK/ERK signalling. Depriving cells of glutamine prevents the activation of p53 by rRNA synthesis inhibitors. Our data reveals a mechanism that cancer cells can exploit to suppress p53-mediated apoptosis during fluctuations in environmental nutrient availability.

cancer biology↗

bioRxiv2021

A root for massive crown-of-thorns starfish outbreaks in the Pacific Ocean

Recurring outbreaks of crown-of-thorns starfish (COTS) severely damage healthy corals in the Western Pacific Ocean. To determine the source of outbreaking COTS larvae and their dispersal routes across the Western Pacific, complete mitochondrial genomes were sequenced from 243 individuals collected in 11 reef regions. Our results indicate that Pacific COTS comprise two major clades, an East-Central Pacific clade (ECP-C) and a Pan-Pacific clade (PP-C). The ECP-C consists of COTS from French Polynesia (FP), Fiji, Vanuatu and the Great Barrier Reef (GBR), and does not appear prone to outbreaks. In contrast, the PP-C, which repeatedly spawns outbreaks, is a large clade comprising COTS from FP, Fiji, Vanuatu, GBR, Papua New Guinea, Vietnam, the Philippines, Japan, Micronesia, and the Marshall Islands. Given the nature of Pacific Ocean currents, the vast area encompassing FP, Fiji, Vanuatu, and the GBR likely supplies larvae for repeated outbreaks, exacerbated by anthropogenic environmental changes, such as eutrophication.

ecology↗

bioRxiv2021

Clomipramine suppresses ACE2-mediated SARS-CoV-2 entry

Myocardial damage caused by the newly emerged coronavirus (SARS-CoV-2) infection is one of key determinants of COVID-19 severity and mortality. SARS-CoV-2 entry to host cells are initiated by binding with its receptor, angiotensin converting enzyme (ACE) 2, and the ACE2 abundance is thought to reflect the susceptibility to infection. Here, we found that clomipramine, a tricyclic antidepressant, potently inhibits SARS-CoV-2 infection and metabolic disorder in human iPS-derived cardiomyocytes. Among 13 approved drugs that we have previously identified as potential inhibitor of doxorubicin-induced cardiotoxicity, clomipramine showed the best potency to inhibit SARS-CoV-2 spike glycoprotein pseudovirus-stimulated ACE2 internalization. Indeed, SARS-CoV-2 infection to human iPS-derived cardiomyocytes (iPS-CMs) and TMPRSS2-expressing VeroE6 cells were dramatically suppressed even after treatment with clomipramine. Furthermore, the combined use of clomipramine and remdesivir was revealed to synergistically suppress SARS-CoV-2 infection. Our results will provide the potentiality of clomipramine for the breakthrough treatment of severe COVID-19.

pharmacology and toxicology↗

bioRxiv2020

Robotic Search for Optimal Cell Culture in Regenerative Medicine

Induced differentiation is one of the most experience- and skill-dependent experimental processes in regenerative medicine, and establishing optimal conditions often takes years. We developed a robotic AI system with a batch Bayesian optimization algorithm that autonomously induces the differentiation of induced pluripotent stem cell-derived retinal pigment epithelial (iPSC-RPE) cells. The system performed 216 forty-day cell culture experiments, with a total experimentation time of 8,640 days. From 200 million possible parameter combinations, the system performed cell culture in 143 different conditions in 111 days, resulting in 88% better iPSC-RPE production than that by the pre-optimized culture in terms of pigmented scores. Our work demonstrates that the use of autonomous robotic AI systems drastically accelerates systematic and unbiased exploration of experimental search space, suggesting immense use in medicine and research.

systems biology↗

bioRxiv2020

Inducing Human Retinal Pigment Epithelium-like Cells from Somatic Tissue

Regenerative medicine relies on basic research to find safe and useful outcomes that are only practical when cost-effective. The human eyeball requires the retinal pigment epithelium (RPE) for support and maintenance that interfaces the neural retina and the choroid at large. Nearly 200 million people suffer from age-related macular degeneration (AMD), a blinding multifactor genetic disease among other retinal pathologies related to RPE degradation. Recently, autologous pluripotent stem cell-derived RPE cells were prohibitively expensive due to time, therefore we developed a new simplified cell reprogramming system. We stably induced RPE-like cells (iRPE) from human fibroblasts by conditional overexpression of broad plasticity and lineage-specific pioneering transcription factors. iRPE cells showed features of modern RPE benchmarks and significant in-vivo integration in transplanted chimeric hosts. Herein, we detail the iRPE system with comprehensive modern single-cell RNA (scRNA) sequencing profiling to interpret and characterize its best cells. We anticipate that our system may enable robust retinal cell induction for regenerative medicine research and affordable autologous human RPE tissue for cell therapy.

bioengineering↗

bioRxiv2020

Efficacy of a novel SARS-CoV-2 detection kit without RNA extraction and purification

Rapid detection of SARS-CoV-2 is critical for the diagnosis of coronavirus disease 2019 (COVID-19) and preventing the spread of the virus. A novel "2019 Novel Coronavirus Detection Kit (nCoV-DK)" halves detection time by eliminating the steps of RNA extraction and purification. We evaluated concordance between the nCoV-DK and direct PCR. The virus was detected in 53/71 fresh samples by the direct method and 55/71 corresponding frozen samples by the nCoV-DK. The overall concordance rate of the virus detection between the two methods was 94.4% (95% CI, 86.2-98.4). Concordance rates were 95.2% (95% CI, 83.8-99.4), 95.5% (95% CI, 77.2-99.9), 85.7% (95% CI, 42.1-99.6) in nasopharyngeal swab, saliva, and sputum samples, respectively. These results indicate that the nCoV-DK effectively detects SARS-CoV-2 in all types of the samples including saliva, while reducing time required for detection, labor, and risk of human error.

microbiology↗