Biology subjects

Hanada, K.

Publications and source records attributed to Hanada, K..

15 recordsLinked to original sources

bioRxiv2026

Early detection of pancreatic ductal adenocarcinoma by single-molecule profiling of pancreatic enzyme activities

Pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer-related mortality, largely due to diagnosis at advanced stages. Early detection through minimally invasive liquid biopsy holds promise for improving patient outcomes. Here, we report a blood-based liquid biopsy platform based on single-molecule enzyme activity profiling (SEAP), which detects proteoform-level alterations in circulating pancreatic enzymes at single-molecule resolution. Using a tissue-centric biomarker discovery strategy, we identified activity signatures of pancreas-specific digestive enzymes associated with PDAC. A combinatorial classifier detected PDAC (stage I-IV) with 95.5% specificity and 75.0% sensitivity (74.2% for stage I-II) across 690 blood samples collected from multiple hospitals and biobanks. Performance was further validated in an independent cohort enriched for early-stage disease, where 54.2% (13/24) of stage IA, 64.3% (9/14) of stage IB, and 21.4% (3/14) of stage 0 lesions were classified as positive. These findings support the clinical potential of SEAP for early detection of PDAC.

biochemistry↗

bioRxiv2025

Concentration-dependent suppression of macrolide resistance in Mycobacterium avium by combination therapy: an improved in vitro time-kill assay and PK/PD modelling study

ObjectivesMacrolides are key drugs in the treatment for Mycobacterium avium pulmonary disease, and combination chemotherapy is essential to preventing macrolide resistance. However, the concentrations of concomitant agents required to suppress resistance emergence remain undefined. This study aimed to quantify the concentration-dependent effects of companion drugs on macrolide resistance using an improved time-kill assay and pharmacokinetic/pharmacodynamic (PK/PD) modelling approach. MethodsTime-kill assays were performed on M. avium ATCC 700898 using azithromycin alone or in combination with ethambutol, rifampicin, amikacin, or clofazimine. Total and resistant bacterial populations were quantified over 28 days. A pharmacodynamic (PD) model describing the dynamics of susceptible and resistant subpopulations was linked to a pharmacokinetic (PK) model incorporating alveolar macrophage concentrations to simulate resistance and bactericidal rates under various dosing regimens. ResultsMacrolide resistance most frequently emerged at 2-4x the minimum inhibitory concentration (MIC) of azithromycin, whereas resistance was less frequent at 8-16x MIC or sub-MIC levels. The addition of companion drugs suppressed resistance emergence even at sub-MIC levels. PK/PD simulations demonstrated that standard-dose ethambutol effectively prevented macrolide resistance, whereas rifampicin provided minimal protection. More frequent dosing schedules enhanced resistance suppression compared with once-daily regimens. ConclusionThis study defines the concentration-dependent contributions of concomitant agents to macrolide resistance suppression in M. avium and establishes a quantitative PK/PD framework for evaluating resistance emergence. These findings provide mechanistic insights to support the optimization of combination dosing strategies for M. avium pulmonary disease.

pharmacology and toxicology↗

bioRxiv2024

Carotenoid pigments enhance rhodopsin-mediated phototrophy by light-harvesting and photocycle-accelerating

Microbial rhodopsins, photoreceptor proteins widely distributed in marine microorganisms, receive large amount of light energy that sustains marine ecosystems. Although rhodopsins generally harbor retinal as their only chromophore, a recent study reported that carotenoid antennae transfer light energy to the retinal in proteorhodopsins, proton pump rhodopsins abundant in marine environments. Here, using marine bacterial isolates, we detected energy transfer from a carotenoid (myxol) to retinal not only in proteorhodopsin but also in the chloride ion-pumping rhodopsin. Carotenoid binding improved the light utilization efficiency of the proteorhodopsin by accelerating the photocycle, together with facilitating light-harvesting. The carotenoid-binding ability is conserved in rhodopsins of the phylum Bacteroidota, which are widely transcribed in the photic zone. These findings suggest that the distribution of carotenoid-binding rhodopsins is more taxon-specific than previously thought, thus underscoring the importance of carotenoid-binding rhodopsins that provide an extended light utilization strategy in the environmental adaptation of marine bacteria.

microbiology↗

bioRxiv2024

Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

Lipogenesis-driven metabolic flux is crucial for differentiation of pathogenic Th17 cells. Although our previous CRISPR-based screening identified PLA2G12A as a key player in this process, it has remained obscure how this secreted phospholipase A2 isoform controls Th17 differentiation. Here we show that global, T cell-specific, or fibroblast-specific deletion of PLA2G12A prevents Th17 differentiation and associated diseases including psoriasis and arthritis. PLA2G12A acts on Th17-derived extracellular vesicles (EVs) to produce lysophospholipids including the ROR{gamma}t activator 1-oleoyl-lysophosphatidylethanolamine. These lysophospholipids are further converted by autotaxin to lysophosphatidic acid (LPA), which assists Th17 differentiation mainly via LPA2 receptor. Moreover, PLA2G12A promotes the secretion and uptake of EVs by Th17 cells and alters their cargo contents. Defective Th17 differentiation by PLA2G12A deficiency is rescued by supplementation with PLA2G12A-modified EVs. Importantly, a PLA2G12A-blocking antibody prevents Th17 differentiation and ameliorates psoriasis and arthritis models. Thus, targeting the PLA2G12A-EV-lysophospholipid axis may be useful for treatment of Th17-related diseases.

immunology↗

bioRxiv2024

Florigen-producing cells express FPF1-LIKE PROTEIN 1 that accelerates flowering and stem growth in long days with sunlight red/far-red ratio in Arabidopsis

Seasonal changes in spring induce flowering by expressing the florigen, FLOWERING LOCUS T (FT), in Arabidopsis. FT is expressed in unique phloem companion cells with unknown characteristics. The question of which genes are co-expressed with FT and whether they have roles in flowering remains elusive. Through tissue-specific translatome analysis, we discovered that under long-day conditions with the natural sunlight red/far-red ratio, the FT-producing cells express a gene encoding FPF1-LIKE PROTEIN 1 (FLP1). The master FT regulator, CONSTANS (CO), controls FLP1 expression, suggesting FLP1s involvement in the photoperiod pathway. FLP1 promotes early flowering independently of FT, is active in the shoot apical meristem, and induces the expression of SEPALLATA 3 (SEP3), a key E-class homeotic gene. Unlike FT, FLP1 facilitates inflorescence stem elongation. Our cumulative evidence indicates that FLP1 may act as a mobile signal. Thus, FLP1 orchestrates floral initiation together with FT and promotes inflorescence stem elongation during reproductive transitions.

plant biology↗

bioRxiv2023

Cytosolic heme metabolism by alternative localization of heme oxygenase 1 in plant cells

Heme, an organometallic molecule, is widely engaged in oxygen transport, electron delivery, enzymatic reactions, and signal transduction. Additionally, heme serves as a precursor to phytochromobilin, the chromophore of plant phytochrome. Heme oxygenase (HO) initiates the first committed step in heme metabolism. Our transcription start site-sequencing (TSS-seq) revealed that HO1 in Arabidopsis thaliana and Oryza sativa (rice) has two TSSs, producing long (HO1L) and short (HO1S) transcripts, with or without an intact N-terminal plastid transit peptide. HO1L and HO1S products localize in plastids and the cytosol, respectively. In Arabidopsis, HO1L is prevalent in light-exposed shoots, while HO1S is clearly detected in roots and etiolated seedlings. During de-etiolation and early development, HO1L ratio gradually rises and HO1S ratio decreases. Light perception via phytochrome and cryptochrome elevates HO1L ratio and reduce HO1S ratio through the functioning of HY5 and HYH transcription factors, and the suppression of DET1, E3 ubiquitin ligase COP1, and PIFs transcription factors. As expected, HO1L product was able to complement the HO1-deficient mutant gun2 (hy1), but surprisingly, HO1S expression could also restore the short hypocotyl phenotype and high pigment content, and make the mutant recover from the gun phenotype. This indicates the formation of functional holo-phytochrome within these lines. Our work highlights the presence of a cytosolic pathway for heme metabolism, especially during etiolation and early development. Furthermore, it supports the hypothesis that a mobile heme signal is involved in the mediation of retrograde signaling from the chloroplast. Significance StatementIn this research, through both TSS-seq and CAGE-seq, we discovered that the HO1 (GUN2 or HY1) gene in both Arabidopsis and rice has two TSSs, generating HO1L and HO1S transcripts. We reveal that the HO1 TSS regulation pathway is the same as the light signaling pathway. Significantly, our study identifies that a cytosolic heme metabolism pathway is existent in plant cells.

plant biology↗

bioRxiv2023

The cholesterol pathway of the Golgi stress response induces cell death and transcription of Golgi-related genes through metabolic dysregulation of phosphatidylinositol-4-phosphate

The Golgi stress response is an important cytoprotective system that enhances Golgi function in response to cellular demand, while cells damaged by prolonged Golgi stress undergo cell death to ensure the survival of organisms. OSW-1, a natural compound with anticancer activity, acts as a potent inhibitor of OSBP that transports cholesterol and phosphatidylinositol-4-phosphate (PI4P) at contact sites between the endoplasmic reticulum and the Golgi apparatus. Previously, we reported that OSW-1 induces the Golgi stress response, resulting in Golgi stress-induced transcription and cell death. However, the underlying molecular mechanism has been unknown. To reveal the mechanism of a novel pathway of the Golgi stress response regulating transcriptional induction and cell death (the cholesterol pathway), we performed a genome-wide knockout screen and found that transcriptional induction as well as cell death induced by OSW-1 was repressed in HeLa cells deficient in factors involved in the PI4P metabolism, such as PITPNB and PI4KB genes. Our data indicate that OSW-1 induces Golgi stress-dependent transcriptional induction and cell death through dysregulation of the PI4P metabolism in the Golgi apparatus.

cell biology↗

bioRxiv2022

Establishment of Measles Virus Receptor-expressing Vero Cells Lacking Functional Poliovirus Receptors

Global efforts are underway to eliminate measles and rubella and active viral surveillance is key to achieving this goal. In addition to this, the World Health Organization announced guidelines for handling materials potentially infectious for poliovirus (PV) to minimize the risk of PV reintroduction and achieve PV eradication. To support the global efforts, we have established new PV-non-susceptible cell lines useful for the isolation of measles virus (MeV) and rubella virus (RuV) (Vero {Delta}PVR1/2 hSLAM+). In the cell lines, MeV and RuV replicated efficiently with no concern about PV replication.

microbiology↗

bioRxiv2022

Fc-modified SARS-CoV-2 neutralizing antibodies with therapeutic effects in two animal models.

The use of therapeutic neutralizing antibodies against SARS-CoV-2 infection has been highly effective. However, there remain few practical antibodies against viruses that are acquiring mutations. In this study, we created 494 monoclonal antibodies from COVID-19-convalescent patients, and identified antibodies that exhibited comparable neutralizing ability to clinically used antibodies in the neutralization assay using pseudovirus and authentic virus including variants of concerns. These antibodies have different profiles against various mutations, which were confirmed by cell-based assay and cryo-electron microscopy. To prevent antibody-dependent enhancement, N297A modification was introduced, and showed a reduction of lung viral RNAs by therapeutic administration in a hamster model. In addition, an antibody cocktail consisting of three antibodies was also administered therapeutically to a macaque model, which resulted in reduced viral titers of swabs and lungs and reduced lung tissue damage scores. These results showed that our antibodies have sufficient antiviral activity as therapeutic candidates.

microbiology↗

bioRxiv2021

Whole-genome sequencing of Vero E6 (C1008) and comparative analysis of four Vero cell sublines

The Vero cell line is an immortalized cell line established from kidney epithelial cells of the African green monkey. A variety of sublines have been established from the original cell line, which display different characteristics. In this study, we determined the whole-genome sequence of Vero E6 (C1008) and performed comparative analysis among Vero JCRB 0111, Vero CCL-81, Vero 76 and Vero E6. Analysis of the copy number changes and loss of heterozygosity revealed that all sublines share a large deletion and loss of heterozygosity on chromosome 12, which harbors type I interferon and CDKN2 gene clusters. We identified a substantial number of genetic differences among the sublines including single nucleotide variants, indels, and copy number variations. The spectrum of single nucleotide variants indicated a close genetic relationship between Vero JCRB0111 and Vero CCL-81, and between Vero 76 and Vero E6, and a considerable genetic gap between the former two and the latter two lines. In contrast, we confirmed the pattern of genomic integration sites of simian endogenous retroviral sequences, which was consistent among the sublines. We identified subline-specific/enriched loss of function and missense variants, which potentially contribute to the differences in response to viral infection among the Vero sublines. In particular, we focused on Vero E6-specific/enriched variants and identified four genes (IL1RAP, TRIM25, RB1CC1, and ATG2A) that contained missense variants specific or enriched in Vero E6. In addition, we found that V739I variants of ACE2, which functions as the receptor for SARS-CoV-2, were heterozygous in Vero JCRB0111, Vero CCL-81, and Vero 76; however, Vero E6 contained the allele with isoleucine, resulting from the loss of one of the X chromosomes.

genomics↗

bioRxiv2021

In vivo hemostatic capability of a novel Tetra-PEG hydrogel

TetraStat is a novel synthetic sealant created with a tetra-armed polyethylene glycol (PEG) hydrogel. It has no risk of infection from biological pathogens and has a hemostatic mechanism independent of the blood coagulation pathway and controllable gelation. We evaluated the hemostatic effect of TetraStat in ex vivo and in vivo experiments for future clinical application. In ex vivo experiments using a circulatory system filled with phosphate-buffered saline under high pressure, needle punctures were astricted with TetraStat and two commercially available hemostatic agents (SURGICEL and TachoSil). For in vivo experiments, rat vena cavae were punctured with 14, 18, and 20 gauge needles, and hemorrhage occurred for several seconds. A porous PEG sponge soaked with TetraStat was applied as a hemostatic system for the massive hemorrhage. In the ex vivo experiment, punctures were sealed completely after 1 min astriction with TetraStat gel; in contrast, SURGICEL and TachoSil failed to seal the hole. In vivo experiments demonstrated that TetraStat successfully caused hemostasis in the punctured vena cava within 1 min of application in a dose-dependent manner. For SURGICEL and TachoSil, successful hemostasis occurred after 5 min astriction but was less frequent after 1 min astriction. Ex vivo and in vivo experiments revealed TetraStats high hemostatic ability under high pressure and in rat vena cava injuries under massive hemorrhage. A porous PEG sponge soaked with TetraStat is a promising advancement in hemostatic systems.

bioengineering↗

bioRxiv2020

Poliovirus-nonsusceptible Vero cell line for the World Health Organization global action plan

Polio or poliomyelitis is a disabling and life-threatening disease caused by poliovirus (PV). As a consequence of global polio vaccination efforts, wild PV serotype 2 has been eradicated, and wild PV serotypes 1- and 3-transmitted cases have been largely eliminated except for in limited regions around the world. However, vaccine-derived PV, pathogenically reverted live PV vaccine strains in vaccinated humans, has become a serious issue. For the global eradication of polio, the World Health Organization is conducting the third edition of the Global Action Plan, which is requesting stringent control of potentially PV-infected materials. To facilitate the mission, we generated a PV-nonsusceptible Vero cell subline, which may serve as an ideal replacement of standard Vero cells to isolate emerging/re-emerging viruses without the risk of generating PV-infected materials.

microbiology↗

bioRxiv2020

Identification of characteristic genomic markers in human hepatoma Huh7 and Huh7.5.1-8 cell lines

The human hepatoma-derived Huh7 cell line and its derivatives (Huh7.5 and Huh7.5.1) have been widely used as a convenient experimental substitute for primary hepatocytes. In particular, these cell lines represent host cells suitable for propagating the hepatitis C virus (HCV) in vitro. The Huh7.5.1-8 cell line, a subline of Huh7.5.1, can propagate HCV more efficiently than its parental cells. To provide genomic information for cells quality control, we performed whole-genome sequencing of Huh7 and Huh7.5.1-8 and identified their characteristic genomic deletions, some of which are applicable to an in-house test for cell authentication. Among the genes related to HCV infection and replication, 53 genes were found to carry missense or loss-of-function mutations likely specific to the Huh7 and/or Huh7.5.1-8. Eight genes, including DDX58 (RIG-I), BAX, EP300, and SPP1 (osteopontin), contained mutations observed only in Huh7.5.1-8 or mutations with higher frequency in Huh7.5.1-8. These mutations might be relevant to phenotypic differences between the two cell lines and may also serve as genetic markers to distinguish Huh7.5.1-8 cells from the ancestral Huh7 cells.

genomics↗

bioRxiv2019

Retrograde Transport of Golgi Enzymes by GOLPH3 Across Maturing Cisternae Regulates Glycan Assembly on Sphingolipids and CellGrowth

Glycans are ubiquitous sugar polymers with major biological functions that are assembled by glyco-enzymes onto cargo molecules during their transport through the Golgi complex. How the Golgi determines glycan assembly is poorly understood. By relying on the Golgi cisternal maturation model and using the glyco-enzyme adaptor and oncoprotein GOLPH3 as a molecular tool, we define the first example of how the Golgi controls glycosylation and associated cell functions. GOLPH3, acting as a component of the cisternal maturation mechanism, selectively binds and recycles a subset of glyco-enzymes of the glycosphingolipid synthetic pathway, hinders their escape to the lysosomes and hence increases their levels through a novel lysosomal degradation-regulated mechanism. This enhances the production of specific growth-inducing glycosphingolipids and reprograms the glycosphingolipid pathway to potentiate mitogenic signaling and cell proliferation. These findings unravel unforeseen organizing principles of Golgi-dependent glycosylation and delineate a paradigm for glycan assembly by the Golgi transport mechanisms. Moreover, they indicate a new role of cisternal maturation as a regulator of glycosylation, and outline a novel mechanism of action for GOLPH3-induced proliferation.

cell biology↗

bioRxiv2019

Colocalization of IQGAP1 with DLC-1 and PLC-δ1; Its potential role in coronary spasm

Phospholipase C (PLC)-{delta}1, activated by p122RhoGTPase-activating protein (GAP)/deleted in liver cancer-1 (p122RhoGAP/DLC-1), contributes to the coronary spastic angina (CSA) pathogenesis. The present study aims to further investigate the p122RhoGAP/DLC-1 protein. We examined molecules assisting this protein and identified a scaffold protein--IQ motif-containing GTPase-activating protein 1 (IQGAP1). IQGAP1-C binds to the steroidogenic acute regulatory-related lipid transfer (START) domain of p122RhoGAP/DLC-1, and PLC-{delta}1 binds to IQGAP1-N, forming a complex. In fluorescence microscopy, small dots of PLC-{delta}1 created fine linear arrays like microtubules, and IQGAP1 and p122RhoGAP/DLC-1 were colocated in the cytoplasm with PLC-{delta}1. Ionomycin induced the raft recruitment of the PLC-{delta}1, IQGAP1, and p122RhoGAP/DLC-1 complex by translocation to the plasma membrane (PM), indicating the movement of this complex is along microtubules with the motor protein kinesin. Moreover, the IQGAP1 protein was elevated in skin fibroblasts obtained from patients with CSA, and it enhanced the PLC activity and peak intracellular calcium concentration in response to acetylcholine. IQGAP1, a novel stimulating protein, forms a complex with p122RhoGAP/DLC-1 and PLC-{delta}1 that moves along microtubules and enhances the PLC activity.

molecular biology↗