Biology subjects

Kasai, H.

Publications and source records attributed to Kasai, H..

12 recordsLinked to original sources

bioRxiv2026

Vorapaxar and aripiprazole suppress hepatitis B virus replication through distinct host signaling pathways

Background & AimsCurrent nucleos(t)ide analogs efficiently suppress hepatitis B virus (HBV) replication but have limited effects on viral transcription from covalently closed circular DNA (cccDNA) and integrated HBV DNA. We aimed to identify clinically applicable compounds that directly inhibit HBV transcription by screening FDA-approved drugs. Approach & ResultsScreening of 1,470 FDA-approved compounds using an HBV enhancer I/X promoter reporter system identified vorapaxar and aripiprazole as potent inhibitors of viral promoter activity. Both compounds suppressed HBV replication in HBV-producing cells, HBV-infected HepG2-hNTCP cells, and primary human hepatocytes. Aripiprazole reduced hepatocyte nuclear factor 4 (HNF4) protein levels through an ERK/JNK-dependent pathway and inhibited HBV core promoter activity, whereas vorapaxar acted independently of HNF4. Both compounds suppressed enhancer I/X promoter activity through inhibition of STAT3 signaling. Vorapaxar inhibited PAR-1-mediated SRC, EGFR, and STAT3 activation, while aripiprazole suppressed SRC-STAT3 signaling independently of EGFR. PAR-1 activation enhanced HBV transcription, whereas PAR-1 knockdown reduced promoter activity and viral RNA expression. Both compounds also reduced HBV replication in human liver chimeric mice at clinically relevant exposure levels without apparent severe toxicity. ConclusionsVorapaxar and aripiprazole suppress HBV transcription and replication through distinct host signaling pathways. These findings identify PAR-1-STAT3 signaling as a previously unrecognized regulator of HBV transcription and suggest that host-targeting approaches may complement current therapies by suppressing viral gene expression from both cccDNA and integrated HBV DNA. Impact and implicationsCurrent nucleos(t)ide analogues effectively suppress HBV reverse transcription but have limited effects on viral transcription from cccDNA and integrated HBV DNA, highlighting the need for therapies targeting viral gene expression. We identify PAR-1- STAT3 signaling as a previously unrecognized regulator of HBV transcription and demonstrate that two clinically approved drugs, vorapaxar and aripiprazole, suppress HBV replication through distinct host signaling pathways. These findings are relevant to researchers developing host-targeting antivirals and to clinicians seeking complementary therapeutic strategies beyond current nucleos(t)ide analogue therapy. Although further clinical validation and combination studies are required, our results provide a rationale for repurposing approved drugs and for developing transcription-targeting therapies that may complement existing treatments for chronic hepatitis B. HighlightsO_LIVorapaxar and aripiprazole suppress HBV through distinct host pathways. C_LIO_LIBoth drugs inhibit HBV replication in vitro and in humanized liver mice. C_LIO_LIPAR-1 inhibition reduces HBV transcription by blocking SRC/EGFR/STAT3 signaling. C_LIO_LIPAR-1-STAT3 signaling is a novel regulator of HBV transcription. C_LIO_LIHost-targeting antiviral therapy complements current HBV treatment. C_LI

pharmacology and toxicology↗

bioRxiv2026

uniSynK: a ratio-optimized bicistronic chemically induced dimerization tool for robust induction of dendritic spine enlargement

Synaptic plasticity is widely implicated in learning and brain-state regulation. Although correlations between synaptic modifications and diverse brain functions have been extensively reported, establishing causality requires tools that can directly and selectively perturb synapses in vivo. Recently, the chemogenetic actuator SYNCit-K (SynK) enabled causal induction of dendritic spine enlargement and synaptic potentiation. However, SynK relies on the co-expression of two independent components delivered by separate viral vectors, making it difficult to reliably achieve appropriate expression levels of both components within the same neuron. Here, using mouse neocortical dissociated culture, we sought to develop a unified, single-vector version of SynK (uniSynK) that enables robust and safe induction of dendritic spine enlargement. We first found that each SynK component induces distinct, concentration-dependent abnormalities in neuronal morphology when overexpressed, underscoring the need for precise control of their expression ratio. To address this challenge, we engineered a single construct in which the two components are linked by internal ribosome entry site (IRES) variants with graded translational efficiencies. Systematic screening of IRES variants identified an optimized configuration that preferentially drives each component within its respective concentration range that preserves normal spine morphology. The optimized uniSynK efficiently induces dendritic spine enlargement, while exhibiting fewer off-target morphological abnormalities than observed in the multi-vector system when expression levels were not properly controlled. By simplifying experimental design, this single-vector, ratio-optimized synaptic chemogenetic tool provides a practical and broadly applicable platform that should facilitate causal interrogation of synaptic plasticity across neural circuits and behavior. Significance StatementSynaptic plasticity is thought to shape learning, brain states, and neuropsychiatric disease, but testing its causal roles requires tools that can directly manipulate synapses with minimal side effects. Here, we developed uniSynK, a single-vector chemogenetic system that induces dendritic spine enlargement by coordinating the expression of two functional components. By defining safe expression ranges and using IRES variants to optimize their ratio, uniSynK achieves robust spine enlargement while reducing unintended morphological abnormalities associated with imbalanced expression. This work provides both a practical tool for synaptic manipulation and a general strategy for optimizing multi-component biological systems.

neuroscience↗

bioRxiv2025

Generation of a HiBiT-expressing recombinant rat hepacivirus supporting both in vivo and in vitro infection

The lack of immuno-competent animal models of hepatitis C virus (HCV) infection has been an obstacle to vaccine development and research on immune responses. Hepacivirus ratti (Norway rat hepacivirus-1: NRHV1) is a virus closely related to HCV that specifically infects the liver and induces hepatocellular carcinoma in rats, making it a promising surrogate model for HCV. NRHV1 expressing a reporter gene serves as a powerful tool for analyzing the in vivo dynamics and pathogenicity mechanisms of NRHV1. In this study, we developed a reporter NRHV1 capable of infection and replication in both mice and cultured cells and established a platform for generating diverse reporter viruses. A reporter virus containing the HiBiT gene in the coding region of NS5A domain III was constructed using circular polymerase extension reaction (CPER). Infection with this reporter virus led to HiBiT activity in infected cells and the activity was correlated with the amount of intracellular viral RNA. In addition, this reporter virus established persistent infection in NOD-SCID mice and led to the generation of HiBiT activity in the livers of infected mice. Furthermore, reporter virus recovered from infected mice could infect and generate HiBiT activity in cultured cells. These results demonstrate that the reporter virus can infect hepatocytes in vivo and in vitro. This platform provides a versatile tool for in vitro quantitative antiviral screening and in vivo pathogenicity studies. Author summaryHepatitis C virus (HCV) infects millions of people and causes severe liver diseases, yet vaccine development has been hindered by the lack of practical small-animal models with intact immunity. Hepacivirus ratti (NRHV1), a close relative of HCV that naturally infects rats, is a promising HCV surrogate, but tools for monitoring NRHV1 infection dynamics have been limited. Here, we developed the first reporter NRHV1 by inserting a small luminescent HiBiT tag into a tolerable region of the NS5A protein. Using a CPER-based reverse genetics system, we generated a recombinant virus that efficiently infected cultured cells and established persistent infection in mice while retaining liver tropism. HiBiT activity closely reflected viral RNA levels, enabling the quantitative monitoring of infection and sensitive evaluation of antiviral drugs and neutralizing antibodies. This reporter NRHV1 system provides a versatile platform for studying hepacivirus pathogenesis and antiviral immunity and will support future efforts toward HCV vaccine development.

microbiology↗

bioRxiv2025

Blockade of associative spine enlargement impairs awake cortical function

Awake cognition is usually attributed to arousal systems and ongoing electrical activity, yet these do not explain how cortical processing is coupled to prior context and memory formation. Associative synaptic plasticity provides a cellular mechanism for such coupling, but is viewed as memory-related rather than an online requirement for cognition. Here we developed SynC, an A/C-activated chemogenetic perturbation that selectively and reversibly blocks associative spine enlargement while sparing non-associative spine dynamics, excitability, synaptic transmission and NMDA receptor-mediated responses. In mice with broad frontoparietal SynC expression, acute A/C reversibly impaired open-field centre exploration, abolished laser-dot chasing, and delayed feeding initiation. During quiet-wake immobile epochs, cortical firing rates and {gamma}-band power were preserved, but pairwise spike correlations were reduced. Mice also intermittently showed sudden behavioural arrest with reduced {gamma} activity but without the robust {delta} elevation of slow-wave sleep. Critically, in vivo two-photon glutamate uncaging induced seconds-scale associative spine enlargement in [~]24% of neocortical spines, acutely blocked by SynC-A/C. These A/C-induced behavioural, circuit and spine-enlargement effects recovered within 1 h. Thus, wake-like firing and {gamma} activity are not sufficient to sustain awake cognitive function. Rapid associative spine enlargement is required to maintain functional cortical coupling and stable wakefulness.

neuroscience↗

bioRxiv2025

Identification of claudin-3 as an entry factor for rat hepacivirus

Approximately 58 million people worldwide are believed to be infected with hepatitis C virus (HCV), a major causative agent of chronic liver diseases. Hepacivirus ratti strain rn-1, which was discovered from Rattus norvegicus (Norway rat) and designated Norway rat hepacivirus 1 (NRHV1), shares similar properties with HCV in terms of genetic homology, target cell tropism, pathogenicity and the immune response. In vivo infection systems for NRHV1 will help overcome the challenges in HCV research for vaccine development. However, the virological characteristics of NRHV1, such as the mechanisms of cell entry, remain largely unexplored, in part owing to a paucity of cell culture systems for NRHV1. Here, we identified the host factors that facilitate NRHV1 entry by profiling the gene expression of two cell lines with different susceptibilities to NRHV1 infection. NRHV1 employs rodent orthologues of HCV entry factors, including scavenger receptor class BI, CD81, and occludin, and utilizes claudin-3 (CLDN3) but not claudin-1. The expression of rat and mouse, but not human, CLDN3 facilitates the entry of NRHV1 into murine cell lines that are insusceptible to NRHV1 infection. The host-specific cell entry of CLDN3 is determined by two amino acid residues, Ile44 and Trp46, in extracellular loop 1. These findings suggest that some hepacivirus species share an HCV-like entry mechanism but show evidence of adaptive evolution of their virus-specific entry factors to host animals.

microbiology↗

bioRxiv2024

HCV infection activates the proteasome via PA28γ acetylation and heptamerization to facilitate the degradation of RNF2, a catalytic component of polycomb repressive complex 1.

We previously reported that hepatitis C virus (HCV) infection or HCV core protein expression induces HOX gene expression by impairing histone H2A monoubiquitination via a proteasome-dependent reduction in the level of RNF2, a key catalytic component of polycomb repressive complex 1 (PRC1) (J. Virol, 2021, 95, e01784-20). In this study, we aimed to investigate the mechanism by which HCV infection accelerates RNF2 degradation. Yeast two-hybrid screening and an immunoprecipitation assay revealed that RNF2 is a PA28{gamma}-binding protein. The proteasome activator PA28{gamma} destabilized the RNF2 protein in a proteasome-dependent manner, since RNF2 degradation was impaired by PA28{gamma} knockout or MG132 treatment. HCV infection or core protein expression reduced the levels of RNF2 and histone H2A K119 monoubiquitination and induced the expression of HOX genes in the presence of PA28{gamma}, while PA28{gamma} knockout reversed these changes. Treatment with a lysine acetyltransferase inhibitor inhibited the acetylation of PA28{gamma} at K195 and the degradation of the RNF2 protein, while treatment with a lysine deacetylase inhibitor accelerated these events in a PA28{gamma}-dependent manner. RNF2 protein degradation was increased by expression of the acetylation mimetic PA28{gamma} mutant but not by expression of the acetylation-defective mutant or the proteasome activation-defective mutant. Furthermore, HCV infection or core protein expression facilitated the interaction between PA28{gamma} and the lysine acetyltransferase CBP/p300 and then accelerated PA28{gamma} acetylation and heptamerization to promote RNF2 degradation. These data suggest that HCV infection accelerates the acetylation-dependent heptamerization of PA28{gamma} to increase the proteasomal targeting of RNF2. IMPORTANCEHCV is a causative agent of HCV-related liver diseases, including hepatic steatosis, cirrhosis and hepatocellular carcinoma. PA28{gamma}, which, in heptameric form, activates the 20S core proteasome for the degradation of PA28{gamma}-binding proteins, is responsible for HCV-related liver diseases. HCV core protein expression or HCV infection accelerates RNF2 degradation, leading to the induction of HOX gene expression via a decrease in the level of H2Aub on HOX gene promoters. However, the mechanism of RNF2 degradation in HCV-infected cells has not been clarified. The data presented in this study suggest that PA28{gamma} acetylation and heptamerization are promoted by HCV infection or by core protein expression to activate the proteasome for the degradation of RNF2 and are responsible for HCV propagation. This study provides novel insights valuable for the development of therapies targeting both HCV propagation and HCV-related diseases.

microbiology↗

bioRxiv2021

Empirical estimation of marine phytoplankton assemblages in coastal and offshore areas using an in situ multi-wavelength excitation fluorometer

Phytoplankton assemblages are important for understanding the quality of primary production in marine ecosystems. Here, we describe development of a methodology for monitoring marine phytoplankton assemblages using an in situ multi-wavelength excitation fluorometer (MEX) and its application for seasonal observations in coastal and offshore areas around Japan. The MEX recorded the fluorescence excited with nine light-emitting diodes, temperature, and sensor depth. We prepared reference datasets comprising temperature, MEX fluorescence, and plant-pigment-based chemotaxonomy phytoplankton assemblages. Target MEX fluorescence was decomposed by reference MEX fluorescence using a linear inverse model for calculating coefficients after the reference data were limited by temperature, followed by reconstruction of plant-pigment-based chemotaxonomy of the target MEX fluorescence using the coefficients and the chemotaxonomy assemblages of the reference data. Sensitivity analysis indicated poor estimation of the proportion and/or chlorophyll a-based abundance of chlorophytes, haptophytes, prasinophytes, and prochlorophytes; however, limiting the estimations to five chemotaxonomic groups [diatoms, dinoflagellates, cryptophytes, cyanobacteria (cyanophytes and prochlorophytes), and other eukaryotes (chlorophytes, haptophytes, and prasinophytes)] resulted in positive correlations of both the proportion and abundances, suggesting that the five taxonomic abundances were well-estimated using the MEX. Additionally, MEX observations denoted spatial and seasonal variations of phytoplankton assemblages, with high contributions from other eukaryotes in every area and season, cyanobacteria highly during the summer in surface Kuroshio and Japan Sea waters, and diatoms in the Oyashio and Oyashio-Kuroshio transition areas and the Okhotsk Sea. Furthermore, ratios of water-column-integrated chlorophyll-based abundances to those on the surface at the chemotaxonomy group level were differed among the areas and groups. These findings suggested that phytoplankton-assemblage monitoring in the vertical direction is essential for evaluation of their current biomass, and that the MEX promotes the acquisition of these observations.

ecology↗

bioRxiv2021

Computational Roles of Intrinsic Synaptic Dynamics

Conventional theories assume that long-term information storage in the brain is implemented by modifying synaptic efficacy. Recent experimental findings challenge this view by demonstrating that dendritic spine sizes, or their corresponding synaptic weights, are highly volatile even in the absence of neural activity. Here we review previous computational works on the roles of these intrinsic synaptic dynamics. We first present the possibility for neuronal networks to sustain stable performance in their presence and we then hypothesize that intrinsic dynamics could be more than mere noise to withstand, but they may improve information processing in the brain. Highlights- Synapses exhibit changes due to intrinsic as well as extrinsic dynamics - Computational frameworks suggest stable network performance despite intrinsic changes - Intrinsic dynamics might be beneficial to information processing

neuroscience↗

bioRxiv2019

Computational characteristics of the striatal dopamine system described by reinforcement learning with fast generalization

Generalization enables applying past experience to similar but nonidentical situations. Therefore, it may be essential for adaptive behaviors. Recent neurobiological observation indicates that the striatal dopamine system achieves generalization and subsequent discrimination by updating corticostriatal synaptic connections in differential response to reward and punishment. To analyze how the computational characteristics in this system affect behaviors, we proposed a novel reinforcement learning model with multilayer neural networks in which the synaptic weights of only the last layer are updated according to the prediction error. We set fixed connections between the input and hidden layers so as to maintain the similarity of inputs in the hidden-layer representation. This network enabled fast generalization, and thereby facilitated safe and efficient exploration in reinforcement learning tasks, compared to algorithms which do not show generalization. However, disturbance in the network induced aberrant valuation. In conclusion, the unique computation suggested by corticostriatal plasticity has the advantage of providing safe and quick adaptations to unknown environments, but on the other hand has the potential defect which can induce maladaptive behaviors like delusional symptoms of psychiatric disorders. Author summaryThe brain has an ability to generalize knowledge obtained from reward- and punishment-related learning. Animals that have been trained to associate a stimulus with subsequent reward or punishment respond not only to the same stimulus but also to resembling stimuli. How does generalization affect behaviors in situations where individuals are required to adapt to unknown environments? It may enable efficient learning and promote adaptive behaviors, but inappropriate generalization may disrupt behaviors by associating reward or punishment with irrelevant stimuli. The effect of generalization here should depend on computational characteristics of underlying biological basis in the brain, namely, the striatal dopamine system. In this research, we made a novel computational model based on the characteristics of the striatal dopamine system. Our model enabled fast generalization and showed its advantage of providing safe and quick adaptation to unknown environments. By contrast, disturbance of our model induced abnormal behaviors. The results suggested the advantage and the shortcoming of generalization by the striatal dopamine system.

neuroscience↗

bioRxiv2019

The minimal behavioral time window for reward conditioning in the nucleus accumbens of mice

The temporal precision of reward-reinforcement learning is determined by the minimal time window of the reward action--theoretically known as the eligibility trace. In animal studies, however, such a minimal time window and its origin have not been well understood. Here, we used head-restrained mice to accurately control the timing of sucrose water as an unconditioned stimulus (US); we found that the reinforcement effect of the US occurred only within 1 s after a short tone of a conditioned stimulus (CS). The conditioning required the dopamine D1 receptor and CaMKII signaling in the nucleus accumbens (NAc). The time window was not reduced by replacing CS with optogenetic stimulation of the synaptic inputs to the NAc, which is in agreement with previous reports on the effective dopamine timing of NAc synapses. Thus, our data suggest that the minimal reward time window is 1 s, and is formed in the NAc.

neuroscience↗

bioRxiv2019

Stringent structural plasticity of dendritic spines revealed by two-photon glutamate uncaging in adult mouse neocortex in vivo

Two-photon uncaging of glutamate is widely utilized to characterize structural plasticity in brain slice preparations in vitro. In this study, we investigated spine plasticity by using, for the first time, glutamate uncaging in the neocortex of adult mice in vivo. Spine enlargement was successfully induced in a smaller fraction of spines in the neocortex (22%) than in young hippocampal slices (95%), even under a low magnesium condition. Once induced, the time course and mean amplitudes of long-term enlargement were the same (81%) as those in vitro. However, low-frequency (1-2 Hz) glutamate uncaging caused spine shrinkage in a similar fraction (34%) of spines as in vitro, but spread to the neighboring spines less frequently than in vitro. Thus, we found that structural plasticity can occur similarly in the adult neocortex in vivo as in the hippocampus in vitro, although it happens stringently in a smaller subset of spines.

neuroscience↗

bioRxiv2019

Intrinsic spine dynamics are critical for recurrent network learning in models with and without autism spectrum disorder

It is often assumed that Hebbian synaptic plasticity forms a cell assembly, a mutually interacting group of neurons that encodes memory. However, in recurrently connected networks with pure Hebbian plasticity, cell assemblies typically diverge or fade under ongoing changes of synaptic strength. Previously assumed mechanisms that stabilize cell assemblies do not robustly reproduce the experimentally reported unimodal and long-tailed distribution of synaptic strengths. Here, we show that augmenting Hebbian plasticity with experimentally observed intrinsic spine dynamics can stabilize cell assemblies and reproduce the distribution of synaptic strengths. Moreover, we posit that strong intrinsic spine dynamics impair learning performance. Our theory explains how excessively strong spine dynamics, experimentally observed in several animal models of autism spectrum disorder, impair learning associations in the brain.

neuroscience↗