Biology subjects

Ishikawa, J.

Publications and source records attributed to Ishikawa, J..

8 recordsLinked to original sources

bioRxiv2026

Structural mechanism of the type IX Retron-Kva2 anti-phage defense system

Retrons are prokaryotic genetic elements that protect bacterial cells from invading phages. The type IX retron comprises a non-coding RNA, a reverse transcriptase (RT), and dual effectors: a putative HEPN nuclease and a winged helix-turn-helix (WH) protein. Here, we show that the type IX retron system from Klebsiella variicola cleaves host rRNAs and tRNAs upon phage infection, mediating anti-phage defense via an abortive-infection mechanism. Cryo-electron microscopy analysis reveals that the RT, HEPN, and WH proteins, along with multicopy single-stranded DNA (msDNA), form a unique, sheet-like supermolecular complex. Notably, the HEPN active site is encircled by the WH and msDNA within the complex, suppressing the nuclease activity prior to phage infection. A phage-encoded exonuclease cleaves the msDNA, likely releasing the HEPN nuclease to cleave host RNAs and induce growth arrest in infected cells. Overall, these findings highlight the structural and functional diversity of prokaryotic anti-phage defense systems.

biochemistry↗

bioRxiv2026

Coordinated synthesis of double-stranded DNA by a dual reverse transcriptase immune system

Defense-associated reverse transcriptase (DRT) systems mediate antiviral immunity through distinct modes of cDNA synthesis: Class 1 DRTs catalyze untemplated synthesis, whereas Class 2 DRTs polymerize noncoding RNA-templated products. However, how these distinct modes drive defense remains unclear. Here, we report that DRT3 immunity arises when Class 1 and Class 2 RT activities cooperate to produce self-complementary double-stranded DNA (dsDNA). DRT3a uses a 5'-ACACAC-3' RNA template to synthesize poly-(dTdG) repeats, whereas DRT3b synthesizes poly-(dCdA) repeats without any nucleic acid template. Cryo-electron microscopy reveals that DRT3b forms a hexamer and uses active-site-adjacent residues as deoxyadenosine and deoxycytidine gates to enforce alternating nucleotide addition, representing a unique example of amino acid-templated DNA polymerization. DRT3 is toxic in cells lacking RecBCD, implicating host recombination machinery in limiting dsDNA accumulation, and the phage-encoded RecBCD inhibitor Gam triggers DRT3-mediated abortive infection. These findings reveal how two polymerases with distinct templating strategies generate complementary DNA for defense.

microbiology↗

bioRxiv2026

2-arm-PEG-oligocations transiently shield the liver sinusoids to mitigate off-target hepatic expression of mRNA lipid nanoparticles

Ionizable lipid nanoparticles (iLNPs) are powerful platforms for mRNA-based vaccines and immunotherapies; however, their intrinsic liver tropism compromises both safety and efficacy. Off-target hepatic protein expression from delivered mRNA raises safety concerns, and hepatic clearance limits efficient iLNP delivery to target organs. In this study, we address these challenges in mouse models by stealth-coating the liver sinusoidal endothelial (LSE) wall, the primary gateway for nanoparticle entry into the liver. Specifically, oligocations conjugated with two-armed PEG (2-arm-PEG-oligocations), a clinically relevant material used in oligonucleotide delivery trials, were employed to transiently anchor PEG to the LSE wall with balanced affinity, ensuring robust coating followed by gradual biliary clearance. This approach reduced hepatic protein expression from iLNPs, subsequently administered either systemically or locally, by more than tenfold. Importantly, the strategy preserved iLNP accumulation in the spleen, a key target organ for vaccines, effectively redirecting iLNPs from the liver to the spleen. Consequently, in vaccine applications, pre-injection of the 2-arm-PEG-oligocation preserved or even enhanced vaccination efficacy while minimizing concerns associated with antigen expression in the liver. In applications involving cytokine mRNA therapy, specifically intratumoral interleukin-12 (IL-12) mRNA administration, systemic pre-injection of the 2-arm-PEG-oligocation successfully reduced off-target hepatic IL-12 expression and subsequent systemic IL-12 exposure, while maintaining antitumor efficacy. Collectively, these results demonstrate that LSE-wall stealth coating is a generalizable strategy to improve both the safety and efficacy of iLNP-based mRNA vaccines and immunotherapies.

bioengineering↗

bioRxiv2025

Episodic experience drives ripple reorganization and synaptic changes in the hippocampus

The hippocampus plays a key role in encoding episodic memory by transforming recent experience into persistent neuronal and synaptic modifications. However, the physiological processes that link real-world experience to coordinated network activity and synaptic reorganization remain incompletely understood. Here, we investigated how distinct types of episodic experience reshape ensemble firing dynamics and synaptic input in the hippocampal CA1 region of freely moving rats. We identified spontaneous super bursts, defined as brief episodes of high-frequency population firing, that emerged preferentially during emotionally salient experiences. These bursts were associated with an increase in ripple firing, defined as short-duration, high-frequency multi-unit spike activity occurring in association with sharp-wave ripples. Analysis of ripple firing patterns revealed experience-dependent diversification, reflected by increased information entropy after episodic experience. Ex vivo whole-cell patch-clamp recordings further demonstrated that miniature excitatory and inhibitory synaptic currents in CA1 pyramidal neurons underwent experience-specific reorganization. Together, these findings support a coordinated cascade in which episodic experience induces population-level ensemble activity, followed by diversification of ripple firing patterns and reorganization of excitatory and inhibitory synaptic inputs in hippocampal CA1. This coordination defines a population-level signature associated with experience-dependent encoding across hippocampal circuits. Key pointsO_LIThe hippocampus is essential for episodic memory, but how real-life experiences change brain activity and synaptic connections remains unclear. C_LIO_LIIn freely moving rats, emotionally salient experiences triggered brief bursts of high-frequency firing involving many neurons in the hippocampal CA1 region ("super bursts"). C_LIO_LIAfter these experiences, short high-frequency firing events linked to memory processing ("ripple firing") became more diverse in their timing and shape. C_LIO_LIRecordings from individual neurons showed that both excitatory and inhibitory synaptic inputs were reorganized in an experience-specific manner. C_LIO_LIThese results suggest that episodic experience is encoded through coordinated changes in population activity, ripple firing patterns, and synaptic inputs in hippocampal CA1. C_LI

neuroscience↗

bioRxiv2025

Combinational adjuvants delivered by ink-jet potentiate naked mRNA vaccines for robust protection against infectious diseases

The use of lipid nanoparticles (LNPs) to enhance delivery and immunogenicity of mRNA vaccines raises safety concerns due to strong reactogenicity. Developing naked mRNA vaccines, which can be delivered through liquid jet-injection to the skin, may offer a safer alternative, but their immunogenicity has been limited. To address this challenge, we screened clinically relevant adjuvants to enhance the potency of naked mRNA jet-injection vaccines in mouse models. Although many adjuvants showed little or inhibitory effects on inducing humoral or cellular immunity--despite their known ability to potentiate conventional non-mRNA vaccine platforms--we identified a combination of aluminum phosphate and CpG oligonucleotide that substantially enhanced both humoral and cellular immune responses with increased IgG2a/IgG1 ratio. This adjuvanted naked mRNA vaccine induced IgG2a and cellular immunity at levels comparable to those achieved with LNPs but with lower local and systemic reactogenicity. Mechanistic investigations revealed a key role for type I interferon signaling during immunization. Notably, this vaccine formulation in mice virtually eliminated infection by both SARS-CoV-2 and influenza A virus (A/California/7/2009 H1N1) in plaque-forming assays. Collectively, the adjuvanted naked mRNA jet-injection vaccine represents a novel, effective, and safe platform against infectious diseases, with potential for clinic-ready translational applications.

bioengineering↗

bioRxiv2025

Sox4 in Treg Cells Suppresses IL-10 Production via c-Maf Degradation and Exacerbates Type 2 Inflammation

The anti-inflammatory cytokine interleukin-10 (IL-10) produced by regulatory T (Treg) cells plays a crucial role in regulating allergic airway inflammation. However, the mechanisms governing IL-10 production in Treg cells remain unclear. In this study, we first identified Sox4 as a gene whose expression is down-regulated in IL-10-producing Treg cells. Comprehensive gene expression profiling of Sox4-deficient Treg cells and Sox4-overexpressing Treg cells revealed specific downregulation of Il10 and Ctla4 by Sox4. Sox4-deficient Treg cells exhibited increased IL-10 production and CTLA-4 expression both in vitro and in vivo and strongly suppressed Th2 cell proliferation compared with Sox4-expressing Treg cells. In a house dust mite (HDM)-induced asthma model, Treg-specific Sox4-deficient (Sox4-cKO) mice exhibited IL-10-dependent reductions in eosinophilic inflammation and Th2 cell infiltration in the airways. A comparison of Sox4-cKO and wild-type Treg cells in the same inflamed environment in mixed bone marrow chimeric mice with HDM-induced asthma revealed that Sox4-mediated suppression of c-Maf is cell-intrinsic. In addition, overexpression of Sox4 in Treg cells repressed the activation of c-Maf-regulated chromatin regions, including those around the Il10 and Ctla4 loci. Moreover, Sox4-induced suppression of IL-10 and CTLA-4 in Treg cells was rescued by forced expression of c-Maf. While Sox4 did not significantly affect Maf mRNA levels, Sox4 degraded c-Maf proteins in Treg cells through its 33 C-terminal residues. Via a domain distinct from its C-terminus, Sox4 physically interacted with c-Maf, subsequently facilitating its degradation through a ubiquitin-proteasome pathway. These findings elucidate a mechanism by which Sox4 influences the asthmatic responses by regulating the c-Maf/IL-10 axis in lung Treg cells.

immunology↗

bioRxiv2022

RNA-triggered protein cleavage and cell death by the RNA-guided type III-E CRISPR-Cas nuclease-protease complex

The type III-E Cas7-11 effector nuclease forms a complex with a CRISPR RNA (crRNA) and the putative caspase-like protease Csx29, catalyzes crRNA-guided target RNA cleavage, and has been used for RNA targeting in eukaryotic cells. Here, we report cryo-electron microscopy structures of the Cas7-11-crRNA-Csx29 complex with and without target RNA, and demonstrate that target RNA binding induces a conformational change in Csx29 and results in the protease activation. Biochemical analysis confirmed that Cas7-11-bound Csx29 cleaves Csx30 in a target RNA-dependent manner. Reconstitution of the system in bacteria uncovered Csx30-dependent cellular toxicity regulated by Csx31, and that Csx29-mediated cleavage produces toxic Csx30 fragments, promoting cell death. We find that Csx30 can bind both Csx31 and the associated sigma factor RpoE, suggesting Csx30 can inhibit RpoE and modulate cellular stress response towards infection. Overall, the RNA-guided nuclease-protease activities of the Cas7-11-Csx29 effector complex facilitate protease-based programmed cell death.

molecular biology↗

bioRxiv2019

A possible coding for experience: ripple-like events and synaptic diversity

The hippocampal CA1 is necessary to maintain experienced episodic memory in many species, including humans. To monitor the temporal dynamics of processing, we recorded multiple-unit firings of CA1 neurons in male rats experiencing one of four episodes for 10 min: restraint stress, social interaction with a female or male, or observation of a novel object. Before an experience, the neurons mostly exhibited sporadic firings with some synchronized (≈ 50 ms) ripple-like firing events in habituated home cage. After experience onset, restraint or social interaction with other rats induced spontaneous high-frequency firings (super bursts) intermittently, while object observation induced the events inconsistently. Minutes after experience initiation, CA1 neurons frequently exhibited ripple-like firings with less-firing silent periods. The number of ripple-like events depended on the episode experienced and correlated with the total duration of super bursts. Experience clearly diversified multiple features of individual ripple-like events in an episode-specific manner, sustained for more than 30 min in the home cage.Ex vivo patch clamp analysis further revealed experience-promoted synaptic plasticity. Compared with unexposed controls, animals experiencing the female, male, or restraint episodes showed cell-dependently increased AMPA- or GABAA receptor– mediated postsynaptic currents, whereas contact with a novel object increased only GABAergic currents. Multivariate ANOVA in multi-dimensional virtual space revealed experience-specific super bursts with subsequent ripple-like events and synaptic plasticity, leading us to hypothesize that these factors are responsible for creating experience-specific memory. It is possible to decipher encrypted experience through the deep learning of the orchestrated ripple-like firings and synaptic plasticity in multiple CA1 neurons.Competing Interest StatementThe authors have declared no competing interest.View Full Text

animal behavior and cognition↗