Biology subjects

Osaki, T.

Publications and source records attributed to Osaki, T..

15 recordsLinked to original sources

bioRxiv2026

Selective Recognition, Covalent Linkage and Chemical Exchange of Artificial Cells through Engineered α-Hemolysin

Bottom-up synthetic biology aims not only to build individual artificial cells but also to connect them into higher-order systems that communicate with each other. Reported artificial cells communicate through partner recognition, physical connection, and chemical signaling that are typically implemented by separate molecular species. Here, we engineer the pore-forming protein -hemolysin (AH) with the SpyTag-SpyCatcher (ST-SC) pair to integrate partner recognition, covalent linkage between cells, and nanopore-mediated connection of their lumens into a single protein system. Fusion of ST or SC to AH preserved heptamer assembly and pore formation, and allowed the two heptamers (AH-ST and AH-SC) to be covalently joined. Cryo-electron microscopy resolved the resulting head-to-head 14-mer assembly at 2.20 [A]. Calcein leakage and single-channel recordings confirmed that the engineered pores remain functional. Mixing ST- and SC-displaying giant unilamellar vesicles (GUVs) resulted in partner-specific association between the two vesicle populations. Finally, photo-triggered release of a newly synthesized caged ethidium bromide (EtBr) in sender GUVs produced EtBr accumulation in receiver GUVs that required direct physical linkage between the two GUVs. This platform provides a modular route toward artificial-cell assemblies in which both connectivity and cargo exchange are programmable.

synthetic biology↗

bioRxiv2026

CHARIOT-AAV: Conjugation of diverse vectors to adeno-associated viruses for delivery of large genes

Systemic, tissue-specific delivery of large transgenes exceeding the packaging capacity of adeno-associated viruses (AAVs) remains a key translational challenge for molecular therapeutics. Vectors with larger capacities, such as lentiviral vectors (LVVs) and lipid nanoparticles (LNPs), often lack adjustable, tissue-specific tropisms. Here we report CHARIOT-AAV (Crosslinked Hybrid Architectures for Robust, Interchangeable, and Organ-specific Targeting with AAV), a platform where diverse delivery vectors are conjugated to AAVs, thereby achieving tissue-specific tropism of AAVs and expanded cargo capacity. AAV-AAV conjugates packaging split SpCas9 constructs in AAV.CAP-B10 capsids demonstrate a [~]2-fold increase in brain gene editing efficiency over unconjugated AAV cocktails after intravenous injection. In addition to AAV-AAV conjugates, AAV-LVV and AAV-LNP conjugates achieve AAV-guided delivery of genetic payloads to target cells. Furthermore, AAV-LNP conjugates enable systemic delivery of mRNAs to brain endothelial cells. CHARIOT-AAV thus provides a modular platform for systemic, tissue-specific delivery of diverse therapeutics beyond the limits of individual vectors.

bioengineering↗

bioRxiv2025

Genome-scale dissection of phase-variable gene function in Campylobacter jejuni using a stabilized phasotype library

Phase variation (PV) enables bacterial pathogens to rapidly alter their surface structures through reversible mutations in simple sequence repeats, promoting immune evasion and environmental adaptation. In Campylobacter jejuni, the stochastic nature of PV has hindered the systematic functional analysis of phase-variable genes (PVGs). Here, we introduce PV-GenShift, a genome-scale screening platform built on a genetically stabilized library of phase-locked C. jejuni variants. By fixing the ON/OFF states of 15 PVGs, PV-GenShift enables reproducible, high-resolution analysis of phasotypes, defined as unique ON/OFF combinations across multiple PVGs, under defined selective pressures. Using models of human serum exposure, murine colonization, and chicken gut passage, we identified distinct phasotypes associated with serum resistance and with enrichment during mouse colonization, particularly involving capsular polysaccharide modifications such as O-methyl phosphoramidation and methylation. In contrast, chicken gut passage resulted in heterogeneous ON/OFF shifts without a dominant phasotype. These findings highlight the combinatorial impact of PVG expression states on bacterial adaptation and establish PV-GenShift as a broadly applicable framework for dissecting PV-driven phenotypic diversity. This approach provides a scalable strategy for exploring genotype-phenotype relationships and offers insights relevant to vaccine design and targeted therapeutics. Significance StatementPhase variation generates phenotypic diversity that enables pathogens to evade immunity and adapt to changing environments; however, its random nature has long obscured functional analysis. This study introduces PV-GenShift, a genome-scale platform that stabilizes phase-variable gene expression in Campylobacter jejuni, allowing the systematic identification of gene combinations that influence survival under selective pressures. Using PV-GenShift, we identified phasotypes associated with serum resistance and enrichment during mouse colonization, while chicken passage produced diverse but non-specific shifts. These results demonstrate how combinatorial ON/OFF states of multiple genes shape bacterial adaptation and provide a generalizable strategy for studying phase variation across pathogens, with implications for vaccine design and targeted therapeutics.

microbiology↗

bioRxiv2025

Identification of KKL-35 as a novel carnosine dipeptidase 2 (CNDP2) inhibitor by in silico screening

Extracellular glutathione (GSH) is degraded on the cell surface, in which the {gamma}-glutamyl residue is removed to generate cysteine-glycine (Cys-Gly) dipeptides that are subsequently transported to the cytoplasm. Carnosine dipeptidase II (CNDP2) is a cytoplasmic enzyme that hydrolyzes Cys-Gly and plays an important role in maintaining intracellular cysteine (Cys) homeostasis. CNDP2-mediated hydrolysis of Cys-Gly promotes Cys mobilization and contributes to the replenishment of intracellular GSH levels. CNDP2 is frequently overexpressed in various cancers and has been implicated in tumor cell proliferation and progression. This mechanism may enhance cancer cell survival by causing resistance to oxidative stress, which indicates that CNDP2 is a potential therapeutic target for cancer treatment. Although bestatin (BES) has been identified as a CNDP2 inhibitor, its limited specificity and suboptimal drug-like properties have limited its therapeutic potential. In this study, we performed an in silico screen of a small-molecule compound library and identified KKL-35 as a novel CNDP2-binding molecule. Molecular dynamics (MD) simulations suggested that KKL-35 interacts within the catalytic pocket. Biochemical assays confirmed that it inhibits CNDP2 enzymatic activity, albeit with lower potency compared with BES. Despite its modest intrinsic activity, KKL-35 exhibits favorable physicochemical and pharmacokinetic properties, which are characterized by a low topological polar surface area (TPSA), reduced molecular flexibility, and well-balanced lipophilicity. This positions it as an attractive and tractable starting point for lead optimization. Taken together, these findings establish KKL-35 as a validated CNDP2 inhibitor and a promising lead compound for the development of more selective therapeutics targeting CNDP2-mediated cancer cell metabolism.

bioinformatics↗

bioRxiv2025

Cell-based multisensor array for detecting cancer biomarkers in human urine

Detecting volatile urinary biomarkers offers a compelling strategy for noninvasive diagnosis of diseases such as cancer. Biohybrid sensors employing sensor cells that express olfactory receptors--with finely tuned molecular recognition capabilities--are garnering increasing attention. However, their application to physiological samples remains challenging due to variability in sensor cell responsiveness and limited signal intensity. Here, we report a volatile biomarker detection platform based on a multisensor array in which insect olfactory receptor- expressing cells are encapsulated in hydrogel and housed within microwells featuring vertical slits. This structural design enables dense three-dimensional cell immobilization and rapid, uniform exposure to target molecules, enhancing the sensitivity of fluorescence responses and minimizing signal variability. Multiple types of sensor cells on a single array enabled the selective and simultaneous detection of potential cancer-related target molecules--acetophenone, phenol, and 6-methyl-5-hepten-2-one. Furthermore, through hexane extraction combined with gas-phase delivery, we reliably detected acetophenone introduced into human urine at micromolar concentrations. This platform offers a compact, reproducible, and scalable solution for volatile biomarker detection using cell-based sensors.

bioengineering↗

bioRxiv2024

miR126-mediated impaired vascular integrity in Rett syndrome

Rett syndrome (RTT) is a neurodevelopmental disorder that is caused by mutations in melty-CpG binding protein 2 (MeCP2). MeCP2 is a non-cell type-specific DNA binding protein, and its mutation influences not only neural cells but also non-neural cells in the brain, including vasculature associated with endothelial cells. Vascular integrity is crucial for maintaining brain homeostasis, and its alteration may be linked to the pathology of neurodegenerative disease, but a non-neurogenic effect, especially the relationship between vascular alternation and Rett syndrome pathogenesis, has not been shown. Here, we recapitulate a microvascular network using Rett syndrome patient-derived induced pluripotent stem (iPS) cells that carry MeCP2[R306C] mutation to investigate early developmental vascular impact. To expedite endothelial cell differentiation, doxycycline (DOX)-inducible ETV2 expression vectors were inserted into the AAVS1 locus of Rett syndrome patient-derived iPS cells and its isogenic control by CRISPR/Cas9. With these endothelial cells, we established a disease microvascular network (Rett-dMVNs) and observed higher permeability in the Rett-dMVNs compared to isogenic controls, indicating altered barrier function by MeCP2 mutation. Furthermore, we unveiled that hyperpermeability is involved in the upregulation of miR126-3p in Rett syndrome patient-derived endothelial cells by microRNA profiling and RNAseq, and rescue of miR126-3p level can recover their phenotype. We discover miR126-3p-mediated vascular impairment in Rett syndrome patients and suggest the potential application of these findings for translational medicine.

cell biology↗

bioRxiv2024

Early differential impact of MeCP2 mutations on functional networks in Rett syndrome patient-derived human cerebral organoids

Human cerebral organoids derived from induced pluripotent stem cells can recapture early developmental processes and reveal changes involving neurodevelopmental disorders. Mutations in the X-linked methyl-CpG binding protein 2 (MECP2) gene are associated with Rett syndrome, and disease severity varies depending on the location and type of mutation. Here, we focused on neuronal activity in Rett syndrome patient-derived organoids, analyzing two types of MeCP2 mutations - a missense mutation (R306C) and a truncating mutation (V247X) - using calcium imaging with three-photon microscopy. Compared to isogenic controls, we found abnormal neuronal activity in Rett organoids and altered network function based on graph theoretic analyses, with V247X mutations impacting functional responses and connectivity more severely than R306C mutations. These changes paralleled EEG data obtained from patients with comparable mutations. Labeling DLX promoter-driven inhibitory neurons demonstrated differences in activity and functional connectivity of inhibitory and excitatory neurons in the two types of mutation. Transcriptomic analyses revealed HDAC2-associated impairment in R306C organoids and decreased GABAA receptor expression in excitatory neurons in V247X organoids. These findings demonstrate mutation-specific mechanisms of vulnerability in Rett syndrome and suggest targeted strategies for their treatment.

bioengineering↗

bioRxiv2024

Polyploidy of MDA-MB-231 cells drives increased extravasation with enhanced cell-matrix adhesion

Metastasis, the leading cause of cancer-related deaths, involves a complex cascade of events, including extravasation. Despite extensive research into metastasis, the mechanisms underlying extravasation remain unclear. Molecular targeted therapies have advanced cancer treatment, yet their efficacy is limited, prompting exploration into novel therapeutic targets. Here, we showed the association of polyploidy in MDA-MB-231 breast cancer cells and their extravasation, using microfluidic systems to reproduce the in vivo microvascular environment. We observed enhanced extravasation in polyploid cells alongside upregulated expression of genes involved in cell-substrate adhesion and cell mechanical dynamics. These findings offer insights into the relationship between polyploidy and extravasation, highlighting potential targets for cancer therapy.

bioengineering↗

bioRxiv2024

Nuclear assembly in giant unilamellar vesicles encapsulating Xenopus egg extract

The reconstitution of a cell nucleus in a lipid bilayer-enclosed synthetic cell makes great strides in bottom-up synthetic biology. In this study, we propose a method for assembling a nucleus in giant unilamellar vesicles (GUVs). To induce reconstitution of the nucleus, we utilize interphase egg extract of African clawed frogs Xenopus laevis, known as a biochemically controllable cell-free system capable of transforming an added sperm chromatin into a nucleus in vitro. We enhanced GUV formation efficiency by the inverted emulsion method through incorporating prolonged waiting time and adding chloroform into lipid-dispersed oil, facilitating subsequent nuclear assembly reactions in the GUVs.Characterization of nucleus-like structures formed in the GUVs revealed the presence of dense DNA and accumulated GFP-NLS in the structure, indicative of functional nuclear import. Immunostaining further validated the presence of nuclear pore complexes on the surfaces of these nucleus-like structures. Our approach offers a versatile platform for constructing artificial cellular systems that closely mimic eukaryotic cells. TeaserA cell nucleus is reconstituted in lipid bilayer-enclosed confinements using egg extract from African clawed frogs.

synthetic biology↗

bioRxiv2023

The A53T mutation in α-synuclein enhances pro-inflammatory activation in human microglia

Parkinsons disease (PD) is characterized by the aggregation of -synuclein into Lewy bodies and Lewy neurites in the brain. Microglia-driven neuroinflammation may contribute to neuronal death in PD, however the exact role of microglia remains unclear and has been understudied. The A53T mutation in the gene coding for -synuclein has been linked to early-onset PD, and exposure to A53T-mutant human -synuclein increases the potential for inflammation of murine microglia. To date, its effect has not been studied in human microglia. Here, we used 2-dimensional cultures of human iPSC-derived microglia and transplantation of these cells into the mouse brain to assess the effects of the A53T mutation on human microglia. We found that A53T-mutant human microglia had an intrinsically increased propensity towards pro-inflammatory activation upon inflammatory stimulus. Additionally, A53T mutant microglia showed a strong decrease in catalase expression in non-inflammatory conditions, and increased oxidative stress. Our results indicate that A53T mutant human microglia display cell-autonomous phenotypes that may worsen neuronal damage in early-onset PD.

neuroscience↗

bioRxiv2022

Mito-FUNCAT-FACS reveals cellular heterogeneity in mitochondrial translation

Mitochondria possess their own genome that encodes components of oxidative phosphorylation (OXPHOS) complexes, and mitochondrial ribosomes within the organelle translate the mRNAs expressed from mitochondrial genome. Given the differential OXPHOS activity observed in diverse cell types, cell growth conditions, and other circumstances, cellular heterogeneity in mitochondrial translation can be expected. Although individual protein products translated in mitochondria have been monitored, the lack of techniques that address the variation in overall mitochondrial protein synthesis in cell populations poses analytic challenges. Here, we adapted mitochondrial-specific fluorescent noncanonical amino acid tagging (FUNCAT) for use with fluorescence-activated cell sorting (FACS) and developed mito-FUNCAT-FACS. The click chemistry-compatible methionine analog L-homopropargylglycine (HPG) enabled the metabolic labeling of newly synthesized proteins. In the presence of cytosolic translation inhibitors, HPG was selectively incorporated into mitochondrial nascent proteins and conjugated to fluorophores via the click reaction (mito-FUNCAT). The application of in situ mito-FUNCAT to flow cytometry allowed us to disentangle changes in net mitochondrial translation activity from those of the organelle mass and detect variations in mitochondrial translation in cancer cells. Our approach provides a useful methodology for examining mitochondrial protein synthesis in individual cells.

molecular biology↗

bioRxiv2021

eIF2B-capturing viral protein NSs suppresses the integrated stress response

Various stressors such as viral infection lead to the suppression of cap-dependent translation and the activation of the integrated stress response (ISR), since the stress-induced phosphorylated eukaryotic translation initiation factor 2 [eIF2(P)] tightly binds to eIF2B to prevent it from exchanging guanine nucleotides on unphosphorylated eIF2. Sandfly fever Sicilian virus (SFSV) evades this cap-dependent translation suppression through the interaction between its nonstructural protein NSs and host eIF2B. Our cryo-electron microscopy (cryo-EM) analysis revealed that SFSV NSs binds to the -subunit of eIF2B in a competitive manner with eIF2(P). Together with SFSV NSs, eIF2B exhibits normal nucleotide exchange activity even in the presence of eIF2(P). A genome-wide ribosome profiling analysis clarified that SFSV NSs in human cultured cells attenuates the ISR. Furthermore, SFSV NSs exhibited neuroprotective effects against the ISR-inducing stress. Since the ISR inhibition is beneficial in various neurological disease models, SFSV NSs is promising as a therapeutic ISR inhibitor.

molecular biology↗

bioRxiv2021

Complex Activity and Short-term Memories in Reciprocally Connected Cerebral Organoids

Macroscopic axonal connections in the human brain distribute information and neuronal activity across the brain. Although brain organoid technologies have recently provided novel avenues to investigate human brain function by replicating small neuronal networks of the brain in vitro, functionality of the macroscopic connections between distant regions has not been investigated with organoids. Here, we describe the neural activity of human cerebral organoids reciprocally connected by a bundle of axons. The connected organoids produced significantly more intense and complex oscillatory activity than conventional cerebral organoids. Optogenetic manipulations revealed that the connected organoids could respond to external stimuli and maintain the elevated frequency of neuronal activity for a short term, indicating that the connected organoids exhibit plasticity as a neuronal network. Our findings highlight functional importance of inter-regional connections and suggest that connected organoids can be used as a powerful tool for investigating the development and functions of macroscopic circuits in the human brain. can allow researchers to develop and test compounds to modulate activity of in vitro neuronal network models in unprecedented ways.

neuroscience↗

bioRxiv2019

A widespread family of heat-resistant obscure (Hero) proteins protect against protein instability and aggregation

Proteins are typically denatured and aggregated by heat. Exceptions to this principle include highly disordered and heat-resistant proteins found in extremophiles, which help these organisms tolerate extreme conditions such as drying, freezing, and high salinity. In contrast, the functions of heat-soluble proteins in non-extremophilic organisms including humans remain largely unexplored. Here we report that heat-resistant obscure (Hero) proteins, which remain soluble after boiling at 95{degrees}C, are widespread in Drosophila and humans. Hero proteins are hydrophilic and highly charged, and function to stabilize various "client" proteins, protecting them from denaturation even under stress conditions such as heat shock, desiccation, and exposure to organic solvents. Hero proteins can also block several different types of pathological protein aggregations in cells and in Drosophila strains that model neurodegenerative diseases. Moreover, Hero proteins can extend lifespan of Drosophila. Our study reveals that organisms naturally use Hero proteins as molecular shields to stabilize protein functions, highlighting their biotechnological and therapeutic potential.

molecular biology↗

bioRxiv2018

Genome-wide association meta-analysis identifies novel GP2 gene risk variants for pancreatic cancer in the Japanese population

The etiology of pancreatic cancer remains largely unknown. Here, we report the results of a meta-analysis of three genome-wide association studies (GWASs) comprising 2,039 pancreatic cancer cases and 32,592 controls, the largest sample size in the Japanese population. We identified 3 (13q12.2, 13q22.1, and 16p12.3) genome-wide significant loci (P<5.0x10-8) and 4 suggestive loci (P<1.0x10-6) for pancreatic cancer. Of these risk loci, 16p12.3 is novel; the lead SNP maps to rs78193826 (odds ratio (OR)=1.46, 95% CI=1.29-1.66, P=4.28x10-9), an Asian-specific, nonsynonymous glycoprotein 2 (GP2) gene variant predicted to be highly deleterious. Additionally, the gene-based GWAS identified a novel gene, KRT8, which is linked to exocrine pancreatic and liver diseases. The identified GP2 gene variants were pleiotropic for multiple traits, including type 2 diabetes, hemoglobin A1c (HbA1c) levels, and pancreatic cancer. Mendelian randomization analyses corroborated causality between HbA1c and pancreatic cancer. These findings suggest that GP2 gene variants are associated with pancreatic cancer susceptibility in the Japanese population, prompting further functional characterization of this locus.

genetics↗