Biology subjects

Miyazaki, K.

Publications and source records attributed to Miyazaki, K..

12 recordsLinked to original sources

bioRxiv2026

A rapidly deployable CRISPR-Cas3 diagnostic platform for emerging RNA viruses

Rapidly converting viral genome information into deployable molecular tests remains a major challenge in outbreak preparedness. We developed CONAN-SWIFT (Simple Workflow for Isothermal Field Testing), a sequence-to-test platform that integrates computational assay design, reverse-transcription loop-mediated isothermal amplification, CRISPR-Cas3 detection, reagent lyophilization and lateral-flow readout. Sequence-guided assays for Andes virus and Bundibugyo virus were established within approximately three weeks and extended to four additional filoviruses. A web-based designer supported crRNA selection, and systematic RT-LAMP primer optimization improved amplification performance. Recombinant Escherichia coli-expressed Cascade enabled standardized preparation of lyophilized Cas3-detection reagents, which were combined with a battery-operated isothermal device. The portable system detected as few as 10 input RNA copies per reaction within approximately 40 min. It also detected viral RNA and biologically contained, replication-incompetent Ebola virus in spiked human blood and concentrated wastewater. These findings establish the analytical feasibility of a rapidly adaptable CRISPR-Cas3 engineering framework for decentralized detection of emerging RNA viruses.

bioengineering↗

bioRxiv2025

Generative AI-based design of hybrid transcriptional activator proteins with new DNA-binding specificity

Transcriptional control arises from the specific recognition of promoter DNA by transcription factors (TFs), forming the basis of cellular information processing and gene regulation. In synthetic biology, TF-promoter interactions are assembled into genetic circuits to program cellular behaviors. To ensure expected circuit performance, most synthetic genetic circuits rely on combinations of well-characterized and orthogonal transcriptional regulations. This reliance minimizes crosstalk but constrains circuit complexity and information integration. Creating hybrid TFs that combine or interpolate promoter specificities could therefore expand the design space of synthetic regulatory systems. However, it remains unclear whether hybrid functions can be created by mixing amino acid sequences, and how such functional integration could be achieved in a principled manner. Here we show that a variational autoencoder (VAE) trained on LuxR-family DNA-binding domains can generate transcription factors with hybrid and partially novel promoter recognition properties. By sampling intermediate regions of the VAE-learned latent space, we designed hybrid TFs that activate both the lux and las promoters. High-throughput sort-seq assays together with individual in vivo assays revealed that a subset of functional variants exhibited dual-responsive behavior while maintaining sequence-selective DNA recognition. Together, these results provide a data-driven strategy for exploring functional intermediate sequences between closely related proteins.

synthetic biology↗

bioRxiv2025

Impaired store-operated Ca2+ entry in mouse epidermis leads to reduced epidermal barrier function via Klk activation.

Store-operated Ca2+ entry (SOCE) is a mechanism by which STIM1/2 detect calcium depletion in the endoplasmic reticulum (ER) and activate ORAI1/TRPC channels on the cell membrane to induce calcium influx into the cytoplasm. This study aimed to investigate SOCEs role in the skin epidermis in vivo, using female mice with epithelial tissue-specific Stim1 and Stim2 knockout (Stim1/2 cKO) and control mice (Stim1/2fl/fl). Keratinocytes from Stim1/2 cKO mice exhibited reduced Stim1 and Stim2 gene expression levels and impaired SOCE function compared with the controls. Histological analysis revealed hyperkeratosis in the Stim1/2 cKO tissues; however, no significant changes were observed in the proliferation or migration of keratinocytes or wound healing of the back skin. RNA-seq analysis indicated altered keratinization and cell-to-cell adhesion in the Stim1/2 cKO mice. Additionally, transepidermal water loss (TEWL) was increased significantly, and a biotinylated reagent diffused from the subcutaneous area into the granular layer, signifying compromised barrier function in the Stim1/2 cKO mice. The Stim1/2 cKO mice exhibited alterations in desmoglein 1 (Dsg1) and elevated levels of Kallikrein-related peptidase (Klk) 6 and Klk7, leading to increased trypsin- and chymotrypsin-like serine protease activities, respectively. These results suggest that SOCE dysfunction leads to hyperkeratosis and impaired epidermal barrier function via Klk activation without affecting skin cell proliferation in vivo. These novel findings improve understanding of the molecular mechanisms underlying calcium signaling in the epidermis barrier and suggest potential avenues for investigating related skin pathologies linked to calcium homeostasis, such as Hailey-Hailey disease (HHD) and Darier disease (DD).

cell biology↗

bioRxiv2025

Single-cell-level digital twins for preterm birth prevention strategies

Digital twin models can accelerate therapeutic development by enabling low-risk testing of candidate interventions. In preterm labor (PTL), a major pregnancy complication where clinical trials face unique ethical and financial barriers, digital twins are especially valuable for evaluating new therapies targeting immune dysfunctions driving PTL. Yet, current models lack single-cell resolution, limiting detection of cell-type-specific mechanisms, off-target effects, and the design of personalized interventions. We present Simulated Immunome Modeling of Clinical Outcomes (SIMCO), a single-cell-level digital twin framework that models immunomodulatory treatment effects on the timing of labor using immunome-wide, single-cell simulations. SIMCOs digital twins are trained and validated on a newly generated mass cytometry atlas of the pregnant immunome exposed to nine candidate drugs preselected for PTL prevention. Applying SIMCO to an independent cohort of pregnant individuals, we simulate treatment effects on gestational length, screening for candidate drugs that delay labor timing and providing system-level mechanistic insight for each drug candidate. Tetrahydrofolate, maprotiline, and the combination of aspirin and lansoprazole emerged as top candidates for PTL prevention, delaying labor onset primarily through enhanced mTOR signaling in innate immune cells and attenuated JAK/STAT signaling in naive CD4 T cells. The codebase is available at https://github.com/ofondeur/SIMCO/.

immunology↗

bioRxiv2025

Functionally-structured Bayesian model for localizing neural activity and information in magnetoencephalography signals

Magnetoencephalography (MEG) is a noninvasive method that can measure human brain activity with high temporal resolution. However, the spatial resolution of MEG is not sufficient to reveal neural mechanisms. Although MEG source estimation overcomes this problem to some extent, the combination of MEG source estimation and multivariate analysis results in "information spreading", where significant predictions are observed in brain areas outside the true signal source location. This paper describes a model that achieves high source estimation accuracy and suppresses information spreading simultaneously. The proposed approach is based on a Bayesian estimation model that utilizes functional structure of the human brain. We compare the performance of the proposed model with simulated data generated under various signal-to-noise ratio conditions. The results show that the functionally-structured Bayesian model achieves source estimation accuracy that is better than that of conventional source estimation models. Additionally, the comparison of information spreading among the models reveals that our model outperforms the conventional ones. These results suggest that information spreading in the MEG source estimation can be suppressed while maintaining high source estimation accuracy.

neuroscience↗

bioRxiv2025

Block-structured Bayesian source estimation model for magnetoencephalography signals

The human brain consists of functionally specialized areas that process specific types of information and interact with one another. Magnetoencephalography (MEG) is a neuroimaging technique that captures brain activity at high temporal resolution. However, its spatial resolution is insufficient to accurately localize neural activation, and thus cannot capture the interactive nature of human brain activity. To resolve this issue, various MEG source estimation models have been proposed. In particular, models incorporating prior information from functional magnetic resonance imaging (fMRI), which offers superior spatial resolution, have improved source estimation accuracy. However, these models typically ignore the similarity of activity across different brain areas, and still fall short in tracking dynamic brain activity pattern at a sufficient spatial scale. In this study, we developed a block-structured model that integrates information about functional areas and the inter-areal relationships of the human brain into MEG source estimation based on a hierarchical Bayesian framework. We evaluated our model performance using simulation data with sequential activation across multiple brain areas. Results showed that our model outperformed conventional approaches in source estimation accuracy, suggesting that incorporating the functional areal information and inter-areal relationships may enhance MEG source estimation, enabling human neuroimaging at high spatiotemporal resolution.

neuroscience↗

bioRxiv2024

Identification and characterization of substrate- and product-selective nylon hydrolases

Enzymes have evolved to rapidly and selectively hydrolyze diverse natural and anthropogenic polymers, but only a limited group of related enzymes have been shown to hydrolyze synthetic polyamides. In this work, we synthesized and characterized a panel of 95 diverse enzymes from the N-terminal nucleophile hydrolase superfamily with 30-50% pairwise amino acid identity. We found that nearly 40% of the enzymes had substantial nylon hydrolase activity, in many cases comparable to that of the best-characterized nylon hydrolase, NylC. There was no relationship between phylogeny and activity, nor any evidence of prior selection for nylon hydrolase activity. Several newly-identified hydrolases showed significant substrate selectivity, generating up to 20-fold higher product titers with Nylon 6,6 versus Nylon 6. Finally, we determined the crystal structure and oligomerization state of a Nylon 6,6-selective hydrolase to elucidate structural factors that could affect activity and selectivity. These new enzymes provide insights into the widespread potential for nylon hydrolase evolution and opportunities for analysis and engineering of improved hydrolases. SignificanceNylons are common industrial polyamides with few recycling options. As an alternative to mechanical or chemical recycling, enzymes may provide a selective and energy-efficient route to deconstruct nylons from mixed waste. Several nylon hydrolases have been identified, most notably NylC, but these enzymes are all closely related and demonstrated similar activity and substrate range. In this work, we investigated a diverse set of enzymes and showed that nylon hydrolase activity is common, providing new insights into the evolution of microbial nylon hydrolysis. Unlike NylC, several enzymes demonstrated unprecedented substrate selectivity, preferentially hydrolyzing Nylon 6,6 compared to Nylon 6. These enzymes can be used to understand substrate selectivity in nylon hydrolysis and to engineer enzymes for nylon recycling from mixed waste.

biochemistry↗

bioRxiv2024

The differential effect of optogenetic serotonergic manipulation on sustained motor actions and stationary waiting for future rewards in mice

Serotonin is an essential neuromodulator that affects behavioral and cognitive functions. Previous studies have shown that activation of serotonergic neurons in the dorsal raphe nucleus (DRN) promotes patience to wait for future rewards. However, it is still unclear whether serotonergic neurons also regulate persistence to act for future rewards. Here we used optogenetic activation and inhibition of DRN serotonergic neurons to examine their effects on sustained motor actions for future rewards. We trained mice to perform stationary waiting and repeated lever-pressing tasks with variable reward delays and tested effects of optogenetic activation and inhibition of DRN serotonergic neurons on task performance. Interestingly, in the lever-pressing task, mice tolerated longer delays as they repeatedly pressed a lever than in the stationary waiting task, suggesting that lever-pressing actions may not simply be costly, but may also be subjectively rewarding. Optogenetic activation of DRN serotonergic neurons prolonged waiting in the stationary waiting task, consistent with previous studies, but it did not affect lever pressing time or numbers. While optogenetic inhibition decreased waiting, it did not affect lever pressing time or numbers. In generalized linear model analysis that incorporated the time during each session and the number of sessions, however, optogenetic activation negatively affected the number and the speed of lever pressing. These results revealed that the necessity of motor actions may increase motivation for delayed rewards and that DRN serotonergic neurons more significantly promote stationary waiting rather than persistent motor actions for future rewards.

animal behavior and cognition↗

bioRxiv2022

Impact of reinfection with SARS-CoV-2 Omicron variants in previously infected hamsters

The diversity of SARS-CoV-2 mutations raises the possibility of reinfection of individuals previously infected with earlier variants, and this risk is further increased by the emergence of the B.1.1.529 Omicron variant. In this study, we used an in vivo, hamster infection model to assess the potential for individuals previously infected with SARS-CoV-2 to be reinfected with Omicron variant and we also investigated the pathology associated with such infections. Initially, Syrian hamsters were inoculated with a lineage A, B.1.1.7, B.1.351, B.1.617.2 or a subvariant of Omicron, BA.1 strain and then reinfected with the BA.1 strain 5 weeks later. Subsequently, the impact of reinfection with Omicron subvariants (BA.1 and BA.2) in individuals previously infected with the BA.1 strain was examined. Although viral infection and replication were suppressed in both the upper and lower airways, following reinfection, virus-associated RNA was detected in the airways of most hamsters. Viral replication was more strongly suppressed in the lower respiratory tract than in the upper respiratory tract. Consistent amino acid substitutions were observed in the upper respiratory tract of infected hamsters after primary infection with variant BA.1, whereas diverse mutations appeared in hamsters reinfected with the same variant. Histopathology showed no acute pneumonia or disease enhancement in any of the reinfection groups and, in addition, the expression of inflammatory cytokines and chemokines in the airways of reinfected animals was only mildly elevated. These findings are important for understanding the risk of reinfection with new variants of SARS-CoV-2. IMPORTANCEThe emergence of SARS-CoV-2 variants and the widespread use of COVID-19 vaccines has resulted in individual differences in immune status against SARS-CoV-2. A decay in immunity over time and the emergence of variants that partially evade the immune response can also lead to reinfection. In this study, we demonstrated that, in hamsters, immunity acquired following primary infection with previous SARS-CoV-2 variants was effective in preventing the onset of pneumonia after reinfection with the Omicron variant. However, viral infection and multiplication in the upper respiratory tract were still observed after reinfection. We also showed that more diverse nonsynonymous mutations appeared in the upper respiratory tract of reinfected hamsters that had acquired immunity from primary infection. This hamster model reveals the within-host evolution of SARS-CoV-2 and its pathology after reinfection, and provides important information for countermeasures against diversifying SARS-CoV-2 variants.

microbiology↗

bioRxiv2021

Genetic perturbation enhances functional heterogeneity in alkaline phosphatase

Enzymes inherently exhibit molecule-to-molecule heterogeneity in catalytic activity or function, which underlies the acquisition of new functions in evolutionary processes. However, correlations between the functional heterogeneity of an enzyme and its multi-functionality or promiscuity remain elusive. In addition, the modulation of functional heterogeneity upon genetic perturbation is currently unexplored. Here, we quantitatively analyzed functional heterogeneity in the wild-type and 69 single-point mutants of Escherichia coli alkaline phosphatase (AP) by employing single-molecule assay with a femtoliter reactor array device. Most mutant enzymes exhibited higher functional heterogeneity than the wild-type enzyme, irrespective of catalytic activity. These results indicated that the wild-type AP minimizes functional heterogeneity, and single-point mutations can significantly expand the span of functional heterogeneity in AP. Moreover, we identified a clear correlation between functional heterogeneity and promiscuous activities. These findings suggest that enzymes can acquire greater functional heterogeneity following marginal genetic perturbations that concomitantly promote catalytic promiscuity.

biochemistry↗

bioRxiv2020

Proton wires mediate the optical signal for ArcLight-type Genetically Encoded Voltage Indicators

The genetically encoded voltage indicators, ArcLight and its derivatives, mediate voltage dependent optical signals by intermolecular, electrostatic interactions between neighboring fluorescent proteins (FPs) via proton wires. A random mutagenesis event placed a negative charge on the exterior of the FP resulting in a greater than 10-fold improvement of the voltage-dependent optical signal. Repositioning this negative charge on the exterior of the FP reversed the polarity of voltage-dependent optical signals suggesting the presence of hot spots capable of interacting with the negative charge on a neighboring FP thereby changing the fluorescent output. To explore the potential effect on the chromophore state, voltage-clamp fluorometry was performed with alternating excitation at 390 nm followed by excitation at 470 nm resulting in several mutants exhibiting voltage-dependent, ratiometric optical signals of opposing polarities. However, the kinetics, voltage ranges, and optimal FP fusion sites were different depending on the wavelength of excitation. These results suggest that the FP has external, electrostatic pathways capable of quenching fluorescence that are wavelength specific. ArcLight-derived GEVIs may therefore offer a novel way to map how conditions external to the {beta}-can structure can affect the fluorescence of the chromophore and transiently manipulate those pathways via conformational changes mediated by whole cell voltage clamp. Statement of SignificanceArcLight-type GEVIs utilize proton pathways that send charge information outside of the FP to the internal chromophore enabling voltage induced conformational changes to affect fluorescence. These pathways are excitation wavelength specific suggesting that different external positions affect the protonated and deprotonated states of the chromophore.

biophysics↗

bioRxiv2018

Dipeptidyl dipeptidase-4 inhibitor, MK-0626, promotes bone marrow-derived endothelial progenitor cell bioactivities for vascular regeneration in diet-induced obese mice

Metabolic syndrome (MS), overlapping type 2 diabetes, hyperlipidemia, and/or hypertension, based on high-fat diet, poses risk for cardiovascular disease. A critical feature associated with such risk is the functional impairment of endothelial progenitor cells (EPCs). Dipeptidyl dipeptidase-4 inhibitors are known not only to inhibit degradation of incretins to control blood glucose levels, but also to improve EPC bioactivity and induce anti-inflammatory effects in tissues. In the present study, we investigated the effects of such an inhibitor, MK-06266, in ischemia model of MS using diet-induced obese (DIO) mice. EPC bioactivity was examined in MK-0626-administered DIO mice and non-treated control group, using an EPC colony-forming assay and bone marrow cKit+ Sca-1+ lineage-cells, and peripheral blood-mononuclear cells. Our results showed that, in vitro, the effect of MK-0626 treatment on EPC bioactivities and differentiation was superior in comparison with non-treatment. Further, in vivo hindlimb ischemia model experiment indicated that microvascular density and pericyte-recruited arteriole number were increased in MK-0626-administered group, but not control group. Lineage profiling of isolated cells from ischemic tissues disclosed that MK-0626 administration has an inhibitory effect on unproductive inflammation. This occurred via a decrease in the influx of total blood cells and pro-inflammatory cells such as neutrophils, total macrophages, M1, total T-cells, cytotoxic T-cells, and B-cells, with a concomitant increase in number of regeneration-associated cells, such as M2/M ratio and Treg/T-helper. Laser Doppler analysis revealed that at day 14 after ischemic injury, blood perfusion in hindlimb was grater in DIO mice treated with MK-0626, but not in control. In conclusion, the dipeptidyl dipeptidase-4 inhibitor has a positive effect on EPC differentiation in MS model of DIO mice. Following ischemic injury, DPP-4 i sharply reduces recruitment of pro-inflammatory cells into ischemic tissue, and triggers regeneration and reparation process. Thus, DPP-4 i is a promising therapeutic agent for MS treatment.

cell biology↗