Biology subjects

Sakai, A.

Publications and source records attributed to Sakai, A..

9 recordsLinked to original sources

bioRxiv2026

Optimization of PURE system composition using automation and active learning

Protein synthesis using recombinant elements (PURE) system has been widely applied in various biological research fields and synthetic cell construction. Optimization efforts to enhance the PURE system performance by adjusting its individual components have remained limited to the expression of single genes with a small number of molecular compositions tested, making it difficult to link component composition to system-level performance across different DNA contexts. Here, we combine automated acoustic liquid handling with an active learning framework to explore broadly the compositional landscape of PURE system. By grouping the 69 individual components (including proteins and tRNAs) into 21 functional sets and iteratively guiding experiments with active learning, we rapidly identify improved compositions and demonstrated up to 3-fold enhancement in protein yield and translation rate for a single reporter gene. We further show that optimization drivers differ between low and high DNA concentrations, revealing that optimal PURE compositions are DNA concentration-dependent. We then apply this optimization strategy to enhance the expression of a 41-kb synthetic chromosome containing 15 genes by maximizing the fluorescence intensities of two reporter proteins. While a 3-fold improvement could be reached on the two gene products guiding learning, a full proteomic analysis revealed that optimization is gene-specific, i.e., changes in PURE system compositions differently impact the amounts of synthesized proteins encoded on the same DNA template. Together, this work establishes active learning as an efficient strategy to navigate the high-dimensional PURE compositional space and provides mechanistic insight into DNA context-dependence of gene expression optimization.

synthetic biology↗

bioRxiv2026

DNA-templated Chiral Metamaterial Array as Information Bits

The growth of digital information demands physically secure information bits that combine intrinsic randomness with multi-layered optical readout. Digital metamaterials with physical unclonable function characteristics are promising, but nearly all examples operate at microwave or terahertz frequencies. Extending digital metamaterials to the visible range requires a physical property that allows binary (0/1) encoding via nanoscale geometry and is orthogonal to intensity-or color-based imaging. Chiral plasmonic metamaterials satisfy these criteria perfectly: their broken mirror symmetry yields chirality polarity in the visible spectrum whose sign and magnitude directly encode bit values ("0", spin-up; "1", spin-down), while remaining invisible under linear polarization. Here, we realize visible-range digital metamaterials by programming self-assembled DNA origami templates with asymmetrically placed gold nanorods to create discrete 3D chiral metamolecules. Using on-surface DNA origami assembly and the Silica Microsphere-Assisted Patterning by Liquid Elimination (SiMPLE) method, we fabricate large-scale bit arrays on optical active glass substrate in solution with [~]1.33 m lattice spacing, [~]86% site occupancy, and [~]12-month stability after silicification. These bottom-up fabricated digital metamaterial array exhibits two independent security layers: (1) a macroscopic spatial pattern only visible by dark-field microscopy, and (2) hidden binary information stored in the single-particle chiroptical response, read out by position-resolved circular dichroism spectroscopy. Quantitative analysis confirms reliable bit encoding and high optical randomness arising from slight structural variations. By leveraging the polarity of plasmonic chirality to translate molecular-scale handedness into robust visible-range digital signals, this work establishes a scalable nanophotonic platform for secure optical information storage, authentication, and encryption.

bioengineering↗

bioRxiv2026

Cryo-EM structure of pro-aggregant P301L/S320F double-mutant tau filaments formed in mouse brains following peripheral AAV delivery

Tauopathies are characterized by the accumulation of abnormally phosphorylated tau filaments, with disease-specific folds revealed by cryo-electron microscopy (cryo-EM). Here, we delivered a recombinant human tau carrying the P301L/S320F double mutation into AppNL-G-F/MAPT double knock-in mice using a blood-brain-barrier-permeable AAV (AAV-PHP.eB), enabling rapid induction of tau aggregation in vivo. The mutant tau was systemically administered via retro-orbital injection, providing a minimally invasive approach for widespread neuronal transduction. Phosphorylated tau aggregation, as well as seeding activity, were observed without the need for exogenous seeds. Our cryo-EM analysis resolved a novel filament fold incorporating residues 279-329, in which the P301L and S320F mutations introduced stabilizing interactions that reinforced filament assembly. This conformation was distinct from previously reported folds, including Alzheimer and Pick tau filaments. Moreover, the filaments adopted a more compact architecture than those observed in patient-derived samples or other model mice. This finding demonstrates that P301L/S320F double-mutant tau adopts a structurally unique, highly aggregation-prone fold, and that this system provides a rapid platform for modeling tau filament formation.

neuroscience↗

bioRxiv2024

Brassinosteroids and Flavonols Confer Temperature Stress Tolerance to Pollen Tube Germination and Growth

Successful reproduction is a prerequisite for the maintenance of species in the era of climate change. In agriculture, temperature has a significant impact on pollen tube elongation and attraction, and consequently, on the efficiency of fruiting and crop yields. Brassinosteroids (BRs) are plant steroid hormones involved in pollen tube germination, elongation, and capacitation. To elucidate the effect of BR signaling in pistils during the reproductive process, transcriptome analysis of BR receptor (BRI1) mutant pistils was performed, which indicated that genes involved in flavonol biosynthesis were downregulated in bri1 pistils. Flavonols are essential for pollen tube growth in some plants and facilitate pollen tube elongation under high-temperature conditions. To investigate the effects of BR and flavonols on pollen tubes under temperature stress, in vitro assays were performed using both the compounds. Brassinolide (BL), which is the most active BR, promoted pollen tube germination at high, low, and optimal temperatures. Germination was further enhanced by the flavonol, quercetin, in the presence of BL especially at low temperatures. in vivo analysis supported the observation that when BL and quercetin were supplied concurrently, the pollen tube guidance rate was rescued at low temperatures. Quercetin enhanced pollen tube elongation under all the conditions tested at an optimal concentration of 15 M. BL elevated reactive oxygen species (ROS) under all temperature conditions, whereas quercetin regulated ROS synergistically with BR. Thus, BRs and flavonols supplied by pistils are suggested to function in pollen tube germination and elongation through the maintenance of ROS homeostasis.

plant biology↗

bioRxiv2024

Simply crushed Zizyphi spinosi semen prevents neurodegenerative diseases and reverses age-related cognitive decline in mice

Neurodegenerative diseases are age-related disorders characterized by the cerebral accumulation of amyloidogenic proteins, and cellular senescence underlies their pathogenesis. Thus, it is necessary for preventing these diseases to remove toxic proteins, repair damaged neurons, and suppress cellular senescence. As a source for such prophylactic agents, we selected Zizyphi spinosi semen (ZSS), a medicinal herb used in traditional Chinese medicine. ZSS hot water extract ameliorated A{beta} and tau pathology and cognitive impairment in mouse models of Alzheimers disease and frontotemporal dementia. Non-extracted ZSS simple crush powder showed stronger effects than the extract and improved -synuclein pathology and cognitive/motor function in Parkinsons disease model mice. Furthermore, when administered to normal aged mice, the ZSS powder suppressed cellular senescence, reduced DNA oxidation, promoted brain-derived neurotrophic factor expression and neurogenesis, and enhanced cognition to levels similar to those in young mice. The quantity of known active ingredients of ZSS, jujuboside A, jujuboside B, and spinosin, was not proportional to the nootropic activity of ZSS. These results suggest that ZSS simple crush powder is a promising dietary material for the prevention of neurodegenerative diseases and brain aging. Impact statement Non-extracted simple crush powder of Zizyphi spinosi semen has not only disease-preventing effects but also brain-rejuvenating effects in mice.

neuroscience↗

bioRxiv2024

Induced systemic resistance by the root colonization of Trichoderma atroviride is independent from the chitin-mediated signaling pathway in Arabidopsis

Beneficial root endophytic fungi induce systemic responses, growth promotion, and induced systemic resistance (ISR) in the colonized host plants. Soil application of chitin, a main component of fungal cell walls, also systemically induces disease resistance. Thus, chitin recognition and its downstream signaling pathway are supposed to mediate ISR triggered by beneficial fungi colonizing the root. This study compared systemic disease resistance and transcriptional responses induced by Trichoderma, a representative beneficial root endophytic fungus, and chitin in Arabidopsis. A significant plant growth promotion was observed under root colonization by the three tested beneficial fungi, Trichoderma atroviride, Serendipita indica, and S. vermifera. Still, only T. atroviride and S. indica triggered ISR against the necrotrophic fungal pathogen Alternaria brassicicola. Induced systemic resistance triggered by T. atroviride was compromised in the chitin-receptor mutant, while systemic resistance caused by soil application of chitin was not. Transcriptome analysis demonstrated that the chitin-regulated genes are mostly shared with those regulated by T. atroviride, but many of the latter were specific. However, the commonly enriched gene ontologies for those regulated genes indicated that T. atroviride inoculation and chitin application systemically control similar transcriptional responses, mainly associated with cell wall functions. Taken together, Trichoderma could trigger ISR primarily independently from the chitin-mediated signaling pathway; however, chitin and Trichoderma would systemically induce similar cellular functions in ISR aboveground.

plant biology↗

bioRxiv2024

Extracellular disposal of nuclear waste by APP: a protective mechanism impaired in Alzheimer's disease

Although the amyloid beta (A{beta}) hypothesis1 has long been central to Alzheimers disease (AD) research, effective therapeutic strategies remain elusive2,3. Here we re-evaluate the functions of amyloid precursor protein (APP) and reveal its critical function in protecting against nuclear impairment-induced cell death and inflammation4,5. Overexpression of APP mitigated etoposide or lamin A knockdown-induced nuclear damage, while APP removal or mutations exacerbated these effects. Interestingly, neurons differentiated from induced pluripotent stem cells (iPSCs) exhibited similar patterns, and notably, familial AD-associated mutant APP failed to confer protection against nuclear impairment. We identify APPs interaction with a cytoplasmic structure of nuclear origin, termed "nuclear waste", and propose its role in extracellular waste disposal. Intriguingly, cells lacking APP showed impaired nuclear waste clearance, leading to abnormal cytoplasmic accumulation of the nuclear waste. Similarly, neuron-specific APP overexpression using adeno-associated virus (AAV) in mice reduced neuronal death and inflammation caused by nuclear damage. Conversely, shRNA-mediated APP exacerbated these effects, and mutant APP associated with familial AD lacked protective effects. Moreover, postmortem analysis of AD brains revealed accumulation of abnormal nuclear waste in the neurocytoplasm, irregular nuclear morphology, and reduced APP levels per neuron. Our data underscore APPs crucial role in disposing of nuclear waste, maintaining cellular homeostasis, and suggest its dysregulation as a potential contributor to AD pathogenesis. Restoring APP waste clearance in AD could be a promising target for disease-modifying therapies.

neuroscience↗

bioRxiv2021

Development of novel potent ligands for GPR85, an orphan G protein-coupled receptor expressed in the brain

GPR85 is a member of the G protein-coupled receptor and is a super-conserved receptor expressed in the brain sub-family (SREB) with GPR27 and GPR173. These three receptors are "orphan receptors"; however, their endogenous ligands have not been identified. SREB has garnered the interest of many scientists because it is expressed in the central nervous system and is evolutionarily conserved. In particular, brain mass is reported to be increased and learning and memory are improved in GPR85 knockout mice (Matsumoto et al., 2008). In this study, we characterized newly synthesized compounds using a GPR85-Gs fusion protein and the [35S]GTP{gamma}S binding assay and identified novel GPR85 inverse-agonists with IC50 values of approximately 1 {micro}M. To analyze the neurochemical character of the compounds and investigate the physiological significance of GPR85, we used cerebellar Purkinje cells expressing GPR85 and an electrophysiological technique. Based on the results, the inverse-agonist compound for GPR85 modulated potassium channel opening. Together with the results of previous gene analysis of GPR85, we expect that the development of the GPR85 ligand will provide new insights into a few types of neurological disorders.

pharmacology and toxicology↗

bioRxiv2020

High-throughput laboratory evolution and evolutionary constraints in Escherichia coli

Understanding the constraints that shape the evolution of antibiotic resistance is critical for predicting and controlling drug resistance. Despite its importance, however, a systematic investigation for evolutionary constraints is lacking. Here, we performed a high-throughput laboratory evolution of Escherichia coli under the addition of 95 antibacterial chemicals and quantified the transcriptome, resistance, and genomic profiles for the evolved strains. Using interpretable machine learning techniques, we analyzed the phenotype-genotype data and identified low dimensional phenotypic states among the evolved strains. Further analysis revealed the underlying biological processes responsible for these distinct states, leading to the identification of novel trade-off relationships associated with drug resistance. We also report a novel constraint that leads to decelerated evolution. These findings bridge the genotypic, gene expression, and drug resistance space and lead to a better understanding of evolutionary constraints for antibiotic resistance.

evolutionary biology↗