Biology subjects

Takeuchi, S.

Publications and source records attributed to Takeuchi, S..

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

Structural basis for Ca2+-dependent, RyR2-selective stabilization by Ryanozole in CPVT

The cardiac ryanodine receptor (RyR2) is a Ca{superscript 2} release channel essential for excitation-contraction coupling. RyR2 mutations cause severe arrhythmogenic disorders, including catecholaminergic polymorphic ventricular tachycardia (CPVT), through gain-of-function (GOF) effects leading to aberrant Ca{superscript 2} release. We have recently developed Ryanozole, a potential therapeutic compound for CPVT, which selectively stabilizes RyR2 in a Ca2+-dependent manner. Here, we define the mechanism of action of Ryanozole by combining high-resolution cryo-electron microscopy, targeted mutagenesis and functional assays. Ryanozole binds to the interface between the Ca{superscript 2}-binding site and the pore-forming S6 helix, interfering with conformational changes required for Ca{superscript 2}-induced channel opening at low Ca2+. We identified key residues for the binding and isoform-specific modulation of Ryanozole. Notably, Ryanozole-bound RyR2 retains its ability to open at high Ca2+ via unique conformational changes. These findings provide a structural basis for CPVT-targeted therapy and redefine the paradigm of small molecule-based regulation of large ion channels.

biochemistry↗

bioRxiv2026

Co-targeting an AMPK--MAPK axis reprograms CAFs and suppresses PDAC

Pancreatic ductal adenocarcinoma (PDAC) is a fatal cancer characterized by limited therapeutic options and a highly treatment-resistant tumor microenvironment. Beyond tumor-intrinsic genetic alterations, growing evidence indicates that host-microbiome interactions influence cancer progression through microbial metabolites. However, how microbiome-derived metabolites influence oncogenic signaling in PDAC remains unclear. Here, integrated profiling revealed a consistent reduction of the microbial metabolite acetic acid in fecal samples from treatment-naive patients with PDAC and in a genetically defined Drosophila model recapitulating key PDAC driver alterations. Acetic acid activates AMP-activated protein kinase, and pharmacological activation of this pathway together with inhibition of mitogen-activated protein kinase signaling suppressed tumor growth in fly and mouse models. Combined pathway targeting restored AMPK activity and suppressed cancer-associated fibroblast activation. These findings identify a microbiome-associated metabolic vulnerability in PDAC and suggest that coordinated targeting of metabolic and oncogenic signaling may restrain tumor progression and improve therapeutic strategies.

cancer biology↗

bioRxiv2025

Meat the Fat: From High Lipid Content to Nutritional Enhancement to Reconstruct Wagyu Fat with Tunable Rheology and Organoleptic Fidelity for Cultivated Meat

Cultivated meat holds promise as a sustainable and ethical alternative to conventional meat, but reproducing the rich flavor, texture, and lipid composition of premium beef, such as Wagyu, remains a major hurdle. In this study, we redefine bovine adipogenesis by systematically optimizing edible biomaterials, fatty acid delivery systems, and culture conditions to engineer high-fat cultivated tissues with authentic sensory properties. Among various candidates, 1% alginate emerged as the optimal scaffold, providing mechanical softness compatible with adipogenic differentiation and mimicking the rheological properties of native fat. Supplementation with oleic acid complexed to edible albumins, especially whey protein (WP), significantly enhanced lipid accumulation and monounsaturated fatty acid content, particularly oleic acid, which reached up to 80.6% of total fatty acids. Engineered fat tissues displayed soft, spreadable textures and organoleptic characteristics consistent with high-quality beef fat. Fatty acid profiling revealed a bovine-like omega-6 signature, dominated by linoleic, arachidonic, and dihomo-{gamma}-linolenic acids, alongside detectable omega-3 subtypes including alpha-linolenic acid and trace levels of EPA and DHA. This composition not only validates the physiological relevance of the engineered fat, but also highlights its potential to deliver bioactive lipids rarely found in terrestrial animal products. Additionally, the WP-oleic acid system supported robust adipogenesis even under lower serum conditions. These findings establish a food-grade, tunable platform for producing physiologically and nutritionally enhanced fat tissues, advancing the realism, functionality, and health potential of next-generation cultivated meat.

bioengineering↗

bioRxiv2025

Antagonistic histone H2A variants and autonomous heterochromatin formation shape epigenomic patterns in Arabidopsis

Heterochromatin formation is pivotal in many eukaryotes with repetitive sequences, such as transposable elements (TEs). However, in plants, where the known de novo DNA methylation mechanism (RdDM) targets euchromatin, how heterochromatin is formed in a region-specific manner remains unclear. We previously reported an RdDM-independent de novo establishment of H3K9me and non-CpG methylation, both of which localize in heterochromatin. Here we show that the mutually exclusive histone H2A variants, H2A.W and H2A.Z, function as guides to initiate heterochromatin formation; H2A.W and H2A.Z promotes and inhibits heterochromatin establishment, respectively, especially in chromosomal arm regions with dispersed TEs. In contrast, pericentromeric TEs demonstrate autonomous heterochromatin formation, less dependently on these H2A variants. Furthermore, H2A.Z protects protein-coding genes from ectopic heterochromatin formation, possibly by preventing its spreading. We propose that the genome indexing mechanism driven by H2A variants, as well as the autonomous formation of pericentromeric heterochromatin, shapes proper epigenomic patterns in Arabidopsis.

genomics↗

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

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↗

bioRxiv2024

Integrated Closed-loop Control of Bio-actuation for Proprioceptive Bio-hybrid Robots

Biohybrid robots are emergent soft robots that combine engineered artificial structures and living biosystems to exploit unique characteristics of biological cells and tissues. Skeletal muscle tissue-based bio-actuators can respond to externally applied stimuli, such as electrical fields. However, current bio-actuation systems rely on open-loop control strategies that lack knowledge of the actuators state. The regulation of output force and position of bio-hybrid robots requires self-sensing control systems that combine bio-actuators with sensors and control paradigms. Here, we propose a soft, fiber-shaped mechanical sensor based on a composite with piezoresistive properties that efficiently integrates with engineered skeletal muscle tissue and senses its contracting states in a cell culture environment in the presence of applied electrical fields. After testing the sensors insulation and biocompatibility, we characterized its sensitivity for typical strains (<1%) and proved its ability to detect motions from contractile skeletal muscle tissue constructs. Finally, we showed that the sensor response can feed an autonomous control system, thus demonstrating the first proprioceptive bio-hybrid robot that can sense and respond to its contraction state. In addition to inspiring intelligent implantable systems, informative biomedical models, and other bioelectronic systems, the proposed technology will encourage strategies to exceed the durability, design, and portability limitations of biohybrid robots and confer them decisional autonomy, thus driving the paradigm shift between bio-actuators and intelligent bio-hybrid robots. One Sentence SummaryIntegrating soft mechanical sensors into engineered skeletal muscle tissue enables bio-hybrid robots with proprioception.

bioengineering↗

bioRxiv2024

PERFORATION-TYPE ANCHORS INSPIRED BY SKIN LIGAMENT FOR THE ROBOTIC FACE COVERED WITH LIVING SKIN

Skin equivalent, a living skin model composed of cells and extracellular matrix, possesses the potential as an ideal covering material for robots due to their biological functionalities. To employ skin equivalents as a coverage material for robots, a secure method for attaching them to the underlying structure is required. In this study, we developed a perforation-type anchor inspired by the structure of skin ligaments as a technique to effectively adhere skin equivalent to robotic surfaces, ensuring a smooth and non-protrusive integration, akin to natural skin adherence. We showed the effectiveness of plasma treatment for collagen-gel penetration within the perforation-type anchor. In characterizing the anchoring system, we investigated the impact of anchor diameter on preventing contraction and changes in holding strength. To showcase the versatility of perforation-type anchors in three-dimensional (3D) coverage applications, a 3D facial mold with intricate surface structure was covered with skin equivalent. Furthermore, we constructed a robotic face covered with dermis equivalent, capable of expressing smiles with the actuation to the skin equivalent through perforation-type anchors. This research introduces an approach for the adhesion and actuation of skin equivalent with perforation-type anchors, potentially contributing to advancements in biohybrid robotics technology.

bioengineering↗

bioRxiv2023

Centimeter-scale perfusable cultured meat with densely packed, highly aligned muscle fibers via hollow fiber bioreactor

The development of in-vitro biofabrication methods for producing cultured meat based on animal cells has been advancing, but replicating the texture of traditional meat in centimeter-scale has been a challenge. To address this, a method using a hollow fiber bioreactor (HFB) has been developed. The HFB contains semipermeable hollow fibers that act as artificial circulatory systems to deliver nutrients and oxygen uniformly to the tissue, along with microfabricated anchors for inducing cell alignment. With active perfusion, the biofabricated centimeter-scale chick muscle tissue shows elevated levels of marker protein expression and sarcomere formation across the whole tissue, along with improved texture and flavor. In the future, further scaling up of this approach using industrial robots has the potential to transform not only the cultured meat industry but also the tissue engineering fields aiming for the formation of large-scale artificial organs.

bioengineering↗

bioRxiv2022

A presynaptic source drives differing levels of surround suppression in two mouse retina ganglion cell types

In early sensory systems, cell-type diversity generally increases from the periphery into the brain, resulting in a greater heterogeneity of responses to the same stimuli. Surround suppression is a canonical visual computation that begins within the retina and is found at varying levels across retinal ganglion cell types. Our results show that divergence in the level of surround suppression occurs subcellularly, at bipolar cell synapses. Using single-cell electrophysiology and serial block-face scanning electron microscopy, we show that two retinal ganglion cell types exhibit very different levels of surround suppression even though they receive input from the same set of bipolar cell types. This divergence of the bipolar cell signal occurs through synapse-specific regulation by amacrine cells at the scale of tens of microns. These findings indicate that each synapse of a single bipolar cell can carry a unique visual signal, expanding the number of possible functional channels at the earliest stages of visual processing.

neuroscience↗

bioRxiv2021

In Vitro Proliferation and Long-Term Preservation of Functional Primary Rat Hepatocytes in Cell Fibers

Primary hepatocytes are essential cellular resource for drug screening and medical transplantation. Since culture systems for them have already succeeded in reconstituting the biomimetic microenvironment, acquiring additional capabilities both to expand primary hepatocytes and to handle them easily would be expected as progress to the next stage. This paper describes a culture system for primary rat hepatocytes that is equipped with scalability and handleability relying on cell fiber technology. Cell fibers are cell-laden core-shell hydrogel microfibers; in the core regions, cells are embedded in extracellular matrix proteins, cultured three-dimensionally, and exposed to soluble growth factors in the culture medium through the hydrogel shells. By encapsulating primary rat hepatocytes within cell fibers, we first demonstrated they increase in number while keeping their viability and their hepatic specific functions for up to thirty days of subsequent culture. Then, we demonstrated the potency of the primary rat hepatocytes that proliferate in cell fibers not only as cell-based sensors to detect drugs that damage hepatic functions and hepatocellular processes but also as transplants to improve the plasma albumin concentrations of congenital analbuminemia. Therefore, our culture system could serve for innovating strategies and promising developments in applying primary hepatocytes to both pharmaceutical and medical fields.

bioengineering↗

bioRxiv2021

Development of ultrafast camera-based imaging of single fluorescent molecules and live-cell PALM

The spatial resolution of fluorescence microscopy has recently been greatly improved. However, its temporal resolution has not been improved much, despite its importance for examining living cells. Here, by developing an ultrafast camera system, we achieved the worlds highest time resolutions for single fluorescent-molecule imaging of 33 (100) {micro}s (multiple single molecules simultaneously) with a single-molecule localization precision of 34 (20) nm for Cy3 (best dye found), and for PALM data acquisition of a view-field of 640x640 pixels at 1 kHz with a single-molecule localization precision of 29 nm for mEos3.2. Both are considered the ultimate rates with available probes. This camera system (1) successfully detected fast hop diffusion of membrane molecules in the plasma membrane, detectable previously only by using less preferable 40-nm gold probes and bright-field microscopy, and (2) enabled PALM imaging of the entire live cell, while revealing meso-scale dynamics and structures, caveolae and paxillin islands in the focal adhesion, proving its usefulness for cell biology research. SummaryAn ultrafast camera developed by Fujiwara et al. allows single fluorescent-molecule imaging every 33 s with a localization precision of 34 nm (every 100 s; 20 nm), and enables ultrafast PALM imaging of whole live cells.

cell biology↗

bioRxiv2021

Focal adhesion membrane is dotted with protein islands and partitioned for molecular hop diffusion

Using the ultrafast camera system and new theories for hop diffusion described in the companion paper, we for the first time demonstrated that membrane molecules undergo hop diffusion among the compartments in the bulk basal plasma membrane (PM), with virtually the same compartment sizes (108 nm) as those in the bulk apical PM and the same dwell lifetimes within a compartment (10 and 24 ms for the phospholipid and transferrin receptor, respectively), suggesting that the basic structures and molecular dynamics are very similar in the bulk regions of the apical and basal PMs. Ultrafast PALM and single-molecule imaging revealed that the focal adhesion (FA) is mostly a fluid membrane, partitioned into [~]74-nm compartments where transferrin receptor and {beta}3 integrin undergo hop diffusion, and that the FA membrane is sparsely dotted with 51-nm diameter paxillin islands, where many other FA proteins probably assemble (compartmentalized archipelago model). {beta}3 integrin intermittently associates with the paxillin islands, dynamically linking them to the extracellular matrix. SummaryAn ultrafast camera with single fluorescent-molecule sensitivities developed by Fujiwara et al. reveals that the focal adhesion membrane is dotted with protein islands and partitioned for molecular hop diffusion, and integrin {beta}3 molecules become temporarily immobilized at the islands.

cell biology↗

bioRxiv2020

Biofabricating murine and human myo-substitutes for rapid volumetric muscle loss restoration

The importance of skeletal muscle tissue is undoubted being the controller of several vital functions including respiration and all voluntary locomotion activities. However, its regenerative capability is limited and significant tissue loss often leads to a chronic pathologic condition known as volumetric muscle loss. Here, we propose a biofabrication approach to rapidly restore skeletal muscle mass, 3D histoarchitecture and functionality. By recapitulating muscle anisotropic organization at the microscale level, we demonstrate to efficiently guide cell differentiation and myobundle formation both in vitro and in vivo. Of note, upon implantation, the biofabricated myo-substitutes support the formation of new blood vessels and neuromuscular junctions - pivotal aspects for cell survival and muscle contractile functionalities - together with an advanced along with muscle mass and force recovery. Together, these data represent a solid base for further testing the myo-substitutes in large animal size and a promising platform to be eventually translated into clinical scenarios.

bioengineering↗