Biology subjects

Takayama, S.

Publications and source records attributed to Takayama, S..

15 recordsLinked to original sources

bioRxiv2026

Evolution of transposons as controlling elements

Transposons were originally described as controlling elements, yet the molecular basis and evolutionary origin of their transcriptional regulatory activities remain elusive. Here, we show that transcriptional regulatory activity is an intrinsic property of transposases (TPases). We demonstrate that the Arabidopsis AtMu1 TPase is sufficient to induce sequence-specific transcriptional activation of cognate copies, revealing a pre-existing cis-regulatory network. We further demonstrate that long-recognized Spm transposon-encoded transcriptional regulator TnpA evolved from an ancestral TPase, linking transcriptional regulators to TPases. Across eukaryotes, we identify multiple domesticated TPase-derived proteins that recurrently lack catalytic residues for transposition, consistent with a one-step regulatory co-option through loss of catalytic activity. Together, our results reveal TPases as autonomous sequence-specific transcriptional regulators and provide a direct evolutionary route from TPases to transcription factors.

genetics↗

bioRxiv2026

HYPER-Net: Physics-Conditioned Self-Supervised Reconstruction for Fourier Light-Field Microscopy

The rapid convergence of optical innovation and machine intelligence is reshaping biological imaging by enabling platforms that jointly advance image formation and computational reconstruction for highspeed, high-resolution volumetric microscopy. However, broadly accessible three-dimensional imaging at high spatiotemporal resolution remains limited by the reliance of existing supervised methods on large modality-matched training datasets, the computational burden of conventional iterative reconstruction, and sensitivity to optical mismatch arising from small deviations in the spatial-angular point spread functions. Here, we introduce HYPER-Net, a physics-conditioned self-supervised framework for Fourier light-field microscopy that integrates scan-free volumetric acquisition with fast, robust three-dimensional reconstruction. HYPER-Net incorporates experiment-specific point-spread functions into the learning process in two complementary roles: as the forward operator that enforces measurement consistency and as a conditioning signal that adaptively modulates intermediate feature representations. This design reduces reliance on paired experimental ground-truth volumes, improves robustness to system variation, and enables generalizable reconstruction across diverse biological contexts. Using human colon organoids, embryonic Xenopus laevis hearts, hiPSC-derived cardiac spheroids, and freely moving Caenorhabditis elegans, we demonstrate high-fidelity volumetric imaging of tissue morphology, cardiac function, calcium-contraction coupling, and locomotion-associated neural and muscular dynamics. These results position HYPER-Net as a versatile framework for rapid volumetric imaging and quantitative analysis of dynamic biological systems across basic research and biomedical applications.

bioengineering↗

bioRxiv2026

Evaluating Evo 2 for plant variant effect prediction

The genomic foundation model Evo 2 enables zero-shot variant effect prediction. Here, we evaluate its performance using Arabidopsis thaliana reproductive barrier genes with experimentally confirmed gain- and loss-of-function variants, and show that Evo 2 distinguishes functionally impactful variants. Together with a sign-reversal amplitude metric that recovers a variant missed by standard scoring, these results highlight the potential of Evo 2 for causal variant prioritization in plant GWAS and QTL mapping.

plant biology↗

bioRxiv2026

A pistil peptide toxin-pollen antidote system for reproductive barrier

Summary paragraphPrezygotic reproductive barriers in angiosperms are crucial for female-mediated selection of compatible male gametes. In this study, we identified a biological activity within the stylar tissues of Arabidopsis thaliana pistils that inhibits the elongation of heterospecific pollen tubes. Using genome-wide association studies, we identified a cysteine-rich peptide predominantly expressed in the style as the responsible barrier. This secreted peptide, named Femme fatale (FEM), inhibited pollen tube elongation of heterospecific species and some conspecific strains, both in vivo and in vitro. Importantly, FEMs function was independent of the known reproductive barrier component SPRI1 that functions in the stigmatic papilla cell surface. This means two factors act spatially in sequence to create a multilayered reproductive barrier system against pollen in pistils. We also identified a secreted protein expressed in pollen, Homme capable (HOM), which is encoded by a gene next to FEM. HOM interacts with FEM to counteract its barrier effect and allow pollen tube elongation. The FEM-HOM locus has undergone frequent tandem duplications during Brassicaceae evolution. Dosage differences between the two factors can lead to reproductive incompatibility among lineages. Together, our findings suggest that FEM and HOM form a toxin-antidote system that helps establish reproductive barriers.

plant biology↗

bioRxiv2024

HIGH THROUGHPUT QUANTITATION OF HUMAN NEUTROPHIL RECRUITMENT AND FUNCTIONAL RESPONSES IN AN AIR-BLOOD BARRIER ARRAY

Dysregulated neutrophil recruitment drives many pulmonary diseases, but most preclinical screening methods are unsuited to evaluate pulmonary neutrophilia, limiting progress towards therapeutics. Namely, high throughput therapeutic screening systems typically exclude critical neutrophilic pathophysiology, including blood-to-lung recruitment, dysfunctional activation, and resulting impacts on the air-blood barrier. To meet the conflicting demands of physiological complexity and high throughput, we developed an assay of 96-well Leukocyte recruitment in an Air-Blood Barrier Array (L-ABBA-96) that enables in vivo-like neutrophil recruitment compatible with downstream phenotyping by automated flow cytometry. We modeled acute respiratory distress syndrome (ARDS) with neutrophil recruitment to 20 ng/mL epithelial-side interleukin 8 (IL-8) and found a dose dependent reduction in recruitment with physiologic doses of baricitinib, a JAK1/2 inhibitor recently FDA-approved for severe COVID-19 ARDS. Additionally, neutrophil recruitment to patient-derived cystic fibrosis sputum supernatant induced disease-mimetic recruitment and activation of healthy donor neutrophils and upregulated endothelial e-selectin. Compared to 24-well assays, the L-ABBA-96 reduces required patient sample volumes by 25 times per well and quadruples throughput per plate. Compared to microfluidic assays, the L-ABBA-96 recruits two orders of magnitude more neutrophils per well, enabling downstream flow cytometry and other standard biochemical assays. This novel pairing of high-throughput in vitro modeling of organ-level lung function with parallel high-throughput leukocyte phenotyping substantially advances opportunities for pathophysiological studies, personalized medicine, and drug testing applications.

bioengineering↗

bioRxiv2024

Stably-Inverted Apical-Out Human Upper Airway Organoids for SARS-CoV-2 Infection and Therapeutic Testing

While breakthroughs with organoids have emerged as next-generation in vitro tools, standardization for drug discovery remains a challenge. This work introduces human airway organoids with reversed biopolarity (AORBs), cultured and analyzed in a high-throughput, single-organoid-per-well format, enabling milestones towards standardization. AORBs exhibit a spatio-temporally stable apical-out morphology, facilitating high-yield direct intact-organoid virus infection. Single-cell RNA sequencing and immunohistochemistry confirm the physiologically relevant recapitulation of differentiated human airway epithelia. The cellular tropism of five severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains along with host response differences between Delta, Washington, and Omicron variants, as observed in transcriptomic profiles, also suggest clinical relevance. Dose-response analysis of three well-studied SARS-CoV-2 antiviral compounds (remdesivir, bemnifosbuvir, and nirmatrelvir) demonstrates that AORBs efficiently predict human efficacy, comparable to gold-standard air-liquid interface cultures, but with higher throughput ([~]10-fold) and fewer cells ([~]100-fold). This combination of throughput and relevance allows AORBs to robustly detect false negative results in efficacy, preventing irretrievable loss of promising lead compounds. While this work leverages the SARS-CoV-2 study as a proof-of-concept application, the standardization capacity of AORB holds broader implications in line with regulatory efforts to push alternatives to animal studies.

bioengineering↗

bioRxiv2023

Comprehensive computational analysis of the SRK-SP11 molecular interaction underlying self-incompatibility in Brassicaceae using improved structure prediction for cysteine-rich proteins

Plants employ self-incompatibility (SI) to promote cross-fertilization. In Brassicaceae, this process is regulated by the formation of a complex between the pistil determinant S receptor kinase (SRK) and the pollen determinant S-locus protein 11 (SP11, also known as S-locus cysteine-rich protein, SCR). In our previous study, we used the crystal structures of two eSRK-SP11 complexes in Brassica rapa S8 and S9 haplotypes and nine computationally predicted complex models to demonstrate that only the SRK ectodomain (eSRK) and SP11 pairs derived from the same S haplotype exhibit high binding free energy. However, predicting the eSRK-SP11 complex structures for the other 100+ S haplotypes and genera remains difficult because of SP11 polymorphism in sequence and structure. Although protein structure prediction using AlphaFold2 exhibits considerably high accuracy for most protein monomers and complexes, 46% of the predicted SP11 structures that we tested showed < 75 mean per-residue confidence score (pLDDT). Here, we demonstrate that the use of curated multiple sequence alignment (MSA) for cysteine-rich proteins significantly improved model accuracy for SP11 and eSRK-SP11 complexes. Additionally, we calculated the binding free energies of the predicted eSRK-SP11 complexes using molecular dynamics (MD) simulations and observed that some Arabidopsis haplotypes formed a binding mode that was critically different from that of B. rapa S8 and S9. Thus, our computational results provide insights into the haplotype-specific eSRK-SP11 binding modes in Brassicaceae at the residue level. The predicted models are freely available at Zenodo, https://doi.org/10.5281/zenodo.8047768.

bioinformatics↗

bioRxiv2023

Liquid plug propagation in computer-controlled microfluidic airway-on-a-chip with semi-circular microchannels

This paper introduces a two-inlet, one-outlet lung-on-a-chip device with semi-circular cross-section microchannels and computer-controlled fluidic switching that enables a broader systematic investigation of liquid plug dynamics in a manner relevant to the distal airways. A leak-proof bonding protocol for micro-milled devices facilitates channel bonding and culture of confluent primary small airway epithelial cells. Production of liquid plugs with computer-controlled inlet channel valving and just one outlet allows more stable long-term plug generation and propagation compared to previous designs. The system also captures both plug speed and length as well as pressure drop concurrently. In one demonstration, the system reproducibly generates surfactant-containing liquid plugs, a challenging process due to lower surface tension that makes the plug formation less stable. The addition of surfactant decreases the pressure required to initiate plug propagation, a potentially significant effect in diseases where surfactant in the airways is absent or dysfunctional. Next, the device recapitulates the effect of increasing fluid viscosity, a challenging analysis due to higher resistance of viscous fluids that makes plug formation and propagation more difficult particularly in airway-relevant length scales. Experimental results show that increased fluid viscosity decreases plug propagation speed for a given air flow rate. These findings are supplemented by computational modeling of viscous plug propagation that demonstrate increased plug propagation time, increased maximum wall shear stress, and greater pressure differentials in more viscous conditions of plug propagation. These results match physiology as mucus viscosity is increased in various obstructive lung diseases where it is known that respiratory mechanics can be compromised due to mucus plugging of the distal airways. Finally, experiments evaluate the effect of channel geometry on primary human small airway epithelial cell injury in this lung-on-a-chip. There is more injury in the middle of the channel relative to the edges highlighting the role of channel shape, a physiologically relevant parameter as airway cross-sectional geometry can also be non-circular. In sum, this paper describes a system that pushes the device limits with regards to the types of liquid plugs that can be stably generated for studies of distal airway fluid mechanical injury.

bioengineering↗

bioRxiv2023

In depth amino acid mutational analysis of the key interspecific incompatibility transmembrane factor Stigmatic Privacy 1

O_LIIn plants, there is an active prezygotic interspecific-incompatibility mechanism to prevent unfavorable hybrids between two species. We previously reported that an uncharacterized protein with four-transmembrane domains, named as Stigmatic Privacy 1 (SPRI1), is responsible for rejecting hetero-specific pollen grains in Arabidopsis thaliana. C_LIO_LIWe have conducted a functional study of the SPRI1 protein, via point-mutational experiments and biochemical analysis. We studied the molecular regulatory mechanisms of SPRI1 and the relationships with its function. C_LIO_LIThe alanine- and glycine-scanning experiments together with the evolutional analysis showed that the structural integrity of the C-terminal regions of the extracellular domain of this protein is important for its function. In addition, we found two cysteines (C67 and C80) within the extracellular domain that may be involved in the formation of intermolecular disulfide bonds. SPRI1 may form homo-multimers and is present as part of a ca. 300 kDa complex. C_LIO_LIOur present study indicates that molecular complex formation ability of SPRI1 may be important to maintain its stability and interspecific incompatibility functions in cells. C_LI

plant biology↗

bioRxiv2023

Manipulation of the nucleoscaffold potentiates cellular reprogramming kinetics

Somatic cell fate is an outcome set by the activities of specific transcription factors and the chromatin landscape and is maintained by gene silencing of alternate cell fates through physical interactions with the nuclear scaffold. Here, we evaluate the role of the nuclear scaffold as a guardian of cell fate in human fibroblasts by comparing the effects of transient loss (knockdown) and mutation (progeria) of functional Lamin A/C, a core component of the nuclear scaffold. We observed that Lamin A/C deficiency or mutation disrupts nuclear morphology, heterochromatin levels, and increases access to DNA in lamina-associated domains. Changes in Lamin A/C were also found to impact the mechanical properties of the nucleus when measured by a microfluidic cellular squeezing device. We also show that transient loss of Lamin A/C accelerates the kinetics of cellular reprogramming to pluripotency through opening of previously silenced heterochromatin domains while genetic mutation of Lamin A/C into progerin induces a senescent phenotype that inhibits the induction of reprogramming genes. Our results highlight the physical role of the nuclear scaffold in safeguarding cellular fate.

cell biology↗

bioRxiv2022

A land plant specific VPS13 mediates polarized vesicle trafficking in germinating pollen

Pollen has an extraordinary ability to convert from a dry state to an extremely rapidly growing state. During pollination, pollen receives water and Ca2+ from the contacting pistil, which will be a directional cue for pollen tube germination. The subsequent rapid activation of directional vesicular transport must support the pollen tube growth, but the molecular mechanism leading to this process is largely unknown. Here we show that a plant-specific VPS13, AtVPS13a, mediates vesicle trafficking during the polarization process in Arabidopsis pollen. AtVPS13a knockout severely affected pollen germination and lipid droplet discharge, while Ca2+-dynamics after pollination was unchanged. Cellular distribution patterns of AtVPS13a and a secretory vesicle marker were synchronized, with a slight delay to the Ca2+-dynamics in polarizing pollen. The absence of AtVPS13a led to reduced cell wall deposition during pollen germination. These results suggest that AtVPS13a mediates pollen polarization, by regulating proper directional vesicular transport following Ca2+-signaling.

plant biology↗

bioRxiv2022

Extended longevity geometrically-inverted proximal tubule organoids for protein uptake studies

While some in vitro platforms have been adapted to study proteinuric kidney disease, organoids have been challenging to study the disease. This is because apical access is historically difficult, and this is the surface on which megalin (LRP2), an endocytic receptor responsible for tubular reabsorption of filtered plasma proteins, resides. Based on our previous geometrically-inverted organoids, this study established high-throughput basal-in and apical-out proximal tubule organoids to study proteinuric kidney disease in a more physiologically consistent manner. Organoids successfully formed around a minimal Matrigel scaffold, and were maintained in culture for 90+ days, the longest reported hanging drop culture to date. The proximal tubule organoids exhibited good polarization, showed upregulation of maturity markers, such as aquaporin-1 and megalin, and experienced less epithelial-to-mesenchymal (EMT) transition compared to 2D cells. To assess protein uptake, fluorescent albumin was placed in the surrounding media, facing the apical surface, and organoids demonstrated functional protein uptake even at 90 days. To mimic proteinuric conditions, organoids were exposed to human serum albumin and released kidney injury molecule-1 (KIM-1), a common biomarker for kidney injury, in both dose- and time-dependent manners. While this study focuses on applications for modeling proteinuric kidney disease conditions, these organoids are envisioned to have broad utility where apical proximal tubule cell access is required.

bioengineering↗

bioRxiv2021

Protocell Arrays for Simultaneous Detection of Diverse Analytes

Simultaneous detection of multiple analytes from a single sample (multiplexing), particularly when at the point of need, can guide complex decision-making without increasing the required sample volume or cost per test. Despite recent advances, multiplexing still typically faces the critical limitation of measuring only one type of molecule per assay platform - for example, only small molecules or only nucleic acids. In this work, we address this bottleneck with a customizable platform that integrates cell-free expression (CFE) with a polymer-based aqueous two-phase system (ATPS) to produce membrane-less "protocells" containing transcription and translation machinery used for analyte detection. Multiple protocells are arrayed in microwells where each protocell droplet performs distinct reactions to detect chemically diverse targets including small molecules, minerals, and nucleic acid sequences, all from the same sample. We demonstrate that these protocell arrays can measure analytes in a human biofluid matrix, maintain function after lyophilization and rehydration, and produce visually interpretable readouts, illustrating its potential for application as a minimal-equipment, field-deployable, multi-analyte detection tool.

synthetic biology↗

bioRxiv2018

Non-adhesive alginate hydrogels support growth of pluripotent stem cell-derived intestinal organoids

Human intestinal organoids (HIOs) represent a powerful system to study human development and are promising candidates for clinical translation as drug-screening tools or engineered tissue. Experimental control and clinical use of HIOs is limited by growth in expensive and poorly defined tumor-cell-derived extracellular matrices, prompting investigation of synthetic ECM-mimetics for HIO culture. Since HIOs possess an inner epithelium and outer mesenchyme, we hypothesized that adhesive cues provided by the matrix may be dispensable for HIO culture. Here, we demonstrate that alginate, a minimally supportive hydrogel with no inherent cell adhesion properties, supports HIO growth in vitro and leads to HIO epithelial differentiation that is virtually indistinguishable from Matrigel-grown HIOs. Additionally, alginate-grown HIOs mature to a similar degree as Matrigel-grown HIOs when transplanted in vivo, both resembling human fetal intestine. This work demonstrates that purely mechanical support from a simple-to-use and inexpensive hydrogel is sufficient to promote HIO survival and development.

developmental biology↗

bioRxiv2016

Pharmaceutical efficacy of human epiphyseal chondrocytes with differential replication numbers for cellular therapy products

The cell-based therapy for cartilage or bone requires a large number of cells serial passages of chondrocytes are, therefore, needed. However, fates of expanded chondrocytes from extra fingers remains unclarified. The chondrocytes from human epiphyses morphologically changed from small polygonal cells, to bipolar elongated spindle cells and to large polygonal cells with degeneration at early passages. Gene of type II collagen was expressed in the cells only at a primary culture (Passage 0) and Passage 1 (P1) cells. The nodules by implantation of P0 to P8 cells were composed of cartilage and perichondrium. The cartilage consisted of chondrocytes with round nuclei and type II collagen-positive matrix, and the perichondrium consisted of spindle cells with type I collage-positive matrix. The cartilage and perichondrium developed to bone with marrow cavity through enchondral ossification. Chondrogenesis and osteogenesis by epiphyseal chondrocytes depended on replication number in culture. It is noteworthy to take population doubling level in correlation with pharmaceutical efficacy into consideration when we use chondrocytes for cell-based therapies.

cell biology↗