Biology subjects

Hayashi, A.

Publications and source records attributed to Hayashi, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Hypothalamic Cell Type Evolution across Vertebrates

The hypothalamus regulates vital functions and is highly conserved among vertebrates occupying diverse niches, but little is known about the evolution of its cell types. Using comparative single-cell transcriptomics across major vertebrate classes, we identified deeply conserved and clade-specific cell types. While homologous cell types express conserved transcription factors and neuropeptides, their regulatory linkages are often rewired across evolution. The paraventricular nucleus largely retains its neuropeptide expression and responds to dehydration with fine cellular resolution from amphibians to mammals, whereas lamina terminalis neurons are specific to terrestrial vertebrates and exhibit gene expression divergence in cell types activated by dehydration and heat. We propose that a conserved set of keystone cell types sustain essential functions across vertebrates, while flexible gene regulatory programs alongside numerous clade-specific cell types offer evolutionary fluidity.

evolutionary biology↗

bioRxiv2026

Dihydropyrimidines sustain aggressive cancer states by stabilizing DPYSL2

Cancer cell states are governed by coordinated signaling and metabolic networks, yet the mechanisms coupling metabolic activity to oncogenic signaling remain poorly understood. Here, we identify dihydropyrimidines (DHPs), metabolites generated during pyrimidine catabolism, as "signaling metabolites" that sustain aggressive cancer cell states. Depletion of DHPs, either by knockout of the pyrimidine catabolic enzyme DPYD or by expression of the DHP-degrading enzyme DPYS, suppressed STAT3 signaling and attenuated mesenchymal and inflammatory transcriptional programs. Mechanistically, DHPs stabilized DPYSL2, a JAK1-interacting adaptor protein required for efficient STAT3 activation. Structural modeling and thermal stability analyses further support a direct interaction between DHPs and DPYSL2. Accordingly, high DPYD expression was associated with EMT, inflammatory signaling, and poor-prognosis breast cancer subtypes across patient cohorts. Ultimately, our findings establish pyrimidine catabolism as a direct regulator of signaling competence and identify DHPs as signaling-active metabolites that couple cellular metabolic state to oncogenic transcriptional programs and mesenchymal identity.

cancer biology↗

bioRxiv2026

POLQ-driven repair scars shape the immunogenic landscape of homologous recombination-deficient pancreatic cancer

Pancreatic cancer (PC) is broadly resistant to immune checkpoint blockade, although a subset of homologous recombination-deficient (HRD) tumors exhibits durable immune engagement. The genomic features that distinguish these immune-responsive tumors from immune-inert HRD tumors remain poorly understood. Here we identify a microhomology-mediated end joining (MMEJ) repair scar, the MMEJ Deletion Footprint (MDF), as a genomic readout of POLQ-associated error-prone repair that enriches for frameshift indels. Across the multi-omic discovery cohort integrating tumor genomics, single-nucleus transcriptomics and spatial immune profiling, MDF-high HRD PC exhibited increased frameshift-indel-derived neoantigens and interferon programs. MDF was further associated with remodeling of the myeloid compartment toward MHC II-high dendritic cell-like antigen-presenting macrophage states and the immune synapse architecture marked by increased spatial interaction between APC-like macrophages and cytotoxic CD8+ T cells. These tissue-level features aligned with a functional trajectory shift of CD8+ T cells, consistent with effective anti-tumor immunity and was associated with favorable clinical outcomes of patients. Together, our findings position MMEJ-linked repair scarring as actionable biology that connects an HRD genotype to immune organization and suggests rational immunotherapy combinations that may enhance antigen presentation and myeloid activation to extend durable benefit in HRD-lineage cancers.

cancer biology↗

bioRxiv2025

Combinational adjuvants delivered by ink-jet potentiate naked mRNA vaccines for robust protection against infectious diseases

The use of lipid nanoparticles (LNPs) to enhance delivery and immunogenicity of mRNA vaccines raises safety concerns due to strong reactogenicity. Developing naked mRNA vaccines, which can be delivered through liquid jet-injection to the skin, may offer a safer alternative, but their immunogenicity has been limited. To address this challenge, we screened clinically relevant adjuvants to enhance the potency of naked mRNA jet-injection vaccines in mouse models. Although many adjuvants showed little or inhibitory effects on inducing humoral or cellular immunity--despite their known ability to potentiate conventional non-mRNA vaccine platforms--we identified a combination of aluminum phosphate and CpG oligonucleotide that substantially enhanced both humoral and cellular immune responses with increased IgG2a/IgG1 ratio. This adjuvanted naked mRNA vaccine induced IgG2a and cellular immunity at levels comparable to those achieved with LNPs but with lower local and systemic reactogenicity. Mechanistic investigations revealed a key role for type I interferon signaling during immunization. Notably, this vaccine formulation in mice virtually eliminated infection by both SARS-CoV-2 and influenza A virus (A/California/7/2009 H1N1) in plaque-forming assays. Collectively, the adjuvanted naked mRNA jet-injection vaccine represents a novel, effective, and safe platform against infectious diseases, with potential for clinic-ready translational applications.

bioengineering↗

bioRxiv2025

Multi-omics profiling with indoor-unmanned phenotyping reveals drought adaptation through constitutive ABF1 expression in wild rice

Improving drought resistance is crucial for stable crop production under climate change. Identifying the mechanisms of drought resistance using diverse genetic resources, including crop wild relatives, would be beneficial for molecular breeding. Here, we developed an indoor, unmanned phenotyping platform that can noninvasively and automatically collect temporal data on plant responses to drought stress. Using this system, we analyzed the phenotypic, transcriptomic, and environmental data of four cultivated rice varieties and five wild relatives. Multi-omics analysis revealed that one wild rice species exhibited drought adaptation through the constitutive expression of ABSCISIC ACID RESPONSIVE ELEMENT-BINDING FACTOR 1 (ABF1), which encodes a transcription factor that regulates drought resistance, before drought stress. Drought testing of introgression lines of cultivated rice with constitutive ABF1 expression revealed higher drought tolerance than in cultivars without a growth penalty. Our findings suggest that constitutive ABF1 expression contributes to drought adaptation in both cultivated and wild rice.

plant biology↗

bioRxiv2025

Evaluation of Conditional Treatment Effect of Salt Stress on Tomato Sugar Content Using Causal Machine Learning: A Pilot Study

Exposing tomatoes to salt stress has been reported to increase the fruit sugar content ({degrees}Brix); however, this treatments causal impact under varying environmental conditions remains unclear. In this pilot study, a causal inference analysis was conducted using a Causal Tree to analyze the factors that influence the effect of salt-stress treatment on improving the {degrees}Brix in tomatoes. Data were collected from a single greenhouse using one cultivar over multiple cultivation periods, totaling 707 fruits. Propensity score matching was applied to reduce the covariate imbalance between the salt (NaCl) treatment and control groups. Using a Causal Tree, conditional average treatment effects were then estimated to assess heterogeneity in treatment impact based on environmental variables, such as temperature, humidity, and illumination. Treatment with NaCl significantly increased {degrees}Brix compared to the control, but the magnitude of the effect varied depending on the environmental conditions. The Causal Tree analysis based on the accumulated cultivation data identified specific combinations of environmental factors under which the impact of NaCl treatment on {degrees}Brix enhancement was more pronounced. For example, the estimated Conditional Average Treatment Effect (CATE) reached as high as 0.88 when humidity was [≥]75%, illuminance was [≥]8144 lx, and categorical variable C3 was F1 or F2. In contrast, the CATE dropped to as low as 0.036 under low humidity (<79%) and high temperature (<24{degrees}C). These findings highlight humidity as the most influential factor, whose impact was modulated by interactions with temperature and light conditions.

plant biology↗

bioRxiv2025

A probiotic bacterium modulates antitumor γδ T-cell responses in lung cancer

The link between the intestinal microflora and cancer outcomes has been recognized for over a decade. Several recent studies have demonstrated that the gut microbiome is associated with the efficiency of T-cell checkpoint blockade therapy for cancer raising interest in strategies to harness this effect via consumption of live microorganisms (probiotics). The probiotic Clostridium butyricum strain MIYAIRI 588 (CBM588) enhances both response rates and overall survival in patients receiving immune checkpoint blockade therapy for non-small cell lung cancer and metastatic renal cell carcinoma but the mechanism underpinning this benefit remains unclear. Here, we show that CBM588 spores induce a population of V{gamma}9V{delta}2 T-cells from the peripheral blood of healthy donors and lung cancer patients. A subset of these T-cells responded to, and directly lysed, cancer cell lines via a butyrophilin 3A-dependant mechanism. In patients taking CBM588 alongside checkpoint blockade, peripheral V{delta}2 T-cells expressed the activation marker CD69 more frequently than those receiving checkpoint blockade alone and the frequency of V{delta}2CD69 cells increased following initiation of CBM588 treatment (p = 0.0041). Our findings provide a potential mechanism by which the manipulation of the intestinal microflora might directly contribute to cancer prognosis by activating immune effector cells with intrinsic anticancer properties.

immunology↗

bioRxiv2025

Single-molecule multimodal timing of in vivo mRNA synthesis

mRNA synthesis requires extensive pre-mRNA maturation, the organisation of which remains unclear. Here, we directly sequence pre-mRNA without metabolic labelling or amplification to resolve transcription and multimodal pre-mRNA processing at single-molecule resolution. Using poly(A) tail measurement, we distinguish transcriptionally engaged pre-mRNA from polyadenylated transcripts, revealing that splicing is substantially delayed compared to previous estimates. Splicing is rare for at least 10 kb behind elongating RNA polymerase II such that thousands of genes remain largely unspliced during transcription, revising the notion that splicing is predominantly co-transcriptional. Progressive splicing becomes apparent only on polyadenylated transcripts, suggesting that splicing is commonly activated after 3' end formation. Unexpectedly, we find abundant m6A on unspliced pre-mRNA, indicating that a substantial portion of RNA methylation precedes splicing. This m6A methylation is suppressed around exon boundaries, indicating that m6A topology is established prior to exon junction complex deposition. Finally, we demonstrate that 3' end cleavage occurs multiple kilobases behind transcription in coordination with efficient transcription termination, and further highlight recursive 3' end formation across hundreds of genes. Conserved from human cells to mouse tissues, we illustrate a revised timeline for mRNA synthesis wherein cleavage, termination, and m6A deposition occur earlier than, or at least partially decoupled from, the bulk of splicing, reframing the sequence of early mammalian gene expression.

genomics↗

bioRxiv2025

Transcriptomic plasticity is a hallmark of metastatic pancreatic cancer

Metastasis is the leading cause of cancer deaths; nonetheless, how tumor cells adapt to vastly different organ contexts is largely unknown. To investigate this question, we generated a transcriptomic atlas of primary tumor and diverse metastatic samples from a patient with pancreatic ductal adenocarcinoma who underwent rapid autopsy. Unsupervised archetype analysis identified both shared and site-specific gene programs, including lipid metabolism and gastrointestinal programs prevalent in peritoneum and stomach wall lesions, respectively. We developed a probabilistic approach for inferring clonal phylogeny from single-cell and matched whole-exome data. Distantly related genetic clones in the peritoneum express the lipid metabolism program, likely due to signaling by the adipocyte-rich peritoneum environment, and cells in most clones express multiple programs, suggesting that transcriptomic plasticity is a prevalent feature of metastatic cells. These deeply annotated analyses using a patient-centric platform provide a model for investigating metastatic mechanisms and plasticity in advanced cancer.

bioinformatics↗

bioRxiv2025

Comparative Extraction of Cellular Features from High-Resolution Volume Imaging

High-resolution volume imaging techniques, such as lattice light-sheet microscopy (LLSM), generate vast and complex datasets that demand advanced analytical approaches to uncover biologically meaningful insights. While LLSMs high spatial and temporal resolution provides critical data for understanding cellular processes, distinguishing subtle differences between cells in distinct states remains challenging. Here, using an adaptive, human-interpretable kernel-based calculation strategy, we developed Machine-Learning-Based Visual Extraction of Structural Features (M-VEST), a method designed to identify and interpret structural differences with high precision. By applying M-VEST to mitotic cells, we uncovered novel functions of the oncogene Aurora kinase A, demonstrating its utility in revealing previously undetected features. Validated using LLSM datasets, M-VEST offers a scalable framework for analyzing large and complex imaging data, advancing insights into cellular dynamics and beyond.

bioinformatics↗

bioRxiv2024

MMM and CLCFM: A 3D point cloud reconstruction method based on photogrammetry for all-around images taken by rotating an individual plant

This research aims to develop a novel technique to acquire a large amount of high-density, high-precision 3D point cloud data for plant phenotyping using photogrammetry technology. The complexity of plant structures, characterized by overlapping thin parts such as leaves and stems, makes it difficult to reconstruct accurate 3D point clouds. One challenge in this regard is occlusion, where points in the 3D point cloud cannot be obtained due to overlapping parts, preventing accurate point capture. Another is the generation of erroneous points in non-existent locations due to image-matching errors along object outlines. To overcome these challenges, we propose a 3D point cloud reconstruction method, CLCFM3 (Closed-Loop Coarse-to-Fine method with Multi-Masked Matching). This method repeatedly executes a process that generates point clouds locally to suppress occlusion (multi-matching) and a process that removes noise points using a mask image (masked matching). Furthermore, we propose the Closed-Loop Coarse-to-Fine Method (CLCFM) to improve the accuracy of structure from motion, which is essential for implementing the proposed point cloud reconstruction method. CLCFM solves loop-closure by performing coarse-to-fine camera position estimation. By facilitating the acquisition of high-density, high-precision 3D data on a large number of plant bodies, as is necessary for research activities, this approach is expected to enable comparative analysis of visible phenotypes in the growth process of a wide range of plant species based on 3D information.

plant biology↗

bioRxiv2024

Genomic evolution of pancreatic cancer at single-cell resolution

We adapted a previously developed targeted single-nucleus DNA sequencing (snDNA-seq) method and constructed a new suite of computational analysis tools to study 137,491 single-nucleus DNA libraries from 24 pancreatic cancers collected under a variety of clinical scenarios including early and late diagnoses, different metastatic sites and before- and after-treatment. We refined the mutational landscape of pancreatic cancer by capturing events missed by bulk sequencing, and validated the evolution pattern of early fixation of driver single-nucleotide variants (SNVs) followed by generation of intratumoral heterogeneity for copy number variations (CNVs). Intertumoral convergent evolution was common, including subclonal inactivation of TGF-{beta} pathway by mutating various components of it; intratumoral convergence was rarely observed, likely due to strong selective force in pancreatic cancer development. Continuous evolution was frequently seen manifesting as CNVs. In the context of non-targeted treatments, no particular pattern was found across metastases or through treatment. In six pancreatic cancers with germline BRCA2 mutation, we discovered varied timing of biallelic inactivation of BRCA2, which sculpted different evolutionary trajectories and could presumably contribute to differential response to treatment. As the first large-scale application of targeted snDNA-seq on pancreatic cancer, this work provides a sample processing and computational analysis pipeline that warrants further clinical utility.

cancer biology↗

bioRxiv2023

In vivo long-term voltage imaging by genetically encoded voltage indicator reveals spatiotemporal dynamics of spinal cord neuronal populations during development

One of the central questions in neural development is how individual neurons assemble functional networks. To address this, it is essential to elucidate how coordinated activity emerges during development. However, tracking the functional maturation of neuronal populations over time remains challenging, as it requires long-term, non-invasive monitoring of membrane potential dynamics. Here, we developed a voltage imaging approach for zebrafish embryos using genetically encoded voltage indicators (GEVIs), enabling fast, direct and cell-type-specific measurements of membrane potentials from defined neuronal populations in a non-invasive manner. Using this approach, we detected coordinated voltage changes in spinal motor neurons with high spatiotemporal resolution. Depolarization and hyperpolarization events were observed at the population, single-cell, and subcellular levels. Notably, long-term voltage imaging revealed the early emergence and progressive maturation of membrane potential dynamics, characterized by increased firing rate, coupling strength and axonal outgrowth. This optical approach constitutes a significant advancement in the study of neural development, providing a powerful tool for investigating the spatiotemporal dynamics of neuronal populations in vivo.

neuroscience↗

bioRxiv2023

GENI: a web server to identify gene set enrichments in tumor samples

The Cancer Genome Atlas (TCGA) and other projects provide informative tumor-associated genomic data for the broad research community. Hence, several useful web-based tools have been generated to ease non-expert users with the analysis and characterization of a specific gene behavior in selected tumors. However, none of the existing tools offer the user the means to evaluate the expression profile of a given gene in the context of the whole transcriptome. Currently, such analyses require prior bioinformatic knowledge and expertise. Therefore, we developed GENI (Gene ENrichment Identifier) as a fast, user-friendly tool to analyze the TCGA expression data for gene set enrichments. GENI analyzes large-scale tumor-associated gene expression datasets and evaluates biological relevance, thus offering researchers a simplified means to analyze cancer patient-derived data. Availability and implementationGENI is a freely available fast web-based tool developed using the R programming language. It is accessible via the URL: https://yoavshaul-lab.shinyapps.io/gsea-geni/. The complete script can be found at https://github.com/ArataHayashi/GENI-Gene-ENrichment-Identifier.

bioinformatics↗

bioRxiv2023

Cutaneous jet-injection of naked mRNA vaccine induces robust immune responses without systemic vaccine spillage

Naked mRNA-based vaccines may reduce the reactogenicity associated with delivery carriers, but their effectiveness has been suboptimal against infectious diseases. Herein, we aimed to enhance their efficacy by using a pyro-drive liquid jet injector that precisely controls pressure to widely disperse mRNA solution in the skin. The jet injection boosted naked mRNA delivery efficiency in the mouse skin. Mechanistic analyses indicate that dendritic cells, upon uptake of antigen mRNA in the skin, migrate to the draining lymph nodes for antigen presentation. Additionally, the jet injector activated innate immune responses in the skin, presumably by inducing physical stress, thus serving as a physical adjuvant. From a safety perspective, our approach, utilizing naked mRNA, restricted mRNA distribution solely to the injection site, preventing systemic pro-inflammatory reactions following vaccination. Ultimately, the jet injection of naked mRNA encoding SARS-CoV-2 spike protein elicited robust humoral and cellular immunity, providing protection against SARS-CoV-2 infection in mice. Furthermore, our approach induced plasma activity of neutralizing SARS-CoV-2 in non-human primates, comparable to that observed in mice, with no detectable systemic reactogenicity.

bioengineering↗

bioRxiv2023

The metabolic enzyme EXT1 is sufficient to induce the epithelial-mesenchymal transition program in cancers

Carcinomas often exhibit aggressive characteristics, such as enhanced migration abilities, through the execution of the epithelial-mesenchymal transition (EMT) program. Heparan sulfate (HS) is a polysaccharide expressed on the surface of aggressive cancer cells, which acts as a co-receptor to stimulate EMT-associated signaling pathways. However, despite HS role in cancer aggressiveness, the mechanisms governing its EMT-dependent biosynthesis remains poorly understood. Here, we characterized the HS chain elongation enzyme, exostosin glycosyltransferase 1 (EXT1), as an essential component of the EMT program. We identified an EMT-dependent expression of EXT1 and its selective upregulation in aggressive tumor subtypes and cell lines. Overexpression of EXT1 in epithelial cells is sufficient to induce HS biosynthesis, cell migration, and invasion, form tumors in mice, and activate the STAT3 pathway. Moreover, its knockout in aggressive cells significantly inhibited their EMT-associated characteristics. These findings demonstrate a cellular mechanism by which metabolic processes regulate signaling pathways to govern cell state.

cancer biology↗

bioRxiv2022

Depletion of FH, an essential TCA cycle enzyme, drives proliferation in a two-step model

Several tumor suppressor genes do not follow the canonical function of cell cycle repressors. For example, fumarate hydratase (FH) is an evolutionary conserved TCA cycle enzyme that reversibly catalyzes the hydration of fumarate to L-malate and has a moonlight function in the DNA damage response (DDR). Interestingly, despite this enzymes essential role in central carbon metabolism, FH is inactive or absent in several tumors, such as hereditary leiomyomatosis and renal cell cancer (HLRCC). Accordingly, FH has a contradictory cellular function, as it is pro-survival through its role in the TCA cycle, yet its loss can drive tumorigenesis. These observations have supported the role of FH as a tumor suppressor. Here, we solved this contradiction by determining the molecular mechanisms that allow the cells to survive and even proliferate upon FH loss. We found that the cells response to FH loss is separated into two distinct time frames based on cell proliferation and DNA damage repair. During the early stages of FH loss, the cells proliferation and DNA damage repair are inhibited. However, over time the cells overcome the FH loss and form knockout clones, indistinguishable from WT cells in their proliferation rate. Due to the FH loss effect on DNA damage repair, we assumed that the recovered cells bear adaptive mutations. Therefore, we applied whole-exome sequencing to identify such mutated genes systematically. Indeed, we identified recurring mutations in genes belonging to the central oncogenic signaling pathways, such as JAK/STAT, which we validated to be impaired in FH-KO clones. Intriguingly, we demonstrated that these adaptive mutations are responsible for FH-KO cell proliferation under TCA cycle malfunction.

cancer biology↗

bioRxiv2022

Application of high-throughput, high-depth, targeted single-nucleus DNA sequencing in pancreatic cancer

Despite insights gained by bulk DNA sequencing of cancer it remains challenging to resolve the admixture of normal and tumor cells, and/or of distinct tumor subclones; high throughput single-cell DNA sequencing circumvents these and brings cancer genomic studies to higher resolution. However, its application has been limited to liquid tumors or a small batch of solid tumors, mainly because of the lack of a scalable workflow to process solid tumor samples. Here we optimized a highly automated nuclei extraction workflow that achieved fast and reliable targeted single-nucleus DNA library preparation of 38 samples from 16 pancreatic adenocarcinoma (PDAC) patients, with an average library yield per sample of 2867 single nuclei. We demonstrate that this workflow not only performs well using low cellularity or low tumor purity samples but reveals novel genomic evolution patterns of PDAC as well.

cancer biology↗