Biology subjects

Hamada, M.

Publications and source records attributed to Hamada, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Alignment-free discovery of conserved RNA structures in the human genome

Many functional RNAs were first found as conserved RNA structures, and their roles in gene regulation were identified later. In humans and other vertebrates, imperfect genome alignments have hindered the discovery of such structures. Here we present RLMHelix, which selects candidate conserved RNA structures from single sequences before any alignment by scoring helices supported by language-model dependencies and thermodynamics. Through distillation, RLMHelix rapidly scans the transcribed human genome and ranks known RNAs higher than conventional thermodynamic scanning. Two-thirds of 15,821 candidates are new to four earlier genome-wide screens, yet agree with in-cell probing, and over half retain structural signal in homologues. In eCLIP data and in exploratory pull-downs, higher RLMHelix scores were associated with more binding by double-stranded RNA-binding proteins. By linking candidates to their binding proteins, RLMHelix yields testable functional hypotheses and illuminates a path from unrecognized human RNA structures to RNA biology and therapeutic targets.

bioinformatics↗

bioRxiv2026

Quantitative host-response mapping enables phenotypic profiling of viruses detected by virome sequencing

Animal-derived viruses have repeatedly caused human outbreaks, motivating efforts to identify and characterize viruses circulating in animals. Virome sequencing has accelerated animal virus discovery, but phenotypic characterization remains difficult to scale. Although generated for viral detection, virome RNA-seq data also capture host transcriptional responses to infection. Here, we developed ViroChart, a comparative transcriptomic framework that quantifies activities across biological pathways and maps diverse mammalian host responses into a common, interpretable low-dimensional space. ViroChart enables clustering of infected samples by host response pattern and quantitative assessment of host response similarity between samples. In virome data sets from wild and farmed animals, some lung samples infected with poorly characterized betacoronaviruses showed host responses resembling those induced by viruses capable of causing fatal disease. ViroChart also revealed host-response heterogeneity in natural infections: among rats with zoonotic rodent-borne viruses, most showed weak responses, consistent with asymptomatic infection, but some showed pronounced inflammation characteristic of acute infection. ViroChart thus enables phenotypic characterization and prioritization of newly detected viruses by leveraging the same RNA-seq data from which their genomes were detected.

microbiology↗

bioRxiv2026

Method-dependent biases in cell type detection between single-cell and single-nucleus RNA sequencing in the photosymbiotic acoel Praesagittifera naikaiensis

Background Comparisons of single-cell and single-nucleus RNA sequencing (scRNA-seq and snRNA-seq) data have been described in some mammalian tissues and, subsequently, in Drosophila, but remain unexplored in most invertebrate lineages. The xenacoelomorphs occupy key phylogenetic positions, yet they differ anatomically from mammals. They have a reduced extracellular matrix, high-salt body fluid, and no circulatory system. Despite these differences, they possess a well-developed nervous system. One of the xenacoelomorphs, the photosymbiotic acoel (Praesagittifera naikaiensis) also harbours symbiotic Tetraselmis algae, whose RNA can be co-captured with host RNA. Results We compared scRNA-seq and snRNA-seq data from whole P. naikaiensis specimens. Both methods yielded high-quality data with comparable gene detection but a larger share of scRNA-seq reads derived from symbiotic algae. Gene-level analyses revealed that neural genes were enriched in snRNA-seq relative to non-neural genes. Cross-method label transfer and integration-based validation identified six snRNA-seq clusters, including some neural populations, that lacked a clear scRNA-seq counterpart. In contrast, three cell populations, including muscle and metabolically active clusters, were reciprocally validated as captured by both methods. Conclusions Our results show that key snRNA-seq advantages, particularly the enhanced recovery of neural transcripts, are recapitulated in our dataset, which is consistent with previous reports in mammals. Furthermore, snRNA-seq reduces symbiont-derived reads and recovers several cell populations underrepresented in scRNA-seq. These findings provide practical guidance for cell atlas construction in non-model, symbiotic invertebrates.

genomics↗

bioRxiv2026

Nationwide multi-omics profiling of Japanese jack mackerel reveals geographic gut microbiome structuring despite host panmixia

Host genetic markers often fail to resolve regional origins in highly connected or panmictic marine species. The Japanese jack mackerel, Trachurus japonicus, is a commercially important fishery species around Japan that shows little or no detectable population structure. Here, we used nationwide multi-omics profiling to compare host genomic variation and gut microbiome composition in wild T. japonicus collected from coastal regions across Japan. We generated MIG-seq data for 43 individuals and 16S rRNA gene profiles for 24 individuals; after quality filtering, 19 individuals remained for matched host-microbiome comparison. Genome-wide host SNP analyses showed weak or absent geographic population structure, consistent with previous evidence of panmixia in Japanese waters. In contrast, gut microbiome composition showed geographic structuring based on Bray-Curtis dissimilarity and PERMANOVA, and this pattern was not explained by proximity to river mouths or host-related variables. Locality- or individual-associated bacterial lineages contributed to the observed differences in the microbiome, while chloroplast-associated and Cyanobacteria-assigned ASVs suggested recent dietary or environmental input. These results indicate that gut microbiome can show regional biological variation not apparent from host genetic markers alone. Our study provides a proof-of-concept example of integrating host genomics and gut microbiome profiling to evaluate regional characteristics and origins in highly connected marine animals.

microbiology↗

bioRxiv2026

BWR-finder: BWT-based de novo interspersed repeat detection for gigantic genomes

We developed BWR-finder (Burrows-Wheeler transform-based Repeat finder), a new software tool for database-free detection of interspersed repeats in large genomes of tens of gigabases. BWR-finder employs a BWT-based seed-and-extend repeat detection algorithm and parallelized extension computation, improving both runtime and memory usage. In benchmarks using the rice and human genomes, BWR-finder reduced runtime compared with RepeatModeler2, HiTE, and REPrise, and reduced memory usage compared with HiTE and REPrise, while maintaining high nucleotide-level repeat detection sensitivity. BWR-finder also enabled whole-genome repeat detection in genomes larger than 10 Gb and identified candidate repeat regions and repeat consensus sequences in the 20.3-Gb Pleurodeles waltl genome that were not associated with existing repeat annotations or libraries.

bioinformatics↗

bioRxiv2026

Polyvinylpyrrolidone enhances AAV transduction in vitro and shows preliminary utility for subretinal delivery

Adeno-associated virus (AAV) vectors are widely used for gene delivery, but inefficient transduction can require high vector doses. We tested whether unmodified linear polyvinylpyrrolidone (PVP), a pharmaceutical excipient, can improve AAV formulation without chemical modification of the vector or polymer. PVP10, PVP40, and PVP360 were evaluated in vitro across HEK293, HeLa, MEF, and CHO cells; selected formulations were tested after intravenous delivery, and 3% PVP40 was tested by subretinal delivery. In vitro, 1-3% PVP increased AAV8- and AAV9-mediated GFP expression across multiple cell lines. PVP360 showed broad activity in the initial cross-cell assay, whereas the HEK293 molecular-weight screen identified PVP40 and PVP360 as the most active formulations. The substantial fold increase observed in CHO cells largely reflected the low baseline transduction of the control group. MTT absorbance declined with concentrated PVP360, whereas PVP40 retained transduction-enhancing activity and was selected for local testing. In the HeLa AAV-DJ assay, the response pattern differed between MOI 1,000 and MOI 100; the largest observed increases in GFP-positive area occurred with 1.5% PVP10 and 1.5% PVP360 at MOI 100. Intravenous AAV9 delivery with PVP did not consistently increase ex vivo organ reporter signal. By contrast, subretinal delivery of AAV-PHP.eB with 3% PVP40 produced a 1.79- fold larger mean DsRed-positive area per retinal section, averaged within each eye (Welchs t-test p = 0.063; Bayesian Pr[{Delta} > 0] = 0.952). These findings support further evaluation of PVP40 for local subretinal AAV delivery.

bioengineering↗

bioRxiv2026

Age-associated erosion of organ-specific endothelial programs compromises tissue function and resilience

Endothelial cells (ECs) express organ-specific gene programs supporting tissue homeostasis and resilience. However, the mechanisms by which aging reshapes these organ-specific endothelial programs and how the resulting changes affect tissue homeostasis, resilience, and disease susceptibility remain largely unknown. Herein, we performed single-cell RNA sequencing of ECs harvested from five organs across the lifespan and found that aging progressively erodes organ-specific endothelial programs while inducing shared interferon-responsive and antigen-presentation programs across organs. Although vascular subtype identity and conserved capillary subset identity were mostly preserved, these organ-specific transcriptional programs were broadly attenuated with aging, indicating erosion of organ-specific endothelial identity to be a fundamental feature of endothelial aging. Importantly, these alterations were associated with declines in specialized EC functions, including alveolar barrier maintenance in the lung, scavenging activity in the liver, angiogenic capacity in the heart, and homeostatic programs in the kidneys and the brain, suggesting that age-related EC alterations compromise tissue homeostasis and resilience in multiple organs. Furthermore, we established a single-cell aging index for alveolar capillary ECs in mice and humans, revealing stress-associated endothelial activation to potentially be an intermediate state linking functional deterioration to cellular senescence, and also demonstrating marked heterogeneity in aging states even among ECs from animals of the same chronological age. Notably, alveolar capillary ECs exhibited progressive functional decline before reaching a senescent-like state, suggesting endothelial dysfunction to precede overt cellular senescence as the organism ages. Collectively, our findings establish progressive erosion of organ-specific endothelial programs as a central feature of vascular aging and provide a conceptual framework for elucidating how endothelial aging contributes to tissue dysfunction and reduced resilience across organs.

physiology↗

bioRxiv2026

A computational framework to model cartilage degeneration induced by mechanoinflammation and cytokine-driven inflammation in post-traumatic osteoarthritis

Knee joint injury is a major risk factor for post-traumatic osteoarthritis (PTOA), often associated with early cartilage degeneration. Mechanical overloading and cytokine-driven inflammation are key drivers of this process, yet the underlying mechanisms and their distinct temporal and spatial contributions to cartilage degradation remain unclear. Here, we present a mechanobiological finite element framework that simulates cartilage degradation through cell-mediated proteolytic activity triggered by mechanoinflammation and cytokine-driven inflammation. The model reproduces experimentally observed depth-dependent loss of collagen and aggrecan, with mechanoinflammation inducing a transient response and cytokine-driven inflammation sustaining prolonged matrix degradation. Sensitivity analysis further shows that mechanoinflammation-driven degradation is governed mainly by protease production per cell, whereas cytokine-driven degradation is more sensitive to the rate of cellular stimulation. Together, this framework provides a mechanistic basis to study proteolytic cartilage degeneration and supports future in silico evaluation of therapeutic strategies aimed at mitigating cartilage degradation in PTOA.

biophysics↗

bioRxiv2026

Integrating spatial and single-cell multi-omics analysis of induced pluripotent stem cell-derived cervical adenocarcinoma model

Human papillomavirus 18 (HPV18) preferentially infects cervical stem cell-like cells and is strongly associated with adenocarcinoma. However, the mechanisms underlying differentiation into cervical adenocarcinoma remain unclear due to the lack of appropriate experimental models. We aimed to establish a model of HPV18-associated cervical adenocarcinoma and elucidate its molecular and cellular differentiation mechanisms. HPV18 E6/E7 were introduced into induced pluripotent stem cell-derived reserve cell-like cells (iRCs) to generate tumor models. Spatial transcriptomics and single-cell multi-omics analyses were performed to integrate histological and molecular data. A distinct component (Gland_A) exhibited morphological and immunohistochemical features of cervical adenocarcinoma and was efficiently induced in iRC-18 tumors. Gland_A showed increased chromatin accessibility and elevated expression of FOXA1, FOXA2, and ALDH1A1. Analysis of clinical samples confirmed enrichment of ALDH1A1 in HPV-associated adenocarcinomas. This model recapitulates key features of HPV18-associated cervical adenocarcinoma and provides insights into its differentiation mechanisms.

cancer biology↗

bioRxiv2026

Establishing genetically controlled, closed 1 colonies of an ascidian

Recent technological advances have made many "non-model" organisms accessible for experimental studies. However, reference inbred strains were not necessarily available, especially in marine invertebrates, and genetic background of organisms used for experiments are often non-uniform. This situation potentially affects experimental reproducibility. Although ascidians, Ciona intestinalis (type A, or C. robusta), are a widely used marine animal for many areas of experimental biology including developmental studies, no reference strains have been obtained despite extensive efforts. As an alternative way to improve reproducibility, we have established and maintained ascidian colonies through intra-population breeding every year from 2016 to 2020, and monitored genomic variants of these colonies. This method does not reduce genetic variations but instead manages and monitors genetic variations in the colonies, providing an easy and cost-effective way of increasing experimental reproducibility. Furthermore, we recently upgraded these genetically isolated, closed colonies that were re-established every year, and have maintained them for more than three years only through intra-population breeding and occasional back-cross using cryopreserved sperm. Genetic variants that we revealed using 3.7 tera-bases of sequence data will help to design future experiments in this species. Our data also show that two wild-populations, which were used to establish the colonies, have maintained distinct genetic backgrounds, although their habitats are directly linked to the Pacific Ocean and only 170 km apart. More importantly, genetic information regarding these colonies will undoubtedly improve experimental reproducibility and traceability, and our method will provide a realistic solution for performing reproducible experiments using non-model organisms.

developmental biology↗

bioRxiv2025

RNA degradation modulates unique aging-related gene expression in naked mole-rats

Gene expression is co-regulated by the rates of RNA synthesis and degradation, and recent evidence has linked their imbalance to the onset of aging. The naked mole-rat (NMR) is a small rodent with an exceptionally long lifespan and markedly delayed aging, yet little is known about its RNA synthesis and degradation characteristics compared to other rodents that age more rapidly. Here, we investigate RNA synthesis and degradation in NMR and mouse skin fibroblasts by monitoring incorporation of a uridine analog, 4-thiouridine. Cross-species analysis showed that the NMR cells have higher overall rates of RNA synthesis and degradation. It further revealed higher RNA degradation rates in aging-related pathways, notably Mtorc1 signaling, likely contributing to reducing the overall expression. Although known aging-associated genes, including Xrcc5, Nudt1, Fen1, and Aptx, were expressed at similar levels in NMR and mouse fibroblasts, the RNA turnover rates were largely altered. To uncover the underlying mechanism enabling differential control of RNA kinetics, we analyzed the transcript feature importance by machine learning and identified key features governing RNA degradation both common and unique in NMR and mouse fibroblasts. Our data highlight a potential role of RNA synthesis and degradation as hidden layers of gene regulation in NMR.

molecular biology↗

bioRxiv2025

Comprehensive and accelerated mapping of driver mutations through single-nucleotide random mutagenesis of target genes

Identification of novel driver mutations is crucial for personalized medicine and drug discovery. However, genome-wide studies based solely on patient-derived data cannot confirm the oncogenic potential of detected mutations. Here, we present a genome editing-based method for comprehensive random mutagenesis of target genes. This approach generates cultured cells harboring random single-nucleotide variants (SNVs) within 10 days of DNA amplicon preparation. To validate its utility, random mutations were introduced into endogenous exons of lung cancer-related oncogenes in HEK293 cells. Spheroid formation assays and xenograft models demonstrated enhanced tumorigenicity, indicating the acquisition of oncogenic potential. Amplicon-sequencing of spheroid transcripts revealed significant enrichment of variants annotated as pathogenic in clinical databases and computational predictions. Furthermore, osimertinib treatment of lung cancer cell lines with random SNVs on EGFR identified 58 novel resistance mutations. These results highlight the effectiveness of our mutagenesis platform, offering a powerful tool to advance cancer research and precision medicine.

bioengineering↗

bioRxiv2025

Blind Prediction of Complex Water and Ion Ensembles Around RNA in CASP16

Biomolecules rely on water and ions for stable folding, but these interactions are often transient, dynamic, or disordered and thus hidden from experiments and evaluation challenges that represent biomolecules as single, ordered structures. Here, we compare blindly predicted ensembles of water and ion structure to the cryo-EM densities observed around the Tetrahymena ribozyme at 2.2-2.3 [A] resolution, collected through target R1260 in the CASP16 competition. 26 groups participated in this solvation cryo-ensemble prediction challenge, submitting over 350 million atoms in total, offering the first opportunity to compare blind predictions of dynamic solvent shell ensembles to cryo-EM density. Predicted atomic ensembles were converted to density through local alignment and these densities were compared to the cryo-EM densities using Pearson correlation, Spearman correlation, mutual information, and precision-recall curves. These predictions show that an ensemble representation is able to capture information of transient or dynamic water and ions better than traditional atomic models, but there remains a large accuracy gap to the performance ceiling set by experimental uncertainty. Overall, molecular dynamics approaches best matched the cryo-EM density, with blind predictions from bussilab_plain_md, SoutheRNA, bussilab_replex, coogs2, and coogs3 outperforming the baseline molecular dynamics prediction. This study indicates that simulations of water and ions can be quantitatively evaluated with cryo-EM maps. We propose that further community-wide blind challenges can drive and evaluate progress in modeling water, ions and other previously hidden components of biomolecular systems.

biophysics↗

bioRxiv2025

Screening and machine-learning assisted prediction of translation-enhancing peptides reducing ribosomal stalling in Escherichia coli

We previously reported that the nascent SKIK peptide enhances translation and alleviates ribosomal stalling caused by arrest peptides (APs) such as SecM and polyproline when positioned immediately upstream of the APs in both Escherichia coli in vivo and in vitro translation systems. In this study, we performed a comprehensive screening of translation-enhancing peptides (TEPs) using a randomized artificial tetrapeptide library. The screening was based on the ability of the peptides to suppress SecM AP-induced translational stalling in E. coli cells. Various TEPs exhibiting a range of translation-enhancing activities were identified. In vitro translation analysis suggested that the fourth amino acid in the tetrapeptide plays a key role in reducing SecM AP-mediated stalling. Furthermore, we developed a machine learning model using a random forest algorithm to predict TEP activity. The predicted values showed a strong correlation with experimentally measured activities. These findings offer a compact peptide toolkit and a data-driven approach for mitigating AP-induced ribosome stalling, with potential applications in synthetic biology.

synthetic biology↗

bioRxiv2025

Title Mafb Deficiency in Myeloid Cells Increases Susceptibility to Mycobacterium tuberculosis Infection in Mice

v-Maf avian musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) is a candidate gene associated with early tuberculosis onset identified by a genome-wide association study. Here, we investigated the role of Mafb in susceptibility to Mycobacterium tuberculosis (Mtb) infection in myeloid-specific Mafb-knockout (Mafb-cKO) mice. We infected bone marrow-derived macrophages (BMMs) from Mafb-cKO mice and Mafb-cKO mice with Mtb. The absence of Mafb promoted Mtb proliferation in BMMs. RNA sequencing (RNA-seq) revealed activation of the metabolic process and impairment of the response to type interferons (IFNs) in Mtb-infected BMMs from Mafb-cKO mice, which conforms to our previous findings in Mtb-infected human macrophages with MAFB knockdown. Mafb deficiency increased mortality and bacterial burden in the lungs and spleens during Mtb infection in mice. RNA-seq revealed weakened leukocyte or lymphocyte chemotaxis in Mtb-infected Mafb-cKO mouse lungs. Flow cytometry demonstrated an alteration in the proportion of immune cells in Mtb-infected mouse lungs due to Mafb deficiency. Together, Mafb in myeloid cells is involved not only in the functional antibacterial process of macrophages but also in immune cell recruitment in the lungs, thereby contributing to host defense against Mtb infection.

immunology↗

bioRxiv2025

Mitotic phosphorylation of ADAR1 regulates its centromeric localization and is required for faithful mitotic progression

Adenosine Deaminase Acting on RNA 1 p110 (ADAR1p110), the constitutively expressed nuclear isoform of the RNA-editing enzyme ADAR1, plays well-established roles in adenosine-to-inosine RNA editing, but its functions during mitosis remain poorly defined. Here, we identify ADAR1p110 as a chromatin-associated factor essential for mitotic chromosome segregation and cell viability. Depletion of ADAR1 caused metaphase arrest, DNA damage accumulation, and apoptosis. Co-immunoprecipitation experiments revealed that ADAR1p110 physically interacts with Structural Maintenance of Chromosome 3 (SMC3), a core component of the cohesin complex. Genome-wide DNA immunoprecipitation sequencing (DIP-seq) showed that ADAR1p110 selectively binds centromeric -satellite DNA during mitosis, a finding validated by DNA immunoprecipitation quantitative polymerase chain reaction (DIP-qPCR). Complementary DNA-RNA immunoprecipitation sequencing (DRIP-seq) revealed that RNA:DNA hybrids (R-loops) are enriched in centromeric regions during mitosis and are further augmented by ADAR1 overexpression. We identified serine 614 (S614) as a key mitosis-specific phosphorylation site on ADAR1p110, and demonstrated that this post-translational modification is essential for its chromatin recruitment, stability, and mitotic function. Rescue experiments using phospho-mimetic mutants (S614D and 3xD) successfully restored mitotic progression in ADAR1-deficient cells, whereas non-phosphorylatable variants failed to do so. These results reveal that ADAR1p110 is phosphorylated in a cell cycle-dependent manner and functions at the intersection of post-transcriptional and post-translational regulation. By coordinating R-loop recognition at centromeres with chromosome cohesion, ADAR1p110 safeguards genome integrity during mitosis. Our findings uncover a previously uncharacterized mechanism through which a canonical RNA-editing enzyme contributes to chromosomal dynamics independent of its deaminase activity.

molecular biology↗

bioRxiv2025

Deciphering the comprehensive relationship between 5'UTR and 3'UTR sequences with deep learning

MotivationRecent advances in mRNA therapeutics have driven further research on untranslated region (UTR) of mRNA. However, prior studies have mainly focused on either the 5' or 3' UTR individually. Increasing evidence suggests potential cooperative effects between these two regions, which remain largely unexplored in computational studies. ResultsWe present a deep learning-based approach to predicting relationships between 5' and 3' UTRs by leveraging latent representations from a pre-trained RNA language model and contrastive learning. Our method effectively identifies highly related UTR pairs, uncovering sequence and expression characteristics that suggest functional interplay. Our analysis revealed that Highly Related UTR pairs (HRUs) are significantly enriched in genes associated with neural development, exhibit distinctive UTR length and secondary structure characteristics, and are involved in cell type-specific regulation of translation efficiency. These findings provide new insights into UTR co-optimization for mRNA therapeutics. AvailabilityThe source code is available for free at https://github.com/hmdlab/utr_pairpred.git. The data and intermediate files used in our analysis are available at https://waseda.box.com/v/utr-pairpred-data.

bioinformatics↗

bioRxiv2025

The integration mechanism of the four positional cues--anterior, posterior, dorsal, and ventral--in axolotl limb regeneration

Axolotls (Ambystoma mexicanum) exhibit a remarkable ability to regenerate limbs after amputation. Classical experiments have suggested that contact between cells derived from distinct orientations--dorsal, ventral, anterior, and posterior--within the regenerating blastema is necessary for accurate limb pattern formation. However, the molecular basis for this requirement has remained largely unknown. Here, we demonstrate that both dorsal and ventral tissues are required for limb formation via induction of Shh expression, which plays a crucial role in limb patterning. Using the accessory limb model (ALM), we induced position-specific blastemas lacking cells derived from a single orientation (anterior, posterior, dorsal, or ventral). Patterned limb formation occurred only in blastemas containing both dorsal- and ventral-derived cells. We further observed that Shh expression requires dorsoventral contact within a blastema, highlighting the necessity of dorsoventral contact for inducing Shh expression. In addition, we identified WNT10B and FGF2 as dorsal- and ventral-mediated signals, respectively, that create the inductive environment for Shh expression. Our findings clarify the role of dorsal and ventral cells in inducing Shh, a mechanism that has rarely been studied in the context of limb regeneration and pattern formation. This model provides new insights into how cells with different positional identities drive the regeneration process.

developmental biology↗