Biology subjects

Kubota, A.

Publications and source records attributed to Kubota, A..

13 recordsLinked to original sources

bioRxiv2026

Click-Prep: An Interactive Data Preparation Tool for Click-qPCR

Click-qPCR is a browser-based application for relative qPCR analysis that requires a tidy-format CSV file containing four columns: sample, group, gene, and Cq. Preparing this input from qPCR instrument output typically requires manual reformatting and calculation of mean Cq values for technical replicates. To simplify this process, we developed Click-Prep (https://kubo-azu.shinyapps.io/Click-Prep/), an interactive web-based application designed specifically to create Click-qPCR input files. Click-Prep imports CSV, TXT, TSV, and XLS/XLSX files and supports skipping of instrument-generated metadata rows, interactive column mapping, and manual assignment of experimental groups. Users can review technical-replicate measurements, exclude selected rows according to predefined quality-control criteria, and calculate mean Cq values for each sample-group-target combination. Missing or nonnumeric Cq values are flagged for review and must be resolved before the mean is calculated. Click-Prep can also combine compatible formatted CSV files, such as datasets obtained from separate qPCR plates. The resulting dataset is exported as a standardized CSV file containing the four fields required by Click-qPCR. By integrating these operations into a guided browser-based workflow, Click-Prep enables users to prepare Click-qPCR input files rapidly and consistently without programming.

bioinformatics↗

bioRxiv2026

Transcriptomic response to histone deacetylase inhibitors in cultured feline cells

ObjectiveIn recent years, the number of cats kept as companion animals has increased, leading to a growing demand for veterinary care. Although some histone deacetylase (HDAC) inhibitors are promising for the treatment of human cancers and neurological diseases, comprehensive systematic research on HDAC inhibitors in domestic cats remains insufficient. Therefore, this study aimed to investigate the effects of HDAC inhibitors on the transcriptome of feline cells. MethodsTwo types of cells derived from domestic cats, Crandell-Rees Feline Kidney (CRFK; kidney-derived) cells and PG-4 cells (astrocyte-derived), were treated with four HDAC inhibitors (panobinostat, trichostatin A, valproic acid, and vorinostat) for 24 h. Transcriptomic changes after treatment were examined using RNA sequencing. ResultsHDAC inhibitor treatment upregulated the expression of intercellular chemical interactions and signal transduction-related genes, similar to observations in human cells. Although HDAC inhibitors did not suppress the expression of cell cycle-related genes in CRFK cells, as observed in human cells, the inhibitors downregulated the expression of organogenesis-related genes. Consistent with observations in human cells, HDAC inhibitors suppressed the expression of cell cycle- and cancer-related genes in PG-4 cells. Importantly, valproic acid, which is thought to be more effective for neurological diseases than for cancer, suppressed the expression of more cancer-related genes in PG-4 cells than the other three HDAC inhibitors. Conclusion and relevanceOur findings revealed that the responses of cells derived from feline organs to various HDAC inhibitors varied considerably depending on the organ of origin and species. Since few studies, including human studies, have comprehensively compared transcriptomic responses to multiple HDAC inhibitor classes across multiple cell types, the results of this study provide a foundation for future research on the treatment and prevention of cancer and neurological diseases in domestic cats and other mammals.

cell biology↗

bioRxiv2026

Multi-omics analysis reveals chromatin and transcriptomic remodeling in hippocampal CA1 following adolescent social isolation

Social isolation (SI) during adolescence is associated with neurobiological alterations; however, its effect on the hippocampal epigenome and transcriptome remains unclear. We performed ATAC-seq and RNA-seq of hippocampal CA1 in an adolescent mouse SI model, with single-cell RNA-seq reference mapping and cell type deconvolution. ATAC-seq identified candidate SI-associated chromatin accessibility alterations, with nominal functional enrichment linking increased enhancer-associated regions to calcium transport and decreased promoter regions to synaptic organization. Motif analysis showed enrichment of the activator protein 1 (AP-1) motif among candidate regions with reduced accessibility. RNA-seq identified 106 differentially expressed genes (DEGs), including upregulated Fosl2 and Hdac9 and downregulated Fbxw7, with enrichment of myelination-related pathways. Although global concordance between chromatin accessibility and transcriptional changes was limited, integrated analysis showed directional concordance between expression and nearby candidate accessibility changes for a subset of DEGs. Cell type analyses suggested that SI-downregulated transcriptional signatures were linked to excitatory neurons, whereas SI-upregulated signatures were enriched in non-neuronal populations, particularly oligodendrocytes. Deconvolution suggested higher estimated oligodendrocyte representation and lower estimated excitatory neuron representation in isolated mice. These findings suggest that adolescent SI is associated with significant transcriptomic remodeling and candidate chromatin accessibility changes within hippocampal CA1, accompanied by distinct neuronal- and glial-associated transcriptional signatures.

neuroscience↗

bioRxiv2026

A practical pipeline for volume rendering of trillion-voxel tomographic data

Recent advancements in tomography produce imaging data of geological materials (rocks and fossils) at trillion-voxel scales with multi-channels. Such high-resolution datasets are potentially keys to unveil evolutionary biological information with various shapes and sizes that have not been ever discovered. Volume rendering is an ideal visualization approach for them because it treats all voxels without relying on user-defined surface boundaries. However, these large-scale real-world tomographic data have rarely been volume-rendered at their native resolution, limiting the examination of rich morphological information. Here, we demonstrate a de facto standard volume-rendering pipeline running on a graphical processing unit (GPU)-equipped supercomputing system toward multi-channel, trillion-voxel tomographic data. Our workflow preserves original resolution, capturing detailed morphological information spanning microscopic to macroscopic scales. Systematic comparison of node types shows that GPU memory, rather than host memory, is the primary bottleneck. Our results establish a baseline for large-scale, multi-channel volume rendering of real tomographic data and demonstrate its applicability to geological samples. This work is presented as a practical demonstration of large-scale volume visualization.

paleontology↗

bioRxiv2026

PANDA: Read-Level Phased Analysis of DNA Amplicons for Methylation Studies

Targeted bisulfite amplicon sequencing is widely used to examine methylation at defined genomic loci, but site-wise methylation estimates can obscure phased patterns among individual reads. We developed PANDA (Phased Analysis for DNA Amplicons), an open-source platform for read-level analysis of targeted bisulfite amplicons generated by Sanger sequencing or amplicon NGS, with graphical and command-line interfaces built on a shared computational core (https://github.com/kubo-azu/PANDA). PANDA integrates reference-coordinate methylation calling and visualization, motif-based haplotype filtering, coordinate linkage of unmerged paired reads, and group comparison. It also implements three complementary heterogeneity metrics: Amplicon PDR (proportion of discordant reads), Window Epipolymorphism, and Amplicon qFDRP (quantitative fraction of discordant read pairs). These metrics use retained read-count weighting and explicit eligibility criteria. Synthetic demonstration datasets illustrated global methylation estimation, sequence-defined haplotype selection, and CpG-level group comparison. The heterogeneity metrics illustrated different aspects of the predefined methylation states. Re-analysis of public chimpanzee and rhesus macaque placental datasets was consistent with the previously reported species-associated methylation contrast and demonstrated coordinate-linked analysis of unmerged paired reads without imputing the unsequenced interval. PANDA provides a reproducible framework for examining phased methylation patterns and within-sample heterogeneity alongside site-wise methylation estimates in amplicon sequencing.

bioinformatics↗

bioRxiv2026

Systems-level Investigation of the Anxiolytic Gut-Brain Interactions induced by Paraprobiotic Lactobacillus brevis SBC8803 in Zebrafish

Anxiety disorders are among the most prevalent mental health conditions worldwide, and interest in psychobiotics--live or inactivated microorganisms that beneficially modulate the microbiota-gut-brain axis--is increasing. Heat-killed Lactobacillus brevis SBC8803 enhances serotonin (5-hydroxytryptamine; 5-HT) signaling and ameliorates stress-related phenotypes in mammals, although the gut-brain pathways mediating these effects remain incompletely defined. Here, we investigated the anxiolytic effects and underlying molecular mechanisms of oral SBC8803 administration in adult zebrafish. Adult male AB-strain zebrafish were fed a diet containing heat-killed SBC8803 for 4 weeks, and anxiety-like behavior was evaluated using the novel tank test. SBC8803-treated fish exhibited a shorter latency to enter the upper half of the tank and more frequent transitions into the upper half, consistent with reduced anxiety-like behavior. To explore underlying mechanisms, we performed brain RNA sequencing and 16S rRNA gene sequencing of intestinal contents, followed by integrative multi-omics analyses. Brain transcriptomic profiling identified differentially expressed genes and enrichment of serotonin receptor, CREB, and oxytocin signaling pathways, suggesting enhanced monoaminergic and plasticity-related signaling. Microbiome functional prediction indicated SBC8803-associated shifts in lipid and vitamin metabolism, including pathways related to riboflavin (vitamin B2) and tryptophan. Gene set variation analysis combined with DIABLO-based data integration revealed coordinated changes between microbial metabolic and brain signaling pathways, consistent with a vitamin B-serotonin-anti-inflammatory axis linking gut metabolism to neural regulation. Furthermore, residual correlation analysis showed innate gut-brain coordination independent of SBC8803 effect, such as the coupling between brain arachidonic acid and gut histidine metabolism. These findings support the biological validity of SBC8803 administration-associated interactions observed in the multi-omics analyses. Collectively, these findings indicate that the paraprobiotic SBC8803 exerts anxiolytic-like effects in zebrafish and reshapes gut-brain network states at behavioral, microbial, and transcriptomic levels, providing a mechanistic framework for considering heat-killed SBC8803 as a candidate psychobiotic for anxiety-related conditions.

microbiology↗

bioRxiv2025

Whole-genome sequencing of CRFK and PG-4 cells to infer the phenotype of the original donors

BackgroundCrandell-Rees Feline Kidney (CRFK; kidney-derived) cells and PG-4 cells (astrocyte-derived) have been in use and have been passaged for decades in laboratories worldwide; however, no detailed information on the genetic background of the donor individuals is available, particularly regarding phenotype characteristics such as coat and iris color. ResultsWe performed whole-genome sequencing of CRFK and PG-4 cells. We analyzed the resulting data to infer the phenotype of the individual from which the cells were derived, specifically for the coat color, coat length, coat pattern, and iris color. Our data suggested that CRFK cells originated from a cat with long black fur lacking stripes and with non-blue irises; PG-4 cells originated from a cat with long bicolored white and black fur without stripes, and with non-blue irises. Analysis of publicly available RNA-seq data confirmed that genes associated with coat phenotype and iris color are expressed in the skin and eyes, as well as in various other organs. ConclusionsVariants of the genes affecting coat phenotype and iris color may influence physiological functions throughout the body. These results shed light on the previously unknown genetic background of commonly used feline cultured cells and the phenotype of the donor individuals. These findings may facilitate more accurate interpretation of data obtained from cultured feline cells and provide guidelines for developing cell lines with domestic cat genotypes exhibiting diverse phenotypes through genome editing. This will help to provide clarity regarding deafness in cats with white fur and blue irises and elucidate the effects of melanocyte destruction caused by KIT gene mutations on the nervous system and the influence of other coat-related factors in organs other than the skin or functions independent of coat formation.

genomics↗

bioRxiv2025

Click-qPCR: an ultra-simple tool for interactive qPCR data analysis

Real-time quantitative PCR (qPCR) is a pivotal technique for analyzing gene expression and DNA copy number variation. However, the limited availability of easy-to-use software tools remains a significant challenge for experimental biologists without programming expertise. To address this issue, we developed Click-qPCR, a user-friendly, web-based Shiny application that requires no local installation. Click-qPCR streamlines {Delta}Cq and {Delta}{Delta}Cq calculations using user-uploaded CSV data files. The application allows users to dynamically select genes and groups, and to perform Welchs t-tests for pairwise comparisons and one-way ANOVA with Dunnetts post-hoc test for multi-group comparisons. Results are immediately visualized as interactive bar plots (mean {+/-} SD with individual data points) and can be downloaded as publication-quality images, along with statistical summaries. Click-qPCR enables researchers to efficiently process, interpret, and visualize their qPCR data regardless of programming experience, thereby accelerating data processing and improving the accessibility of routine analysis. The Shiny application of Click-qPCR is freely available at https://kubo-azu.shinyapps.io/Click-qPCR/. The source code and user guide for Click-qPCR are openly available on GitHub at https://github.com/kubo-azu/Click-qPCR.

bioinformatics↗

bioRxiv2025

Housing environment bilaterally alters transcriptomic profile in the rat hippocampal CA1 region

Brain asymmetry is a fundamental feature of neural organization. However, the molecular basis of hippocampal lateralization in response to environmental stimuli remains poorly understood. Here, we examined the transcriptomic profiles of the left and right hippocampal CA1 regions in rats reared under isolated or enriched housing conditions to elucidate hemisphere-specific responses and shared molecular adaptations. RNA-sequencing analysis revealed lateralized differences in the number and identity of differentially expressed genes, accompanied by distinct biological themes, as indicated by overrepresentation and gene set enrichment analysis. The left CA1 region was prominently engaged in pathways related to synaptic organization and mitochondrial function, whereas the right CA1 region exhibited enrichment in transcriptional regulation and RNA metabolic processes. Despite these asymmetries, co-expression and protein-protein interaction network analyses revealed shared molecular architectures. Immediate early genes formed consistent central hubs across both hemispheres, and a common Mecp2-Grin2b-Cdkl5-Tet3 protein interaction cluster was identified as a potential integrative regulatory module. Additional enrichment analysis of differentially expressed genes shared between hemispheres further highlighted conserved responses, particularly in synaptic plasticity and cell-cell communication. Together, these findings demonstrate that the left and right CA1 regions employ distinct yet partially convergent transcriptional programs to adapt to environmental stimuli. This coordinated molecular asymmetry provides novel insights into hippocampal lateralization and its role in experience-dependent brain plasticity.

neuroscience↗

bioRxiv2024

A florigen-expressing subpopulation of companion cells expresses other small proteins and reveals a nitrogen-sensitive FT repressor

The precise onset of flowering is crucial to ensure successful plant reproduction. The gene FLOWERING LOCUS T (FT) encodes florigen, a mobile signal produced in leaves that initiates flowering at the shoot apical meristem. In response to seasonal changes, FT is induced in phloem companion cells located in distal leaf regions. Thus far, a detailed molecular characterization of the FT-expressing cells has been lacking. Here, we used bulk nuclei RNA-seq and single nuclei RNA (snRNA)-seq to investigate gene expression in FT-expressing cells and other phloem companion cells. Our bulk nuclei RNA-seq demonstrated that FT-expressing cells in cotyledons and true leaves showed differences especially in FT repressor genes. Within the true leaves, our snRNA-seq analysis revealed that companion cells with high FT expression form a unique cluster in which many genes involved in ATP biosynthesis are highly upregulated. The cluster also expresses other genes encoding small proteins, including the flowering and stem growth inducer FPF1-LIKE PROTEIN 1 (FLP1) and the anti-florigen BROTHER OF FT AND TFL1 (BFT). In addition, we found that the promoters of FT and the genes co-expressed with FT in the cluster were enriched for the consensus binding motifs of NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1 (NIGT1). Overexpression of the paralogous NIGT1.2 and NIGT1.4 repressed FT expression and significantly delayed flowering under nitrogen-rich conditions, consistent with NIGT1s acting as nitrogen-dependent FT repressors. Taken together, our results demonstrate that major FT-expressing cells show a distinct expression profile that suggests that these cells may produce multiple systemic signals to regulate plant growth and development.

plant biology↗

bioRxiv2021

Evolutionary history of mammalian UDP-glucuronosyltransferase (UGT)1 and UGT2 families: the emergence of UGT2B subfamily in eutherians after the diversification of flowering plants

The UDP-glucuronosyltransferase (UGT) gene family is responsible for the transfer of glucuronic acid to exogenous and endogenous chemicals. Based on the highly diversified number of genes, the mammalian UGT1A and UGT2B subfamily genes are believed to be involved in the conjugation reactions of xenobiotic metabolism. However, it is speculated that the UGT2 family genes are not involved in the xenobiotic metabolism of avian species due to the less diverse number of genes. In this study, we aimed to investigate the evolutionary history of mammalian UGT1 and UGT2 family genes and determine when the diversification of UGT2B genes occurred. We also attempted to identify the main factors responsible for the diversification of UGT genes and the effect of the selection pressure on the structure of the UGT isozymes. By examining the genomic information and feeding habits of 67 species representing each mammalian family, we discovered that the UGT2B genes emerged in the Eutheria on or after Cretaceous period and that their number were higher in plant-eating mammals (herbivore or omnivore) than in carnivorous mammals. We also found that the UGT2B genes in some herbivorous mammals underwent positive selection. In contrast, the diversity of the UGT1 family genes was inherited from the common ancestor of birds and mammals. Furthermore, by predicting 3D structure of UGT enzymes, estimating selection pressure on amino acid sites, and performing molecular dynamics simulations, we showed that UGT2B and some UGT2A isozymes, which have increasing gene numbers in each mammalian species, have in common that a portion of the -helix loosens to form a hinge-like structure, that the amino acid site at which the -helix loosens is under positive selection, and that the -helix loosening increases the fluctuations of the UGT2B proteins. Thus, our findings suggest that the emergence of angiosperms (flowering plants) and the occurrence of "animal-plant warfare" influenced the evolution of this gene family involved in the xenobiotic metabolism of eutherians. Furthermore, future research investigating the marsupials and birds that do not possess UGT2B genes is required to elucidate the mechanisms underlying the metabolism of chemical substances in these species.

evolutionary biology↗

bioRxiv2018

Dawn and photoperiod sensing by phytochrome A

In plants, light receptors play a pivotal role in photoperiod sensing, enabling them to track seasonal progression. Photoperiod sensing arises from an interaction between the plants endogenous circadian oscillator and external light cues. Here, we characterise the role of phytochrome A (phyA) in photoperiod sensing. Our meta-analysis of functional genomic datasets identified phyA as a principal transcriptional regulator of morning-activated genes, specifically in short photoperiods. We demonstrate that PHYA expression is under the direct control of the PHYTOCHROME INTERACTING FACTOR transcription factors, PIF4 and PIF5. As a result, phyA protein accumulates during the night, especially in short photoperiods. At dawn phyA activation by light results in a burst of gene expression, with consequences for anthocyanin accumulation. The combination of complex regulation of PHYA transcript and the unique molecular properties of phyA protein make this pathway a sensitive detector of both dawn and photoperiod.\n\nSignificance statementThe changing seasons subject plants to a variety of challenging environments. In order to deal with this, many plants have mechanisms for inferring the season by measuring the duration of daylight in a day. A number of well-known seasonal responses such as flowering are responsive to daylength or photoperiod. Here, we describe how the photoreceptor protein phytochrome A senses short photoperiods. This arises from its accumulation during long nights, as happens during winter, and subsequent activation by light at dawn. As a result of this response, the abundance of red anthocyanin pigments is increased in short photoperiods. Thus, we describe a mechanism underlying a novel seasonal phenotype in an important model plant species.

plant biology↗

bioRxiv2017

Time-resolved Interaction Proteomics of the Putative Scaffold Protein GIGANTEA in Arabidopsis thaliana

The large, plant-specific protein GIGANTEA (GI) is involved in many physiological processes, mediating rhythmic, post-translational regulation in part through circadian and light regulation of GI RNA expression. GI binds several proteins implicated in the circadian clock, the control of photoperiodic flowering, and abiotic stress responses, and has co-chaperone activity. By extension, further interaction partners might mediate the less well-understood roles of GI but the number and rhythmicity of these interactors is unknown. Here, we seek potential interactors in a time-specific manner, using quantitative proteomics from a time series study of transgenic Arabidopsis thaliana plants constitutively expressing an epitope-tagged GI protein. Previously-identified, direct and indirect interactors of GI were detected but no further F-box proteins related to known GI partners ZTL/FKF1/LKP2. The predominantly non-rhythmic, interacting proteins were implicated in protein folding or degradation, metabolism and chromatin modification, including a small set of partners shared with other clock-related proteins. A transcription factor homologue that we name CYCLING DOF FACTOR 6 (CDF6) was shown to interact both with GI and the ZTL/FKF1/LKP2 proteins and to control photoperiodic flowering. Our results indicate the biochemical pathways, beyond circadian and flowering regulation, that might be affected by GIGANTEAs rhythmic, post-translational control.\n\nSignificance StatementSignificance statement of up to two sentences of no more than 75 words total;\n\nThe GIGANTEA protein of Arabidopsis was known for circadian and flowering functions, mediated by the FKF1/LKP2/ZTL family of GI-interacting, F-box proteins, then for a co-chaperone activity of unknown scope. We performed time-resolved, interaction proteomics, identifying CDF6 (At1g26790) as a morning-specific GI interactor that controls flowering time. Unlike FKF1 and CDF proteins, most of the 240 candidate partners were not rhythmically enriched. They link GI to proteostasis and metabolic functions that might mediate GIs physiological functions.

plant biology↗