Biology subjects

Makino, K.

Publications and source records attributed to Makino, K..

5 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

Massively multiplex multimodal chemical screens at single-cell resolution

Recent applications of scRNA-seq for massively multiplexed chemical screens have enabled comprehensive profiling of drug responses at unprecedented scale and resolution. However, current assays remain limited to RNA readouts, lacking information on other phenotypic and mechanistic layers such as chromatin accessibility, protein abundance and post-translational modifications. Here, we introduce a scalable framework for multimodal chemical screens, combining parallel small-molecule perturbations with multimodal readouts. We extend existing experimental platforms into icCITE-plex and DOGMA-plex, enabling joint profiling of RNA, protein, and epigenomic responses to thousands of chemical perturbations in parallel. To systematically decode the regulatory circuitry underlying these responses, we develop MoCAVI, a contrastive analysis framework that disentangles the effect of small molecules from control variation in multimodal measurements, and PERCISTRA, a pipeline that infers causal links between chromatin accessibility and gene expression. Applied across ~410,000 primary T cells under ~2,800 conditions, our approach resolves compound-specific mechanisms, highlights off-target effects, and links chromatin accessibility changes to transcription factor networks in primary T cells. Our results establish a generalizable platform for profiling and analyzing cellular responses to chemical perturbations across multiple modalities.

genomics↗

bioRxiv2025

Joint single-cell measurements of surface proteins, intracellular proteins and gene expression with icCITE-seq

The development of single-cell RNA-sequencing assays has transformed our understanding of cellular and tissue heterogeneity, yielding significant insights into disease biology and its underlying mechanisms. In this work, we describe icCITE-seq (intracellular cellular indexing of transcriptomes and epitopes), a scalable method that simultaneously measures surface and intracellular protein levels alongside gene expression across thousands of cells. We validate the specificity of intracellular staining and demonstrate the utility of this multi-omic approach in interrogating phenotypic cellular states through targeted genetic perturbations in primary human T cells. icCITE-seq enables systematic profiling of gene expression, coupled with cytoplasmic, nuclear and PTM epitopes, providing an integrated approach towards understanding cellular identity, complexity and disease regulatory mechanisms.

immunology↗

bioRxiv2024

Inhibition of microtubule polymerization and dynein impairs the nuclear localization of the ependymoma-associated ZFTA-RELA fusion protein and NF-κB activation

Ependymomas are rare and chemotherapy-resistant gliomas. One subclass of supratentorial ependymomas, ST-EPN-ZFTA, expresses a fusion protein consisting of a nuclear protein, zinc finger translocation associated (ZFTA), and v-rel reticuloendotheliosis viral oncogene homolog A (RELA), an effector transcription factor of the nuclear factor-{kappa}B (NF-{kappa}B) pathway (ZFTA-RELA). Constitutive localization of ZFTA-RELA to the nucleus hyperactivates the oncogenic NF-{kappa}B signaling pathway, thereby contributing to the pathogenesis of ST-EPN-ZFTA. To identify compounds that inhibit NF-{kappa}B activity induced by ZFTA-RELA, we established a high-throughput screening system using the NF-{kappa}B-responsive luciferase reporter cell line 6E8, which expresses ZFTA-RELA in a doxycycline-dependent manner. A chemical library of 9600 compounds selected for their structural diversity was screened, and a colchicine derivative was identified. Among colchicine and six of its derivatives, the IC50 on ZFTA-RELA-dependent NF-{kappa}B-responsive luciferase activity in 6E8 cells was found to be the lowest for colchicine at 90 nM. Interestingly, microtubule polymerization inhibitors (colchicine and vinblastine) and dynein inhibitors (ciliobrevin D and dynarrestin) impaired ZFTA-RELAs nuclear localization and NF-{kappa}B activity in 6E8 cells. These findings indicate that microtubule polymerization and dynein play pivotal roles in activating the NF-{kappa}B pathway in ST-EPN-ZFTA by promoting the nuclear localization of ZFTA-RELA. Consequently, inhibition of microtubule polymerization may be a therapeutic strategy for ST-EPN-ZFTA.

cell biology↗

bioRxiv2021

Liver ductal organoids reconstruct intrahepatic biliary trees in decellularized liver grafts.

Three-dimensional scaffolds decellularized from native organs are a promising technique to establish engineered liver grafts and overcome the current shortage of donor organs. However, limited sources of bile duct cells and inappropriate cell distribution in bioengineered liver grafts have hindered their practical application. Organoid technology is anticipated to be an excellent tool for the advancement of regenerative medicine. In the present study, we reconstructed intrahepatic bile ducts in a rat decellularized liver graft by recellularization with liver ductal organoids. Using an ex vivo perfusion culture system, we demonstrated the biliary characteristics of repopulated mouse liver organoids, which maintained bile duct markers and reconstructed biliary tree-like networks with luminal structures. We also established a method for the co-recellularization with engineered bile ducts and primary hepatocytes, revealing the appropriate cell distribution to mimic the native liver. We then utilized this model in human organoids to demonstrate the reconstructed bile ducts. Our results show that liver ductal organoids are a potential cell source for bile ducts from bioengineered liver grafts using three-dimensional scaffolds.

bioengineering↗