Biology subjects

Oda, H.

Publications and source records attributed to Oda, H..

16 recordsLinked to original sources

bioRxiv2025

Cell-based multisensor array for detecting cancer biomarkers in human urine

Detecting volatile urinary biomarkers offers a compelling strategy for noninvasive diagnosis of diseases such as cancer. Biohybrid sensors employing sensor cells that express olfactory receptors--with finely tuned molecular recognition capabilities--are garnering increasing attention. However, their application to physiological samples remains challenging due to variability in sensor cell responsiveness and limited signal intensity. Here, we report a volatile biomarker detection platform based on a multisensor array in which insect olfactory receptor- expressing cells are encapsulated in hydrogel and housed within microwells featuring vertical slits. This structural design enables dense three-dimensional cell immobilization and rapid, uniform exposure to target molecules, enhancing the sensitivity of fluorescence responses and minimizing signal variability. Multiple types of sensor cells on a single array enabled the selective and simultaneous detection of potential cancer-related target molecules--acetophenone, phenol, and 6-methyl-5-hepten-2-one. Furthermore, through hexane extraction combined with gas-phase delivery, we reliably detected acetophenone introduced into human urine at micromolar concentrations. This platform offers a compact, reproducible, and scalable solution for volatile biomarker detection using cell-based sensors.

bioengineering↗

bioRxiv2024

Nanoscale visualization of Drosophila E-cadherin ectodomain fragments and their interactions using DNA origami nanoblocks

The adhesive function of cell surface proteins can be visually assessed through direct observation; however, the underlying structures that mediate adhesion typically remain invisible at the nanoscale level. This hinders knowledge on the diversity of molecular architectures responsible for cell-cell adhesion. Drosophila E-cadherin (DE-cadherin), a classical cadherin with a unique domain structure, demonstrates adhesive function; however, it lacks a structural model that explains its adhesion mechanism. In this study, we present a novel application of DNA origami technology to create a cell-free, flat environment in which full DE-cadherin ectodomains are anchored using SNAP-tags and biotin-streptavidin interactions. DNA origami was assembled into a 120 nm long block, bearing 5 or 14 biotin:streptavidin sites that were evenly spaced on one lateral face. DE-cadherin ectodomain fragments were attached via biotinylated SNAP-tags. These decorated DNA origami nanoblocks were subjected to transmission electron and high-speed atomic force microscopy, which revealed a hinge-like site that separated the membrane-distal and -proximal portions of the DE-cadherin ectodomain, suggesting a role in mechanical flexibility. We also observed interactions between DE-cadherin ectodomains via their membrane-distal portions on single DNA origami nanoblocks. We reconstituted an adhesion-like process via pairing DNA origami nanoblocks using DE-cadherin ectodomain interactions. Homophilic associations of functional DE-cadherin ectodomains between the paired DNA origami nanoblocks were visualized at the nanoscale, displaying strand-like molecular configurations, likely representing the extracellular cadherin repeats without regular arrays of structural elements. This study introduces a DNA origami-based platform for reconstituting and visualizing cadherin ectodomain interactions, with potential applications for a broader range of adhesion molecules. HighlightsO_LIDNA origami technology was applied to perform a structure-function study of cadherin. C_LIO_LIDNA origami nanoblocks decorated with DE-cadherin ectodomains were observed by TEM/HS-AFM. C_LIO_LIA hinge-like site that separated the membrane-distal and -proximal portions of the DE-cadherin ectodomain was revealed. C_LIO_LIAn adhesion-like process was mimicked via pairing two nanoblocks using DE-cadherin ectodomain interactions. C_LIO_LIHomophilic associations of DE-cadherin ectodomains between the nanoblocks were visualized at the nanoscale level. C_LI

molecular biology↗

bioRxiv2024

Local DNA compaction creates TF-DNA clusters that enable transcription

Transcription factor (TF) clusters have been suggested to facilitate transcription. The mechanisms driving the formation of TF clusters and their impact on transcription, however, remain largely unclear. This is mostly due to the lack of a tractable system. Here, we exploit the transcriptional activation of mir430 in zebrafish embryos to simultaneously follow the dynamic formation of a large Nanog cluster, the underlying DNA, and transcription output by live imaging at high temporal and spatial resolution. We find that the formation of a Nanog cluster that can support transcription requires local DNA compaction. This brings more Nanog-binding sites into the cluster, and therefore more Nanog. Importantly, we find that Nanog stabilizes this TF-DNA cluster, which emphasizes the interdependent relationship between TFs and DNA dynamics in cluster formation. Once the Nanog-DNA cluster at the mir430 locus reaches a maximum amount of Nanog, transcription begins. This maximum is a locus-intrinsic feature, which shows that the locus self-regulates the recruitment of an optimal amount of Nanog. Our study supports a model in which endogenous TF clusters positively impact transcription and form through a combination of DNA binding and local DNA compaction.

cell biology↗

bioRxiv2024

Reconstruction of artificial nuclei with nuclear import activity in living mouse oocytes

The cell nucleus is a dynamic structure repeating disassembly and reformation during mitosis. Reformation of the nucleus is essential for cell proliferation, and therefore, the factors required for nuclear reformation are fundamental for eukaryotes. Although various factors have been identified in in vitro systems using frog egg extracts and in vivo imaging of somatic cells, little is known about the factors required for the formation of functional nuclear structures in living mouse eggs. To identify such factors, we used a reconstruction approach to construct an artificial nucleus around DNA in mouse eggs. T4 phage DNA (166 kbp) was microinjected into living mouse oocytes. Amounts of DNA injected and injection timing were examined to determine the conditions appropriate for the formation of functional nuclei. Microinjection of 100-500 ng/{micro}l DNA during metaphase through telophase of the second meiosis, but not the subsequent interphase, was important for the formation of artificial nucleus. This T4 DNA-derived artificial nucleus had the structure of nuclear lamina and nuclear pore complexes, and nuclear transport activity, similar to natural nuclei. These results suggest that exogenous DNA can form a functional nucleus in mouse oocytes, regardless of the sequence or the source of DNA.

cell biology↗

bioRxiv2024

Morphological Study of Left-right Head Asymmetry in Doubledaya bucculenta (Coleoptera: Erotylidae: Languriinae)

Left-right asymmetry in paired organs is well-documented across various species, including the claws of fiddler crabs and snail-eating snakes dentition. However, the mechanisms underlying these asymmetries remain largely elusive. This study investigates Doubledaya bucculenta (Coleoptera: Erotylidae), a lizard beetle species known for pronounced left-sided asymmetry in adult female mandible and gena. Given that insect mouthparts comprise multiple functionally significant appendages, we aimed to clarify the degree of asymmetry extending beyond the mandibles and genae. Phenotypic morphology was assessed through trait measurement and asymmetry index calculations. Our detailed morphometric analyses revealed left-longer asymmetry not only in mandibles and genae but also in maxillae and labium. Notably, the degree of asymmetry in other mouthparts was generally less pronounced compared to outer mandibles, suggesting a potential influence of left mandible development on other mouthparts. Additionally, male mandibles exhibited region-specific asymmetry, potentially indicative of constrained evolutionary adaptations. This study enhances a comprehensive understanding of adult phenotype morphology and offers insights into the developmental basis of asymmetrical mouthparts.

zoology↗

bioRxiv2024

PERFORATION-TYPE ANCHORS INSPIRED BY SKIN LIGAMENT FOR THE ROBOTIC FACE COVERED WITH LIVING SKIN

Skin equivalent, a living skin model composed of cells and extracellular matrix, possesses the potential as an ideal covering material for robots due to their biological functionalities. To employ skin equivalents as a coverage material for robots, a secure method for attaching them to the underlying structure is required. In this study, we developed a perforation-type anchor inspired by the structure of skin ligaments as a technique to effectively adhere skin equivalent to robotic surfaces, ensuring a smooth and non-protrusive integration, akin to natural skin adherence. We showed the effectiveness of plasma treatment for collagen-gel penetration within the perforation-type anchor. In characterizing the anchoring system, we investigated the impact of anchor diameter on preventing contraction and changes in holding strength. To showcase the versatility of perforation-type anchors in three-dimensional (3D) coverage applications, a 3D facial mold with intricate surface structure was covered with skin equivalent. Furthermore, we constructed a robotic face covered with dermis equivalent, capable of expressing smiles with the actuation to the skin equivalent through perforation-type anchors. This research introduces an approach for the adhesion and actuation of skin equivalent with perforation-type anchors, potentially contributing to advancements in biohybrid robotics technology.

bioengineering↗

bioRxiv2023

Mono-ADP-ribosyltransferase 1 (Artc1)-deficiency decreases tumorigenesis, increases inflammation, decreases cardiac contractility, and reduces survival

Arginine-specific mono-ADP-ribosylation is a reversible post-translational modification; arginine-specific, cholera toxin-like mono-ADP-ribosyltransferases (ARTCs) transfer ADP-ribose from NAD+ to arginine, followed by cleavage of ADP-ribose-(arginine)protein bond by ADP-ribosylarginine hydrolase 1 (ARH1), generating unmodified (arginine)protein. ARTC1 has been shown to enhance tumorigenicity as does Arh1 deficiency. In this study, Artc1-KO and Artc1/Arh1-double-KO mice showed decreased spontaneous tumorigenesis and increased age-dependent, multi-organ inflammation with upregulation of pro-inflammatory cytokine TNF-. In a xenograft model using tumorigenic Arh1-KO mouse embryonic fibroblasts (MEFs), tumorigenicity was decreased in Artc1-KO and heterozygous recipient mice, with tumor infiltration by CD8+ T cells and macrophages, leading to necroptosis, suggesting that ARTC1 promotes the tumor microenvironment. Furthermore, Artc1/Arh1-double-KO MEFs showed decreased tumorigenesis in nude mice, showing that tumor cells as well as tumor microenvironment require ARTC1. By echocardiography and MRI, Artc1-KO and heterozygous mice showed male-specific, reduced myocardial contractility. Furthermore, Artc1-KO male hearts exhibited enhanced susceptibility to myocardial ischemia-reperfusion-induced injury with increased receptor-interacting protein kinase 3 (RIP3) protein levels compared to WT mice, suggesting that ARTC1 suppresses necroptosis. Overall survival rate of Artc1-KO was less than their Artc1-WT counterparts, primarily due to enhanced immune response and inflammation. Thus, anti-ARTC1 agents may reduce tumorigenesis but may increase multi-organ inflammation and decrease cardiac contractility. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/527366v1_ufig1.gif" ALT="Figure 1"> View larger version (84K): org.highwire.dtl.DTLVardef@1cded54org.highwire.dtl.DTLVardef@9851ceorg.highwire.dtl.DTLVardef@22cd8aorg.highwire.dtl.DTLVardef@e42e7a_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2022

Targeted disruption of transcription bodies causes widespread activation of transcription

The localization of transcriptional activity in specialized transcription bodies is a hallmark of gene expression in eukaryotic cells. It remains unclear, however, if and how they affect gene expression. Here, we disrupted the formation of two prominent endogenous transcription bodies that mark the onset of zygotic transcription in zebrafish embryos and analysed the effect on gene expression using enriched SLAM-Seq and live-cell imaging. We find that the disruption of transcription bodies results in downregulation of hundreds of genes, providing experimental support for a model in which transcription bodies increase the efficiency of transcription. We also find that a significant number of genes are upregulated, counter to the suggested stimulatory effect of transcription bodies. These upregulated genes have accessible chromatin and are poised to be transcribed in the presence of the two transcription bodies, but they do not go into elongation. Live-cell imaging shows that the disruption of the two large transcription bodies enables these poised genes to be transcribed in ectopic transcription bodies, suggesting that the large transcription bodies sequester a pause release factor. Supporting this hypothesis, we find that CDK9, the kinase that releases paused polymerase II, is highly enriched in the two large transcription bodies. Importantly, overexpression of CDK9 in wild type embryos results in the formation of ectopic transcription bodies and thus phenocopies the removal of the two large transcription bodies. Taken together, our results show that transcription bodies regulate transcription genome-wide: the accumulation of transcriptional machinery creates a favourable environment for transcription locally, while depriving genes elsewhere in the nucleus from the same machinery.

cell biology↗

bioRxiv2022

Actin filaments accumulate in the nucleus during interphase and remain in the vicinity of condensing chromosomes during prophase to metaphase in zebrafish early embryos

In the cytoplasm, filamentous actin (F-actin) plays a critical role in cell regulation, including cell migration, stress fiber formation, and cytokinesis. Recent studies have shown that actin filaments that form in the nucleus are associated with diverse functions. Here, using live imaging of an F-actin-specific probe, superfolder GFP-tagged utrophin (UtrCH-sfGFP), we demonstrated the dynamics of nuclear actin in zebrafish (Danio rerio) embryos. In early zebrafish embryos up to around the high stage, UtrCH-sfGFP increasingly accumulated in nuclei during the interphase and reached a peak during the prophase. After nuclear envelope breakdown (NEBD), patches of UtrCH-sfGFP remained in the vicinity of condensing chromosomes during the prometaphase to metaphase. When zygotic transcription was inhibited by injecting -amanitin, the nuclear accumulation of UtrCH-sfGFP was still observed at the sphere and dome stages, suggesting that zygotic transcription may induce a decrease in nuclear F-actin. The accumulation of F-actin in nuclei may contribute to proper mitotic progression of large cells with rapid cell cycles in zebrafish early embryos, by assisting in NEBD, chromosome congression, and/or spindle assembly. Summary statementFilamentous actin accumulates in the nucleus of zebrafish early embryos and forms patches associating with condensing chromosomes during prophase.

cell biology↗

bioRxiv2022

Nanog organizes transcription bodies

The localization of transcriptional activity in specialized transcription bodies is a hallmark of gene expression in eukaryotic cells. How proteins of the transcriptional machinery come together to form such bodies, however, is unclear. Here, we take advantage of two large, isolated, and long-lived transcription bodies that reproducibly form during early zebrafish embryogenesis, to characterize the dynamics of transcription body formation. Once formed, these transcription bodies are enriched for initiating and elongating RNA polymerase II, as well as the transcription factors Nanog and Sox19b. Analyzing the events leading up to transcription, we find that Nanog and Sox19b cluster prior to transcription, and independently of RNA accumulation. The clustering of transcription factors is sequential; Nanog clusters first, and this is required for the clustering of Sox19b and the initiation of transcription. Mutant analysis revealed that both the DNA-binding domain, as well as one of the two intrinsically disordered regions of Nanog are required to organize the two bodies of transcriptional activity. Taken together, our data suggests that the clustering of transcription factors dictates the formation of transcription bodies. HIGHLIGHTSO_LITranscription factors cluster prior to, and independently of transcription C_LIO_LINanog organizes transcription bodies: it is required for the clustering of Sox19b as well as RNA polymerase II C_LIO_LIThis organizing activity requires its DNA binding domain as well as one of its intrinsically disordered regions C_LIO_LITranscription elongation results in the disassembly of transcription factor clusters C_LI

cell biology↗

bioRxiv2022

Lineage-specific, fast-evolving GATA-like gene regulates zygotic gene activation to promote endoderm specification and pattern formation in the Theridiidae spider

BackgroundThe process of early development varies across the species-rich phylum Arthropoda. Owing to the limited research strategies for dissecting lineage-specific processes of development in arthropods, little is known about the variations in early arthropod development at molecular resolution. The Theridiidae spider, Parasteatoda tepidariorum, has its genome sequenced and could potentially to contribute to dissecting early embryonic processes. ResultsWe present genome-wide identification of candidate genes that exhibit locally restricted expression in germ-disc forming stage embryos of P. tepidariorum, based on comparative transcriptomes of isolated cells from different regions of the embryo. A subsequent pilot screen by parental RNA interference identifies three genes required for body axis formation. One of them is a GATA-like gene that has been fast evolving after duplication and divergence from a canonical GATA family gene. This gene is designated fuchi nashi (fuchi) after its knockdown phenotypes, where the cell movement toward the formation of a germ disc was reversed. fuchi expression occurs in cells outside a forming germ disc and persists in the endoderm. Transcriptome and chromatin accessibility analyses of fuchi pRNAi embryos suggest that early fuchi activity regulates chromatin state and zygotic gene activation to promote endoderm specification and pattern formation. We also show that there are many uncharacterized genes regulated by fuchi. ConclusionsOur genome-based research using an arthropod phylogenetically distant from Drosophila identifies a lineage-specific, fast-evolving gene with key developmental roles in one of the earliest, genome-wide regulatory events, and allows for molecular exploration of the developmental variations in early arthropod embryos.

developmental biology↗

bioRxiv2020

Regulation of human development by ubiquitin chain editing of chromatin remodelers

Embryonic development occurs through commitment of pluripotent stem cells to differentiation programs that require highly coordinated changes in gene expression. Chromatin remodeling of gene regulatory elements is a critical component of how such changes are achieved. While many factors controlling chromatin dynamics are known, mechanisms of how different chromatin regulators are orchestrated during development are not well understood. Here, we describe LINKED (LINKage-specific-deubiquitylation-deficiency-induced Embryonic Defects) syndrome, a novel multiple congenital anomaly disorder caused by hypomorphic hemizygous missense variants in the deubiquitylase OTUD5/DUBA. Studying LINKED mutations in vitro, in mouse, and in models of neuroectodermal differentiation of human pluripotent stem cells, we uncover a novel regulatory circuit that coordinates chromatin remodeling pathways during early differentiation. We show that the K48-linkage-specific deubiquitylation activity of OTUD5 is essential for murine and human development and, if reduced, leads to aberrant cell-fate specification. OTUD5 controls differentiation through preventing the degradation of multiple chromatin regulators including ARID1A/B and HDAC2, mutation of which underlie developmental syndromes that exhibit phenotypic overlap with LINKED patients. Accordingly, loss of OTUD5 during early differentiation leads to less accessible chromatin at neural and neural crest enhancers and thus aberrant rewiring of gene expression networks. Our work identifies a novel mechanistic link between phenotypically related developmental disorders and an essential function for linkagespecific ubiquitin editing of substrate groups (i.e. chromatin remodeling complexes) during early cellfate decisions - a regulatory concept, we predict to be a general feature of embryonic development.

genetics↗

bioRxiv2019

Attenuated Salmonella typhimurium cancer therapy has direct effects on the tumor epithelium in colorectal cancer

Bacterial cancer therapy (BCT) shows great promise for treatment of solid tumors, yet basic mechanisms of bacterial-induced tumor suppression remain undefined. The intestinal epithelium is the natural route of infection for Salmonella and thus harbors innate immune defenses which protect against infection. Attenuated strains of Salmonella enterica serovar Typhimurium (STm) have commonly been used in mouse models of BCT, largely with the use of xenograft and orthotopic transplant cancer models. We aimed to better understand the tumor epithelium-targeted mechanisms of BCT by using mouse models of intestinal tumorigenesis and tumor organoid cultures to assess the effectiveness and mechanisms of treatment with aromatase A-deficient STm (STm{Delta}aroA). STm{Delta}aroA delivered by oral gavage could significantly reduce tumor burden and tumor load in both a colitis-associated colon cancer model (CAC) and in a spontaneous intestinal cancer model, Apcmin/+ mice. STm{Delta}aroA colonization of tumors caused alterations in transcription of mRNAs associated with epithelial-mesenchymal transition as well as metabolic and cell cycle-related transcripts. Metabolomic analysis of tumors demonstrated alteration in the metabolic environment of STm{Delta}aroA-treated tumors, suggesting STm{Delta}aroA imposes metabolic competition on the tumor. Use of tumor organoid cultures in vitro demonstrated that STm{Delta}aroA can directly affect the tumor epithelium with alterations in transcripts and metabolites similar to in vivo-treated tumors. Thereby, we demonstrate that bacterial cancer therapy is efficacious in autochthonous intestinal cancer models, that BCT imposes metabolic competition, and that BCT has direct effects on the tumor epithelium, which have not previously been appreciated.\n\nOne Sentence SummaryAttenuated Salmonella enterica serovar Typhimurium can home to gastrointestinal tumors and directly affect the tumor epithelium, inducing transcriptional and metabolic changes that lead to reduced tumor burden in mice.

cancer biology↗

bioRxiv2019

Quantitative measurements of chromatin modification dynamics during zygotic genome activation

Histone posttranslational modifications (PTMs) are key gene expression regulators, but their rapid dynamics during development remain difficult to capture. We describe an approach that images fluorescent antibody fragments to quantify PTM enrichment and active transcription at defined loci during zygotic genome activation in living zebrafish embryos. The technique reveals a drastic increase in histone H3 Lys27 acetylation (H3K27ac) before genome activation both at a specific locus and globally.

cell biology↗

bioRxiv2018

A genetically encoded probe for imaging HA-tagged protein translation, localization, and dynamics in living cells and animals

To expand the toolbox of imaging in living cells, we have engineered a new single chain variable fragment (scFv) that binds the classic linear HA epitope with high affinity and specificity in vivo. The resulting probe, which we call the HA frankenbody, is capable of lighting up in multiple colors HA-tagged nuclear, cytoplasmic, and membrane proteins in diverse living cell types. The HA frankenbody also enables state-of-the-art single-molecule experiments, which we demonstrate by tracking single mRNA translation dynamics in living U2OS cells and neurons. In combination with the SunTag, we track two mRNA species simultaneously to demonstrate comparative single-molecule studies of translation can now be done with genetically encoded tools alone. Finally, we use the HA frankenbody to precisely quantify the expression of HA tagged proteins in developing zebrafish embryos. The versatility of the HA frankenbody makes it a powerful new tool for imaging protein dynamics in vivo.\n\nOne-sentence summaryA genetically encodable intracellular single-chain variable fragment that selectively binds the HA epitope (YPYDVPDYA) with high affinity in living cells and organisms can be used to quantify HA-tagged protein translation, localization, and dynamics.

cell biology↗

bioRxiv2017

Chromatin tethering to the nuclear envelope by nuclear actin filaments: a novel role of the actin cytoskeleton in the Xenopus blastula

The Xenopus oocyte is known to accumulate filamentous or F-actin in the nucleus, but it is currently unknown whether F-actin also accumulates in embryo nuclei. Using fluorescence-labeled actin reporters, we examined the actin distribution in Xenopus embryonic cells and found that F-actin accumulates in nuclei during the blastula stage but not during the gastrula stage. To further investigate nuclear F-actin, we devised a Xenopus egg extract that reproduces the formation of nuclei in which F-actin accumulates. Using this extract, we found that F-actin accumulates primarily at the sub-nuclear membranous region and is essential to maintain chromatin binding to the nuclear envelope in well-developed nuclei. We also provide evidence that nuclear F-actin increases the structural stability of nuclei and contributes to chromosome alignment on the mitotic spindle at the following M phase. These results suggest the physiological importance of nuclear F-actin accumulation in rapidly dividing, large Xenopus blastula cells.

cell biology↗