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Biology subjects

Ono, T.

Publications and source records attributed to Ono, T..

12 recordsLinked to original sources

Condensin II collaborates with cohesin to establish and maintain interphase chromosome territories

Despite the well-established role of condensin II in mitotic chromosome assembly, its function in interphase chromosome organization remains poorly understood. Here, we applied multiscale fluorescence in situ hybridization (FISH) techniques to human cell lines engineered for single or double depletion of condensin II and cohesin, and examined their functional interplay at two distinct stages of the cell cycle. Our results demonstrate that a functional "handover" from condensin II to cohesin during the mitosis-to-G1 transition is critical for establishing chromosome territories (CTs) in the newly assembling nucleus. During G2 phase, condensin II and cohesin cooperate to maintain global CT morphology, although they act at different genomic scales. Strikingly, double depletion of both complexes causes CTs to collapse and accumulate abnormally at the nucleolar periphery. Based on these findings, we will discuss how the condensin and cohesin complexes act in an orderly and cooperative manner to orchestrate chromatin dynamics across genomic scales, thereby supporting higher-order chromosome organization throughout the cell cycle.

cell biology↗

Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

Lipogenesis-driven metabolic flux is crucial for differentiation of pathogenic Th17 cells. Although our previous CRISPR-based screening identified PLA2G12A as a key player in this process, it has remained obscure how this secreted phospholipase A2 isoform controls Th17 differentiation. Here we show that global, T cell-specific, or fibroblast-specific deletion of PLA2G12A prevents Th17 differentiation and associated diseases including psoriasis and arthritis. PLA2G12A acts on Th17-derived extracellular vesicles (EVs) to produce lysophospholipids including the ROR{gamma}t activator 1-oleoyl-lysophosphatidylethanolamine. These lysophospholipids are further converted by autotaxin to lysophosphatidic acid (LPA), which assists Th17 differentiation mainly via LPA2 receptor. Moreover, PLA2G12A promotes the secretion and uptake of EVs by Th17 cells and alters their cargo contents. Defective Th17 differentiation by PLA2G12A deficiency is rescued by supplementation with PLA2G12A-modified EVs. Importantly, a PLA2G12A-blocking antibody prevents Th17 differentiation and ameliorates psoriasis and arthritis models. Thus, targeting the PLA2G12A-EV-lysophospholipid axis may be useful for treatment of Th17-related diseases.

immunology↗

Fusion of histone variants to Cas9 enhances homology-directed repair

As a versatile genome editing tool, the CRISPR-Cas9 system induces DNA double-strand breaks at targeted sites to activate mainly two DNA repair pathways: HDR which allows precise editing via recombination with a homologous template DNA, and NHEJ which connects two ends of the broken DNA, which is often accompanied by random insertions and deletions. Therefore, how to enhance HDR while suppressing NHEJ is a key to successful applications that require precise genome editing. Histones are small proteins with a lot of basic amino acids that generate electrostatic affinity to DNA. Since H2A.X is involved in DNA repair processes, we fused H2A.X to Cas9 and found that this fusion protein could improve the HDR/NHEJ ratio. As various post-translational modifications of H2A.X play roles in the regulation of DNA repair, we also fused H2A.X mimicry variants to replicate these post-translational modifications including phosphorylation, methylation, and acetylation. However, none of them were effective to improve the HDR/NHEJ ratio. We further fused other histone variants to Cas9 and found that H2A.1 exhibited the improved HDR/NHEJ ratio better than H2A.X. Thus, the fusion of histone variants to Cas9 is a promising option to enhance precise genome editing.

cell biology↗

Evidence of the simultaneous replications of active viruses in specimens positive for multiple respiratory viruses

Genetic diagnostic assays for the detection of respiratory viruses sometimes show simultaneous multiple infections with low copy numbers. In such cases, the disease is considered caused by a single etiologic agent and others are nonspecific reactions and/or contaminations. Interferon-dependent interference is seen in dual infections of influenza and respiratory syncytial virus, which are the main causes of respiratory infections. Virus isolation is one of the solutions in detecting other active viruses present in specimens, and the air-liquid interface culture of human bronchial/tracheal epithelial cells (HBTEC-ALI) is optimal for the isolation of respiratory viruses owing to its wide range of susceptibility. In this study, we successfully confirmed the replications of various viruses from specimens with low copy numbers and passed 2-3 viruses simultaneously using HBTEC-ALI cultures, mainly including human bocavirus 1 and/or human rhinovirus.

microbiology↗

A mechanism of melanogenesis mediated by E-cadherin downregulation and its involvement in solar lentigines

BackgroundIntensive studies have revealed pleiotropic melanocytic factors for age spot formation. In particular, dysfunctional keratinocyte differentiation is thought to be an upstream cause of age spot formation. Although keratinocyte differentiation is mediated by a cell-cell contact factor, E-cadherin, its involvement in age spots remains unknown. To find the origin of age spots and an integrated solution, we focused on E-cadherin. MethodsImmunofluorescent staining with cutaneous tissues and cultured cells was performed. Keratinocytes treated with siRNAs were cocultured with melanocytes. With the supernatants of the keratinocyte culture, secretion factors were identified using proteomic analysis. For the activity of melanogenesis and the ingredient screening, a quantitative PCR was performed. For the behavioral analysis of melanocytes, time-lapse imaging of melanocytes was done by confocal laser scanning microscopy. ResultsIn age spots, E-cadherin expression in the epidermis was downregulated, suggesting that E-cadherin is implicated in age spot formation. E-cadherin knockdown (E-cad-KD) keratinocytes not only promoted the secretion of melanocytic/inflammatory factors, but also increased melanogenesis by upregulating the expression of melanogenesis factors. Furthermore, live imaging showed E-cadherin downregulation detained melanocyte dynamics and accelerated melanin-uptake. Finally, we identified Rosa multiflora fruit extract as a solution for upregulating E-cadherin in keratinocytes. ConclusionOur findings showed that E-cadherin downregulation triggers various downstream melanocytic processes such as secretion of melanocytic factors and melanogenesis. Additionally, we showed that Rosa multiflora fruit extract upregulates E-cadherin expression in keratinocytes.

cell biology↗

In Vivo Delivery of Therapeutic Molecules by Transplantation of Genome-Edited Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (iPSCs) have already been used in transplantation therapies. Currently, cells from healthy people are transplanted into patients with diseases. With the rapid evolution of genome editing technology, genetic modification could be applied to enhance the therapeutic effects of iPSCs, such as the introduction of secreted molecules to make the cells a drug delivery system. Here, we addressed this possibility by utilizing a Fabry disease mouse model, as a proof of concept. Fabry disease is caused by the lack of -Galactosidase A (GLA). We previously developed an immunotolerant therapeutic molecule, modified -N-acetylgalactosaminidase (mNAGA). We confirmed that secreted mNAGA from genome-edited iPSCs compensated for the GLA activity in GLA-deficient cells using an in vitro co-culture system. Moreover, iPSCs transplanted into Fabry model mice secreted mNAGA and supplied GLA activity to the liver. This study demonstrates the great potential of genome-edited iPSCs secreting therapeutic molecules.

cell biology↗

NF-κB Decoy ODN-Loaded Poly lactic-co-glycolic Acid Nanospheres Inhibit Alveolar Ridge Resorption

Residual ridge resorption combined with dimensional loss resulting from tooth extraction has a prolonged correlation with early excessive inflammation. Nuclear factor-kappa B (NF-{kappa}B) decoy oligodeoxynucleotide (ODN) is a member of a group of double-stranded DNA capable of downregulating the expression of downstream genes of the NF-{kappa}B pathway. The healing action of its embellished effect combined with poly(lactic-co-glycolic acid) (PLGA) nanospheres on tooth extraction socket still remains unknown. Hence, the aim of this study was to investigate the therapeutic effect of NF-{kappa}B decoy ODN-loaded PLGA nanospheres (PLGA-NfD) transfected into extraction sockets in Wistar/ST rats. Micro-computed tomography and trabecular bone analysis following treatment with PLGA-NfD demonstrated inhibition of vertical alveolar bone loss with increased bone volume, smoother trabecular bone surface, thicker trabecular bone, larger trabecular number and separation, and fewer bone porosities. Histomorphometric and reverse transcription-quantitative polymerase chain reaction analysis revealed reduced tartrate-resistant acid phosphatase-expressing osteoclasts, interleukin-1{beta}, tumor necrosis factor-, receptor activator of NF-{kappa}B ligand, turnover rate and increased transforming growth factor-{beta}1 immunopositive reactions and relative gene expressions. These data demonstrate that local delivery of PLGA-NfD could be used as a substantial suppressor of inflammation during the healing process in a tooth extraction socket, with the potential of accelerated new bone formation.

immunology↗

Myogenetic oligodeoxynucleotide (myoDN) complexed with berberine promotes differentiation of chicken skeletal muscle myoblasts

Skeletal muscle myoblasts are myogenic precursors that develop into myofibers during muscle formation. Improvement of myoblast differentiation is important for advancing meat production by domestic animals. We recently identified novel oligodeoxynucleotides (ODNs) termed myogenetic ODNs (myoDNs) that promote the differentiation of mammalian myoblasts. An isoquinoline alkaloid, berberine, forms a complex with one of the myoDNs, iSN04, and enhances its activities. This study investigated the effects of myoDNs on chicken myoblasts to elucidate their species-specific actions. Seven myoDNs (iSN01-iSN07) were found to facilitate the differentiation of chicken myoblasts into myosin heavy chain (MHC)-positive myocytes. The iSN04-berberine complex exhibited a higher myogenetic activity than iSN04 alone, which was shown to enhance the differentiation of myoblasts into myocytes, myotube formation, and myogenic gene expression (MyoD, myogenin, MHC, and myomaker). These data indicate that myoDNs promoting chicken myoblast differentiation may be used as potential feed additives in broiler diets.

cell biology↗

Reward-associated configural cues elicit theta oscillations of rat retrosplenial neurons phase-locked to LFP theta cycle

Previous behavioral studies implicated the retrosplenial cortex (RSC) in stimulus-stimulus associations, and also in retrieval of remote associative memory based on EEG theta oscillations. To investigate the neural mechanisms underlying these processes, RSC neurons and local field potentials (LFPs) were simultaneously recorded from well-trained rats performing a cue-reward association task. In the task, simultaneous presentation of two multimodal conditioned stimuli (configural CSs) predicted a reward outcome opposite to that associated with individual presentation of each elemental CS. Here, we show neurophysiological evidence that the RSC is involved in stimulus-stimulus association where configural CSs are discriminated from each elementary CS that is a constituent of the configural CSs, and that memory retrieval of rewarding CSs is associated with theta oscillation of RSC neurons during CS presentation, which is phase-locked to LFP theta cycles.

neuroscience↗

Lipopolysaccharide preconditioning augments phagocytosis of malaria-parasitized red blood cells through induced bone marrow-derived macrophages in the liver, thereby increasing the murine survival after Plasmodium yoelii infection

Malaria remains a grave concern for humans, as effective medical countermeasures for malaria infection have yet to be developed. Phagocytic clearance of parasitized red blood cells (pRBCs) by macrophages is an important front-line innate host defense against malaria infection. We previously showed that repeated injections of low-dose lipopolysaccharide (LPS) prior to bacterial infection, called LPS preconditioning, strongly augmented phagocytic/bactericidal activity in murine macrophages. However, how LPS preconditioning prevents murine malaria infection is unclear. We investigated the protective effects of LPS preconditioning against lethal murine malaria infection, focusing on CD11bhigh F4/80low liver macrophages, which are increased by LPS preconditioning. Mice were subjected to LPS preconditioning by intraperitoneal injections of low-dose LPS for 3 consecutive days, and 24 h later, they were intravenously infected with pRBCs of Plasmodium yoelii 17XL. LPS preconditioning markedly increased the murine survival and reduced parasitemia, while it did not reduce TNF secretions, only delaying the peak of plasma IFN-{gamma} after malaria infection in mice. An in vitro phagocytic clearance assay of pRBCs showed that the CD11bhigh F4/80low liver macrophages of the LPS-preconditioned mice had significantly augmented phagocytic activity against pRBCs. The adoptive transfer of CD11bhigh F4/80low liver macrophages from LPS-preconditioned mice to control mice significantly improved the survival after malaria infection. We conclude that LPS preconditioning stimulated CD11bhigh F4/80low liver macrophages to augment the phagocytic clearance of pRBCs, which may play a central role in resistance against malaria infection. LPS preconditioning may be an effective tool for preventing malaria infection.

immunology↗

Lab-on-a-graphene-FET detection of key molecular events underpinning influenza virus infection and effect of antiviral drugs

Small solid-state devices are candidates for accelerating biomedical assays/drug discovery, however their potential remains unfulfilled. Here, we demonstrate that graphene-field effect transistors (FET) can be used to successfully detect the key molecular events underlying viral infections and the effect of antiviral drugs. Our device success is achieved by bio-mimicking the host-cell surface during an influenza infection at the graphene channel. In-situ AFM confirms the biological interactions at the sialic acid-functionalized graphene: viral hemagglutinin (HA) binds to sialic acid, and neuraminidase (NA) reacts with the sialic acid-HA complex. The graphene-FET detects HA binding to sialic acid, and NA cleavage of sialic acid. The inhibitory effect of the drug "zanamivir" on NA-sialic acid interactions is monitored in real-time; the reaction rate constant of NA-sialic acid reaction was successfully determined. We demonstrate that graphene-FETs are powerful platforms for measurement of biomolecular interactions and contribute to future deployment of solid-state devices in drug discovery/biosensing.

bioengineering↗

Ki-67 and condensins support the integrity of mitotic chromosomes through distinct mechanisms

Although condensins play essential roles in mitotic chromosome assembly, Ki-67, a protein localizing to the periphery of mitotic chromosomes, had also been shown to make a contribution to the process. To examine their respective roles, we generated a set of HCT116-based cell lines expressing Ki-67 and/or condensin subunits that were fused with an auxin-inducible degron for their conditional degradation. Both the localization and the dynamic behavior of Ki-67 on mitotic chromosomes were not largely affected upon depletion of condensin subunits, and vice versa. When both Ki-67 and SMC2 (a core subunit of condensins) were depleted, ball-like chromosome clusters with no sign of discernible thread-like structures were observed. This severe defective phenotype was distinct from that observed in cells depleted of either Ki-67 or SMC2 alone. Our results show that Ki-67 and condensins, which localize to the external surface and the central axis of mitotic chromosomes, respectively, have independent yet cooperative functions in supporting the structural integrity of mitotic chromosomes.\n\nList of Abbreviations used

cell biology↗