Biology subjects

Tanimoto, K.

Publications and source records attributed to Tanimoto, K..

9 recordsLinked to original sources

bioRxiv2026

Liver organoid-mediated cyclophosphamide neurotoxicity in CNS organoids in a multi-organ microphysiological system

Cyclophosphamide (CP) is a widely used alkylating agent whose cytotoxic activity depends on hepatic CYP450-mediated bioactivation. While CP-associated neurotoxicity and cognitive impairment are recognized clinically, the mechanisms of secondary organ damage through metabolic cross-talk remain poorly understood due to limitations of conventional monoculture models. Here we employ a multi-organ microphysiological system (MPS) connecting stem cell derived liver and CNS organoids via microfluidic channels to model inter-organ drug metabolism and secondary toxicity. Liver organoids were treated with CP (0-200 {micro}M) for 48 hours, and connected CNS organoids were assessed for secondary damage by confocal Z-stack imaging of DNA damage ({gamma}H2AX), neuronal identity (NeuN), and nuclear content (DAPI). We observe dose-dependent reduction in NeuN expression and {gamma}H2AX signal in connected CNS organoids, consistent with neurotoxic metabolite transfer from liver. Critically, CNS-to-CNS control connections show no comparable damage at equivalent CP concentrations, confirming that hepatic metabolism is required for CNS toxicity. These findings validate the MPS platform for modelling multi-organ drug toxicity and provide direct evidence that liver-derived CP metabolites drive secondary neurotoxicity through inter-organ metabolic communication.

bioengineering↗

bioRxiv2025

Identification of a novel small molecule facilitating HIV elimination by the "Shock-and-Kill" approach

The development of antiretroviral therapy (ART) has significantly improved the prognosis of people living with HIV (PLWH). However, reservoirs of latent viruses represent a major barrier to achieving a complete cure of HIV infection. To overcome the burden of viral latency, the "Shock-and-Kill" strategy employs latency-reversing agents (LRAs) to reactivate the virus and thus render it susceptible to elimination. Existing LRAs are not optimal and there is an unmet need for improvement. By screening a small molecule library, we identified the novel LRA candidate 2-HSB (2-hydrido-2,2-spirobi(1,3,2-benzodioxaphosphole)) which not only reactivates latent HIV provirus but also induces specific cytopathic effects in both monocytic and T lymphocytic latent HIV reservoirs. Thus, this novel compound represents a dual "shock and kill" agent, unlike known LRAs which are limited to the "shock". The probable involvement of HIV-1 tat protein may account for its selectivity. Although both shock and kill effects seemed to be regulated by spleen tyrosine kinase, they were independent of each other, indicating that neither activation-induced cell death nor cell death-induced reactivation was occurring. Crucially, 2-HSB induced viral transcription in ART-suppressed PLWH samples in vitro. Our findings present 2-HSB as a promising dual-action compound for the "Shock and Kill" strategy, demonstrating efficacy in both transformed cell lines and clinically-relevant primary samples. These results raise the possibility of distinguishing the mode of action of the two effects, potentially leading to the development of a single agent causing both HIV-1 reactivation and elimination and contributing to the improvement of treatment.

microbiology↗

bioRxiv2025

Functional impact of pathogenic Runt domain mutations in Runx2 in vivo: Insights into the skeletal and dental anomalies of cleidocranial dysplasia

Runt-related transcription factor 2 (RUNX2) is essential for skeletogenesis, and mutations in its gene cause cleidocranial dysplasia (CCD), an autosomal dominant skeletal disorder. The evolutionarily conserved 128-amino acid Runt homology domain (RHD) of human RUNX2 is essential for DNA binding and heterodimerization, and serves as a mutation hotspot associated with severe CCD phenotypes. To elucidate the functional impact of pathogenic RHD mutations in vivo, we generated two novel mouse lines: one carrying a missense mutation, c.695G>A (p.R232Q) (Runx2m/+), corresponding to the human RUNX2 c.674G>A (p.R225Q), and the other harboring a frameshift mutation, c.697_698delGA (p.E233TfsTer9) (Runx2112/+), causing a premature stop codon. Homozygous Runx2m/mand Runx2112/112 mice lacked membranous ossification, whereas heterozygous Runx2m/+ and Runx2112/+ mice displayed typical CCD-like skeletal features, including an open anterior fontanelle and clavicle hypoplasia. Unexpectedly, heterozygotes carrying pathogenic mutations in RHD developed an accessory root-like protrusion at the furcation of three-rooted maxillary first molars, representing a previously unrecognized dental phenotype during root development. Dual luciferase assays revealed impaired transactivation of the p.R232Q mutant Runx2 on the osteocalcin enhancer/promoter. Wild-type Runx2 was robustly expressed in osteoblasts and hypertrophic chondrocytes during bone formation, but the mutant Runx2 exhibited reduced expression in hypertrophic chondrocytes and partially impaired nuclear localization, resulting in arrested osteoblast and chondrocyte maturation. Our mutant mouse model provides a valuable in vivo platform to study CCD pathogenesis, mechanisms of tooth root furcation, and therapeutic interventions targeting dysfunctional RHD.

genetics↗

bioRxiv2023

Sclerostin modulates the degree of mineralization and the stiffness profile of the fibrocartilaginous enthesis for mechanical tissue integrity

Fibrocartilaginous entheses consist of four graded tissue layers including tendon, the unmineralized and mineralized fibrocartilage, and subchondral bone with varying degrees of stiffness. Here we examined the functional role of sclerostin that is expressed in mature mineralized fibrochondrocytes. Following rapid mineralization of the unmineralized fibrocartilage and parallel replacement of epiphyseal hyaline cartilage by bone, the unmineralized fibrocartilage re-expanded after a decline in alkaline phosphatase activity at the mineralization front. Sclerostin was co-expressed with osteocalcin in the bottom of the mineralized fibrocartilage adjacent to subchondral bone. In Scx deficient mice with less mechanical loading due to defects of the Achilles tendon, the number of sclerostin+ fibrochondrocytes was significantly reduced in the defective enthesis where chondrocyte maturation was markedly impaired in both fibrocartilage and hyaline cartilage. Loss of the Sost gene, coding for sclerostin, caused increased mineral density in the mineralized zones of the fibrocartilaginous enthesis. Atomic force microscopy analysis revealed the higher stiffness of fibrocartilage. These lines of evidence suggest that sclerostin in mature mineralized fibrochondrocytes acts as a modulator for mechanical tissue integrity of the fibrocartilaginous enthesis.

developmental biology↗

bioRxiv2023

Homeostatic Regulation of Seizure Susceptibility and Cognitive Function by ER Quality Controller Derlin-1 through Maintenance of Adult Neurogenesis

Dysfunction of organelle is closely associated with neurological diseases involving disruption of adult neurogenesis. However, the role of the endoplasmic reticulum (ER)-related molecules in this process remains largely unexplored. Here we show that Derlin-1, an ER quality controller, maintains adult neurogenesis in a spatiotemporal manner. Deletion of Derlin-1 in the mouse central nervous system induces ectopic localization of newborn neurons and impairs neural stem cells (NSCs) transition from active to quiescent states, resulting in early depletion of hippocampal NSCs. As a result, Derlin-1- deficient mice exhibit phenotypes of increased seizure susceptibility and impaired cognitive function. Reduced expression of signal transducer and activator of transcription 5b (Stat5b) was found to be responsible for the impairment of adult neurogenesis in Derlin-1-deficient NSCs. Remarkably, the inhibition of histone deacetylase activity ameliorated seizure susceptibility and cognitive dysfunction in Derlin-1-deficient mice by increasing Stat5b expression and restoring abnormal neurogenesis. Overall, our findings demonstrate that Derlin-1, as its characteristic function, plays an essential role in the homeostasis of adult neurogenesis via Stat5b expression, thus regulating seizure susceptibility and cognitive function.

neuroscience↗

bioRxiv2022

Enterococcal linear plasmids adapt to Enterococcus faecium and spread within multidrug-resistant clades.

Antimicrobial resistance (AMR) of bacterial pathogens, including enterococci, is a global concern, and plasmids are crucial for spreading and maintaining AMR genes. Plasmids with linear topology were recently identified in clinical multidrug-resistant enterococci. The enterococcal linear-form plasmids, such as pELF1, confer resistance to clinically important antimicrobials, including vancomycin; however, little information exists about their epidemiological and physiological effects. In this study, we identified several lineages of enterococcal linear plasmids that are structurally conserved and occur globally. pELF1-like linear plasmids show plasticity in acquiring and maintaining AMR genes, often via transposition with the mobile genetic element IS1216E. This linear plasmid family has several characteristics enabling long-term persistence in the bacterial population, including high horizontal self-transmissibility, low-level transcription of plasmid-encoded genes, and a moderate effect on the Enterococcus faecium genome alleviating fitness cost and promoting vertical inheritance. Combined with its broad host range, the linear plasmid is an important factor in the spread and maintenance of AMR genes among enterococci.

microbiology↗

bioRxiv2021

Inhibiting SARS-CoV-2 infection in vitro by suppressing its receptor, angiotensin-converting enzyme 2, via aryl-hydrocarbon receptor signal

Since understanding molecular mechanisms of SARS-CoV-2 infection is extremely important for developing effective therapies against COVID-19, we focused on the internalization mechanism of SARS-CoV-2 via ACE2. Although cigarette smoke is generally believed to be harmful to the pathogenesis of COVID-19, cigarette smoke extract (CSE) treatments were surprisingly found to suppress the expression of ACE2 in HepG2 cells. We thus tried to clarify the mechanism of CSE effects on expression of ACE2 in mammalian cells. Because RNA-seq analysis suggested that suppressive effects on ACE2 might be inversely correlated with induction of the genes regulated by aryl hydrocarbon receptor (AHR), the AHR agonists 6-formylindolo(3,2-b)carbazole (FICZ) and omeprazole (OMP) were tested to assess whether those treatments affected ACE2 expression. Both FICZ and OMP clearly suppressed ACE2 expression in a dose-dependent manner along with inducing CYP1A1. Knock-down experiments indicated a reduction of ACE2 by FICZ treatment in an AHR-dependent manner. Finally, treatments of AHR agonists inhibited SARS-CoV-2 infection into Vero E6 cells as determined with immunoblotting analyses detecting SARS-CoV-2 specific nucleocapsid protein. We here demonstrate that treatment with AHR agonists, including CSE, FICZ, and OMP, decreases expression of ACE2 via AHR activation, resulting in suppression of SARS-CoV-2 infection in mammalian cells.

molecular biology↗

bioRxiv2018

Effect of continuous compressive force on the expression of RANKL, OPG, and VEGF in MC3T3-E1 and MLO-Y4 cells

Osteocytes, known to have mechano-sensory functions, influence the regulation of bone remodeling. However, the mechanism by which osteocytes regulate bone metabolism when mechanical forces are being applied is still unclear. Osteoclastogenesis is mainly regulated by receptor activator of nuclear factor kappa-B ligand (RANKL); the protein osteoprotegerin (OPG) and angiogenesis also play important roles in osteogenesis. RANKL, OPG, and vascular endothelial growth factor (VEGF) are thought to be key factors for bone metabolism. In this study, we examined the effect of a continuous compressive force (CF) on the expression of RANKL, OPG, and VEGF in osteoblastic murine osteocytes (MLO-Y4) and osteoblastic (MC3T3-E1) cells. Gene and protein expression levels of RANKL, OPG, and VEGF in MLO-Y4 and MC3T3-E1 cells were quantitatively determined by real-time PCR and enzyme-linked immunosorbent assay (ELISA). Both cell types were also subjected to a CF of 1.0 g/cm2 for 1, 3, 6, and 12 hours. Furthermore, the effect of a stretch-activated (S-A) channel was examined by gadolinium (Gd3+) administration. The ratio of gene and protein expressions of RANKL, VEGF, and RANKL/OPG in MLO-Y4 cells were significantly higher than in MC3T3-E1 cells, while the expression of OPG was significantly lower. After CF application, both cell types showed significant increases in RANKL and VEGF expression as well as the RANKL/OPG ratio. Additionally, the upregulated gene and protein levels of these factors were reduced by Gd3+ administration.\n\nThese findings suggest that osteocytes play more important roles in bone metabolism and angiogenesis than osteoblasts. Osteocytes regulate the expression of RANKL, OPG, and VEGF via the S-A channel through the response to mechanical stress.

cell biology↗

bioRxiv2018

No evidence for transvection in vivo by a superenhancer:promoter pair integrated into identical open chromatin at the Rosa26 locus

Long-range associations between enhancers and their target gene promoters have been shown to play critical roles in executing genome function. Recent variations of chromosome capture technology have revealed a conprehensive view of intra- and inter-chromosomal contacts between specific genomic sites. The locus control region of the {beta}-globin genes ({beta}-LCR) is a super-enhancer that is capable of activating all of the {beta}-like globin genes within the locus in cis through physical interaction by forming DNA loops. CTCF helps to mediate loop formation between LCR-HS5 and 3HS1 in the human {beta}-globin locus, in this way thought to contribute to the formation of a \"chromatin hub\". The {beta}-globin locus is also in close physical proximity to other erythrocyte-specific genes located long distances away on the same chromosome. In this case, erythrocyte-specific genes gather together at a shared \"transcription factory\" for co-transcription. Theoretically, enhancers could also activate target gene promoters on different chromosomes in trans, a phenomenon originally described as transvection in Drosophilla. Although close physical proximity has been reported for the {beta}-LCR and the {beta}-like globin genes when integrated at the mouse homologous loci in trans, their structural and functional interactions were found to be rare, possibly because a lack of suitable regulatory elements that might facilitate trans interactions. Therefore, we re-evaluated presumptive transvection-like enhancer-promoter communication by introducing CTCF binding sites and erythrocyte-specific transcription units into both LCR-enhancer and {beta}-promoter alleles, each inserted into the mouse ROSA26 locus on separate chromosomes. Following cross-mating of mice to place the two mutant loci at the identical chromosomal position and into active chromation in trans, their transcriptional output was evaluated. The results demonstrate that there was no significant functional association between the LCR and the {beta}-globin gene in trans even in this idealized experimental context.

genomics↗