Biology subjects

Nakao, Y.

Publications and source records attributed to Nakao, Y..

9 recordsLinked to original sources

bioRxiv2026

Stromal CTHRC1 protects the valvular interstitium from macrophage-associated inflammatory remodeling and calcification

Background: Calcific aortic valve disease (CAVD) is characterized by progressive inflammatory and fibrocalcific remodeling. Although valvular interstitial cells (VICs) are generally considered to drive fibrosis and osteogenic remodeling, whether injury-activated VICs mount endogenous protective responses that preserve the valvular interstitial microenvironment and restrain calcification remains unknown. Methods: We performed spatial transcriptomic profiling of aortic valves in a mouse model of endothelial injury-induced CAVD to define early injury-responsive programs within the valvular interstitium. The cellular origin and spatial distribution of candidate protective factors were examined by immunohistochemistry and lineage tracing, and their relevance to human disease was assessed using stenotic aortic valves. The functional role of CTHRC1 was investigated using genetic Cthrc1 deficiency combined with longitudinal hemodynamic assessment, histological analysis, and spatial transcriptomic profiling. Results: Spatial transcriptomics identified Cthrc1 as a prominent component of an early injury-induced stromal response in the expanding valvular interstitium. CTHRC1 was strongly expressed in activated VICs within thickened murine valve leaflets and human stenotic aortic valves. Lineage tracing demonstrated that the expanded VIC population arose predominantly from PDGFR{beta}+ resident interstitial cells, with minimal endothelial contribution. Despite comparable early hemodynamic responses to endothelial injury, Cthrc1 deficiency exacerbated chronic valvular calcification. Spatial profiling of Cthrc1-deficient valves revealed pronounced interstitial accumulation of galectin-3+ foamy macrophages, accompanied by mitochondrial respiratory-chain signature loss and cell death-associated pathway activation. These findings indicate that transient CTHRC1 induction after endothelial injury defines an endogenous stromal protective response that preserves the valvular interstitial microenvironment and limits macrophage-associated tissue injury and subsequent dystrophic calcification. Conclusions: Injury-activated VICs are not merely effectors of pathological remodeling, but can engage an endogenous tissue-protective response through CTHRC1. These findings identify a previously unrecognized stromal defense mechanism linking endothelial injury to macrophage-associated inflammatory remodeling and dystrophic calcification and suggest CTHRC1-dependent stromal protection as a potential therapeutic axis for limiting CAVD progression.

molecular biology↗

bioRxiv2026

Cytotoxicity-based High-throughput Screening System for CAR T Cell

Some chimeric antigen receptor (CAR) T cell therapies have shown strong clinical efficacy, yet systematic screening of new CAR designs remains constrained by labor-intensive, low-throughput evaluation methods. To address this limitation, we developed a cytotoxicity-centered, high-throughput screening platform that integrates single-cell pooled screening with fully automated arrayed screening enabling both large-scale library handling and quantitative functional resolution for systematic CAR design exploration. Using a mutation-based CAR design approach guided by protein fitness prediction, we generated a 4-1BB-based CAR library with approximately 10 theoretical variants while minimizing the prevalence of low-activity designs. In pooled screening, CAR T cells were evaluated at the single-cell level based on cytotoxicity and proliferation, enabling rapid enrichment of high-performing variants from a highly diverse library. Subsequent automated arrayed screening quantitatively measured cytotoxicity with high reproducibility, providing high-resolution functional data suitable for comparative ranking. Selected CAR variants demonstrated superior antitumor efficacy in a leukemia xenograft model compared with a template CAR. Furthermore, systematic analysis of mutation sites from an enhanced CAR variant identified essential mutation combinations underlying functional enhancement. Together, this study establishes a cytotoxicity-focused screening framework that provides a robust approach for optimizing CAR architectures and accelerating the development of CAR T-cell therapies.

synthetic biology↗

bioRxiv2025

Osteochondrogenic Transdifferentiation of Vascular Smooth Muscle Cells and Microenvironmental Dynamics in Medial Arterial Calcification

BackgroundVascular calcification, particularly medial arterial calcification (MAC), considerably affects cardiovascular mortality. Current treatments are limited because of the unclear molecular mechanisms of MAC. This study aimed to establish an MAC mouse model using O-ring-induced transverse aortic constriction (OTAC) and to identify critical genes, pathways, and cellular interactions involved in MAC formation by combining lineage tracing technology and single-cell RNsA sequencing (scRNA-seq). MethodsWe developed an OTAC mouse model to mimic MAC. Adult C57BL/6J male mice underwent OTAC, and subsequent analyses were performed at various intervals. Histological and immunohistochemical evaluations were conducted to observe changes in vascular smooth muscle cells (VSMCs). Lineage tracing was used to confirm the origin of osteochondrogenic cells in the tunica media. Additionally, scRNA-seq was performed to capture the dynamic changes in VSMCs and other cell types involved in MAC development. ResultsOsteochondrogenic cells and the subsequent MAC in OTAC mice were observed using histological evaluations. Immunohistochemistry showed time-dependent decreases in -SMA expression and increases in SOX9 and RUNX2 expression in VSMCs. Lineage tracing using Myh11CreERT2;ROSA26-EGFP mice confirmed that osteochondrogenic cells originated from contractile VSMCs. scRNA-seq confirmed osteochondrogenic transdifferentiation of VSMCs and highlighted dynamic changes in surrounding cells involved in calcification-related and inflammatory processes. ConclusionThe OTAC method enabled a comprehensive understanding of the microenvironmental dynamics during MAC progression at the single-cell level. This powerful model provides a robust platform for future therapeutic intervention research. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/684711v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@c64634org.highwire.dtl.DTLVardef@12b0b9aorg.highwire.dtl.DTLVardef@106c5b4org.highwire.dtl.DTLVardef@19dd4f4_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Efficient biallelic knock-in in mouse embryonic stem cells by in vivo-linearization of donor and transient inhibition of DNA Polymerase θ/DNA-PK

CRISPR/Cas9-mediated homology-directed repair (HDR) is used for error-free targeted knock-in of foreign donor DNA. However, the low efficiency of HDR-mediated knock-in hinders establishment of knock-in clones. Double-strand breaks (DSBs) induced by CRISPR/Cas9 are preferentially repaired by non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) before HDR can occur, thereby preventing HDR-mediated knock-in. NHEJ/MMEJ also cause random integrations, which give rise to false-positive knock-in events, or silently disrupt the genome. In this study, we optimized an HDR-mediated knock-in method for mouse embryonic stem cells (mESCs). We succeeded in improving efficiency of HDR-mediated knock-in of a plasmid donor while almost completely suppressing NHEJ/MMEJ-based integration by combining in vivo-linearization of the donor plasmid, transient knockdown of DNA Polymerase {theta}, and chemical inhibition of DNA-dependent protein kinase (DNA-PK) by M3814. This method also dramatically improved the efficiency of biallelic knock-in; at the Rosa26a locus, 95% of HDR-mediated knock-in clones were biallelic. We designate this method BiPoD (Biallelic knock-in assisted by Pol {theta} and DNA-PK inhibition). BiPoD achieved simultaneous efficient biallelic knock-in into two loci. BiPoD, therefore, enables rapid and easy establishment of biallelic knock-in mESC lines.

molecular biology↗

bioRxiv2020

Intravenous edaravone plus therapeutic hypothermia offers limited neuroprotection in the hypoxic-ischaemic newborn piglet

Therapeutic hypothermia is a standard therapy for neonatal hypoxic-ischaemic encephalopathy. One potential additional therapy is the free radical scavenger edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one). To compare the neuroprotective effects of edaravone plus therapeutic hypothermia with those of therapeutic hypothermia alone after a hypoxic-ischaemic insult in the newborn piglet, anaesthetized piglets were subjected to 40 min of hypoxia (3-5% inspired oxygen) and cerebral ischaemia was assessed by cerebral blood volume. Body temperature was maintained at 38.5 {degrees}C in the normothermia group (NT, n = 8) and at 34 {degrees}C (24 h after the insult) in the therapeutic hypothermia (TH, n = 7) and therapeutic hypothermia plus edaravone (3 mg intravenous every 12 h for 3 days after the insult; TH+EV, n = 6) groups under mechanical ventilation. Five days after the insult, the mean (standard deviation) neurological scores were 10.9 (5.7) in the NT group, 17.0 (0.4) in the TH group (p = 0.025 vs. NT) and 15.0 (3.9) in the TH+EV group. The histopathological score of the TH+EV group showed no significant improvement compared with that of the other groups. In conclusion, edaravone plus therapeutic hypothermia had no additive neuroprotective effects after hypoxia-ischaemia in neurological and histopathological assessments.

neuroscience↗

bioRxiv2020

Histone modification dynamics as revealed by a multicolor immunofluorescence-based single-cell analysis

Post-translational modifications on histones can be stable epigenetic marks and transient signals that can occur in response to internal and external stimuli. Levels of histone modifications fluctuate during the cell cycle and vary among different cell types. Here we describe a simple system to monitor the levels of multiple histone modifications in single cells by multicolor immunofluorescence using directly labeled modification-specific antibodies. We first analyzed histone H3 and H4 modifications during the cell cycle. Levels of active marks, such as acetylation and H3K4 methylation, were increased during the S phase, in association with chromatin duplication. By contrast, levels of some repressive modifications gradually increased during the G2 and the next G1 phases. We applied this method to validate the target modifications of various histone demethylases in cells using a transient overexpression system. We also screened chemical compounds in marine organism extracts that affect histone modifications and identified psammaplin A, which was previously reported to inhibit histone deacetylases. Thus, the method presented here is a powerful and convenient tool for analyzing the changes in histone modifications.

cell biology↗

bioRxiv2019

Epigenetic Effects of Exposure to Insecticide on Early Differentiation of Mouse Embryonic Stem Cells

Increasing evidence indicates that insecticides induce various diseases via DNA methylation. DNA methylation plays an important role during cell differentiation and exhibits its greatest vulnerability to environmental factors during embryogenesis. Therefore, it is important to evaluate the effects on DNA methylation at the early stage of cell differentiation to understand developmental toxicity. However, DNA methylation induced by insecticides and the associated effects on cell differentiation are unclear. In this research, we introduced a high-content approach utilizing mouse embryonic stem cells harboring enhanced green fluorescent protein fused with methyl CpG-binding protein to evaluate global DNA methylation induced by various insecticides. DNA methylation was assessed in 22 genes after pesticide exposure to investigate the relationships with biological processes such as cell cycle, cell apoptosis, and cell differentiation. Exposure to acetamiprid, imidacloprid, carbaryl, and o,p'-DDT increased the granular intensity, indicating their global DNA-methylating effects. Exposure to imidacloprid decreased DNA methylation in genes such as Cdkn2a, Dapk1, Cdh1, Mlh1, Timp3, and Rarb, indicating the potential influence of the DNA methylation pattern on cell differentiation. We developed a promising approach for evaluating global DNA methylation, and our findings suggested that imidacloprid might exhibit developmental effects through DNA methylation pattern.

pharmacology and toxicology↗

bioRxiv2019

Cultivating in situ triggers growth of previously uncultivated microorganisms via a growth initiation factor in nature

Most microorganisms resist cultivation under standard laboratory conditions. On the other hand, to cultivate microbes in a membrane-bound device incubated in nature (in situ cultivation) is an effective approach. In the present study, we applied in situ cultivation to isolate diverse previously uncultivated marine sponge-associated microbes and comparatively analyzed this methods efficiencies with those of the conventional method. Then, we attempted to clarify the key and unknown mechanism of in situ cultivation by focusing on growth triggering via growth initiation factor. We hypothesized that majority of environmental microorganisms are in nongrowing state and requiring \"growth initiation factor\" for the recovery and that can be provided from environments. Consequently, significantly more novel and diverse microbial types were isolated via in situ cultivation than by standard direct plating (SDP). Next, the effect of the sponge extract on starvation recovery was compared between strains derived from in situ and SDP cultivation. Adding small amounts of the sponge extracts to the medium elevated the colony-formation efficiencies of the in situ strains at the starvation recovery step, while it showed no positive effect on that of SDP strains. Conversely, specific growth rates or carrying capacities of all tested strains were not positively affected. These results indicate that, 1) the sponge extract contains chemical compounds that facilitate starvation recovery, these substances selectively worked on the in situ strains, and 2) growth initiation factor in the sponge extract did not continuously promote growth activity but worked as triggers for regrowth (resuscitation from dormancy).\n\nImportanceMost microbial species resist cultivation under laboratory condition. This is critical impediment for both academic and applied microbiology, and thus clarification of the mechanism of microbial uncultivability is highly demanded. Several evidences have been reported that to cultivate microbes in a membrane-bound device incubated in nature (in situ cultivation) is an effective approach. However, the mechanism behind this approach has not been clarified. The present study shows the evidence that 1) initiating growth is a key for cultivating previously uncultivated microbes rather than simple growth promotion, and 2) growth initiation factor (signaling-like compounds) in natural environments stimulate microbial resuscitation from a nongrowing state. Since no study has focused on growth initiation for cultivation of previously uncultivated microorganisms, the discovery shown in the present study provides a new insight into microorganisms previously considered uncultivable and a microbial growth controlling system in nature.

microbiology↗

bioRxiv2018

Structural instability of IκB kinase β promotes autophagic degradation through enhancement of Keap1 binding

IKK{beta}, an essential kinase of NF-[kcy]B signaling, is composed of an N-terminal kinase domain (KD) and a C-terminal scaffolding domain, containing a ubiquitin-like domain (ULD). The Hsp90 chaperon has special responsibility for folding of protein kinases including IKK{beta}. Here, we found that Hsp90 inhibition induced IKK{beta} degradation, which is partially mediated by Keap1. Geldanamycin (GA), a Hsp90 inhibitor, enhances association of IKK{beta} with Keap1 through the binding site in KD, and translocates IKK{beta} to detergent-insoluble fractions leading its autophagic degradation. An electrophile tBHQ suppressed Keap1-mediated proteasomal Nrf2 degradation but not autophagic IKK{beta} degradation. Substitution mutation of Leu353 to Ala in the ULD destabilizes IKK{beta}, enhances its association with Keap1, translocates it to detergent-insoluble fractions, and causes its autophagic degradation. These results suggest that Keap1 is involved in the degradation of structural destabilized IKK{beta} and negative regulation of NF-[kcy]B under proteotoxic stress.

biochemistry↗