Biology subjects

Sakamoto, H.

Publications and source records attributed to Sakamoto, H..

13 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↗

bioRxiv2025

Synaptic active zones are ordered nanostructures designed by supramolecular block copolymers

Neurotransmitters are released at presynaptic active zones, where a conserved cytomatrix exhibits nanoscale order first visualized by electron microscopy over half a century ago. However, the physical principles underlying this architecture have remained unclear. Here we show that active zone scaffolds act as protein block copolymers, whose nanoscale microphase separation produces hexagonally packed nanodot arrays. Super-resolution imaging combined with block-copolymer theory identifies ELKS and RIM as the minimal pair sufficient to reproduce this ordered lattice, mediated by orthogonal interactions between ELKS coiled-coil domains and RIM intrinsically disordered regions. Additional active zone proteins, including RIMBP and Liprin-, modulate lattice spacing and morphology in a stoichiometry-dependent manner, coupling nanoscale order to neurotransmission. More broadly, block-copolymer microphase separation emerges as a design principle for constructing functional nanostructures in living systems.

biophysics↗

bioRxiv2024

Suppressive effects of oroxylin A on intracellular proliferation of Toxoplasma gondii via host cell ERK phosphorylation inhibition

Toxoplasma gondii poses a significant threat to immunocompromised patients, resulting in high mortality rates. Considering the side effects of anti-Toxoplasma drugs, we focused on a potential candidate, oroxylin A (OA), a common component extracted from Astragalus membranaceus and Scutellaria baicalensis which suppressed the growth of T. gondii in vitro and in vivo. Our result demonstrated that OA suppressed T. gondii intracellular proliferation and downregulated phosphorylation of ERK1/2 of T. gondii-infected host cells very similar to the MEK1 specific inhibitor PD98059. Our in silico analysis showed that OA interacts sufficiently with the mammalian MEK1 region where established MEK1 inhibitors like PD98059 and trametinib bind. Moreover, OA improved the survival rate in T. gondii-infected mice. This study proposes that host MEK1 is a novel potential target for anti-Toxoplasma drugs with a new mechanism of action.

microbiology↗

bioRxiv2022

Aquaporin regulates cell rounding through vacuole formation during endothelial-to-hematopoietic transition

Endothelial-to-hematopoietic transition (EHT) is crucial for hematopoietic stem cell (HSC) generation. During EHT, the morphology of hemogenic endothelial cells (HECs) changes from flat and adherent to spherical HSCs, which detach from the dorsal aorta. HECs attain a rounded shape in a mitosis-independent manner before cell adhesion termination, suggesting an atypical cell-rounding mechanism. However, the direct mechanisms underlying this change in cell morphology during EHT remain unclear. Here, we show that large vacuoles were transiently formed in HECs and that aquaporin-1 (AQP1) was localized in the vacuole and plasma membranes. Overexpression of AQP1 in non-HECs induced ectopic vacuole expansion, cell rounding, and subsequent cell detachment from the endothelium into the bloodstream, mimicking EHT. Loss of redundant AQP functions by CRISPR/Cas9 gene editing in HECs impeded the morphological EHT. Our findings provide the first evidence indicating that morphological segregation of HSCs from endothelial cells is regulated by water influx into vacuoles. These findings provide important insights for further exploration of the mechanisms underlying cell/tissue morphogenesis through water-adoptive cellular responses. SUMMARY STATEMENTHemogenic endothelial cells transiently form large vacuoles during endothelial-to-hematopoietic transition. Aquaporin water channels regulate cell rounding and detachment of emerging hematopoietic stem cells through vacuole formation.

developmental biology↗

bioRxiv2022

RIM-BP2 is required for rapid neurotransmitter release through regulation of Ca2+ channel clustering at hippocampal mossy fiber terminals

Synaptic vesicles dock and fuse at the presynaptic active zone (AZ), the specialized site for transmitter release. AZ proteins play multiple roles such as recruitment of Ca2+ channels as well as synaptic vesicle docking, priming and fusion. However, the precise role of each AZ protein type remains unknown. In order to dissect the role of RIM-BP2 at mammalian cortical synapses having low release probability, we applied direct electrophysiological recording and super-resolution imaging to hippocampal mossy fiber terminals of RIM-BP2 KO mice. By using direct presynaptic recording, we found the reduced Ca2+ currents. The measurements of EPSCs and presynaptic capacitance suggested that the initial release probability was lowered because of the reduced Ca2+ influx and impaired fusion competence in RIM-BP2 KO. Nevertheless, larger Ca2+ influx restored release partially. Consistent with presynaptic recording, STED microscopy suggested less abundance of P/Q-type Ca2+ channels at AZs deficient in RIM-BP2. Our results suggest that the RIM-BP2 regulates both Ca2+ channel abundance and transmitter release at mossy fiber synapses.

neuroscience↗

bioRxiv2022

Synapse type-specific molecular nanoconfigurations of the presynaptic active zone in the hippocampus identified by systematic nanoscopy

Neurotransmitters are released by exocytosis from synaptic vesicles at the active zone in the presynaptic terminal. The scaffold of the active zone consists of only a few evolutionarily conserved proteins, including RIM, CAST/ELKS, and RIM-BP, and tethers Munc13 and Ca2+ channels. The molecular principles that enable these proteins to mediate synaptic diversity have remained unclear. Here, we identified synapse type-specific molecular nanoconfigurations in the active zone by systematic quantification of active zone proteins using nanoscopy at two types of excitatory synapses and two types of inhibitory synapses in the rat hippocampal CA3 region. Quantitative analysis revealed that Munc13 content was particularly varied among the various synapse types and that the physical proximity of Ca2+ channels to the active zone scaffolds correlated with the efficacy of neurotransmitter release. We propose that the active zone is a flexible supramolecular assembly that can tune its composition and spatial configuration to adjust neurotransmitter release.

neuroscience↗

bioRxiv2021

NDR1 and the Arabidopsis Plasma Membrane ATPase AHA5 are Required for Processes that Converge on Drought Tolerance and Immunity

NON-RACE-SPECIFIC DISEASE RISISTANCE1 (NDR1) is a key component of plant immune signaling, required for defense against the bacterial pathogen Pseudomonas syringae. Plant stress responses have overlapping molecular, physiological, and cell biology signatures, and given the central role of NDR1 during biotic stress perception and signaling, we hypothesized that NDR1 also functions in abiotic stress responses, including in a role that mediates signaling at the plasma membrane (PM) - cell wall (CW) continuum. Here, we demonstrate that NDR1 is required for the induction of drought stress responses in plants, a role that couples stress signaling in an abscisic acid-dependent manner. We show that NDR1 physically associates with the PM-localized H+-ATPases AHA1, AHA2, and AHA5 and is required for proper regulation of H+-ATPase activity and stomatal guard cell dynamics, providing a mechanistic function of NDR1 during drought responses. In the current study, we demonstrate that NDR1 functions in signaling processes associated with both biotic and abiotic stress response pathways, a function we hypothesize represents NDR1s role in the maintenance of cellular homeostasis during stress. We propose a role for NDR1 as a core transducer of signaling between cell membrane processes and intercellular stress response activation.

plant biology↗

bioRxiv2021

Immunoelectron microscopic characterization of vasopressin neurons in macaques by use of formaldehyde-fixed tissues stored for several years

Translational research often requires the testing of experimental therapies in primates, but research in non-human primates is now stringently controlled by law around the world. Tissues fixed in formaldehyde without glutaraldehyde have been thought to be inappropriate for use in electron microscopic analysis, particularly those of the brain. Here we report the immunoelectron microscopic characterization of arginine vasopressin (AVP)-producing neurons in macaque hypothalamo-pituitary axis tissues fixed with 4% formaldehyde and stored at -25{degrees}C for several years. The size difference of dense-cored vesicles between magnocellular and parvocellular AVP neurons was detectable in their cell bodies and perivascular nerve endings located, respectively, in the posterior pituitary and median eminence. Furthermore, glutamate and the vesicular glutamate transporter 2 were colocalized with AVP in perivascular nerve endings of both the posterior pituitary and the external layer of the median eminence, suggesting that both magnocellular and parvocellular AVP neurons are glutamatergic in primates. Both ultrastructure and immunoreactivity can therefore be sufficiently preserved in macaque brain tissues stored long-term for light microscopy. Taken together, these results suggest that this methodology could be applied to the human post-mortem brain and be very useful in translational research.

neuroscience↗

bioRxiv2020

Revisiting the gastrin-releasing peptide/bombesin system: A reverse-evolutionary study considering Xenopus

Gastrin-releasing peptide (GRP), first isolated from the porcine stomach, is a neuropeptide that modulates the autonomic system in mammals and has previously been considered to be the mammalian equivalent of bombesin, a fourteen amino acid peptide first isolated from the skin of the European fire-bellied toad, Bombina bombina. Bombesin-like peptides and the related neuromedin B (NMB) have since been identified in mammals. However, the orthologous relationships among GRP/NMB/bombesin and their receptors in vertebrates are still not well understood. Our studies have focused on the GRP system that is widely conserved among vertebrates. We have used phylogenetic analysis and reverse transcription-PCR, quantitative PCR, immunohistochemistry, and Western blotting experiments to examine the expression of both GRP and its receptor (GRPR) in a clawed frog (Xenopus tropicalis) and to understand the derivation of GRP system in the ancestor of mammals. We demonstrate, by phylogenetic and synteny analyses, that GRP is not a mammalian counterpart of bombesin and also that, whereas the GRP system is widely conserved among vertebrates, the NMB/bombesin system has diversified in certain lineages, in particular in frog species. In Xenopus, we found the expression of the mRNA for both GRP and GRPR in the brain and stomach. In addition, our quantitative PCR analysis shows that, in Xenopus, the expression of GRP mRNA is highest in the brain, whereas expression of GRPR mRNA is highest in the spinal cord. Our immunohistochemical analysis shows that GRP-immunoreactive cell bodies and fibers are distributed in several telencephalic, diencephalic, and rhombencephalic regions and spinal cord of Xenopus. Our Western blotting analysis also indicates the presence of GRPR protein in the brain and spinal cord of Xenopus. We conclude that GRP peptides and their receptors have evolved to play multiple roles in both the gut and brain of amphibians as one of the gut-brain peptide systems. Author SummaryBombesin is a putative antibacterial peptide isolated from the skin of the frog, Bombina bombina. Two related (bombesin-like) peptides, gastrin-releasing peptide (GRP) and neuromedin B (NMB) have been found in mammals. The history of GRP/bombesin discovery has caused little attention to be paid to the evolutionary relationship of GRP/bombesin and their receptors in vertebrates. We have classified the peptides and their receptors from the phylogenetic viewpoint using a newly established genetic database and bioinformatics. We demonstrate, by phylogenetic and synteny analyses, that GRP is not a mammalian counterpart of bombesin and also that, whereas the GRP system is widely conserved among vertebrates, the NMB/bombesin system has diversified in certain lineages, in particular in frogs. Gene expression analyses combined with immunohistochemistry and Western blotting experiments indicate that GRP peptides and their receptors have evolved from ancestral (GRP) homologues to play multiple roles in both the gut and the brain as one of the gut-brain peptide systems of vertebrates, which is distinct from the frog bombesin lineage.

evolutionary biology↗

bioRxiv2020

Impaired expression of chloroplast HSP90C chaperone activates plant defense responses leading to a disease symptom-like phenotype

RNA-seq analysis of a transgenic tobacco plant, i-hpHSP90C, in which chloroplast HSP90C genes can be silenced in an artificially inducible manner resulting in the development of chlorosis, revealed the up- and down-regulation of 2746 and 3490 genes, respectively. Gene Ontology analysis of these differentially expressed genes indicated the upregulation of ROS-responsive genes, the activation of the innate immunity and cell death pathways, and the downregulation of genes involved in photosynthesis, plastid organization, and cell cycle. Cell death was confirmed by trypan blue staining and electrolyte leakage assay and the H2O2 production by diaminobenzidine staining. The upregulation of ER stress-responsive genes suggested the interplay between ER protein quality control and chloroplast or immune response. The results collectively suggest that the reduced levels of HSP90C chaperone leads the plant to develop chlorosis primarily through the global downregulation of chloroplast and photosynthesis-related genes and additionally through the light-dependent production of ROS, followed by the activation of immune responses including the cell death. HighlightInduced silencing of HSP90C gene caused the upregulation of stress-responsive genes and the activation of innate immune response, which resulted in the chlorosis development accompanying cell death.

plant biology↗

bioRxiv2019

Evolutionary Origins of Recurrent Pancreatic Cancer

Surgery is the only curative option for Stage I/II pancreatic cancer, nonetheless most patients will recur after surgery and die of their disease. To identify novel opportunities for management of recurrent pancreatic cancer we performed whole exome or targeted sequencing of 10 resected primary cancers and matched intrapancreatic recurrences or distant metastases. We identified that adjuvant or first-line platinum therapy corresponds to an increased mutational burden of recurrent disease. Recurrent disease is enriched for mutations that activate Mapk/Erk and PI3K/AKT signaling and develops from a monophyletic or polyphyletic origin. Treatment induced genetic bottlenecks lead to a modified genetic landscape and subclonal heterogeneity for driver gene alterations in part due to intermetastatic seeding. In one patient what was believed to be recurrent disease was an independent (second) primary tumor. These findings advocate for combination therapies with immunotherapy and routine post-treatment sampling as a component of management of recurrent pancreatic cancer.

cancer biology↗

bioRxiv2019

The Genetic Basis of Transcriptional and Spatial Heterogeneity of Squamous Features in Pancreatic Ductal Adenocarcinoma

Recent studies indicate that pancreatic cancer expression profiles are variable and largely reflect a classical or basal-type phenotype. We performed genetic sequencing, RNA-seq, and histologic review of multiregion sampled pancreatic cancers and found that squamous and squamoid features, indicators of poor prognosis, correlate with a "basal-like" expressional type. Cancers with squamous features were more likely to have truncal mutations in chromatin modifier genes and intercellular heterogeneity for MYC amplification that was associated with entosis. In most patients the basal phenotype coexisted with a glandular component, and phylogenetic studies indicated that it arose from a subclonal population in the tumor. These data provide a unifying paradigm for understanding the interrelationship of basal-type features, squamous histology, and somatic mutations in chromatin modifier genes in the context of the clonal evolution of pancreatic cancer.

cancer biology↗

bioRxiv2018

Genome-wide association meta-analysis identifies novel GP2 gene risk variants for pancreatic cancer in the Japanese population

The etiology of pancreatic cancer remains largely unknown. Here, we report the results of a meta-analysis of three genome-wide association studies (GWASs) comprising 2,039 pancreatic cancer cases and 32,592 controls, the largest sample size in the Japanese population. We identified 3 (13q12.2, 13q22.1, and 16p12.3) genome-wide significant loci (P<5.0x10-8) and 4 suggestive loci (P<1.0x10-6) for pancreatic cancer. Of these risk loci, 16p12.3 is novel; the lead SNP maps to rs78193826 (odds ratio (OR)=1.46, 95% CI=1.29-1.66, P=4.28x10-9), an Asian-specific, nonsynonymous glycoprotein 2 (GP2) gene variant predicted to be highly deleterious. Additionally, the gene-based GWAS identified a novel gene, KRT8, which is linked to exocrine pancreatic and liver diseases. The identified GP2 gene variants were pleiotropic for multiple traits, including type 2 diabetes, hemoglobin A1c (HbA1c) levels, and pancreatic cancer. Mendelian randomization analyses corroborated causality between HbA1c and pancreatic cancer. These findings suggest that GP2 gene variants are associated with pancreatic cancer susceptibility in the Japanese population, prompting further functional characterization of this locus.

genetics↗