This study aimed to evaluate the effects of long-term administration of Ninjin'yoeito, a traditional Japanese Kampo medicine, on age-related salivary hypofunction in mice.
Methods
Male senescence-accelerated mice (SAMP1) were divided into two groups and fed either a control diet, or a diet containing 3 % Ninjin'yoeito extract for four months. Salivary gland function was assessed by measuring the flow rate after muscarinic stimulation of the submandibular glands perfused ex vivo. Additional analysis included histological and immunohistochemical evaluations, blood tests, and real-time PCR to investigate gland morphology, immune cell profiles, gene expression, and inflammation/aging markers.
Results
Long-term Ninjin'yoeito administration significantly increased salivary secretion after stimulation and decreased the number of vacuoles in acinar cells as compared to the controls. Ninjin'yoeito improved the plasma albumin levels and increased the lymphocyte ratio in the white blood cell count. Furthermore, real-time PCR revealed decreased levels of inflammatory cytokine and senescence-associated genes. No significant changes were detected in the expression or localization of the main salivary secretion-related channels, such as transmembrane protein 16A and aquaporin 5.
Conclusions
Ninjin'yoeito enhances salivary secretion from the submandibular glands of aged mice by improving nutritional status, modulating immune function, suppressing chronic inflammation, and attenuating cellular aging. These results suggest that Ninjin'yoeito has potential applications as a complementary therapy for age-related dry mouth.
{"title":"Ninjin'yoeito enhances saliva secretion in aged mice via nutritional, immune, anti-inflammatory, and anti-aging effects","authors":"Tomoki Kurakata, Yusuke Kondo, Akihiro Nakamura, Yui Hirata Obikane, Tomotaka Nodai, Takashi Munemasa, Taro Mukaibo, Ryuji Hosokawa, Chihiro Masaki","doi":"10.1016/j.job.2026.100739","DOIUrl":"10.1016/j.job.2026.100739","url":null,"abstract":"<div><h3>Objectives</h3><div>This study aimed to evaluate the effects of long-term administration of Ninjin'yoeito, a traditional Japanese Kampo medicine, on age-related salivary hypofunction in mice.</div></div><div><h3>Methods</h3><div>Male senescence-accelerated mice (SAMP1) were divided into two groups and fed either a control diet, or a diet containing 3 % Ninjin'yoeito extract for four months. Salivary gland function was assessed by measuring the flow rate after muscarinic stimulation of the submandibular glands perfused ex vivo. Additional analysis included histological and immunohistochemical evaluations, blood tests, and real-time PCR to investigate gland morphology, immune cell profiles, gene expression, and inflammation/aging markers.</div></div><div><h3>Results</h3><div>Long-term Ninjin'yoeito administration significantly increased salivary secretion after stimulation and decreased the number of vacuoles in acinar cells as compared to the controls. Ninjin'yoeito improved the plasma albumin levels and increased the lymphocyte ratio in the white blood cell count. Furthermore, real-time PCR revealed decreased levels of inflammatory cytokine and senescence-associated genes. No significant changes were detected in the expression or localization of the main salivary secretion-related channels, such as transmembrane protein 16A and aquaporin 5.</div></div><div><h3>Conclusions</h3><div>Ninjin'yoeito enhances salivary secretion from the submandibular glands of aged mice by improving nutritional status, modulating immune function, suppressing chronic inflammation, and attenuating cellular aging. These results suggest that Ninjin'yoeito has potential applications as a complementary therapy for age-related dry mouth.</div></div>","PeriodicalId":45851,"journal":{"name":"Journal of Oral Biosciences","volume":"68 1","pages":"Article 100739"},"PeriodicalIF":2.3,"publicationDate":"2026-01-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145976126","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-12DOI: 10.1016/j.job.2026.100736
Naoyuki Mitarai , Chihiro Nakatomi , Chia-Chien Hsu , Hiroka Yasuda , Mari Fukuzaki , Tomoki Shimada , Ai Orimoto , Chiaki Kitamura , Kentaro Ono
Objectives
Although pharyngolaryngeal injury impairs water-evoked swallowing, its influence on nociceptive swallowing reflexes remains unclear. Therefore, this study aimed to determine the effects of pharyngolaryngeal injury on nociceptive receptor-dependent swallowing reflexes.
Methods
Adult male rats underwent endoscopic evaluation of swallowing under anesthesia. The number, latency, and swallowing intervals were quantified. Distilled water, capsaicin (TRPV1 agonist), and allyl isothiocyanate (AITC, TRPA1 agonist) were infused onto the pharyngolaryngeal surface via a syringe pump at a constant flow for 10 s. Blue-dyed water confirmed gastric delivery. Pharyngolaryngeal injury was induced by topical application of 10 % acetic acid. The nodose and petrosal/jugular-complex ganglia were analyzed for TRPV1 and TRPA1 gene expression using quantitative RT-PCR.
Results
Gastric delivery from the pharyngolaryngeal region was confirmed with blue-dyed water, although aspiration was observed, indicating the reliability of the endoscopic swallowing evaluation. Pharyngolaryngeal injury significantly suppressed water-evoked swallowing, consistent with previous findings. Contrastingly, capsaicin robustly evoked swallowing post-injury, with attenuated desensitization upon repeated stimulation. AITC did not change the number of swallows but prolonged latency. Quantitative RT-PCR revealed significant upregulation of TRPV1 gene expression in the petrosal/jugular-complex ganglia, whereas TRPA1 expression remained unchanged in both ganglia.
Conclusion
Nociceptive stimuli elicit swallowing via mechanisms that are distinct from those activated by water. Reduced desensitization of capsaicin-evoked swallowing after pharyngolaryngeal injury suggests facilitation through TRPV1-dependent pathways, potentially driven by increased TRPV1 expression in petrosal/jugular-complex afferents.
{"title":"Effects of pharyngolaryngeal injury on nociceptive swallowing reflexes","authors":"Naoyuki Mitarai , Chihiro Nakatomi , Chia-Chien Hsu , Hiroka Yasuda , Mari Fukuzaki , Tomoki Shimada , Ai Orimoto , Chiaki Kitamura , Kentaro Ono","doi":"10.1016/j.job.2026.100736","DOIUrl":"10.1016/j.job.2026.100736","url":null,"abstract":"<div><h3>Objectives</h3><div>Although pharyngolaryngeal injury impairs water-evoked swallowing, its influence on nociceptive swallowing reflexes remains unclear. Therefore, this study aimed to determine the effects of pharyngolaryngeal injury on nociceptive receptor-dependent swallowing reflexes.</div></div><div><h3>Methods</h3><div>Adult male rats underwent endoscopic evaluation of swallowing under anesthesia. The number, latency, and swallowing intervals were quantified. Distilled water, capsaicin (TRPV1 agonist), and allyl isothiocyanate (AITC, TRPA1 agonist) were infused onto the pharyngolaryngeal surface via a syringe pump at a constant flow for 10 s. Blue-dyed water confirmed gastric delivery. Pharyngolaryngeal injury was induced by topical application of 10 % acetic acid. The nodose and petrosal/jugular-complex ganglia were analyzed for TRPV1 and TRPA1 gene expression using quantitative RT-PCR.</div></div><div><h3>Results</h3><div>Gastric delivery from the pharyngolaryngeal region was confirmed with blue-dyed water, although aspiration was observed, indicating the reliability of the endoscopic swallowing evaluation. Pharyngolaryngeal injury significantly suppressed water-evoked swallowing, consistent with previous findings. Contrastingly, capsaicin robustly evoked swallowing post-injury, with attenuated desensitization upon repeated stimulation. AITC did not change the number of swallows but prolonged latency. Quantitative RT-PCR revealed significant upregulation of TRPV1 gene expression in the petrosal/jugular-complex ganglia, whereas TRPA1 expression remained unchanged in both ganglia.</div></div><div><h3>Conclusion</h3><div>Nociceptive stimuli elicit swallowing via mechanisms that are distinct from those activated by water. Reduced desensitization of capsaicin-evoked swallowing after pharyngolaryngeal injury suggests facilitation through TRPV1-dependent pathways, potentially driven by increased TRPV1 expression in petrosal/jugular-complex afferents.</div></div>","PeriodicalId":45851,"journal":{"name":"Journal of Oral Biosciences","volume":"68 1","pages":"Article 100736"},"PeriodicalIF":2.3,"publicationDate":"2026-01-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145976192","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Periostin is an extracellular matrix protein that is highly expressed in hard tissue-associated regions, including the periosteum and periodontal ligament, where it is essential for bone formation and craniofacial development. In this study, the aim was to clarify its functions in skeletal maintenance during aging by comparing periostin-deficient (Postn−/−) and wild-type (Postn+/+) mice.
Methods
Comprehensive micro-computed tomography analyses were performed on Postn−/− and Postn+/+ mice at 18 and 62 weeks of age to assess age-related changes in craniofacial and femoral morphologies.
Results
At 18 weeks, there were craniofacial abnormalities in Postn−/− mice, including the absence of the foramen-like structure on the ventral mandible, increased incisor curvature, and a reduction in pulp cavity size. With age, these alterations became more pronounced, with significant differences in the angulation of the upper incisors, vertical height of the mandibular body, progressive enamel defects that expanded across the surface of the maxillary and mandibular incisors, and further narrowing of the pulp cavity. Additionally, in Postn+/+ mice, there was an expected age-dependent reduction in bone volume fraction in the femur, but not in Postn−/− mice at 18 or 62 weeks, suggesting suppression of age-related trabecular deterioration. In contrast, aged Postn−/− mice had ectopic bone formation in the distal femur and around the knee joint, indicating aberrant ossification in the absence of periostin.
Conclusions
Periostin deficiency induces alterations in craniofacial, dental, and skeletal morphologies during aging. Periostin is also required for normal trabecular remodeling. These findings underscore its essential role in skeletal integrity and provide insights into bone biology and aging-related tissue remodeling.
{"title":"Periostin deficiency leads to age-dependent craniofacial, dental, and skeletal morphological alterations in mice","authors":"Akihiko Fujita , Masahiro Chatani , Yurie Sato , Natsuhiro Takahashi , Yuki Azetsu , Akiko Karakawa , Nobuhiro Sakai , Shugo Haga , Haruhisa Nakano , Masamichi Takami","doi":"10.1016/j.job.2026.100734","DOIUrl":"10.1016/j.job.2026.100734","url":null,"abstract":"<div><h3>Objectives</h3><div>Periostin is an extracellular matrix protein that is highly expressed in hard tissue-associated regions, including the periosteum and periodontal ligament, where it is essential for bone formation and craniofacial development. In this study, the aim was to clarify its functions in skeletal maintenance during aging by comparing periostin-deficient (<em>Postn</em><sup>−/−</sup>) and wild-type (<em>Postn</em><sup>+/+</sup>) mice.</div></div><div><h3>Methods</h3><div>Comprehensive micro-computed tomography analyses were performed on <em>Postn</em><sup>−/−</sup> and <em>Postn</em><sup>+/+</sup> mice at 18 and 62 weeks of age to assess age-related changes in craniofacial and femoral morphologies.</div></div><div><h3>Results</h3><div>At 18 weeks, there were craniofacial abnormalities in <em>Postn</em><sup>−/−</sup> mice, including the absence of the foramen-like structure on the ventral mandible, increased incisor curvature, and a reduction in pulp cavity size. With age, these alterations became more pronounced, with significant differences in the angulation of the upper incisors, vertical height of the mandibular body, progressive enamel defects that expanded across the surface of the maxillary and mandibular incisors, and further narrowing of the pulp cavity. Additionally, in <em>Postn</em><sup>+/+</sup> mice, there was an expected age-dependent reduction in bone volume fraction in the femur, but not in <em>Postn</em><sup>−/−</sup> mice at 18 or 62 weeks, suggesting suppression of age-related trabecular deterioration. In contrast, aged <em>Postn</em><sup>−/−</sup> mice had ectopic bone formation in the distal femur and around the knee joint, indicating aberrant ossification in the absence of periostin.</div></div><div><h3>Conclusions</h3><div>Periostin deficiency induces alterations in craniofacial, dental, and skeletal morphologies during aging. Periostin is also required for normal trabecular remodeling. These findings underscore its essential role in skeletal integrity and provide insights into bone biology and aging-related tissue remodeling.</div></div>","PeriodicalId":45851,"journal":{"name":"Journal of Oral Biosciences","volume":"68 1","pages":"Article 100734"},"PeriodicalIF":2.3,"publicationDate":"2026-01-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145976193","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-10DOI: 10.1016/j.job.2026.100737
Yohann Flottes , Elisabeth Dursun
Objective
To investigate the dose-dependent effects of Bisphenol A-glycidyl methacrylate (Bis-GMA), a monomer released from dental resin composites, on the proteome of human gingival fibroblasts.
Methods
Primary human gingival fibroblasts were exposed to different concentrations of Bis-GMA (0.002 mM, 0.02 mM, and 0.2 mM) for 72 h, and protein extracts were analyzed using two-dimensional electrophoresis coupled with liquid chromatography-tandem mass spectrometry. Differentially abundant proteins were identified and functionally classified using STRING and Gene Ontology enrichment analysis.
Results
A total of 45 proteins were differentially abundant across the three exposure conditions. At 0.002 mM, there were indicators of an adaptive response, with upregulation of proteins related to mitochondrial function and cytoskeletal integrity. At 0.02 mM, there were indicators of cellular stress, including alterations in nuclear transport, cytoskeletal disorganization, and energy metabolism. At 0.2 mM, there was a marked shift towards protein underabundance, which may indicate disruption of proteostasis, oxidative stress, and endoplasmic reticulum stress. The proteomic profiles reflect the progressive loss of homeostasis, culminating in a cellular state compatible with apoptosis or senescence.
Conclusions
These findings raise concerns regarding low-level exposure to Bis-GMA, which is often encountered after dental restorations. These results support the necessity to adopt clinical procedures that minimize release of monomers and to develop safer monomers.
{"title":"Proteomic analysis of fibroblasts exposed to Bis-GMA","authors":"Yohann Flottes , Elisabeth Dursun","doi":"10.1016/j.job.2026.100737","DOIUrl":"10.1016/j.job.2026.100737","url":null,"abstract":"<div><h3>Objective</h3><div>To investigate the dose-dependent effects of Bisphenol A-glycidyl methacrylate (Bis-GMA), a monomer released from dental resin composites, on the proteome of human gingival fibroblasts.</div></div><div><h3>Methods</h3><div>Primary human gingival fibroblasts were exposed to different concentrations of Bis-GMA (0.002 mM, 0.02 mM, and 0.2 mM) for 72 h, and protein extracts were analyzed using two-dimensional electrophoresis coupled with liquid chromatography-tandem mass spectrometry. Differentially abundant proteins were identified and functionally classified using STRING and Gene Ontology enrichment analysis.</div></div><div><h3>Results</h3><div>A total of 45 proteins were differentially abundant across the three exposure conditions. At 0.002 mM, there were indicators of an adaptive response, with upregulation of proteins related to mitochondrial function and cytoskeletal integrity. At 0.02 mM, there were indicators of cellular stress, including alterations in nuclear transport, cytoskeletal disorganization, and energy metabolism. At 0.2 mM, there was a marked shift towards protein underabundance, which may indicate disruption of proteostasis, oxidative stress, and endoplasmic reticulum stress. The proteomic profiles reflect the progressive loss of homeostasis, culminating in a cellular state compatible with apoptosis or senescence.</div></div><div><h3>Conclusions</h3><div>These findings raise concerns regarding low-level exposure to Bis-GMA, which is often encountered after dental restorations. These results support the necessity to adopt clinical procedures that minimize release of monomers and to develop safer monomers.</div></div>","PeriodicalId":45851,"journal":{"name":"Journal of Oral Biosciences","volume":"68 1","pages":"Article 100737"},"PeriodicalIF":2.3,"publicationDate":"2026-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145976188","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Secreted modular calcium-binding proteins (SMOCs), including SMOC1 and SMOC2, are secreted protein acidic and rich in cysteine family matricellular proteins that regulate cell–matrix interactions and growth factor signaling across development and disease. Recent studies positioned SMOC1 and SMOC2 as direct downstream targets of Runt-related transcription factor 2 (Runx2), the master transcription factor for osteoblast differentiation, and revealed dose-dependent modulation of bone morphogenetic protein 2 (BMP2) signaling by SMOC2.
Highlight
SMOC1/2 exhibit distinct yet complementary roles in skeletal morphogenesis, extracellular matrix assembly, and mineralization. At lower concentrations, SMOC2 enhances BMP2 signaling, whereas at higher concentrations, it inhibits receptor engagement, explaining context-dependent effects on osteogenesis. Genetic evidence from mouse models and human variants (e.g., SMOC2 Leu359Arg) links disrupted SMOC–ECM interactions to growth-plate defects, delayed ossification, and craniofacial phenotypes. Beyond the skeleton, SMOC proteins participate in angiogenesis, ocular development, kidney and testis biology, and platelet thrombin responses.
Conclusion
The Runx2–SMOC axis provides a mechanistic bridge between transcriptional programs and matricellular control of BMP2 signaling and ECM dynamics. Clarifying isoform-specific functions and ECM anchoring mechanisms will inform therapeutic strategies for skeletal disorders and broader pathologies in which SMOC proteins are implicated.
{"title":"The Runx2-SMOC axis in skeletal development: Expanding roles of Smoc1 and Smoc2 in development and disease","authors":"Yoshifumi Takahata , Mitsuki Urushizaki , Kanta Wakamori , Miho Kakiuchi , Tomohiko Murakami , Maiko Omi-Sugihara , Kenji Hata , Riko Nishimura","doi":"10.1016/j.job.2026.100738","DOIUrl":"10.1016/j.job.2026.100738","url":null,"abstract":"<div><h3>Background</h3><div>Secreted modular calcium-binding proteins (SMOCs), including SMOC1 and SMOC2, are secreted protein acidic and rich in cysteine family matricellular proteins that regulate cell–matrix interactions and growth factor signaling across development and disease. Recent studies positioned SMOC1 and SMOC2 as direct downstream targets of Runt-related transcription factor 2 (Runx2), the master transcription factor for osteoblast differentiation, and revealed dose-dependent modulation of bone morphogenetic protein 2 (BMP2) signaling by SMOC2.</div></div><div><h3>Highlight</h3><div>SMOC1/2 exhibit distinct yet complementary roles in skeletal morphogenesis, extracellular matrix assembly, and mineralization. At lower concentrations, SMOC2 enhances BMP2 signaling, whereas at higher concentrations, it inhibits receptor engagement, explaining context-dependent effects on osteogenesis. Genetic evidence from mouse models and human variants (e.g., SMOC2 Leu359Arg) links disrupted SMOC–ECM interactions to growth-plate defects, delayed ossification, and craniofacial phenotypes. Beyond the skeleton, SMOC proteins participate in angiogenesis, ocular development, kidney and testis biology, and platelet thrombin responses.</div></div><div><h3>Conclusion</h3><div>The Runx2–SMOC axis provides a mechanistic bridge between transcriptional programs and matricellular control of BMP2 signaling and ECM dynamics. Clarifying isoform-specific functions and ECM anchoring mechanisms will inform therapeutic strategies for skeletal disorders and broader pathologies in which SMOC proteins are implicated.</div></div>","PeriodicalId":45851,"journal":{"name":"Journal of Oral Biosciences","volume":"68 1","pages":"Article 100738"},"PeriodicalIF":2.3,"publicationDate":"2026-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145938326","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The interaction between osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) regulates osteoclast differentiation and bone resorption. Although their binding affinity has been analyzed using purified proteins in biochemical assays, including surface plasmon resonance, measurements under living-cell conditions remain limited. In this study, a bioluminescence-based assay using Gaussia luciferase (GLase/GLuc) was adapted to quantify the binding affinity between OPG and RANKL in living cells.
Methods
OPG and OPG–GLase fusion proteins (OPG-GLase) were purified from conditioned media of 293T cells. Osteoclast differentiation assays were performed to determine whether OPG-GLase retained the biological activity of OPG. Purified proteins were then incubated with CHO–K1 cells expressing RANKL, and saturation binding assays were performed to calculate dissociation constants (Kd). The same assay was performed using pgsD-677 cells, a CHO–K1 mutant line that lacks heparan sulfate proteoglycan synthesis.
Results
Immunofluorescence and blocking experiments confirmed that OPG-GLase specifically binds to RANKL on the surface of CHO–K1 cells. OPG-GLase retained the ability to inhibit osteoclast differentiation to a similar extent as OPG. Saturation binding assays and non-linear regression analysis yielded a Kd of 11.0 nM in CHO–K1 cells. A parallel assay using pgsD-677 cells produced a comparable Kd of 12.6 nM, indicating that heparan sulfate proteoglycans contributed minimally to OPG–RANKL affinity under the experimental conditions used in this study.
Conclusions
This bioluminescence-based method provides a simple and accessible platform for evaluating interactions between soluble and membrane-bound proteins in living cells, with potential application in the development of drugs targeting the RANKL–RANK–OPG axis.
{"title":"Determination of the binding affinity between osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) utilizing Gaussia luciferase in living cells","authors":"Yuto Shibata , Hisayo Nishida-Fukuda , Hironao Nakayama , Yudai Izumi , Reina Suzuki , Satoru Yokawa , Takuma Sato , Tadahide Furuno , Mamoru Matsubara , Ken Miyazawa , Shinji Fukuda , Takahiro Suzuki","doi":"10.1016/j.job.2025.100727","DOIUrl":"10.1016/j.job.2025.100727","url":null,"abstract":"<div><h3>Objectives</h3><div>The interaction between osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) regulates osteoclast differentiation and bone resorption. Although their binding affinity has been analyzed using purified proteins in biochemical assays, including surface plasmon resonance, measurements under living-cell conditions remain limited. In this study, a bioluminescence-based assay using <em>Gaussia</em> luciferase (GLase/GLuc) was adapted to quantify the binding affinity between OPG and RANKL in living cells.</div></div><div><h3>Methods</h3><div>OPG and OPG–GLase fusion proteins (OPG-GLase) were purified from conditioned media of 293T cells. Osteoclast differentiation assays were performed to determine whether OPG-GLase retained the biological activity of OPG. Purified proteins were then incubated with CHO–K1 cells expressing RANKL, and saturation binding assays were performed to calculate dissociation constants (Kd). The same assay was performed using pgsD-677 cells, a CHO–K1 mutant line that lacks heparan sulfate proteoglycan synthesis.</div></div><div><h3>Results</h3><div>Immunofluorescence and blocking experiments confirmed that OPG-GLase specifically binds to RANKL on the surface of CHO–K1 cells. OPG-GLase retained the ability to inhibit osteoclast differentiation to a similar extent as OPG. Saturation binding assays and non-linear regression analysis yielded a Kd of 11.0 nM in CHO–K1 cells. A parallel assay using pgsD-677 cells produced a comparable Kd of 12.6 nM, indicating that heparan sulfate proteoglycans contributed minimally to OPG–RANKL affinity under the experimental conditions used in this study.</div></div><div><h3>Conclusions</h3><div>This bioluminescence-based method provides a simple and accessible platform for evaluating interactions between soluble and membrane-bound proteins in living cells, with potential application in the development of drugs targeting the RANKL–RANK–OPG axis.</div></div>","PeriodicalId":45851,"journal":{"name":"Journal of Oral Biosciences","volume":"68 1","pages":"Article 100727"},"PeriodicalIF":2.3,"publicationDate":"2026-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145938461","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-07DOI: 10.1016/j.job.2025.100730
Yi Lu , Yuya Su , Yuto Nagatomo , Chenyang Zhang , Shigenori Nagai , Naoto Nishii , Hiroyuki Harada , Peiya Lin , Sayaka Katagiri , Miyuki Azuma
Objectives
Aging induces senescence-related immune changes that affect antitumor immunity and treatment efficacy. Immune checkpoint inhibitors (ICIs) targeting programmed cell death-1 (PD-1) are approved for recurrent and metastatic head and neck squamous cell carcinoma (SCC). We aimed to investigate how aging alters the tumor immune microenvironment and modulates ICI efficacy in a murine SCC model.
Methods
We analyzed SCCVII tumor growth and phenotypes of tumor-draining lymph node (TDLN) cells and tumor-infiltrating leukocytes in young, middle-aged, and aged mice. We examined the effects of antibodies against PD-1 ligand-1 (PD-L1) in young and aged mice.
Results
Tumor growth accelerated with age and was accompanied with increased CD206+ M2-like tumor-associated macrophage (TAM) accumulation. In intact LNs and TDLNs, CD8+ T cells (CD8T), interferon (IFN)-γ, and PD-1 expression increased with age. The CD8T phenotype in the tumor microenvironment (TME) differed between young and aged mice; however, CD8T and regulatory T cells preferentially recruited to the TME across all age groups. PD-1 expression was significantly impaired in TME of aged mice. PD-1−IFN-γ− CD8T predominated in aged mice, whereas PD-1+IFN-γ− CD8T predominated in young mice. Anti-PD-L1 treatment in aged mice enhanced antitumor immunity, but preferentially increased PD-1+ CD8T in both TDLNs and TME.
Conclusions
In the SCCVII model, aging impaired the antitumor immune responses, associated with early recruitment of M2-like TAMs. PD-1 expression in aged TME CD8T was impaired, but the PD-L1 blockade increased PD-1 expression, suggesting that the site of action for PD-L1 blockade differs between young and aged mice.
{"title":"Impaired PD-1 expression in tumor-infiltrating senescent CD8+ T cells is reversed by PD-L1 blockade in a murine squamous cell carcinoma model","authors":"Yi Lu , Yuya Su , Yuto Nagatomo , Chenyang Zhang , Shigenori Nagai , Naoto Nishii , Hiroyuki Harada , Peiya Lin , Sayaka Katagiri , Miyuki Azuma","doi":"10.1016/j.job.2025.100730","DOIUrl":"10.1016/j.job.2025.100730","url":null,"abstract":"<div><h3>Objectives</h3><div>Aging induces senescence-related immune changes that affect antitumor immunity and treatment efficacy. Immune checkpoint inhibitors (ICIs) targeting programmed cell death-1 (PD-1) are approved for recurrent and metastatic head and neck squamous cell carcinoma (SCC). We aimed to investigate how aging alters the tumor immune microenvironment and modulates ICI efficacy in a murine SCC model.</div></div><div><h3>Methods</h3><div>We analyzed SCCVII tumor growth and phenotypes of tumor-draining lymph node (TDLN) cells and tumor-infiltrating leukocytes in young, middle-aged, and aged mice. We examined the effects of antibodies against PD-1 ligand-1 (PD-L1) in young and aged mice.</div></div><div><h3>Results</h3><div>Tumor growth accelerated with age and was accompanied with increased CD206<sup>+</sup> M2-like tumor-associated macrophage (TAM) accumulation. In intact LNs and TDLNs, CD8<sup>+</sup> T cells (CD8T), interferon (IFN)-γ, and PD-1 expression increased with age. The CD8T phenotype in the tumor microenvironment (TME) differed between young and aged mice; however, CD8T and regulatory T cells preferentially recruited to the TME across all age groups. PD-1 expression was significantly impaired in TME of aged mice. PD-1<sup>−</sup>IFN-γ<sup>−</sup> CD8T predominated in aged mice, whereas PD-1<sup>+</sup>IFN-γ<sup>−</sup> CD8T predominated in young mice. Anti-PD-L1 treatment in aged mice enhanced antitumor immunity, but preferentially increased PD-1<sup>+</sup> CD8T in both TDLNs and TME.</div></div><div><h3>Conclusions</h3><div>In the SCCVII model, aging impaired the antitumor immune responses, associated with early recruitment of M2-like TAMs. PD-1 expression in aged TME CD8T was impaired, but the PD-L1 blockade increased PD-1 expression, suggesting that the site of action for PD-L1 blockade differs between young and aged mice.</div></div>","PeriodicalId":45851,"journal":{"name":"Journal of Oral Biosciences","volume":"68 1","pages":"Article 100730"},"PeriodicalIF":2.3,"publicationDate":"2026-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145938310","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Serum is an essential supplement of basal cell culture media, providing growth factors, adhesion molecules, hormones, lipids, and minerals that support cell survival and function. This study was conducted to investigate the effects of ultracentrifugation-separated fractions of fetal bovine serum (FBS) on the proliferation and differentiation of dental epithelial cells with the aim of identifying the key serum-derived factors that regulate cellular activities.
Methods
FBS was fractionated by ultracentrifugation at 100,000×g for 18 h at 4 °C into three layers, designated as the FBS-1st (top), -2nd (middle), and 3rd (bottom) fractions. Each fraction was added at 10 % (v/v) to the culture medium of rat dental epithelial HAT-7 cells. Cell proliferation was assessed by cell counting and 5-bromo-2′-deoxyuridine incorporation, and differentiation was evaluated by ameloblastin immunostaining. The involvement of lactoferrin and its receptors was examined using recombinant protein stimulation, reverse transcription polymerase chain reaction, small interfering RNA knockdown, and western blotting.
Results
FBS-3rd significantly promoted cell proliferation, whereas FBS-1st and -2nd inhibited it. Ameloblastin staining revealed no significant differences between FBS-1st and the control; however, it was suppressed in the FBS-2nd and -3rd. Proteomic analysis identified lactoferrin as the most abundant protein in FBS-3rd, and exogenous lactoferrin enhanced HAT-7 cell proliferation. The lactoferrin receptor, low-density lipoprotein receptor-related protein 1 (Lrp1), was expressed in HAT-7 cells, and Lrp1 knockdown stopped lactoferrin-induced proliferation. Lactoferrin induced phosphorylation of extracellular signal-regulated kinase 1/2, but not Akt serine/threonine kinase, and mitogen-activated protein kinase kinase inhibition with U0126 suppressed the proliferative effect of lactoferrin.
Conclusion
Lactoferrin, which was enriched in the FBS-3rd fraction, may contribute to the proliferation of dental epithelial cells via the Lrp1-ERK signaling pathway.
{"title":"Ultracentrifuged serum fractions reveal promotion of dental epithelial proliferation by lactoferrin","authors":"Yoshihito Yamakawa , Kimiko Yamaguchi-Ueda , Asuna Sugimoto , Yuki Akazawa , Tomokazu Hasegawa , Yumiko Nakashima , Rika Kurogoushi , Manami Tanaka , Muhammad Dhiaulfikri Nauval Hadiana , Nodoka Ato , Akihito Yamamoto , Tomonori Iwasaki , Tsutomu Iwamoto","doi":"10.1016/j.job.2025.100728","DOIUrl":"10.1016/j.job.2025.100728","url":null,"abstract":"<div><h3>Objectives</h3><div>Serum is an essential supplement of basal cell culture media, providing growth factors, adhesion molecules, hormones, lipids, and minerals that support cell survival and function. This study was conducted to investigate the effects of ultracentrifugation-separated fractions of fetal bovine serum (FBS) on the proliferation and differentiation of dental epithelial cells with the aim of identifying the key serum-derived factors that regulate cellular activities.</div></div><div><h3>Methods</h3><div>FBS was fractionated by ultracentrifugation at 100,000×<em>g</em> for 18 h at 4 °C into three layers, designated as the FBS-1st (top), -2nd (middle), and 3rd (bottom) fractions. Each fraction was added at 10 % (v/v) to the culture medium of rat dental epithelial HAT-7 cells. Cell proliferation was assessed by cell counting and 5-bromo-2′-deoxyuridine incorporation, and differentiation was evaluated by ameloblastin immunostaining. The involvement of lactoferrin and its receptors was examined using recombinant protein stimulation, reverse transcription polymerase chain reaction, small interfering RNA knockdown, and western blotting.</div></div><div><h3>Results</h3><div>FBS-3rd significantly promoted cell proliferation, whereas FBS-1st and -2nd inhibited it. Ameloblastin staining revealed no significant differences between FBS-1st and the control; however, it was suppressed in the FBS-2nd and -3rd. Proteomic analysis identified lactoferrin as the most abundant protein in FBS-3rd, and exogenous lactoferrin enhanced HAT-7 cell proliferation. The lactoferrin receptor, low-density lipoprotein receptor-related protein 1 (Lrp1), was expressed in HAT-7 cells, and <em>Lrp1</em> knockdown stopped lactoferrin-induced proliferation. Lactoferrin induced phosphorylation of extracellular signal-regulated kinase 1/2, but not Akt serine/threonine kinase, and mitogen-activated protein kinase kinase inhibition with U0126 suppressed the proliferative effect of lactoferrin.</div></div><div><h3>Conclusion</h3><div>Lactoferrin, which was enriched in the FBS-3rd fraction, may contribute to the proliferation of dental epithelial cells via the Lrp1-ERK signaling pathway.</div></div>","PeriodicalId":45851,"journal":{"name":"Journal of Oral Biosciences","volume":"68 1","pages":"Article 100728"},"PeriodicalIF":2.3,"publicationDate":"2026-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145938325","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Organic acids contribute significantly to the flavor of fermented foods by imparting sourness. Although mice generally avoid sour taste, previous studies have reported greater consumption of l-lactic acid than its d-enantiomer, suggesting enantiomer-specific recognition. This behavior is hypothesized to involve TAS1Rs, which consists of sweet/umami receptors. However, it remains unclear whether TAS1Rs additionally contribute to the recognition of other chiral organic acids. This study aimed to evaluate the role of TAS1Rs, particularly TAS1R3, in the modulation of enantiomer-dependent behavioral responses to organic acids in mice.
Methods
Behavioral responses were evaluated using 48-h and 1-h 2-bottle tests. Binding of organic acids to TAS1Rs was investigated by differential scanning fluorimetry (DSF) with the ligand-binding domain (LBD) of medaka Tas1r2a/Tas1r3.
Results
Wild-type mice consumed more d-malic acid than l-malic acid in the 48-h test, whereas Tas1r3-KO mice showed no such difference. This pattern was not observed in the short-term 1-h test, which minimized the contribution of post-ingestion and learned effects. DSF analysis revealed no binding of any of the tested organic acids to the LBD of medaka Tas1r2a/Tas1r3.
Conclusions
Organic acids may elicit TAS1R3-dependent post-ingestion signals that contribute to enantiomer-selective consumption in mice. Electrostatic interactions and hydrogen-bonding networks within the orthosteric pocket of TAS1Rs may account for the differences in binding affinity to the LBD of medaka Tas1r2a/Tas1r3 between organic acids and L-alanine, a known ligand.
目的有机酸通过赋予发酵食品酸味对其风味有重要贡献。虽然老鼠一般都不吃酸味，但之前的研究表明，它们对l-乳酸的摄入比d-对映体要多，这表明它们对对映体有特异性识别。这种行为被认为与TAS1Rs有关，TAS1Rs由甜/鲜味受体组成。然而，目前尚不清楚TAS1Rs是否也有助于识别其他手性有机酸。本研究旨在评估TAS1Rs，特别是TAS1R3在小鼠对有机酸依赖的对映体行为反应的调节中的作用。方法采用48 h和1 h 2瓶试验评价行为反应。利用medaka Tas1r2a/Tas1r3的配体结合结构域（LBD），用差示扫描荧光法（DSF）研究了有机酸与TAS1Rs的结合。结果野生型小鼠在48 h内摄入的d-苹果酸多于l-苹果酸，而Tas1r3-KO小鼠则无此差异。这种模式在短期1-h测试中没有观察到，这最小化了摄入后和学习效应的贡献。DSF分析显示，所有被测有机酸均未与medaka Tas1r2a/Tas1r3的LBD结合。结论有机酸可能引发tas1r3依赖性食后信号，促进小鼠对映体的选择性消耗。静电相互作用和TAS1Rs正位口袋内的氢键网络可能解释了有机酸和l -丙氨酸（一种已知的配体）与medaka Tas1r2a/Tas1r3的LBD结合亲和力的差异。
{"title":"Insights into the taste of organic acids via TAS1Rs","authors":"Yuko Yamase , Katsuki Takebe , Kengo Horie , Yoshihiro Mitoh , Atsuko Yamashita , Ryusuke Yoshida","doi":"10.1016/j.job.2025.100731","DOIUrl":"10.1016/j.job.2025.100731","url":null,"abstract":"<div><h3>Objectives</h3><div>Organic acids contribute significantly to the flavor of fermented foods by imparting sourness. Although mice generally avoid sour taste, previous studies have reported greater consumption of <span>l</span>-lactic acid than its <span>d</span>-enantiomer, suggesting enantiomer-specific recognition. This behavior is hypothesized to involve TAS1Rs, which consists of sweet/umami receptors. However, it remains unclear whether TAS1Rs additionally contribute to the recognition of other chiral organic acids. This study aimed to evaluate the role of TAS1Rs, particularly TAS1R3, in the modulation of enantiomer-dependent behavioral responses to organic acids in mice.</div></div><div><h3>Methods</h3><div>Behavioral responses were evaluated using 48-h and 1-h 2-bottle tests. Binding of organic acids to TAS1Rs was investigated by differential scanning fluorimetry (DSF) with the ligand-binding domain (LBD) of medaka Tas1r2a/Tas1r3.</div></div><div><h3>Results</h3><div>Wild-type mice consumed more <span>d</span>-malic acid than <span>l</span>-malic acid in the 48-h test, whereas <em>Tas1r3</em>-KO mice showed no such difference. This pattern was not observed in the short-term 1-h test, which minimized the contribution of post-ingestion and learned effects. DSF analysis revealed no binding of any of the tested organic acids to the LBD of medaka Tas1r2a/Tas1r3.</div></div><div><h3>Conclusions</h3><div>Organic acids may elicit TAS1R3-dependent post-ingestion signals that contribute to enantiomer-selective consumption in mice. Electrostatic interactions and hydrogen-bonding networks within the orthosteric pocket of TAS1Rs may account for the differences in binding affinity to the LBD of medaka Tas1r2a/Tas1r3 between organic acids and L-alanine, a known ligand.</div></div>","PeriodicalId":45851,"journal":{"name":"Journal of Oral Biosciences","volume":"68 1","pages":"Article 100731"},"PeriodicalIF":2.3,"publicationDate":"2026-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145938324","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Many bacteriome studies employing next-generation sequencing (NGS) have focused on the partial sequencing of the 16S rRNA gene (rDNA). This limits both species-level resolution and quantitative interpretation of bacterial abundance, which have been addressed in this study.
Methods
Nanopore sequencing was used to analyze nearly the entire 16S rDNA sequence for species-level classification, and quantitative PCR for bacterial quantification. This approach was applied in a small-cohort feasibility study to investigate oral bacterial changes in orthodontic patients. Saliva samples were collected from 10 patients before and 1, 3, and 6 months after the initiation of orthodontic treatment.
Results
Dental caries was not detected during the study, and periodontal pockets ≥4 mm were rarely observed. However, all patients exhibited changes in bleeding on probing (BOP), which is indicative of early stage gingivitis. Although the compositional analysis did not reveal any significant association between specific bacterial species and BOP changes, the quantitative analysis showed a positive correlation between BOP changes and two bacterial species in the phyla Candidatus Saccharibacteria: Candidatus Saccharimonas aalborgensis and Candidatus Saccharibacteria bacterium oral taxon TM7x. No correlation was observed with representative periodontal disease-associated bacteria.
Conclusions
These findings support the feasibility of species-level and quantitative bacteriome analysis in a small cohort and highlight the bacterial species potentially linked to early stage gingivitis.
{"title":"Broad-range 16S rDNA sequencing and quantitative bacteriome profiling: a small-cohort feasibility study in orthodontic patients with early gingivitis","authors":"Tadaharu Yokogawa , Keiji Nagano , Hiroshi Miyakawa , Mari Fujita , Chen-Hsuan Chiu , Masahiro Iijima","doi":"10.1016/j.job.2025.100732","DOIUrl":"10.1016/j.job.2025.100732","url":null,"abstract":"<div><h3>Objectives</h3><div>Many bacteriome studies employing next-generation sequencing (NGS) have focused on the partial sequencing of the 16S rRNA gene (rDNA). This limits both species-level resolution and quantitative interpretation of bacterial abundance, which have been addressed in this study.</div></div><div><h3>Methods</h3><div>Nanopore sequencing was used to analyze nearly the entire 16S rDNA sequence for species-level classification, and quantitative PCR for bacterial quantification. This approach was applied in a small-cohort feasibility study to investigate oral bacterial changes in orthodontic patients. Saliva samples were collected from 10 patients before and 1, 3, and 6 months after the initiation of orthodontic treatment.</div></div><div><h3>Results</h3><div>Dental caries was not detected during the study, and periodontal pockets ≥4 mm were rarely observed. However, all patients exhibited changes in bleeding on probing (BOP), which is indicative of early stage gingivitis. Although the compositional analysis did not reveal any significant association between specific bacterial species and BOP changes, the quantitative analysis showed a positive correlation between BOP changes and two bacterial species in the phyla Candidatus <em>Saccharibacteria</em>: Candidatus <em>Saccharimonas aalborgensis</em> and Candidatus <em>Saccharibacteria</em> bacterium oral taxon TM7x. No correlation was observed with representative periodontal disease-associated bacteria.</div></div><div><h3>Conclusions</h3><div>These findings support the feasibility of species-level and quantitative bacteriome analysis in a small cohort and highlight the bacterial species potentially linked to early stage gingivitis.</div></div>","PeriodicalId":45851,"journal":{"name":"Journal of Oral Biosciences","volume":"68 1","pages":"Article 100732"},"PeriodicalIF":2.3,"publicationDate":"2025-12-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145883337","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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