Protein kinase C delta enhances the diagnostic performance of hepatocellular carcinoma.

BACKGROUND: The conventional markers for hepatocellular carcinoma (HCC), α-fetoprotein (AFP) and des-γ-carboxy prothrombin (DCP), have several limitations; both have low sensitivity in patients with early-stage HCC; low sensitivity for AFP with HCC after eliminating hepatitis C virus (HCV); low specificity for DCP in patients with non-viral HCC, which is increasing worldwide; low specificity for AFP in patients with liver injury; and low specificity for DCP in patients treated with warfarin. To overcome these issues, the identification of novel biomarkers is an unmet need. OBJECTIVE: This study aimed to assess the usefulness of serum protein kinase C delta (PKCδ) for detecting these HCCs. METHODS: PKCδ levels were measured using a sandwich enzyme-linked immunosorbent assay in 363 chronic liver disease (CLD) patients with and without HCC. RESULTS: In both viral and non-viral CLD, PKCδ can detect HCCs with high sensitivity and specificity, particularly in the very early stages. Notably, the value and sensitivity of PKCδ were not modified by HCV elimination status. Liver injury and warfarin administration, which are known to cause false-positive results for conventional markers, did not modify PKCδ levels. CONCLUSIONS: PKCδ is an enhanced biomarker for the diagnosis of HCC that compensates for the drawbacks of conventional markers.

Three-dimensional changes in the cranial base associated with soft-diet feeding.

BACKGROUND: Masticatory activity affects the morphology of the maxillo-mandibular complex, however, its influence on the cranial base remains to be elucidated. The recent integration of quantitative morphometric analysis with 3D imaging enabled a comprehensive and high-resolution morphological characterization of the craniofacial complex. We aimed to investigate the influence of masticatory activity on the morphology of the growing cranial base by three-dimensional (3D) geometric morphometric approach using micro-CT. METHODS: The micro-CT data was reanalyzed to illustrate the 3D shape of the cranial base, and wireframe models were generated by connecting landmarks on the images. In the original study, mice were fed a soft diet (SD) of powdered pellets or a conventional hard diet (HD) for 6 weeks from 3 to 9 weeks of age, immediately after weaning. A principal component (PC) analysis analyzed shape variations and assessed their significance, while canonical variate (CV) analysis facilitated the comparison and differentiation of groups based on shape, unveiling meaningful shape distinctions. RESULTS: Three PCs were extracted that significantly separated the SD and HD groups among those explaining variations in shape. These PCs were related to the length of the sphenoid bone, the width of the anterior part of the sphenoid bone, and the length of the cranial base. Furthermore, one CV effectively distinguished SD from HD, and CV analysis showed that the sphenoid was shortened in the length and narrowed at the border of the temporal bone in SD mice. CONCLUSIONS: Masticatory loading affects the skeletal development of the cranial base. The morphology of the sphenoid bone was affected in both the sagittal and transverse axes.

Novel prediction models for pharyngeal-airway volume based on the cranial-base and midsagittal cross-sectional area of the airway in the pharyngeal region: A cephalometric and magnetic resonance imaging study.

OBJECTIVE: The objective of the study was to elucidate the association between cranial base (Bjork-Jarabak analysis), midsagittal cross-sectional area of the airway in the pharyngeal region (MCSA-PR) data and pharyngeal-airway volume (PAV) and develop a model that could help clinicians predict PAV using two-dimensional (2D) data (Bjork polygon and MCSA-PR). MATERIALS AND METHODS: Pre-treatment lateral cephalometric radiographs and magnetic resonance imaging (MRI) scans of 82 women were categorized into three anteroposterior skeletal groups based on ANB angle: Class I (n = 29), 1.5° ≤ ANB≤5.1°; Class II (n = 26), ANB >5.1°; Class III (n = 27), ANB <1.5°. The Bjork polygon, MCSA-PR data from cephalograms and PAV data from MRI scans were examined. Intergroup comparisons were performed using the Kruskal-Wallis test and one-way analysis of variance (ANOVA), with pairwise comparisons conducted using the Bonferroni-corrected Mann-Whitney U-test for the Kruskal-Wallis test and Bonferroni-corrected multiple comparison test for one-way ANOVA. Forward multiple linear regression was used to create model equations for predicting PAV. RESULTS: MCSA-PR and anterior (N-S) and posterior (S-Ar) cranial-base lengths were positively correlated with the PAV. We developed four models; three operated at the group level, and one encompassed the entire sample. Notably, all models could effectively explain the variance in the PAV data. The model for the Class I group was the strongest (adjusted R2 = 0.77). CONCLUSION: Our findings indicate the remarkable potential of the MCSA-PR, N-S and Bjork sum angles (BSA) as predictors of the PAV and the relevance of 2D cephalometric and cranial-base parameters in predicting the three-dimensional (3D) pharyngeal-airway size.

Bundling of collagen fibrils influences osteocyte network formation during bone modeling.

Osteocytes form a cellular network by gap junctions between their cell processes. This network is important since intercellular communication via the network is essential for bone metabolism. However, the factors that influence the formation of this osteocyte network remain unknown. As the early stage of osteocyte network formation occurs on the bone surface, we observed a newly formed trabecular bone surface by orthogonal focused ion beam-scanning electron microscopy. The embedding late osteoblast processes tended to avoid bundled collagen fibrils and elongate into sparse collagen fibrils. Then, we examined whether the inhibition of bundling of collagen fibrils using a potent lysyl oxidase inhibitor, ß-aminopropionitrile (BAPN) changed the cellular network of the chick calvaria. The osteocyte shape of the control group was spindle-shape, while that of the BAPN group was sphere-shaped. In addition, the osteocyte processes of the control group were elongated vertically to the long axis of the cell body, whereas the osteocyte processes of the BAPN group were elongated radially. Therefore, it was suggested that the bundling of collagen fibrils influences normal osteocyte network formation during bone modeling.

Low geriatric nutritional risk index predicts poor prognosis in patients with cirrhosis: a retrospective study.

Aim: Malnutrition, which increases the risk of liver disease-related events and mortality, is a serious complication in cirrhosis. This study aimed to investigate whether the geriatric nutritional risk index (GNRI) could predict the long-term prognosis in patients with cirrhosis. Methods: We retrospectively evaluated 266 patients with cirrhosis and classified them into two groups based on baseline GNRI scores: risk (≤98, n = 104) and no-risk groups (>98, n = 162). The cumulative survival rates were compared between the two groups in patients with compensated and decompensated cirrhosis, respectively. Cox proportional hazards regression analysis was used to identify significant and independent factors associated with mortality. Results: The median observation period was 54.9 (33.6-61.7) months and 65 (24.4%) liver disease-related deaths occurred during the follow-up period. The GNRI scores significantly and inversely correlated with Child-Pugh score (r = -0.579), model for end-stage liver disease score (r = -0.286), and Mac-2 binding protein glycosylation isomer (r = -0.494). Multivariate analysis identified low GNRI as a significant and independent factor associated with mortality [overall cohort: hazard ratio (HR), 0.926; p < 0.001; compensated cirrhosis: HR, 0.947; p = 0.003; decompensated cirrhosis: HR, 0.923; p < 0.001]. The risk group demonstrated significantly lower cumulative survival rates than the no-risk group in overall cohort, and patients with compensated and decompensated cirrhosis (p < 0.001, <0.001, and = 0.013, respectively). Conclusion: Low GNRI was associated with poor long-term prognosis in both patients with compensated and decompensated cirrhosis. Therefore, the GNRI is a simple and useful tool for predicting prognosis and modifying the nutritional status in patients with cirrhosis.

DYRK2 promotes chemosensitivity via p53-mediated apoptosis after DNA damage in colorectal cancer.

Dual-specificity tyrosine-regulated kinase 2 (DYRK2) is a protein kinase that phosphorylates p53-Ser46 and induces apoptosis in response to DNA damage. However, the relationship between DYRK2 expression and chemosensitivity after DNA damage in colorectal cancer has not been well investigated. The aim of the present study was to examine whether DYRK2 could be a novel marker for predicting chemosensitivity after 5-fluorouracil- and oxaliplatin-induced DNA damage in colorectal cancer. Here we showed that DYRK2 knockout decreased the chemosensitivity to 5-fluorouracil and oxaliplatin in p53 wild-type colorectal cancer cells, whereas the chemosensitivity remained unchanged in p53-deficient/mutated colorectal cancer cells. In addition, no significant differences in chemosensitivity to 5-fluorouracil and oxaliplatin between scramble and siDYRK2 p53(-/-) colorectal cancer cells were observed. Conversely, the combination of adenovirus-mediated overexpression of DYRK2 with 5-fluorouracil or oxaliplatin enhanced apoptosis and chemosensitivity through p53-Ser46 phosphorylation in p53 wild-type colorectal cancer cells. Furthermore, DYRK2 knockout decreased chemosensitivity to 5-fluorouracil and oxaliplatin in p53 wild-type xenograft mouse models. Taken together, these findings demonstrated that DYRK2 expression was associated with chemosensitivity to 5-fluorouracil and oxaliplatin in p53 wild-type colorectal cancer, suggesting the importance of evaluating the p53 status and DYRK2 expression as a novel marker in therapeutic strategies for colorectal cancer.

Ruxolitinib altered IFN-ß induced necroptosis of human dental pulp stem cells during osteoblast differentiation.

OBJECTIVE: This study aimed to evaluate the role of ruxolitinib in the interferon beta (IFN-ß) mediated osteoblast differentiation using human dental pulp stem cells (hDPSCs). DESIGN: hDPSCs from five deciduous teeth of healthy patients were stimulated by adding human recombinant IFN-ß protein (1 or 2 ng/ml) to the osteogenic differentiation induction medium. Substrate formation was determined using Alizarin Red staining, calcium concentration, and osteoblast marker expression levels. Ruxolitinib was used to inhibit the Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathway. Apoptosis was detected using terminal deoxynucleotidyl nick-end labeling (TUNEL) staining, and necroptosis was detected using propidium iodide staining and phosphorylated mixed lineage kinase domain-like protein (pMLKL) expression. RESULTS: In the IFN-ß-treated group, substrate formation was inhibited by a reduction in alkaline phosphatase (ALP) expression in a concentration-dependent manner. Although the proliferation potency was unchanged between the IFN-ß-treated and control groups, the cell number was significantly reduced in the experimental group. TUNEL-positive cell number was not significantly different; however, the protein level of necroptosis markers, interleukin-6 (IL-6) and pMLKL were significantly increased in the substrate formation. Cell number and ALP expression level were improved in the group administered ruxolitinib, a JAK-STAT inhibitor. Additionally, ruxolitinib significantly suppressed IL-6 and pMLKL levels. CONCLUSION: Ruxolitinib interfered with the IFN-ß-mediated necroptosis and osteogenic differentiation via the JAK-STAT pathway.

Dyrk2 gene transfer suppresses hepatocarcinogenesis by promoting the degradation of Myc and Hras.

Background & Aims: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and has a poor prognosis. However, the molecular mechanisms underlying hepatocarcinogenesis and progression remain unknown. In vitro gain- and loss-of-function analyses in cell lines and xenografts revealed that dual-specificity tyrosine-regulated kinase 2 (DYRK2) influences tumour growth in HCC. Methods: To investigate the role of Dyrk2 during hepatocarcinogenesis, we developed liver-specific Dyrk2 conditional knockout mice and an in vivo gene delivery system with a hydrodynamic tail vein injection and the Sleeping Beauty transposon. The antitumour effects of Dyrk2 gene transfer were investigated in a murine autologous carcinogenesis model. Results: Dyrk2 expression was reduced in tumours, and that its downregulation was induced before hepatocarcinogenesis. Dyrk2 gene transfer significantly suppressed carcinogenesis. It also suppresses Myc-induced de-differentiation and metabolic reprogramming, which favours proliferative, and malignant potential by altering gene profiles. Dyrk2 overexpression caused Myc and Hras degradation at the protein level rather than at the mRNA level, and this degradation mechanism was regulated by the proteasome. Immunohistochemical analyses revealed a negative correlation between DYRK2 expression and MYC and longer survival in patients with HCC with high-DYRK2 and low-MYC expressions. Conclusions: Dyrk2 protects the liver from carcinogenesis by promoting Myc and Hras degradation. Our findings would pave the way for a novel therapeutic approach using DYRK2 gene transfer. Impact and Implications: Hepatocellular carcinoma (HCC) is one of the most common cancers, with a poor prognosis. Hence, identifying molecules that can become promising targets for therapies is essential to improve mortality. No studies have clarified the association between DYRK2 and carcinogenesis, although DYRK2 is involved in tumour growth in various cancer cells. This is the first study to show that Dyrk2 expression decreases during hepatocarcinogenesis and that Dyrk2 gene transfer is an attractive approach with tumour suppressive activity against HCC by suppressing Myc-mediated de-differentiation and metabolic reprogramming that favours proliferative and malignant potential via Myc and Hras degradation.

Mechanical stretching determines the orientation of osteoblast migration and cell division.

Osteoblasts alignment and migration are involved in the directional formation of bone matrix and bone remodeling. Many studies have demonstrated that mechanical stretching controls osteoblast morphology and alignment. However, little is known about its effects on osteoblast migration. Here, we investigated changes in the morphology and migration of preosteoblastic MC3T3-E1 cells after the removal of continuous or cyclic stretching. Actin staining and time-lapse recording were performed after stretching removal. The continuous and cyclic groups showed parallel and perpendicular alignment to the stretch direction, respectively. A more elongated cell morphology was observed in the cyclic group than in the continuous group. In both stretch groups, the cells migrated in a direction roughly consistent with the cell alignment. Compared to the other groups, the cells in the cyclic group showed an increased migration velocity and were almost divided in the same direction as the alignment. To summarize, our study showed that mechanical stretching changed cell alignment and morphology in osteoblasts, which affected the direction of migration and cell division, and velocity of migration. These results suggest that mechanical stimulation may modulate the direction of bone tissue formation by inducing the directional migration and cell division of osteoblasts.

Electrochemical control of bone microstructure on electroactive surfaces for modulation of stem cells and bone tissue engineering.

Controlling stem cell behavior at the material interface is crucial for the development of novel technologies in stem cell biology and regenerative medicine. The composition and presentation of bio-factors on a surface strongly influence the activity of stem cells. Herein, we designed an electroactive surface that mimics the initial process of trabecular bone formation, by immobilizing chondrocyte-derived plasma membrane nanofragments (PMNFs) on its surface for rapid mineralization within 2 days. Moreover, the electroactive surface was based on the conducting polymer polypyrrole (PPy), which enabled dynamic control of the presentation of PMNFs on the surface via electrochemical redox switching, further resulting in the formation of bone minerals with different morphologies. Furthermore, bone minerals with contrasting surface morphologies had differential effects on the differentiation of human bone marrow-derived stem cells (hBMSCs) cultured on the surface. Together, this electroactive surface showed multifunctional characteristics, not only allowing dynamic control of PMNF presentation but also promoting the formation of bone minerals with different morphologies within 2 days. This electroactive substrate could be valuable for more precise control of stem cell growth and differentiation, and further development of more suitable microenvironments containing bone apatite for housing a bone marrow stem cell niche, such as biochips/bone-on-chips.

Treatment of Severe Open Bite Malocclusion with Four-Piece Segmental Horseshoe Le Fort I Osteotomy.

Appropriate operations in severe anterior open bite (AOB) cases are extremely complicated to perform because of the multiple surgical procedures involved, the difficulty of predicting posttreatment aesthetics, and the high relapse rate. We herein report a 16-year-old girl with skeletal Class II, severe AOB malocclusion, and crowding with short roots, and aesthetic and functional problems. Four-piece segmental Le Fort I osteotomy with horseshoe osteotomy was performed for maxillary intrusion, and bilateral sagittal split ramus osteotomy (SSRO) and genioplasty were performed for mandibular advancement. The malocclusion and skeletal deformity were significantly improved by the surgical orthodontic treatment. Functional and aesthetic occlusion with an improved facial profile was established, and no further root shortening was observed. Acceptable occlusion and dentition were maintained after a two-year retention period. This strategy of surgical orthodontic treatment with a complicated operative procedure might be effective for correcting certain severe AOB malocclusion cases.

Pharmacologic inhibition of PARP5, but not that of PARP1 or 2, promotes cytokine production and osteoclastogenesis through different pathways.

OBJECTIVES: PARPs, which are members of the poly(ADP-ribose) polymerase superfamily, promote tumorigenesis and tumour-associated inflammation and are thus therapeutic targets for several cancers. The aim of the present study is to investigate the mechanistic insight into the roles PARPs for inflammation. METHODS: Primary murine macrophages were cultured in the presence or absence of the PARP5 inhibitor NVP-TNKS656 to examine the role of PARP5 for cytokine production. RESULTS: In contrast to the roles of other PARPs for induction of inflammation, we found in the present study that pharmacologic inhibition of PARP5 induces production of inflammatory cytokines in primary murine macrophages. We found that treatment with the PARP5 inhibitor NVP-TNKS656 in macrophages enhanced steady-state and LPS-mediated cytokine production through degradation of IκBα and subsequent nuclear translocation of NF-κB. We also found that pharmacologic inhibition of PARP5 stabilises the adaptor protein 3BP2, a substrate of PARP5, and that accelerated cytokine production induced by PARP5 inhibition was rescued in 3BP2-deleted macrophages. Additionally, we found that LPS increases the expression of 3BP2 and AXIN1, a negative regulator of ß-catenin, through suppression of PARP5 transcripts in macrophages, leading to further activation of cytokine production and inhibition of ß-catenin-mediated cell proliferation, respectively. Lastly, we found that PARP5 inhibition in macrophages promotes osteoclastogenesis through stabilisation of 3BP2 and AXIN1, leading to activation of SRC and suppression of ß-catenin, respectively. CONCLUSIONS: Our results show that pharmacologic inhibition of PARP5 against cancers unexpectedly induces adverse autoinflammatory side effects through activation of innate immunity, unlike inhibition of other PARPs.

Important roles of odontoblast membrane phospholipids in early dentin mineralization.

The objective of this study was to first identify the timing and location of early mineralization of mouse first molar, and subsequently, to characterize the nucleation site for mineral formation in dentin from a materials science viewpoint and evaluate the effect of environmental cues (pH) affecting early dentin formation. Early dentin mineralization in mouse first molars began in the buccal central cusp on post-natal day 0 (P0), and was first hypothesized to involve collagen fibers. However, elemental mapping indicated the co-localization of phospholipids with collagen fibers in the early mineralization area. Co-localization of phosphatidylserine and annexin V, a functional protein that binds to plasma membrane phospholipids, indicated that phospholipids in the pre-dentin matrix were derived from the plasma membrane. A 3-dimensional in vitro biomimetic mineralization assay confirmed that phospholipids from the plasma membrane are critical factors initiating mineralization. Additionally, the direct measurement of the tooth germ pH, indicated it to be alkaline. The alkaline environment markedly enhanced the mineralization of cell membrane phospholipids. These results indicate that cell membrane phospholipids are nucleation sites for mineral formation, and could be important materials for bottom-up approaches aiming for rapid and more complex fabrication of dentin-like structures.

Imaging of Molecular Interaction Between CCN Protein and Its Binding Partners: An In Situ Proximity Ligation Assay of Interaction Between CCN2 and Rab14 in Chondrocytes.

An in situ proximity ligation assay (PLA) enables visualization of protein interactions in fixed cells. It is a powerful method for investigating protein-protein binding of endogenously expressed proteins. To confirm binding between CCN2 and Rab14 GTPase (Rab14) in chondrocytes, we performed a PLA using chondrocytic HCS-2/8 cells. The protocol in this chapter introduces an optimized technique for visualizing intracellular interactions of CCN2 and Rab14 in fixed cells using a PLA.

A morphometric analysis of the osteocyte canaliculus using applied automatic semantic segmentation by machine learning.

INTRODUCTION: Osteocytes play a role as mechanosensory cells by sensing flow-induced mechanical stimuli applied on their cell processes. High-resolution imaging of osteocyte processes and the canalicular wall are necessary for the analysis of this mechanosensing mechanism. Focused ion beam-scanning electron microscopy (FIB-SEM) enabled the visualization of the structure at the nanometer scale with thousands of serial-section SEM images. We applied machine learning for the automatic semantic segmentation of osteocyte processes and canalicular wall and performed a morphometric analysis using three-dimensionally reconstructed images. MATERIALS AND METHODS: Six-week-old-mice femur were used. Osteocyte processes and canaliculi were observed at a resolution of 2 nm/voxel in a 4 × 4 µm region with 2000 serial-section SEM images. Machine learning was used for automatic semantic segmentation of the osteocyte processes and canaliculi from serial-section SEM images. The results of semantic segmentation were evaluated using the dice similarity coefficient (DSC). The segmented data were reconstructed to create three-dimensional images and a morphological analysis was performed. RESULTS: The DSC was > 83%. Using the segmented data, a three-dimensional image of approximately 3.5 µm in length was reconstructed. The morphometric analysis revealed that the median osteocyte process diameter was 73.8 ± 18.0 nm, and the median pericellular fluid space around the osteocyte process was 40.0 ± 17.5 nm. CONCLUSION: We used machine learning for the semantic segmentation of osteocyte processes and canalicular wall for the first time, and performed a morphological analysis using three-dimensionally reconstructed images.

Investigation of the molecular causes underlying physical abnormalities in Diamond-Blackfan anemia patients with RPL5 haploinsufficiency.

Diamond-Blackfan anemia (DBA) is a genetic disorder caused by mutations in genes encoding ribosomal proteins and characterized by erythroid aplasia and various physical abnormalities. Although accumulating evidence suggests that defective ribosome biogenesis leads to p53-mediated apoptosis in erythroid progenitor cells, little is known regarding the underlying causes of the physical abnormalities. In this study, we established induced pluripotent stem cells from a DBA patient with RPL5 haploinsufficiency. These cells retained the ability to differentiate into osteoblasts and chondrocytes. However, RPL5 haploinsufficiency impaired the production of mucins and increased apoptosis in differentiated chondrocytes. Increased expression of the pro-apoptotic genes BAX and CASP9 further indicated that RPL5 haploinsufficiency triggered p53-mediated apoptosis in chondrocytes. Murine double minute 2 (MDM2), the primary negative regulator of p53, plays a crucial role in erythroid aplasia in DBA patient. We found the phosphorylation level of MDM2 was significantly decreased in RPL5 haploinsufficient chondrocytes. In stark contrast, we found no evidence that RPL5 haploinsufficiency impaired osteogenesis. Collectively, our data support a model in which RPL5 haploinsufficiency specifically induces p53-mediated apoptosis in chondrocytes through MDM2 inhibition, which leads to physical abnormalities in DBA patients.

High-resolution image-based simulation reveals membrane strain concentration on osteocyte processes caused by tethering elements.

Osteocytes are vital for regulating bone remodeling by sensing the flow-induced mechanical stimuli applied to their cell processes. In this mechanosensing mechanism, tethering elements (TEs) connecting the osteocyte process with the canalicular wall potentially amplify the strain on the osteocyte processes. The ultrastructure of the osteocyte processes and canaliculi can be visualized at a nanometer scale using high-resolution imaging via ultra-high voltage electron microscopy (UHVEM). Moreover, the irregular shapes of the osteocyte processes and the canaliculi, including the TEs in the canalicular space, should considerably influence the mechanical stimuli applied to the osteocytes. This study aims to characterize the roles of the ultrastructure of osteocyte processes and canaliculi in the mechanism of osteocyte mechanosensing. Thus, we constructed a high-resolution image-based model of an osteocyte process and a canaliculus using UHVEM tomography and investigated the distribution and magnitude of flow-induced local strain on the osteocyte process by performing fluid-structure interaction simulation. The analysis results reveal that local strain concentration in the osteocyte process was induced by a small number of TEs with high tension, which were inclined depending on the irregular shapes of osteocyte processes and canaliculi. Therefore, this study could provide meaningful insights into the effect of ultrastructure of osteocyte processes and canaliculi on the osteocyte mechanosensing mechanism.

O-GlcNAcylation drives calcium signaling toward osteoblast differentiation: A bioinformatics-oriented study.

This study aimed to reveal the possible mechanisms by which O-linked-N-acetylglucosaminylation (O-GlcNAcylation) regulates osteoblast differentiation using a series of bioinformatics-oriented experiments. To examine the influence of O-GlcNAcylation levels on osteoblast differentiation, osteoblastic MC3T3-E1 cells were treated with O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) inhibitors. Correlations between the levels of O-GlcNAcylation and the expression of osteogenic markers as well as OGT were evaluated by qPCR and western blotting. The O-GlcNAcylated proteins assumed to correlate with Runx2 expression were retrieved from several public databases and used for further bioinformatics analysis. Following the findings of the bioinformatics analysis, intracellular calcium ([Ca2+ ]i ) was monitored in the cells treated with OGT and OGA inhibitors using a confocal laser-scanning microscope (CLS). The interaction effect between O-GlcNAcylation and [Ca2+ ]i on osteogenic marker expression was determined using stable OGT knockdown MC3T3-E1 cells. O-GlcNAcylation was positively associated with osteoblast differentiation. The time-course profile of global O-GlcNAcylated proteins showed a distinctive pattern with different molecular weights during osteoblast differentiation. The expression pattern of several O-GlcNAcylated proteins was significantly similar to that of Runx2 expression. Bioinformatic analysis of the retrieved Runx2-related-O-GlcNAcylated-proteins revealed the importance of [Ca2+ ]i . CLS showed that alteration of O-GlcNAcylation rapidly changed [Ca2+ ]i in MC3T3-E1 cells. O-GlcNAcylation and [Ca2+ ]i showed an interaction effect on the expression of osteogenic markers. OGT knockdown disrupted the [Ca2+ ]i -induced expression changes of osteogenic markers. O-GlcNAcylation interacts with [Ca2+ ]i and elicits osteoblast differentiation by regulating the expression of osteogenic markers.

Extracellular vesicles of P. gingivalis-infected macrophages induce lung injury.

Periodontal diseases are common inflammatory diseases that are induced by infection with periodontal bacteria such as Porphyromonas gingivalis (Pg). The association between periodontal diseases and many types of systemic diseases has been demonstrated; the term "periodontal medicine" is used to describe how periodontal infection/inflammation may impact extraoral health. However, the molecular mechanisms by which the factors produced in the oral cavity reach multiple distant organs and impact general health have not been elucidated. Extracellular vesicles (EVs) are nano-sized spherical structures secreted by various types of cells into the tissue microenvironment, and influence pathophysiological conditions by delivering their cargo. However, a detailed understanding of the effect of EVs on periodontal medicine is lacking. In this study, we investigated whether EVs derived from Pg-infected macrophages reach distant organs in mice and influence the pathophysiological status. EVs were isolated from human macrophages, THP-1 cells, infected with Pg. We observed that EVs from Pg-infected THP-1 cells (Pg-inf EVs) contained abundant core histone proteins such as histone H3 and translocated to the lungs, liver, and kidneys of mice. Pg-inf EVs also induced pulmonary injury, including edema, vascular congestion, inflammation, and collagen deposition causing alveoli destruction. The Pg-inf EVs or the recombinant histone H3 activated the NF-κB pathway, leading to increase in the levels of pro-inflammatory cytokines in human lung epithelial A549 cells. Our results suggest a possible mechanism by which EVs produced in periodontal diseases contribute to the progression of periodontal medicine.