Automated sample preparation for electrospray ionization mass spectrometry based on CLOCK-controlled autonomous centrifugal microfluidics.

We report a centrifugal microfluidic device that automatically performs sample preparation under steady-state rotation for clinical applications using mass spectrometry. The autonomous microfluidic device was designed for the control of liquid operation on centrifugal hydrokinetics (CLOCK) paradigm. The reported device was highly stable, with less than 7% variation with respect to the time of each unit operation (sample extraction, mixing, and supernatant extraction) in the preparation process. An agitation mechanism with bubbling was used to mix the sample and organic solvent in this device. We confirmed that the device effectively removed the protein aggregates from the sample, and the performance was comparable to those of conventional manual sample preparation procedures that use high-speed centrifugation. In addition, probe electrospray ionization mass spectrometry (PESI-MS) was performed to compare the device-treated and manually treated samples. The obtained PESI-MS spectra were analyzed by partial least squares discriminant analysis, and the preparation capability of the device was found to be equivalent to that of the conventional method.

New Diagnostic Modality Combining Mass Spectrometry and Machine Learning for the Discrimination of Malignant Intraductal Papillary Mucinous Neoplasms.

BACKGROUND: An intraductal papillary mucinous neoplasm (IPMN) is a pancreatic tumor with malignant potential. Although we anticipate a sensitive method to diagnose the malignant conversion of IPMN, an effective strategy has not yet been established. The combination of probe electrospray ionization-mass spectrometry (PESI-MS) and machine learning provides a promising solution for this purpose. METHODS: We prospectively analyzed 42 serum samples obtained from IPMN patients who underwent pancreatic resection between 2020 and 2021. Based on the postoperative pathological diagnosis, patients were classified into two groups: IPMN-low grade dysplasia (n = 17) and advanced-IPMN (n = 25). Serum samples were analyzed by PESI-MS, and the obtained mass spectral data were converted into continuous variables. These variables were used to discriminate advanced-IPMN from IPMN-low grade dysplasia by partial least square regression or support vector machine analysis. The areas under receiver operating characteristics curves were obtained to visualize the difference between the two groups. RESULTS: Partial least square regression successfully discriminated the two disease classes. From another standpoint, we selected 130 parameters from the entire dataset by PESI-MS, which were fed into the support vector machine. The diagnostic accuracy was 88.1%, and the area under the receiver operating characteristics curve was 0.924 by this method. Approximately 10 min were required to perform each method. CONCLUSION: PESI-MS combined with machine learning is an easy-to-use tool with the advantage of rapid on-site analysis. Here, we show the great potential of our system to diagnose the malignant conversion of IPMN, which would be a promising diagnostic tool in clinical settings.

Extracellular acidification augments sclerostin and osteoprotegerin production by Ocy454 mouse osteocytes.

Osteocytes sense the microenvironmental stimuli, including mechanical stress, and regulate bone resorption by osteoclasts and bone formation by osteoblasts. Diabetes and cancer metastasis to bone raise l-lactic acid in the bone tissue, causing acidification. Here, we investigated the effects of l-lactic acid and extracellular acidification on the function of mouse Ocy454 osteocytes. L- and d-lactic acid with low chiral selectivity and acidification of the medium raised the production of sclerostin and osteoprotegerin by Ocy454 cells. The mRNA expression of their genes increased after either treatment of L- and d-lactic acid or acidification of the medium. Furthermore, the conditioned medium of Ocy454 cells cultured in an acidic environment suppressed the induction of alkaline phosphatase activity in MC3T3-E1 cells, which was recovered by the anti-sclerostin antibody. While it is reported that HDAC5 inhibits the transcription of the sclerostin gene, extracellular acidification reduced the nuclear localization of HDAC5 in Ocy454 cells. While calmodulin kinase II (CaMKII) is known to phosphorylate and induce extranuclear translocation of HDAC5, KN-62, an inhibitor of CaMKII lowered the expression of the sclerostin gene in Ocy454 cells. Collectively, extracellular acidification is a microenvironmental factor that modulates osteocyte functions.

Probe electrospray ionization mass spectrometry-based rapid diagnosis of liver tumors.

BACKGROUND AND AIM: Prompt differential diagnosis of liver tumors is clinically important and sometimes difficult. A new diagnostic device that combines probe electrospray ionization-mass spectrometry (PESI-MS) and machine learning may help provide the differential diagnosis of liver tumors. METHODS: We evaluated the diagnostic accuracy of this new PESI-MS device using tissues obtained and stored from previous surgically resected specimens. The following cancer tissues (with collection dates): hepatocellular carcinoma (HCC, 2016-2019), intrahepatic cholangiocellular carcinoma (ICC, 2014-2019), and colorectal liver metastasis (CRLM, 2014-2019) from patients who underwent hepatic resection were considered for use in this study. Non-cancerous liver tissues (NL) taken from CRLM cases were also incorporated into the analysis. Each mass spectrum provided by PESI-MS was tested using support vector machine, a type of machine learning, to evaluate the discriminatory ability of the device. RESULTS: In this study, we used samples from 91 of 139 patients with HCC, all 24 ICC samples, and 103 of 202 CRLM samples; 80 NL from CRLM cases were also used. Each mass spectrum was obtained by PESI-MS in a few minutes and was evaluated by machine learning. The sensitivity, specificity, and diagnostic accuracy of the PESI-MS device for discriminating HCC, ICC, and CRLM from among a mix of all three tumors and from NL were 98.9%, 98.1%, and 98.3%; 87.5%, 93.1%, and 92.6%; and 99.0%, 97.9%, and 98.3%, respectively. CONCLUSION: This study demonstrated that PESI-MS and machine learning could discriminate liver tumors accurately and rapidly.

8-Nitro-cGMP suppresses mineralization by mouse osteoblasts.

Nitric oxide and reactive oxygen species regulate bone remodeling, which occurs via bone formation and resorption by osteoblasts and osteoclasts, respectively. Recently, we found that 8-nitro-cGMP, a second messenger of nitric oxide and reactive oxygen species, promotes osteoclastogenesis. Here, we investigated the formation and function of 8-nitro-cGMP in osteoblasts. Mouse calvarial osteoblasts were found to produce 8-nitro-cGMP, which was augmented by tumor necrosis factor-α (10 ng/ml) and interleukin-1ß (1 ng/ml). These cytokines suppressed osteoblastic differentiation in a NO synthase activity-dependent manner. Exogenous 8-nitro-cGMP (30 µmol/L) suppressed expression of osteoblastic phenotypes, including mineralization, in clear contrast to the enhancement of mineralization by osteoblasts induced by 8-bromo-cGMP, a cell membrane-permeable analog of cGMP. It is known that reactive sulfur species denitrates and degrades 8-nitro-cGMP. Mitochondrial cysteinyl-tRNA synthetase plays a crucial role in the endogenous production of RSS. The expression of osteoblastic phenotypes was suppressed by not only exogenous 8-nitro-cGMP but also by silencing of the Cars2 gene, indicating a role of endogenous 8-nitro-cGMP in suppressing the expression of osteoblastic phenotypes. These results suggest that 8-nitro-cGMP is a negative regulator of osteoblastic differentiation.

Zoledronate promotes inflammatory cytokine expression in human CD14-positive monocytes among peripheral mononuclear cells in the presence of γδ T cells.

Bisphosphonates distributed to bone exert toxic effects specifically towards osteoclasts. On the other hand, intravenous administration of a nitrogen-containing bisphosphonate (N-BP) such as zoledronate induces acute-phase reactions (APRs), including influenza-like fever 1 day later, indicating an interaction with immunocompetent cells circulating blood. Although it has been reported that activation of γδ T cells is pivotal to induce an APR following treatment with zoledronate, downstream events, including the production of inflammatory cytokines after activation of γδ T cells, remain obscure. We investigated the effects of zoledronate on inflammatory cytokine expression in human peripheral blood mononuclear cells (PBMCs) in vitro. While zoledronate induced mRNA expressions of tumour necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6 and interferon-γ (IFN-γ) in PBMC, depletion of γδ T cells abolished that zoledronate-induced expression of those cytokines, indicating the necessity of γδ T cells for expression induction by zoledronate. However, which types of cells were responsible for the production of those cytokines in blood remained unclear. As it is generally accepted that monocytes and macrophages are primary sources of inflammatory cytokines, CD14+ cells from PBMC were exposed to zoledronate in the presence of PBMC, which resulted in induced expression of mRNAs for IL-1ß, IL-6 and IFN-γ, but not for TNF-α. These results indicate that CD14+ cells are responsible, at least in part, for the production of IL-1ß, IL-6 and IFN-γ in blood exposed to zoledronate. This suggests that CD14+ cells play an essential role in the occurrence of APRs following N-BP administration.

A new rapid diagnostic system with ambient mass spectrometry and machine learning for colorectal liver metastasis.

BACKGROUND: Probe electrospray ionization-mass spectrometry (PESI-MS) can rapidly visualize mass spectra of small, surgically obtained tissue samples, and is a promising novel diagnostic tool when combined with machine learning which discriminates malignant spectrum patterns from others. The present study was performed to evaluate the utility of this device for rapid diagnosis of colorectal liver metastasis (CRLM). METHODS: A prospectively planned study using retrospectively obtained tissues was performed. In total, 103 CRLM samples and 80 non-cancer liver tissues cut from surgically extracted specimens were analyzed using PESI-MS. Mass spectra obtained by PESI-MS were classified into cancer or non-cancer groups by using logistic regression, a kind of machine learning. Next, to identify the exact molecules responsible for the difference between CRLM and non-cancerous tissues, we performed liquid chromatography-electrospray ionization-MS (LC-ESI-MS), which visualizes sample molecular composition in more detail. RESULTS: This diagnostic system distinguished CRLM from non-cancer liver parenchyma with an accuracy rate of 99.5%. The area under the receiver operating characteristic curve reached 0.9999. LC-ESI-MS analysis showed higher ion intensities of phosphatidylcholine and phosphatidylethanolamine in CRLM than in non-cancer liver parenchyma (P < 0.01, respectively). The proportion of phospholipids categorized as monounsaturated fatty acids was higher in CRLM (37.2%) than in non-cancer liver parenchyma (10.7%; P < 0.01). CONCLUSION: The combination of PESI-MS and machine learning distinguished CRLM from non-cancer tissue with high accuracy. Phospholipids categorized as monounsaturated fatty acids contributed to the difference between CRLM and normal parenchyma and might also be a useful diagnostic biomarker and therapeutic target for CRLM.

Lipidome-based rapid diagnosis with machine learning for detection of TGF-ß signalling activated area in head and neck cancer.

BACKGROUND: Several pro-oncogenic signals, including transforming growth factor beta (TGF-ß) signalling from tumour microenvironment, generate intratumoural phenotypic heterogeneity and result in tumour progression and treatment failure. However, the precise diagnosis for tumour areas containing subclones with cytokine-induced malignant properties remains clinically challenging. METHODS: We established a rapid diagnostic system based on the combination of probe electrospray ionisation-mass spectrometry (PESI-MS) and machine learning without the aid of immunohistological and biochemical procedures to identify tumour areas with heterogeneous TGF-ß signalling status in head and neck squamous cell carcinoma (HNSCC). A total of 240 and 90 mass spectra were obtained from TGF-ß-unstimulated and -stimulated HNSCC cells, respectively, by PESI-MS and were used for the construction of a diagnostic system based on lipidome. RESULTS: This discriminant algorithm achieved 98.79% accuracy in discrimination of TGF-ß1-stimulated cells from untreated cells. In clinical human HNSCC tissues, this approach achieved determination of tumour areas with activated TGF-ß signalling as efficiently as a conventional histopathological assessment using phosphorylated-SMAD2 staining. Furthermore, several altered peaks on mass spectra were identified as phosphatidylcholine species in TGF-ß-stimulated HNSCC cells. CONCLUSIONS: This diagnostic system combined with PESI-MS and machine learning encourages us to clinically diagnose intratumoural phenotypic heterogeneity induced by TGF-ß.

Rapid automated diagnosis of primary hepatic tumour by mass spectrometry and artificial intelligence.

BACKGROUND AND AIMS: Complete surgical resection with negative margin is one of the pillars in treatment of liver tumours. However, current techniques for intra-operative assessment of tumour resection margins are time-consuming and empirical. Mass spectrometry (MS) combined with artificial intelligence (AI) is useful for classifying tissues and provides valuable prognostic information. The aim of this study was to develop a MS-based system for rapid and objective liver cancer identification and classification. METHODS: A large dataset derived from 222 patients with hepatocellular carcinoma (HCC, 117 tumours and 105 non-tumours) and 96 patients with mass-forming cholangiocarcinoma (MFCCC, 50 tumours and 46 non-tumours) were analysed by Probe Electrospray Ionization (PESI) MS. AI by means of support vector machine (SVM) and random forest (RF) algorithms was employed. For each classifier, sensitivity, specificity and accuracy were calculated. RESULTS: The overall diagnostic accuracy exceeded 94% in both the AI algorithms. For identification of HCC vs non-tumour tissue, RF was the best, with 98.2% accuracy, 97.4% sensitivity and 99% specificity. For MFCCC vs non-tumour tissue, both algorithms gave 99.0% accuracy, 98% sensitivity and 100% specificity. CONCLUSIONS: The herein reported MS-based system, combined with AI, permits liver cancer identification with high accuracy. Its bench-top size, minimal sample preparation and short working time are the main advantages. From diagnostics to therapeutics, it has the potential to influence the decision-making process in real-time with the ultimate aim of improving cancer patient cure.

Nephronectin Expression is Inhibited by Inorganic Phosphate in Osteoblasts.

Nephronectin (Npnt), an extracellular matrix protein, is known to be a ligand of integrin α8ß1, and it has also been known to play critical roles as various organs. In the present study, elevated extracellular inorganic phosphate (Pi) strongly inhibited the expression of Npnt in MC3T3-E1 cells, while the existence of extracellular calcium (Ca) was indispensable for its effect. Furthermore, Pi-induced inhibition of Npnt gene expression was recovered by inhibitors of both sodium-dependent Pi transporter (Pit) and fibroblast growth factor receptors (Fgfrs). These results demonstrated that Npnt gene expression is regulated by extracellular Pi via Pit and Fgfrs.

Detection of potential new biomarkers of atherosclerosis by probe electrospray ionization mass spectrometry.

INTRODUCTION: Atherosclerotic diseases are the leading cause of death worldwide. Biomarkers of atherosclerosis are required to monitor and prevent disease progression. While mass spectrometry is a promising technique to search for such biomarkers, its clinical application is hampered by the laborious processes for sample preparation and analysis. METHODS: We developed a rapid method to detect plasma metabolites by probe electrospray ionization mass spectrometry (PESI-MS), which employs an ambient ionization technique enabling atmospheric pressure rapid mass spectrometry. To create an automatic diagnosis system of atherosclerotic disorders, we applied machine learning techniques to the obtained spectra. RESULTS: Using our system, we successfully discriminated between rabbits with and without dyslipidemia. The causes of dyslipidemia (genetic lipoprotein receptor deficiency or dietary cholesterol overload) were also distinguishable by this method. Furthermore, after induction of atherosclerosis in rabbits with a cholesterol-rich diet, we were able to detect dynamic changes in plasma metabolites. The major metabolites detected by PESI-MS included cholesterol sulfate and a phospholipid (PE18:0/20:4), which are promising new biomarkers of atherosclerosis. CONCLUSION: We developed a remarkably fast and easy method to detect potential new biomarkers of atherosclerosis in plasma using PESI-MS.

Hepatitis B virus prevents excessive viral production via reduction of cell death-inducing DFF45-like effectors.

The relationship between hepatitis B virus (HBV) infection and lipid accumulation remains largely unknown. In this study, we investigated the effect of HBV propagation on lipid droplet growth in HBV-infected cells and HBV-producing cell lines, HepG2.2.15 and HBV-inducible Hep38.7-Tet. The amount of intracellular triglycerides was significantly reduced in HBV-infected and HBV-producing cells compared with HBV-lacking control cells. Electron and immunofluorescent microscopic analyses showed that the average size of a single lipid droplet (LD) was significantly less in the HBV-infected and HBV-producing cells than in the HBV-lacking control cells. Cell death-inducing DFF45-like effectors (CIDEs) B and C (CIDEB and CIDEC), which are involved in LD expansion for the improvement of lipid storage, were expressed at a significantly lower level in HBV-infected or HBV-producing cells than in HBV-lacking control cells, while CIDEA was not detected in those cells regardless of HBV production. The activity of the CIDEB and CIDEC gene promoters was impaired in HBV-infected or HBV-producing cells compared to HBV-lacking control cells, while CIDEs potentiated HBV core promoter activity. The amount of HNF4α, that can promote the transcription of CIDEB was significantly lower in HBV-producing cells than in HBV-lacking control cells. Knockout of CIDEB or CIDEC significantly reduced the amount of supernatant HBV DNA, intracellular viral RNA and nucleocapsid-associated viral DNA, while the expression of CIDEB or CIDEC recovered HBV production in CIDEB- or CIDEC-knockout cells. These results suggest that HBV regulates its own viral replication via CIDEB and CIDEC.

Extracellular matrix loss in chondrocytes after exposure to interleukin-1ß in NADPH oxidase-dependent manner.

Osteoarthritis is a degenerative joint disease caused by excessive death of chondrocytes and loss of the extracellular matrix (ECM) in articular cartilage. We previously reported that reactive oxygen species (ROS) generated by the NADPH oxidase (NOX) isoform NOX-2 are involved in chondrocyte death induced by interleukin-1ß (IL-1ß). In this study, we investigate the role of NOX-2 in the production and degradation of ECM by chondrocytes. Although IL-1ß lowered the mRNA expression of type II collagen (Col2a1) and aggrecan (Acan) in mouse chondrocyte-like ATDC5 cells, RNA silencing of Nox2 did not change the mRNA expression of these major components of the ECM of cartilage. Hence, NOX-2 is not involved in the IL-1ß-induced suppression of ECM production. On the other hand, the NOX inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), the ROS scavenger N-acetylcysteine and an antisense oligodeoxynucleotide for Nox2 prevented the loss of proteoglycan induced by IL-1ß in highly differentiated ATDC5 cells. Furthermore, AEBSF did not affect the expression of hyaluronidase-1 and -2, whereas it suppressed hyaluronidase activity in culture medium. IL-1ß-induced intra- and extracellular acidification was also suppressed by AEBSF, as was the antisense oligodeoxynucleotide for Nox2. Since hyaluronidase activity is known to be higher under acidic conditions, NOX-2 probably contributes to ECM loss by the activation of hyaluronidase through acidification.

In vivo endoscopic mass spectrometry using a moving string sampling probe.

At present, endoscopy relies almost exclusively on optical microscopy and the accurate analysis such as MS interrogation is performed ex situ using biopsy. In this work, a novel probing system is developed to perform in situ and in vivo endoscopic mass spectrometry using a moving string for the sampling and transportation of material. A prototype of a mass spectrometric endoscope is constructed using an industrial endoscope and a commercial mass spectrometer. The sampling system consists of a moving cotton thread driven by motorized pulleys. When the target surface is touched by the sampling probe, the cotton thread "wipes" and transports the adhered sample to the ion source. Depending on the target analytes, desorption electrospray and atmospheric pressure chemical ionization sources are employed interchangeably for the desorption and ionization. The surface under analysis is not subjected to heat, organic solvents, high voltage or charged droplets. In situ endoscopic MS of a living mouse and surface analysis inside a volunteer subject's mouth are demonstrated.

Nanoindentation time-dependent deformation/recovery suggestive of methylglyoxal induced glycation in calcified nodules.

Although empirical findings have indicated increase in bone fracture risk in type 2 diabetes patients, that has yet to be proven by results obtained at the material level. Here, we report evidence showing nanoscale time-dependent deformation/recovery of in vitro calcified nodules mimicking bone turnover in type 2 diabetes in respect to methylglyoxal (MG)-induced glycation. Nanoindentation test results revealed that calcified nodules cultured with MG did not show adequate dimensional recovery, despite a large creep rate during constant load indentation testing. This lesser recovery is likely based on the linear matrix polymerization network formed by advanced glycation end products (AGEs) as a secondary product of MG. Since elevated serum MG and abnormal bone turnover related to the amount of AGEs are observed in cases of type 2 diabetes, this time-dependent behavior may be one of the factors of the bone fracture mechanism at the material level in affected patients.

Enhanced and suppressed mineralization by acetoacetate and ß-hydroxybutyrate in osteoblast cultures.

It is known that diabetes aggravates alveolar bone loss associated with periodontitis. While insulin depletion increases the blood concentration of ketone bodies, i.e., acetoacetate and ß-hydroxybutyrate, their roles in bone metabolism have not been much studied until today. We investigated the effects of acetoacetate and ß-hydroxybutyrate on mineralization of extracellular matrix in cultures of mouse osteoblastic MC3T3-E1 cells and primary mouse osteoblasts in the presence and absence of bone morphogenetic protein-2. Acetoacetate potentiated alkaline phosphatase activity in MC3T3-E1 cells in a concentration-dependent manner, ranging from physiological to pathological concentrations (0.05-5 mmol/L). In contrast, ß-hydroxybutyrate lowered it in the same experimental settings. Mineralization in cultures of these cells was also up-regulated by acetoacetate and down-regulated by ß-hydroxybutyrate. Similar results were obtained in cultures of mouse primary osteoblasts. Neither alkaline phosphatase mRNA nor its protein expression in MC3T3-E1 cells was affected by acetoacetate or ß-hydroxybutyrate, indicating that these ketone bodies control the enzyme activity of alkaline phosphatase in osteoblasts and hence their mineralization bi-directionally. Finally, either gene silencing of monocarboxylate transporter-1, a major transmembrate transporter for ketone bodies, nullified the effects of ketone bodies on alkaline phosphatase activity in MC3T3-E1 cells. Collectively, we found that ketone bodies bidirectionally modulates osteoblast functions, which suggests that ketone bodies are important endogenous factors that regulate bone metabolism in both physiological and pathological situations.

Porphyromonas gingivalis-derived lysine gingipain enhances osteoclast differentiation induced by tumor necrosis factor-α and interleukin-1ß but suppresses that by interleukin-17A: importance of proteolytic degradation of osteoprotegerin by lysine gingipain.

Periodontitis is a chronic inflammatory disease accompanied by alveolar bone resorption by osteoclasts. Porphyromonas gingivalis, an etiological agent for periodontitis, produces cysteine proteases called gingipains, which are classified based on their cleavage site specificity (i.e. arginine (Rgps) and lysine (Kgps) gingipains). We previously reported that Kgp degraded osteoprotegerin (OPG), an osteoclastogenesis inhibitory factor secreted by osteoblasts, and enhanced osteoclastogenesis induced by various Toll-like receptor (TLR) ligands (Yasuhara, R., Miyamoto, Y., Takami, M., Imamura, T., Potempa, J., Yoshimura, K., and Kamijo, R. (2009) Lysine-specific gingipain promotes lipopolysaccharide- and active-vitamin D3-induced osteoclast differentiation by degrading osteoprotegerin. Biochem. J. 419, 159-166). Osteoclastogenesis is induced not only by TLR ligands but also by proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-17A, in inflammatory conditions, such as periodontitis. Although Kgp augmented osteoclastogenesis induced by TNF-α and IL-1ß in co-cultures of mouse osteoblasts and bone marrow cells, it suppressed that induced by IL-17A. In a comparison of proteolytic degradation of these cytokines by Kgp in a cell-free system with that of OPG, TNF-α and IL-1ß were less susceptible, whereas IL-17A and OPG were equally susceptible to degradation by Kgp. These results indicate that the enhancing effect of Kgp on cytokine-induced osteoclastogenesis is dependent on the difference in degradation efficiency between each cytokine and OPG. In addition, elucidation of the N-terminal amino acid sequences of OPG fragments revealed that Kgp primarily cleaved OPG in its death domain homologous region, which might prevent dimer formation of OPG required for inhibition of receptor activator of nuclear factor κB ligand. Collectively, our results suggest that degradation of OPG by Kgp is a crucial event in the development of osteoclastogenesis and bone loss in periodontitis.

Bropirimine inhibits osteoclast differentiation through production of interferon-ß.

Bropirimine is a synthetic agonist for toll-like receptor 7 (TLR7). In this study, we investigated the effects of bropirimine on differentiation and bone-resorbing activity of osteoclasts in vitro. Bropirimine inhibited osteoclast differentiation of mouse bone marrow-derived macrophages (BMMs) induced by receptor activator of nuclear factor κB ligand (RANKL) in a concentration-dependent manner. Furthermore, it suppressed the mRNA expression of nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1), a master transcription factor for osteoclast differentiation, without affecting BMM viability. Bropirimine also inhibited osteoclast differentiation induced in co-cultures of mouse bone marrow cells (BMCs) and mouse osteoblastic UAMS-32 cells in the presence of activated vitamin D3. Bropirimine partially suppressed the expression of RANKL mRNA in UAMS-32 cells induced by activated vitamin D3. Finally, the anti-interferon-ß (IFN-ß) antibody restored RANKL-dependent differentiation of BMMs into osteoclasts suppressed by bropirimine. These results suggest that bropirimine inhibits differentiation of osteoclast precursor cells into osteoclasts via TLR7-mediated production of IFN-ß.

Localization and osteoblastic differentiation potential of neural crest-derived cells in oral tissues of adult mice.

In embryos, neural crest cells emerge from the dorsal region of the fusing neural tube and migrate throughout tissues to differentiate into various types of cells including osteoblasts. In adults, subsets of neural crest-derived cells (NCDCs) reside as stem cells and are considered to be useful cell sources for regenerative medicine strategies. Numerous studies have suggested that stem cells with a neural crest origin persist into adulthood, especially those within the mammalian craniofacial compartment. However, their distribution as well as capacity to differentiate into osteoblasts in adults is not fully understood. To analyze the precise distribution and characteristics of NCDCs in adult oral tissues, we utilized an established line of double transgenic (P0-Cre/CAG-CAT-EGFP) mice in which NCDCs express green fluorescent protein (GFP) throughout their life. GFP-positive cells were scattered like islands throughout tissues of the palate, gingiva, tongue, and buccal mucosa in adult mice, with those isolated from the latter shown to form spheres, typical cell clusters composed of stem cells, under low-adherent conditions. Furthermore, GFP-positive cells had markedly increased alkaline phosphatase (a marker enzyme of osteoblast differentiation) activity and mineralization as shown by alizarin red staining, in the presence of bone morphogenetic protein (BMP)-2. These results suggest that NCDCs reside in various adult oral tissues and possess potential to differentiate into osteoblastic cells. NCDCs in adults may be a useful cell source for bone regeneration strategies.