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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.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.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.This study demonstrated that PESI-MS and machine learning could discriminate liver tumors accurately and rapidly.

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.

Key Points Allele-specific methylation of the ASNS gene is associated with asparaginase sensitivity and therapeutic outcome in T-ALL.Pediatric T-ALL patients with poor prognostic SPI1 fusion exclusively exhibited ASNS hypomethylation status., Visual Abstract, Asparaginase therapy is a key component of chemotherapy for patients with T-cell acute lymphoblastic leukemia (T-ALL). Asparaginase depletes serum asparagine by deamination into aspartic acid. Normal hematopoietic cells can survive due to asparagine synthetase (ASNS) activity, whereas leukemia cells are supposed to undergo apoptosis due to silencing of the ASNS gene. Because the ASNS gene has a typical CpG island in its promoter, its methylation status in T-ALL cells may be associated with asparaginase sensitivity. Thus, we investigated the significance of ASNS methylation status in asparaginase sensitivity of T-ALL cell lines and prognosis of childhood T-ALL. Sequencing of bisulfite polymerase chain reaction products using next-generation sequencing technology in 22 T-ALL cell lines revealed a stepwise allele-specific methylation of the ASNS gene, in association with an aberrant methylation of a 7q21 imprinted gene cluster. T-ALL cell lines with ASNS hypermethylation status showed significantly higher in vitro l-asparaginase sensitivity in association with insufficient asparaginase-induced upregulation of ASNS gene expression and lower basal ASNS protein expression. A comprehensive analysis of diagnostic samples from pediatric patients with T-ALL in Japanese cohorts (N = 77) revealed that methylation of the ASNS gene was associated with an aberrant methylation of the 7q21 imprinted gene cluster. In pediatric T-ALL patients in Japanese cohorts (n = 75), ASNS hypomethylation status was significantly associated with poor therapeutic outcome, and all cases with poor prognostic SPI1 fusion exclusively exhibited ASNS hypomethylation status. These observations show that ASNS hypomethylation status is associated with asparaginase resistance and is a poor prognostic biomarker in childhood T-ALL.

We report a centrifugal microfluidic device that automatically executes sample preparation at steady-state rotation for clinical applications using mass spectrometry. The autonomous microfluidic device was designed for the control of liquid operation based on centrifugal hydrokinetics (CLOCK) paradigm. The reported device was highly stable with less than 7% variation in the time of each unit operation (sample extraction, mixing, and supernatant extraction) with respect to the preparation process. Mixing of the sample and organic solvent in this device was realized using an agitation mechanism with bubbling. We confirmed that the device effectively removed protein aggregates from the sample, and the performance was comparable to that of conventional manual sample preparation procedure that uses high-speed centrifuge. 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, confirming that the preparation capability of the device was equivalent to that of the conventional method.

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.

Down’s syndrome is one of the most common human congenital genetic diseases and affected patients have increased risk of periodontal disease. To examine involvement of the disease with periodontal disease development, we established immortalized periodontal ligament cells obtained from a Down’s syndrome patient by use of SV40T-Ag and hTERT gene transfection. Expressions of SV40T-Ag and hTERT were observed in periodontal ligament cell-derived immortalized cells established from healthy (STPDL) and Down’s syndrome patient (STPDLDS) samples. Primary cultured periodontal ligament cells obtained from a healthy subject (pPDL) had a limited number of population doublings (DSCR-1 in STPDLDS is greater than that in STPDL. These results suggest that the newly established STPDLDS cell line may be a useful tool for study of periodontal disease in Down’s syndrome patients.

Background and Aim Preoperative histological evaluation of pancreatic neoplasms is important for guiding the resection strategy and preventing postoperative adverse events. However, conventional endoscopic methods have technical limitations that reduce the accuracy of the histopathological examination. Probe electrospray ionization mass spectrometry (PESI‐MS) may be a useful technique for rapidly evaluating small specimens. Methods This single‐center prospective study included patients with pancreatic neoplasms between October 2018 and December 2019. Pancreatic ductal adenocarcinoma (PDAC) specimens were obtained via endoscopic ultrasound‐guided fine needle aspiration (EUS‐FNA) and non‐neoplastic tissue was obtained via surgery. Specimens were subjected to PESI‐MS and the mass spectra were analyzed using partial least squares regression‐discriminant analysis. Results The study included 40 patients with 20 nonneoplastic specimens and 19 PDAC specimens (1 case of neuroendocrine carcinoma was omitted). All nonneoplastic specimens were sufficient for PESI‐MS analysis, although only 7 of 19 PDAC specimens were sufficient for PESI‐MS analysis because of poor sample quality or insufficient quantity (, Endoscopic ultrasound‐guided fine needle aspiration is a common technique for obtaining specimens for histopathological evaluation, although it does not always provide a sufficient specimen quantity and quality to support an accurate diagnosis. However, the specimens might be evaluable using probe electrospray ionization mass spectrometry (PESI‐MS). The present study revealed PESI‐MS might be a useful technique for evaluating pancreatic specimens and guiding the diagnosis and treatment, even in cases where only small specimens are available.

BackgroundProbe 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).MethodsA 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.ResultsThis 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 P ConclusionThe 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.

Background: Most pancreatic cancers are found at progressive stages when they cannot be surgically removed. Therefore, a highly accurate early detection method is urgently needed. Methods: This study analyzed serum from Japanese patients who suffered from pancreatic ductal adenocarcinoma (PDAC) and aimed to establish a PDAC-diagnostic system with metabolites in serum. Two groups of metabolites, primary metabolites (PM) and phospholipids (PL), were analyzed using liquid chromatography/electrospray ionization mass spectrometry. A support vector machine was employed to establish a machine learning-based diagnostic algorithm. Results: Integrating PM and PL databases improved cancer diagnostic accuracy and the area under the receiver operating characteristic curve. It was more effective than the algorithm based on either PM or PL database, or single metabolites as a biomarker. Subsequently, 36 statistically significant metabolites were fed into the algorithm as a collective biomarker, which improved results by accomplishing 97.4% and was further validated by additional serum. Interestingly, specific clusters of metabolites from patients with preoperative neoadjuvant chemotherapy (NAC) showed different patterns from those without NAC and were somewhat comparable to those of the control. Conclusion: We propose an efficient screening system for PDAC with high accuracy by liquid biopsy and potential biomarkers useful for assessing NAC performance.

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)‐1β, 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‐1β, 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‐1β, 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.

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.

Background Several pro-oncogenic signals, including transforming growth factor beta (TGF-β) 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-β signalling status in head and neck squamous cell carcinoma (HNSCC). A total of 240 and 90 mass spectra were obtained from TGF-β-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-β1-stimulated cells from untreated cells. In clinical human HNSCC tissues, this approach achieved determination of tumour areas with activated TGF-β 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-β-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-β.

The articular cartilage is an avascular tissue, and oxygen tensions in its superficial and deeper zones are estimated to be 6% and 1%. Degeneration of the articular cartilage begins from the surface zone in osteoarthritis. We previously reported that monocarboxylate transporter-1, a transmembrane transporter for monocarboxylates, played an essential role in the interleukin-1β-induced expression of NADPH oxidase-2, a reactive oxygen species-producing enzyme, and reactive oxygen species-dependent death of mouse chondrogenic ATDC5 cells cultured in a normal condition (20% oxygen). Here, we investigated the effect of oxygen tension on interleukin-1β-induced events described above in ATDC5 cells. Interleukin-1β induced the death of ATDC5 cells under 20% and 6% oxygen but did not under 2% and 1% oxygen. Interleukin-1β induced Mct1 (monocarboxylate transporter-1 gene) and Nox2 (NADPH oxidase-2 gene) mRNAs' expression under 20% oxygen in 24 h, respectively, but not under 2% oxygen. On the other hand, a 24-h incubation with interleukin-1β upregulated the expression of Nos2 (inducible nitric oxide synthase gene) mRNA irrespective of oxygen tension. Furthermore, inhibition of I-κB kinase suppressed the interleukin-1β-induced expression of Mct1 mRNA in the cells cultured under 20% and 2% oxygen, indicating NF-κB plays an essential role in the induction of the Mct1 gene expression. The results suggest that interleukin-1β induces monocarboxylate transporter-1 in an oxygen tension-dependent manner required for cell death in ATDC5 cells. These results might explain some part of the degenerative process of the articular cartilage, which begins from its superficial zone in the pathogenesis of osteoarthritis.

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-1β (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

Biomarkers may be of value for the early detection of gastric cancer (GC) and the preoperative identification of tumor characteristics to guide treatment strategies. The present study analyzed the expression levels of phospholipids in plasma from patients with GC using liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) to detect reliable biomarkers for GC. Furthermore, combining the results with a machine learning strategy, the present study attempted to establish a diagnostic system for GC. A total of 20 plasma samples from preoperative patients with GC and 16 plasma samples from tumor-free patients (controls) were selected from our biobank named 'SHINGEN (Yamanashi Biobank of Gastroenterological Cancers)', which includes a total of 1,592 plasma samples, and were analyzed by LC/ESI-MS. The obtained data were discriminated using a machine learning-based diagnostic algorithm, whose discriminant ability was confirmed through leave-one-out cross-validation. Using LC/ESI-MS, the levels of 236 lipid molecules were determined. Biomarker analysis revealed that a few lipids that were downregulated in the GC group could discriminate between the GC and control groups. Whole lipid composition analysis using partial least squares regression revealed good discrimination ability between the GC and control groups. Integrative analysis of all molecules using the aforementioned machine learning method exhibited a diagnostic accuracy of 94.4% (specificity, 93.8%; sensitivity, 95.0%). In conclusion, the outcomes of the present study suggested the potential future application of the aforementioned system in clinical settings. By accumulating more reliable data, the present system will be able to detect early-stage cancer and will be capable of predicting the efficacy of each therapeutic strategy.

A moving string sampling probe and a new ESI based ionization source that can be readily incorporated into the existing endoscopes are developed for performing in vivo mass spectrometry during the endoscopic procedure. The medical-grade silk suture driven by a stepping motor is used to perform the sampling on the region of interest when the probe head is brought gently into contact with the surface of the gastrointestinal tissue. The tissues and the compounds adhered to the sampling string are transported to an ionization region inside the ion inlet tube in which they are extracted and ionized by the charging droplets generated from an electrospray outside the ion inlet. Since the extraction/ionization and sampling processes are isolated, organic solvents, high voltage (HV), and heating can be used for the optimization of ionization without compromising the biocompatibility of the sampling probe. The demonstration of the in vivo analysis of the gastric mucosa of a mouse is performed using a 2 m long gastrointestinal endoscope.

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ratios (m/z), which are useful to deduce molecular weights and structures. While it is essentially a destructive analytical method, recent advancements in the ambient ionization technique have enabled us to acquire data while leaving tissue in a relatively intact state in terms of integrity. Probe electrospray ionization (PESI) is a so-called direct method because it does not require complex and time-consuming pretreatment of samples. A fine needle serves as a sample picker, as well as an ionization emitter. Based on the very sharp and fine property of the probe tip, destruction of the samples is minimal, allowing us to acquire the real-time molecular information from living things in situ. Herein, we introduce three applications of PESI-MS technique that will be useful for biomedical research and development. One involves the application to solid tissue, which is the basic application of this technique for the medical diagnosis. As this technique requires only 10 mg of the sample, it may be very useful in the routine clinical settings. The second application is for in vitro medical diagnostics where human blood serum is measured. The ability to measure fluid samples is also valuable in various biological experiments where a sufficient volume of sample for conventional analytical techniques cannot be provided. The third application leans toward the direct application of probe needles in living animals, where we can obtain real-time dynamics of metabolites or drugs in specific organs. In each application, we can infer the molecules that have been detected by MS or use artificial intelligence to obtain a medical diagnosis.

Although many variants of the parathyroid hormone 1 receptor (PTH1R) gene are known to be associated with primary failure of eruption (PFE), the mechanisms underlying the link remains poorly understood. We here performed functional analyses of PTH1R variants reported in PFE patients—namely, 356C>T (P119L), 395C>T (P132L), 439C>T (R147C), and 1148G>A (R383Q)—using HeLa cells with a lentiviral vector-mediated genetic modification. Two particular variants, P119L and P132L, had severe reduction in a level of N-linked glycosylation when compared with wild-type PTH1R, whereas the other 2 showed modest alteration. PTH1R having P119L or P132L showed marked decrease in the affinity to PTH1-34, which likely led to severely impaired cAMP accumulation upon stimulation in cells expressing these mutants, highlighting the importance of these 2 amino acid residues for ligand-mediated proper functioning of PTH1R. To further gain insights into PTH1R functions, we established the induced pluripotent stem cell (iPSC) lines from a patient with PFE and the heterozygous P132L mutation. When differentiated into osteoblastic-lineage cells, PFE-iPSCs showed no abnormality in mineralization. The mRNA expression of RUNX2, SP7, and BGLAP, the osteoblastic differentiation-related genes, and that of PTH1R were augmented in both PFE-iPSC-derived cells and control iPSC-derived cells in the presence of bone morphogenetic protein 2. Also, active vitamin D3 induced the expression of RANKL, a major key factor for osteoclastogenesis, equally in osteoblastic cells derived from control and PFE-iPSCs. In sharp contrast, exposure to PTH1-34 resulted in no induction of RANKL mRNA expression in the cells expressing P132L variant PTH1R, consistent with the idea that a type of heterozygous PTH1R gene mutation would spoil PTH-dependent response in osteoblasts. Collectively, this study demonstrates a link between PFE-associated genetic alteration and causative functional impairment of PTH1R, as well as a utility of iPSC-based disease modeling for future elucidation of pathogenesis in genetic disorders, including PFE.

Osteocytes regulate bone remodeling, especially in response to mechanical loading and unloading of bone, with nitric oxide reported to play an important role in that process. In the present study, we found that 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP), a second messenger of nitric oxide in various types of cells, was produced by osteocytes in bone tissue as well as cultured osteocytic Ocy454 cells. The amount of 8-nitro-cGMP in Ocy454 cells increased during incubation with parathyroid hormone or prostaglandin E2, both of which are known to upregulate receptor activator of nuclear factor-κB ligand (RANKL) mRNA expression in osteocytes. On the other hand, exogenous 8-nitro-cGMP did not have effects on either the presence or absence of these bioactive substances. Furthermore, neither an inhibitor of nitric oxide synthase nor 8-bromo-cGMP, a cell-permeable analog of cGMP, showed remarkable effects on mRNA expression of sclerostin or RANKL. These results indicate that neither nitric oxide nor its downstream compounds, including 8-nitro-cGMP, alone are sufficient for induction of functional changes in osteocytes.

Nephronectin (Npnt), an extracellular matrix protein, is known to be a ligand of integrin α8β1, 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.

Distant metastasis remarkably worsens the prognoses of malignant melanoma patients. Toll-like receptors (TLRs) recognize molecules derived from many types of pathogens and activate the innate intravital immune system. In this study, we examined the effects of R848, a TLR7 ligand, on bone invasion by malignant melanoma cells. Mice underwent transplantation with cells of a malignant melanoma cell line B16F10, and were also administered R848 every three days. Hindlimbs were obtained 13 days after transplantation and invasion of bone marrow by B16F10 cells was evaluated. ELISA was used to determine the concentrations of cytokines in mouse serum and in the culture medium from bone marrow macrophages (BMMs) in the presence or absence of R848. In addition, MTS assays were used to examine the effects of media from BMM cultures on the proliferation of B16F10 cells. The rate of infiltration by B16F10 cells and the area of invasion were significantly reduced with R848 administration. Furthermore, serum levels of IL-6, IL-12, and IFN-γ were significantly increased in mice administered R848, with the same trend observed in the culture medium of BMMs treated with R848. In addition, B16F10 cell proliferation was suppressed by the addition of medium from cultured BMMs treated with R848. Neutralization by antibodies against IL-6, IL-12, and IFN-γ abrogated the suppression of proliferation of B16F10 cells by culture medium from BMMs treated with R848. Our results suggest that R848 drives the production of IL-6, IL-12, and IFN-γ in BMMs, which reduces proliferation and bone invasion by B16F10 cells.

Osteoclasts are multinucleated giant cells differentiated from monocyte-macrophage-lineage cells under stimulation of receptor activator of nuclear factor κ-B (RANK) ligand (RANKL) produced by osteoblasts and osteocytes. Although it has been reported that nitric oxide (NO) and reactive oxygen species (ROS) are involved in this process, the mechanism by which these labile molecules promote osteoclast differentiation are not fully understood. In this study, we investigated the formation and function of 8-nitro-cGMP, a downstream molecule of NO and ROS, in the process of osteoclast differentiation in vitro. 8-Nitro-cGMP was detected in mouse bone marrow macrophages and osteoclasts differentiated from macrophages in the presence of RANKL. Inhibition of NO synthase suppressed the formation of 8-nitro-cGMP as well as RANKL-induced osteoclast differentiation from macrophages. On the other hand, RANKL-induced osteoclast differentiation was promoted by addition of 8-nitro-cGMP to the cultures. In addition, 8-nitro-cGMP enhanced the mRNA expression of RANK, the receptor for RANKL. However, 8-bromo-cGMP, a membrane-permeable derivative of cGMP, did not have an effect on either RANKL-induced osteoclast differentiation or expression of the RANK gene. These results suggest that 8-nitro-cGMP is a novel positive regulator of osteoclast differentiation, which might help to explain the roles of NO and ROS in osteoclast differentiation.