The stiffness and collagen control differentiation of osteoclasts with an altered expression of c-Src in podosome.

Osteoclasts are hematopoietic cells attached to the bones containing type I collagen-deposited hydroxyapatite during bone resorption. Two major elements determine the stiffness of bones: regular calcified bone (bone that is resorbable by osteoclasts) and un-calcified osteoid bone (bone that is un-resorbable by osteoclasts). The osteolytic cytokine RANKL promotes osteoclast differentiation; however, the roles of the physical interactions of osteoclasts with calcified and un-calcified bone at the sealing zones and the subsequent cellular signaling remain unclear. In this study, we investigated podosomes, actin-rich adhesion structures (actin-ring) in the sealing zone that participates in sensing hard stiffness with collagen in the physical environment during osteoclast differentiation. RANKL-induced osteoclast differentiation induction was promoted when Raw264.7 cells were cultured on collagen-coated plastic dishes but not on non-coated plastic dishes, which was associated with the increased expression of podosome-related genes and Src. In contrast, when cells were cultured on collagen gel, expression of podosome-related genes and Src were not upregulated. The induction of podosome-related genes and Src requires hard stiffness with RGD-containing substratum and integrin-mediated F-actin polymerization. These results indicate that osteoclasts sense both the RGD sequence and stiffness of calcified collagen through their podosome components regulating osteoclast differentiation via the c-Src pathway.

Effects of 4'-Demethylnobiletin and 4'-Demethyltangeretin on Osteoclast Differentiation In Vitro and in a Mouse Model of Estrogen-Deficient Bone Resorption.

Citrus nobiletin (NOB) and tangeretin (TAN) show protective effects against disease-related bone destruction. We achieved demethylation of NOB and TAN into 4'-demethylnobiletin (4'-DN) and 4'-demethyltangeretin (4'-DT) using enzyme-manufacturing methods. In this study, we examined the effects of 4'-DN and 4'-DT on in vitro osteoclast differentiation, and on in vivo osteoporotic bone loss in ovariectomized (OVX) mice. 4'-DN and 4'-DT clearly suppressed the osteoclast differentiation induced by interleukin IL-1 or RANKL treatment. 4'-DN and 4'-DT treatments resulted in higher inhibitory activity in osteoclasts in comparison to NOB or TAN treatments. RANKL induced the increased expression of its marker genes and the degradation of IκBα in osteoclasts, while these were perfectly attenuated by the treatment with 4'-MIX: a mixture of 4'-DN and 4'-DT. In an in silico docking analysis, 4'-DN and 4'-DT directly bound to the ATP-binding pocket of IKKß for functional inhibition. Finally, the intraperitoneal administration of 4'-MIX significantly protected against bone loss in OVX mice. In conclusion, 4'-DN, 4'-DT and 4'-MIX inhibited the differentiation and function of bone-resorbing osteoclasts via suppression of the NF-κB pathway. Novel 4'-DN, 4'-DT and 4'-MIX are candidates for maintaining bone health, which may be applied in the prevention of metabolic bone diseases, such as osteoporosis.

Establishment of a Triple Quadrupole HPLC-MS Quantitation Method for Dystrophin Protein in Mouse and Human Skeletal Muscle.

Duchenne muscular dystrophy (DMD) is the most common type of neuromuscular disease caused by mutations in the DMD gene encoding dystrophin protein. To quantitively assess human dystrophin protein in muscle biopsy samples, it is imperative to consistently detect as low as 0.003% of the dystrophin protein relative to the total muscle protein content. The quantitation of dystrophin protein has traditionally been conducted using semiquantitative immunoblotting or immunohistochemistry; however, there is a growing need to establish a more precise quantitative method by employing liquid chromatography-mass spectrometry (LC-MS) to measure dystrophin protein. In this study, a novel quantification method was established using a mouse experiment platform applied to the clinical quantification of human dystrophin protein. The method using a spike-in approach with a triple quadrupole LC-MS quantitated the amount of dystrophin in wild-type and human DMD transgenic mice but not in DMD-null mice. In conclusion, we established a quantitating method of dystrophin using HPLC-LC-MS with a novel spike-in approach. These results indicate that our methodology could be applied to several LC-MS devices to enable the accurate measurement of dystrophin protein in patients with DMD.

Influence of technique used to attach the infusion set to peristaltic finger smart-pumps on dispensing time: an experimental study.

BACKGROUND: Infusion sets designed for peristaltic finger smart pumps (PFSPs) are necessary for the pumps' accurate handling. We previously found that medication dispensing is occasionally incomplete following the calculated infusion time when using certain combinations of PFSPs and infusion sets at a Japanese hospital. Thus, in this study, we investigated the cause of this observed delay by determining the effect of infusion set attachment technique on dispensing time using a combination of three kinds of PFSPs and five kinds of polyvinyl chloride (PVC) and polybutadiene (PB) infusion sets. METHODS: PFSPs with their exclusive infusion sets were used. The PVC and PB infusion sets were either not stretched or stretched to 1-3 cm and attached to the PFSP's liquid delivery system. PFSP dispensing rates were set at 25-400 mL/h. The primary outcome was the time required to dispense 100 mL of saline in a volumetric flask. RESULTS: The complete dispensing time correlated with the input time for all equipment combinations when the infusion sets were not stretched before attachment to the PFSP (R2 = 0.9998-1.0000). When stretched, the complete dispensing time was longer than the input time (P < 0.01-0.05, analysis of variance with Tukey-Kramer multiple comparisons). The maximum dispensing time extension ratio for the PVC and PB infusion sets was 141.8% and 113.0%, respectively. CONCLUSION: Certain attachment techniques for infusion sets can adversely prolong drug dispensing time. As such, pharmacists should provide medical staff with information about the devices used to administer drugs, as well as about the drugs themselves.

A randomized phase II study of gemcitabine plus Z-360, a CCK2 receptor-selective antagonist, in patients with metastatic pancreatic cancer as compared with gemcitabine plus placebo.

BACKGROUND: We investigated the efficacy and safety of 60, 120, or 240 mg of Z-360, which is a highly potent cholecystokinin2-receptor-selective antagonist, combined with gemcitabine in patients with metastatic pancreatic cancer. METHODS: Patients were randomly assigned in a 1:1:1:1 ratio to one of four treatment groups. Patients received 1000 mg/m2 gemcitabine for each cycle and Z-360 tablets of 60 mg (GZ 60 mg group), 120, 240 mg or placebo tablets (Gem group) orally twice daily. The primary endpoint was overall survival (OS). RESULTS: The median OS was 1.3 months longer in the GZ 60 mg group compared with the Gem group (8.5 vs. 7.2 months) and the risk of death was reduced by 19% compared with the Gem group, although there were no statistically significant differences. The study treatments were well tolerated. CONCLUSIONS: In this Phase II study, no statistically significant differences between the GZ groups and Gem group were detected in any analysis. However, Z-360 in dose of 60 mg tends to improve OS in patients with metastatic pancreatic cancer with low toxic effect. Further exploratory trials with other agents such as gemcitabine plus nab-paclitaxel might be beneficial.