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Arbutin, a natural polyphenol, inhibits OS cell viability, invasion and migration by down‐regulating MTHFD1L expression. miR‐338‐3p directly targets MTHFD1L. This regulatory action is associated with the inactivation of the AKT/mTOR signaling pathway., Arbutin, a glycoside extracted from the plant Arctostaphylos uva‐ursi, has been previously reported to possess antioxidant, anti‐inflammatory and anticancer effects. Here, we investigated whether arbutin affects the proliferation of the cells of the osteosarcoma (OS) cell lines MG‐63 and SW1353. Arbutin suppressed OS cell viability in a dose‐ and time‐dependent manner, as shown by Cell Counting Kit‐8 assay. Furthermore, arbutin exposure decreased the protein levels of MTHFD1L, CCND1 and phosphorylated‐protein kinase B (AKT)/phosphorylated‐mammalian target of rapamycin (mTOR). Potential upstream miRNAs of MTHFD1L were predicted using TargetScan, PICTAR5, miRanda and miRWalk. We performed luciferase activity assays to show that miR‐338‐3p directly targets and negatively regulates the expression of MTHFD1L. Knockdown of miR‐338‐3p promoted cell invasion, migration and proliferation in arbutin‐treated OS cells via MTHFD1L. In summary, our data suggest that arbutin inhibits OS cell proliferation, migration and invasion via miR‐338‐3p/MTHFD1L and by inactivating the AKT/mTOR pathway.

The aim of the present study was to investigate the role of microRNA-21 (miR-21) in regulating the classical WNT/β-catenin signaling pathway by targeting low-density lipoprotein-related receptor 6 (LRP6) in non-alcoholic fatty liver disease (NAFLD). For this purpose, we established a NAFLD model by feeding C57BL/6J mice a methionine-choline-deficient diet. Antagomir-21 was then injected via the tail vein, and the expression levels of WNT/β-catenin signaling pathway-related proteins, such as LRP6, glycogen synthase kinase-3β (GSK3β), p-β-catenin, β-catenin and the downstream protein, peroxisome proliferator-activated receptor γ (PPAR-γ), and lipid metabolism-related genes, including sterol regulatory element-binding transcription factor 1c (SREBP1c), fatty acid synthase (FAS), carnitine palmitoyl transferase 1α (CPT1α) and adenosine 5-monophosphate (AMP)-activated protein kinase α (AMPKα), were detected. The results revealed that in the NAFLD model, LRP6 expression was negatively associated with miR-21 expression. After antago-nizing the expression of miR-21, the protein level of LRP6 was increased. In addition, the WNT/β-catenin signaling pathway was activated, and lipid accumulation and inflammation were alleviated in the liver. However, the expression of PPAR-γ was not inhibited following the upregulation of the WNT signaling pathway. Taken together, the results of this study demonstrate that the inhibition of miR-21 expression can alleviate NAFLD by targeting LRP6 to activate the WNT/β-catenin signaling pathway.

Osteosarcoma (OS) is the most frequent primary malignancy initially in bone with multiple genomic aberrations. Methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L) is linked with the progression of diverse tumors. However, its function in OS is not understood completely. The expression pattern and prognostic significance of MTHFD1L in OS tissues were analyzed based on GEO database. The expression level of MTHFD1L in OS cell lines was explored by qRT-PCR. The cell proliferation, colony formation ability, invasion as well as migration in OS cells after MTHFD1L knockdown were determined using cell counting kit 8 (CCK-8) assay, colony formation and transwell methods. GSEA analysis was performed to predict the underlying mechanisms of MTHFD1L in OS development. Furthermore, the western blot was utilized to study the influence of MTHFD1L on AKT/mTOR pathway. Our results indicated that MTHFD1L expression was significantly up-regulated in OS tissues and cells compared with normal tissues and cells. High expression of MTHFD1L could lead to poor prognosis of OS patients. Cell proliferation, colony formation ability, migration and invasion were blocked because of reduced MTHFD1L

Konjac glucomannan (KGM) electrogels were successfully prepared under alternating current (AC) in the presence of potassium chloride (KCl). The structure of the gels was studied using Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, and scanning electron microscopy (SEM). A single-factor experiment was performed to optimize the preparation of the gels. Our results showed that KGM was degraded under AC and partially deacetylated. KGM and KCl formed the structure , and electrogels with porous structures retained some acetyl groups. Furthermore, as the KCl concentrations, voltages, time, and KGM concentrations increased, the viscoelastic moduli of the gels increased; the moduli decreased when the KCl concentrations, voltages, and time exceeded critical values.

This chapter introduces the basic principle of biomineralization and the theory of biological mineralization interface control, and the progress of natural bone formation and mineralization. Natural bone is composed of mineralized collagen (MC) by a series of self-assembly and molecular rearrangement and subsequent processing and modification of the formation. The natural bone formation and biological mineralization are closely related. Preliminary mineralization of collagen and its surface nucleation sites. Biomimetic preparation of hydroxyapatite MC fibers are a long-sought bone graft material in many laboratories because of their composition, structure, and properties. Natural bones are similar and have good bioactivity and biodegradability. There are many reports on the structure, properties, synthesis mechanism and application of collagen/HA and with the increasing acceptance of biomimetic materials, several collagen and hydroxyapatite composite materials have gained market access permits, and more and more doctors are accepting them.

Extensive evidence indicates that oxidative stress plays a pivotal role in the development of osteoporosis. We show that naringin, a natural antioxidant and anti-inflammatory compound, effectively protects human adipose-derived mesenchymal stem cells (hADMSCs) against hydrogen peroxide (H2O2)-induced inhibition of osteogenic differentiation. Naringin increased viability of hAMDSCs and attenuated H2O2-induced cytotoxicity. Naringin also reversed H2O2-induced oxidative stress. Oxidative stress induced by H2O2 inhibits osteogenic differentiation by decreasing alkaline phosphatase (ALP) activity, calcium content and mRNA expression levels of osteogenesis marker genes RUNX2 and OSX in hADMSCs. However, addition of naringin leads to a significant recovery, suggesting the protective effects of naringin against H2O2-induced inhibition of osteogenic differentiation. Furthermore, the H2O2-induced decrease of protein expressions of β-catenin and clyclin D1, two important transcriptional regulators of Wnt-signaling, was successfully rescued by naringin treatment. Also, in the presence of Wnt inhibitor DKK-1, naringin is no longer effective in stimulating ALP activity, increasing calcium content and mRNA expression levels of RUNX2 and OSX in H2O2-exposed hADMSCs. These data clearly demonstrates that naringin protects hADMSCs against oxidative stress-induced inhibition of osteogenic differentiation, which may involve Wnt signaling pathway. Our work suggests that naringin may be a useful addition to the treatment armamentarium for osteoporosis and activation of Wnt signaling may represent attractive therapeutic strategy for the treatment of degenerative disease of bone tissue.

The objective of this study was to investigate the prevalence of the cfr gene in Escherichia coli isolates from domestic animals in Northeast China and to characterize the cfr-containing plasmids. Between June 2015 and April 2016, 370 E. coli isolates were collected from pigs, chickens, and dairy cows in Northeast China. Among these, 111 were florfenicol resistant, including 109 isolates carrying the floR gene and 6 positives for cfr. The prevalence of cfr in E. coli isolates from the four northeast provinces in China was 1.6% (6/370), which was higher than that previously reported (0.08% and 0.5%). All six cfr-containing E. coli isolates were highly resistant to florfenicol (100%), cefotaxime (100%), and fosfomycin (100%). Complete sequence analysis of two cfr-carrying plasmids revealed high homology of the IncX4-type pEC14cfr plasmid with two other cfr-harboring plasmids, pSD11 and pGXEC6, found in swine E. coli isolates from southern China. pEC14cfr-like plasmids have been isolated in five provinces in southern and northern China. The isolation sites were up to 2700 kilometers apart, implying that pEC14cfr-like plasmids are likely to be national epidemic cfr-carrying plasmids that mediate the dissemination of cfr in China. Moreover, the genetic structure (IS26-IS26-cfr-rec-pre/mob-ramA-IS26) of the second cfr-carrying plasmid, IncF14:A-:B- pEC295cfr, represents a novel genetic environment for cfr identified for the first time in the present study. Sequence homology analysis indicated that the cfr-carrying element was most likely introduced into a cfr-negative pEC12 plasmid backbone, which evolved into the cfr-carrying vector, pEC295cfr. Moreover, isolation of the IncF14:A-:B- pEC295cfr plasmid harboring cfr suggests that IncFII plasmids maybe have become additional effective vehicles for cfr dissemination. These results highlight the importance of surveying the prevalence of IncX4 and IncFII plasmids in gram-negative bacteria, especially in swine E. coli isolates.

At the time of implanting bone-related implants into human body, a variety of biological responses to the material surface occur with respect to surface chemistry and physical state. The commonly used biomaterials (e.g. titanium and its alloy, Co-Cr alloy, stainless steel, polyetheretherketone, ultra-high molecular weight polyethylene and various calcium phosphates) have many drawbacks such as lack of biocompatibility and improper mechanical properties. As surface modification is very promising technology to overcome such problems, a variety of surface modification techniques have been being investigated. This review paper covers recent advances in surface modification techniques of bone-related materials including physicochemical coating, radiation grafting, plasma surface engineering, ion beam processing and surface patterning techniques. The contents are organized with different types of techniques to applicable materials, and typical examples are also described.

The collagenase cathepsin K has been shown important in the pathogenesis of rheumatoid arthritis (RA). Icariin is the major pharmacologically active flavonol diglycoside of Herba Epimedii, an herb used in Chinese traditional medicine to treat arthritis. We investigated whether icariin can inhibit the protease activity of cathepsin K and its effects on a murine model of collagen-induced arthritis (CIA). Six-week old female BALB/C mice were immunized with type II collagen and treated with vehicle alone icariin (25mg/kg) for 21 days; a control remained untreated. Serum concentrations of type I collagen C-terminal telopeptide (CTX-I) and cartilage oligomeric matrix protein (COMP) and urinary concentrations of deoxypyridinoline (DPD) were measured, and disease severity was assessed. Compared with immunized, untreated mice, immunized icariin-treated mice had significantly lower urinary DPD (~25%, p

A simple, rapid, and selective method to determine the concentration of mildronate in human plasma and urine using ultra performance liquid chromatography–tandem mass spectrometry (UPLC-MS-MS) was developed and validated. The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring mode via electrospray ionization at m/z 147.2–58.0 for mildronate and m/z 147.2–87.8 for the internal standard, carbachol. The UPLC separation was carried out with a UPLC BEH HILIC column. The mobile phase consisted of 0.08% formic acid in 30 mM ammonium acetate solution and acetonitrile (23:77, v/v). Plasma samples were extracted from plasma by protein precipitation and urine samples were diluted with the mobile phase. The analysis time was 3.5 min for each sample. Linear calibration curves ranged from 0.10 to 100.00 μg mL−1 in human plasma and 0.50 to 600.00 μg mL−1 in urine. The method had been successfully applied to a pharmacokinetic study in healthy volunteers. After single intravenously administration of 250, 500, and 750 mg mildronate, the elimination half-life (t 1/2) were (2.74 ± 0.67), (4.86 ± 0.82) and (5.16 ± 0.77) h, respectively. The t 1/2 for the 250 mg dose did vary significantly with other dosages (P

A solid-phase extraction–liquid chromatographic–tandem mass spectrometry method for the determination of nalbuphine concentrations in human plasma has been developed. Samples (1 mL) were extracted using a Strata™-X solid phase extraction cartridges. Chromatographic separation of nalbuphine and naloxone (internal standard) was achieved on a Phenomenex Kinetex PFP (2.6 μm, 100 A, 100 × 2.1 mm) column using a mobile phase consisting of 0.1% formic acid, 15 mM ammonium acetate in deionized water and acetonitrile (60:40, v/v). The flow rate was 0.3 mL/min and the total run time was 2 min. Detection of the analytes was achieved using positive ion electrospray ionization via multiple reactions monitoring mode. The mass transitions were m/z 358 340 for nalbuphine and m/z 328 310 for naloxone. The assay was linear over the concentration range 0.50–500.00 ng/mL, with correlation coefficients ≥0.995. The lower limit of quantitation was set at 0.5 ng/mL plasma based on an average signal-to-noise ratio of 44.79. The intra- and inter-day precision was less than 8.07% in terms of relative standard deviation and accuracy ranged from 94.97 to 106.29% at all quality control levels. The method was applied successfully to determine nalbuphine concentrations in human plasma samples obtained from subjects receiving intravenous administration of nalbuphine. The method is rapid, sensitive, selective and directly applicable to human pharmacokinetic studies involving nalbuphine. Copyright © 2011 John Wiley & Sons, Ltd.

According to physiological and biochemical characteristics of Leptospirillum ferriphilum, a strain of object bacteria was isolated successfully. Bacteria were enriched by selective liquid medium and plated on designed single-layered agar solid medium. Colony was cultured and bacteria were collected. The morphologies of the object bacteria were observed using crystal violet staining, scanning electron microscope(SEM) and transmission electron microscope (TEM). The result of 16S rDNA identification shows that this bacterium belongs to Leptospirillum ferriphilum and it is named as Leptospirillum ferriphilum strain D1. These results indicate that this new single-layered agar solid medium is efficient and simple for isolation of Leptospirillum ferriphilum. Additionally, physiological-biochemical characteristics show that the optimum initial pH value and its growth temperature are 1.68 and 40 °C, respectively. The culture of it is used to leach a complex concentrate chalocpyrite, the leaching efficiencies of copper and iron are 1.93% and 13.74%, respectively, and it is more effective than the A. ferrooxidans culture in the leaching of the complex concentrate chalcopyrite.

Bacterial ghosts offer a new avenue for the study of inactivated vaccines. However, for many years the mechanism of genetic inactivation was controversial. To obtain mouse monoclonal antibodies (mAbs) against protein E will allow the observation of its location and dynamic expression to expand understanding of the lysis mechanism. In this study, a His-tagged ΔE fusion protein expressed in bacteria was used as the immunogen, and mAbs targeting protein E were produced by the hybridoma technique and selected by enzyme-linked immunosorbent assay using GST-E and GST-ΔE as coating proteins. Purified mAbs from mouse ascites were screened against a phage-displayed random dodecapeptide library (Ph.D.-12). After three rounds of biopanning, 30 phage clones were randomly selected and sequenced, and their amino acids were deduced. One epitope showed a good match with protein E at 57–63aa (KPLN--R) and the synthetic decapeptide Epep (VRLKPLNCSR) strongly inhibited combination of E-A5 with E fusion proteins. Immunofluorescence microscopy indicated specific immunoreactivity of E-A5 with Escherichia coli-expressed native protein E. The novel mAbs may be of great potential value in analysis of the function and lysis pathway of protein E and other relative phages and in evaluation of E as potential marker of bacterial ghosts.

Purpose: To develop a sensitive and rapid method for the simultaneous determination of curcumin and piperine in human plasma.Methods: The method was based on high-performance liquid chromatography (HPLC) with electrospray ionization tandem mass spectrometer (ESI-MS/MS) detection in positive ionization mode. The analytes and internal standard were isolated from acidified plasma using liquid–liquid extraction (LLE). The organic extracts were evaporated, reconstituted in mobile phase and injected into the HPLC-MS/MS system. The analytes were chromatographed on a XB-C8 analytical column and MS-MS detection was performed on an electrospray ionization tandem mass spectrometer operated in multiple reaction monitoring (MRM) mode. Precursor→product combinations of m/z 369.9→177.0, 286.3→201.1 and 285.6→193.1 were used to quantify curcumin, piperine and the internal standard (IS), respectively. The assay was validated in the concentration range of 1.0 – 100.0 ng/ml for curcumin and 0.5 – 800.0 ng/ml for piperine using 0.5 ml of plasma.Results: The lowest limit of quantification (LLOQ) for curcumin and piperine was 1.00 and 0.50 ng/ml, respectively. The precision of the assay (expressed as coefficient of variation, CV) was < 12.6 % at all concentrations within the standard curve range, with adequate assay accuracy. Stability data revealed that the drugs were stable in plasma under various test conditions.Conclusion: The method is highly selective and rugged for the estimation of curcumin and piperine in human plasma and would be applicable to toxicokinetic, pharmacokinetic, bioavailability, and bioequivalence studies.Keywords: Curcumin, Piperine; HPLC-MS/MS, Simultaneous determination

The expression and regulation of the MHC class II and interleukin-1 genes was studied during the differentiation of bone marrow stem cells into macrophages. Bone marrow cells were cultured in the presence of CSF-1 and allowed to differentiate for 3, 5, 7, and 9 d. Cells were stimulated with either medium, endotoxin (1 gg/ml) or interferon-r (IFN-r, 100 U/ml) 24 hr prior to the end of the culture period. Upon completion of culture, total cytoplasmic RNA was isolated from the adherent bone marrow-derived macrophages (BMDM) and subjected to Northern blot analysis. The A and E mRNA was constitutively expressed by medium treated BMDM after 3, g, and/d, but by 9 d the mRNA had declined to values lower than those observed on d 3. IFN-r treatment augmented A and E mRNA at each time point, whereas endotoxin treatment decreased values after 5 and 7 d. Additional studies demonstrated that IFN-r was not being produced by either the non-adherent or adherent cells nor was the production of PGE responsible for the decline in MHC class II gene expression. IL-1 mRNA was only detected in those cultures stimulated with endotoxin; medium alone or IFN-r treatment did not induce IL-1 mRNA transcription. Antigen-primed lymph node T cells and T cell hybridomas demonstrated that the ability of BMDM to function as antigen presenting cells was limited by the differentiation-specific regulation of both class II and IL-1 gene products. Exogenous Eα and Aβ genes were inserted into bone marrow stem cells using either a modified calcium phosphate transfection or an electroporation procedure and their expression was monitored using Northern blot and immunofluorescence analysis. However, there was no alteration in the expression of membrane IL-1 following trans-fection. We currently are examining the expression of regulatory factors which are responsible for the differentiation-specific regulation of MHC class II and IL-1 gene expression.