ELISA array-based immunoassay for multiplex detection of osteoporosis-relevant biomarkers.

Aim: This study aimed to develop an ELISA array-based immunoassay for the simultaneous detection of osteoporosis-relevant biomarkers. Methods: ELISA array was constructed and its performance was evaluated using commercial antigens. The validity was further confirmed using traditional ELISA. Results: The constructed ELISA array showed good repeatability. The linear ranges and limits of detection for the four markers mentioned above were 0.01-8 ng/ml (1 pg/ml), 5.0-1000 ng/ml (1 ng/ml), 0.5-400 ng/ml (0.1 ng/ml) and 1-100 ng/ml (1 ng/ml), respectively. The measured concentrations of serum samples by ELISA array showed high correlation with those from traditional ELISA. Conclusion: ELISA array-based immunoassay provided a feasible and productive method to multiplex measure markers for osteoporosis.

Trifluoperazine as an alternative strategy for the inhibition of tumor growth of colorectal cancer.

The development of cancer in patients with schizophrenia is affected by genetic and environmental factors and antipsychotic medication. Several studies found that schizophrenia was associated with decreased risk of some cancers, and the neuroleptic medication might help to reduce the risk of colorectal cancer (CRC). Phenothiazine drugs including trifluoperazine (TFP) are widely used antipsychotic drugs and showed some antitumor effects, we here investigated the potential application of TFP in the treatment of colon cancer. A series doses of TFP were treated to the colon cancer cell line HCT116 and the inhibitory concentration (IC50 ) of TFP for HCT116 was determined by cell counting kit-8. The results indicated that the treatment of TFP impaired the cell vitality of HCT116 in a dose- and time-dependent manner. Meanwhile, the Edu assay demonstrated that the proliferation was also inhibited by TFP, which was accompanied with the induction of apoptosis and autophagy. The expression of CCNE1, CDK4, and antiapoptosis factor BCL-2 was downregulated but the proapoptosis factor BAX was upregulated. The autophagy inhibitor chloroquine could significantly reverse the TFP-induced apoptosis. Moreover, the ability of migration and invasion of HCT116 was found to be suppressed by TFP, which was associated with the inhibition of epithelial-mesenchymal transition (EMT). The function of TFP in vivo was further confirmed. The results showed that the administration of TFP remarkably abrogated the tumor growth with decreased tumor volume and proliferation index Ki-67 level in tumor tissues. The EMT phenotype was also confirmed to be inhibited by TFP in vivo, suggesting the promising antitumor effects of TFP in CRC.

Chemical synthesis, microbial transformation and biological evaluation of tetrahydroprotoberberines as dopamine D1/D2 receptor ligands.

Dopamine D1/D2 receptors are important targets for drug discovery in the treatment of central nervous system diseases. To discover new and potential D1/D2 ligands, 17 derivatives of tetrahydroprotoberberine (THPB) with various substituents were prepared by chemical synthesis or microbial transformation using Streptomyces griseus ATCC 13273. Their functional activities on D1 and D2 receptors were determined by cAMP assay and calcium flux assay. Seven compounds showed high activity on D1/D2 receptor with low IC50 values less than 1 µM. Especially, top compound 5 showed strong antagonistic activity on both D1 and D2 receptor with an IC50 of 0.391 and 0.0757 µM, respectively. Five compounds displayed selective antagonistic activity on D1 and D2 receptor. The SAR studies revealed that (1) the hydroxyl group at C-9 position plays an important role in keeping a good activity and small or fewer substituents on ring D of THPBs may also stimulate their effects, (2) the absence of substituents at C-9 position tends to be more selective for D2 receptor, and (3) hydroxyl substitution at C-2 position and the substitution at C-9 position may facilitate the conversion of D1 receptor from antagonist to agonist. Molecular docking simulations found that Asp 103/Asp 114, Ser 107/Cys 118, and Trp 285/ Trp 386 of D1/ D2 receptors are the key residues, which have strong interactions with the active D1/D2 compounds and may influence their functional profiles.

Preparation and evaluation of dextran-grafted mixed-mode chromatography adsorbents.

In the present work, mixed-mode chromatography adsorbents grafted with dextran of different molecular weight were prepared. Compared to non-grafted adsorbent (BA-6B), the morphology of dextran-grafted adsorbents (BA-6B-Ts) did not change significantly, while the water content (ω), porosity (P) and pore volume (V) decreased. Static adsorption, salt tolerance and dynamic adsorption behaviors of adsorbents BA-6B-Ts against BSA/IgG were studied. Compared to adsorbent BA-6B, adsorbents BA-6B-Ts had higher saturated adsorption capacity (Qm) toward BSA/IgG, especially for BA-6B-T20, the Qm values toward BSA and IgG increased by 32.8% and 39.5%, respectively. Adsorbents BA-6B-Ts showed enhanced salt tolerance toward BSA, and relatively weaker salt tolerance toward IgG. The relative adsorption capacity of BA-6B-T20 was 66.7% at 0.1 mol/L NaCl and 39.2% at 0.5 mol/L NaCl, higher than those of BA-6B. Moreover, for both BSA and IgG, the effective pore diffusivity (De) of BA-6B-Ts was higher than that of BA-6B. De values of BA-6B-T20 toward BSA and IgG increased by 77% and 139% compared to BA-6B, respectively. Dynamic binding capacities (DBCs) of BA-6B-Ts were also improved. DBC of BSA and IgG on BA-6B-T20 were 25.6% and 46.5% higher than those on BA-6B. The possible mechanisms for the above difference in adsorption behavior of dextran-grafted adsorbents toward BSA/IgG were discussed, providing some insights into protein adsorption and mass transport for dextran-grafted MMC resins.

Design and activity study of a melittin-thanatin hybrid peptide.

The unique antimicrobial mechanism of antimicrobials make them a promising substitute for antibiotics for fighting drug-resistant bacteria. Both melittin and thanatin have antimicrobial bioactivity. However, thanatin does not inhibit the growth of Staphylococcus aureus. Melittin can inhibit S. aureus and has strong hemolytic activity. In the present study, the mutant fragments of melittin and thanatin were combined by flexible peptides to form a novel hybrid peptide, which was synthesized based on the secondary and tertiary structure prediction. The hybrid peptide inhibited S. aureus with a hemolytic concentration of above 45 µmol/L and inhibition rate in SMMC-7721 cells of 19.14%. The hybrid antimicrobial peptide, which was designed by the combination of α-helix and ß-lamellar antimicrobial peptides, showed that both types of peptides did not interact with each either on spatial structure or biological activities, thereby providing a novel idea for the design of artificial antimicrobial peptides.

Molecular dynamics perspective on the thermal stability of mandelate racemase.

Mandelate racemase from Pseudomonas putida is a promising candidate for the dynamic kinetic resolution of α-hydroxy carboxylic acids. In the present study, the thermal stability of mandelate racemase was investigated through molecular dynamics simulations in the temperature range of 303-363 K, which can guide the design of mandelate racemase with higher stability. The basic features such as radius of gyration, surface accessibility, and secondary structure content suggested the instability of mandelate racemase at high temperatures. With increase in temperature, α-helix content reduced significantly, especially the α-helices exposed to the environment. At the simulation time scale considered, intra-protein hydrogen bonds, hydrogen bonds between protein and water decreased at 363 K, while the number of salt-bridges increased. The long-distance networks remarkably changed at 363 K. A considerable number of long-lived (percentage existence time higher than 90%) hydrogen bonds and Cα contacts were lost. Root mean square fluctuation analysis revealed regions with high fluctuation, which should be helpful in the reengineering of mandelate racemase for enhanced thermal stability.

Effect of Montmorillonite on 4-Nonylphenol Enrichment in Zebrafish.

The aim of this study was to investigate the effect of montmorillonite on nonylphenol (4-nonylphenol, 4-NP) enrichment in a zebrafish model. The AB strain zebrafish were used as the animal subjects, and three concentration gradients were set for both nonylphenol and montmorillonite, according to their actual concentrations in aquaculture water in Huzhou City. A group treated with nonylphenol alone was also set, adding up to 12 experimental groups. Concentrations of nonylphenol enriched in the liver, muscle and gills of zebrafish were detected by solid phase microextraction⁻high performance liquid chromatography at Days 7, 15 and 30, respectively. Additionally, the relative enzymatic activity of superoxide dismutase (SOD) and the glutathione S-transferase (GST) were also detected, and the data were statistically analyzed. The results showed that the concentrations of nonylphenol in zebrafish peaked at Day 7 and gradually decreased afterwards for all the experimental groups. The montmorillonite reduces short-term accumulation of nonylphenol in gills, and the high concentration of nonylphenol facilitates its enrichment in liver and muscle, while the low concentration of nonylphenol does not. Meanwhile, the low concentration of nonylphenol in liver exerts an influence on the inductive effect of SOD and GST, while the high concentration of nonylphenol shows the inhibiting effect of SOD and GST.

Sodium Phenylbutyrate Inhibits Tumor Growth and the Epithelial-Mesenchymal Transition of Oral Squamous Cell Carcinoma In Vitro and In Vivo.

Sodium phenylbutyrate (SPB) as a salt of 4-phenylbutyric acid (4-PBA) has been reported to be an ammonia scavenger, histone deacetylase inhibitor, and an endoplasmic reticulum stress inhibitor in various diseases, including neurological diseases, inflammatory disorders, and carcinogenesis. Although phenylbutyrate showed effective antitumor properties in many cancers, its role in oral squamous cell carcinoma (OSCC) remains further characterized. Thus, the OSCC cell lines CAL27, HSC3, and SCC4 were treated with a series of doses of SPB for different times. The IC50 of three cell lines for SPB was determined to be 4.0, 3.7, and 3.0 mM. The CCK-8 assay indicated that the treatment of SPB induced continuous inhibition of cell vitality of three cell lines. Apoptosis was assessed by Hoechst assay that showed that SPB could significantly promote cell apoptosis. Moreover, the apoptosis-related pathway was analyzed, and the results showed that the expression of antiapoptosis factor BCL-2 was downregulated by SPB but the cleavage of caspase-3 was increased. Meanwhile, it was found that SPB also impaired the migration and invasion of OSCC cells in vitro. Mechanistically, the transforming growth factor-ß (TGFB) related epithelial-mesenchymal transition (EMT) was inhibited by SPB with decreased mesenchymal marker N-cadherin and increased epithelial marker E-cadherin. Furthermore, the antitumor effect of SPB in vivo was also demonstrated. The administration of SPB induced remarkably tumor regression with decreased tumor volume, and the TGFB level and EMT phenotype in vivo were also inhibited. These data demonstrated that the treatment of SPB could function as antitumor therapeutics for OSCC.