A selective molecularly imprinted polymer for immobilization of acetylcholinesterase (AChE): an active enzyme targeted and efficient method

In the present study, we immobilized acetylcholinesterase (AChE) enzyme onto acetylcholine removed imprinted polymer and acetylcholine containing polymer. First, the polymers were produced with acetylcholine, substrate of AChE, by dispersion polymerization. Then, the enzyme was immobilized onto the polymers by using two different methods: In the first method (method A), acetylcholine was removed from the polymer, and then AChE was immobilized onto this polymer (acetylcholine removed imprinted polymer). In the second method (method B), AChE was immobilized onto acetylcholine containing polymer by affinity. In method A, enzyme‐specific species (binding sites) occurred by removing acetylcholine from the polymer. The immobilized AChE reached 240% relative specific activity comparison with free AChE because the active enzyme molecules bounded onto the polymer. Transmission electron microscopy results were taken before and after immobilization of AChE for the assessment of morphological structure of polymer. Also, the experiments, which include optimum temperature (25–65°C), optimum pH (3–10), thermal stability (4–70°C), kinetic parameters, operational stability and reusability, were performed to determine the characteristic of the immobilized AChE. Copyright © 2015 John Wiley & Sons, Ltd.     

A Novel Role of Matrix Metalloproteinase-8 in Macrophage Differentiation and Polarization

Matrix metalloproteinase-8 (MMP8) has been shown to influence various cellular functions. As monocytes and macrophages (Mφ) express MMP8, we investigated if MMP8 played a role in macrophage differentiation and polarization. MMP8 expression was significantly increased during monocyte differentiation into Mφ. Monocyte-derived Mφ from MMP8-deficient mice expressed higher levels of M1-Mφ markers but lower levels of M2-Mφ markers than monocyte-derived Mφ from wild-type mice. Although Mφ from either MMP8-deficient or wild-type mice were inducible by interferon-γ into M1-Mφ, only wild-type Mφ but not MMP8-deficient Mφ could be induced into M2-Mφ by interleukin-4. However, MMP8-deficient Mφ exposed to conditioned culture media of wild-type Mφ developed a M2-Mφ phenotype. Compared with conditioned culture media of wild-type Mφ, conditioned culture media of MMP8-deficient Mφ contained a lower concentration of active transforming growth factor-β (TGF-β), an M2-Mφ inducer. Moreover, evidence also showed that the degradation of the TGF-β sequester, fibromodulin, was modulated by MMP8. The data indicate a previously unknown role of MMP8 in M2-Mφ polarization by cleaving fibromodulin and therefore increasing the bioavailability of the M2-Mφ inducer TGF-β.     

Clinical significance of miR-140-5p and miR-193b expression in patients with breast cancer and relationship to IGFBP5

The functional role of IGFBP5 in breast cancer is complicated. Experimental and bioinformatics studies have shown that IGFBP5 is targeted by miR-140-5p and miR-193b, although this has not yet been proven in clinical samples. The aim of this study was to evaluate the expression of miR-140-5p and miR-193b in breast cancer and adjacent normal tissue and assess its correlation with IGFBP5 and the clinicopathological characteristics of the tumors. IGFBP5 protein expression was analyzed immunohistochemically and IGFBP5, miR-140 and miR-193b mRNA expression levels were analyzed with real-time RT-PCR. Tumor tissue had higher miR-140-5p expression than adjacent normal tissue (p = 0.015). Samples with no immunohistochemical staining for IGFBP5 showed increased miR-140-5p expression (p = 0.009). miR-140-5p expression was elevated in invasive ductal carcinomas (p = 0.002), whereas basal-like tumors had decreased expression of miR-140-5p compared to other tumors (p = 0.008). Lymph node-positive samples showed an approximately 13-fold increase in miR-140-5p expression compared to lymph node-negative tissue (p = 0.049). These findings suggest that miR-140-5p, but not miR-193b, could be an important determinant of IGFBP5 expression and clinical phenotype in breast cancer patients. Further studies are needed to clarify the expressional regulation of IGFBP5 by miR-140-5p.     

A new impedimetric biosensor utilizing VEGF receptor-1 (Flt-1): early diagnosis of vascular endothelial growth factor in breast cancer

A new impedimetric biosensor, based on the use of vascular endothelial growth factor receptor-1 (VEGF-R1), was developed for the determination of vascular endothelial growth factor (VEGF). VEGF-R1 was immobilized through covalent coupling with 3-mercaptopropionic acid which formed a self-assembled monolayer on gold electrodes. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy techniques were employed to characterize the immobilization process and to detect VEGF. To successfully construct the biosensor current, experimental parameters were optimized. Kramers-Kronig Transform was performed on the experimental impedance data. The obtained results provided a linear response range from 10 to 70 pg/mL human VEGF. The applicability of the developed biosensor in the determination of VEGF in a spiked artificial human serum sample was experienced, yielding average recovery of 101%, in that order, with an average relative deviation value less than 5%.     

Conformational regulation of the EGFR kinase core by the juxtamembrane and C-terminal tail: a molecular dynamics study

The catalytic domain of epidermal growth factor receptor (EGFR) is activated by dimerization, which requires allosteric coupling between distal dimerization and catalytic sites. Although crystal structures of EGFR kinases, solved in various conformational states, have provided important insights into EGFR activation by dimerization, the atomic details of how dimerization signals are dynamically coupled to catalytic regions of the kinase core are not fully understood. In this study, we have performed unrestrained and targeted molecular dynamics simulations on the active and inactive states of EGFR, followed by principal component analysis on the simulated trajectories, to identify correlated motions in the EGFR kinase domain upon dimerization. Our analysis reveals that the conformational changes associated with the catalytic functions of the kinase core are highly correlated with motions in the juxtamembrane (JM) and C-terminal tail, two flexible structural elements that play an active role in EGFR kinase activation and dimerization. In particular, the opening and closing of the ATP binding lobe relative to the substrate binding lobe is highly correlated with motions in the JM and C-terminal tail, suggesting that ATP and substrate binding can be coordinated with dimerization through conformational changes in the JM and C-terminal tail. Our study pinpoints key residues involved in this conformational coupling, and provides new insights into the role of the JM and C-terminal tail segments in EGFR kinase functions.     

Ways of coping as predictors of satisfaction with curriculum and academic success in medical school

The purpose of the present study was to determine the coping strategies of medical students and to investigate the effects of coping strategies on student satisfaction and academic achievement with different instruction methods. A total of 152 medical students was followed throughout the first 2 yr of medical education between 2008 and 2010. Students completed a sociodemographic questionnaire and revised form of the Ways of Coping questionnaire both at the beginning of the first year and at the end of the second year. These forms provided data about the characteristics and main coping strategies (problem focused or emotion focused) of the students and revealed the change over time. At the end of the second year, participants also completed a satisfaction questionnaire asking their satisfaction with lectures, problem-based learning, and practicals. The authors used block, problem-based learning, and practical exam scores of the students attained in the past 2 yr as academic achievement indicators. No sociodemographic variable was related to coping strategy. The majority of students (80.9%) adopted problem-focused coping. A shift occurred in the main coping strategies of some students in both sides. Problem-focused coping scores decreased over time. Problem-focused coping positively correlated with satisfaction with practicals and practical exam scores, whereas emotion-focused coping showed the same correlation negatively. The main coping strategy also predicted satisfaction and exam success in practicals. In conclusion, a main coping strategy may be helpful to predict student satisfaction and academic achievement with some student-centered instruction methods. Determining undesired coping strategies may provide an opportunity for intervention to prevent relevant dissatisfaction and failure.