Microarray-based resequencing by apyrase-mediated allele-specific extension

We have developed an array-based resequencing method to determine genetic alterations in putative cancer genes. The method relies on that the specificity of DNA polymerase in allele-specific extensions can be enhanced by terminating the extension reactions with apyrase and that a tiling set of primers are synthesized covering the investigated gene sequence. We report on such apyrase-mediated allele-specific primer extension (AMASE) assay as a method suitable for high-throughout resequencing and mutation detection in tumor suppressor genes and oncogenes. In the experimental setup, primers complementary to codons 12, 13 and codon 61 of the N-ras proto-oncogene were spotted onto glass slides. Overlapping sense and anti-sense primers were designed so that complementary primers for all possible mutations in each base position were investigated. The extension reactions were performed in a single step following hybridization of target DNA to the immobilized primers on the array surface. Mutation detection limits and the possibility of quantifying the mutations were investigated using synthetic oligonucleotides. In addition, 64 clinical samples were sequenced and 16 of these showed mutations in the N-ras gene.     

Measurement of electrostatic interactions in protein folding with the use of protein charge ladders

This paper describes a new method for the measurement of the role of interactions between charged groups on the energetics of protein folding. This method uses capillary electrophoresis (CE) and protein charge ladders (mixtures of protein derivatives that differ incrementally in number of charged groups) to measure, in a single set of electrophoresis experiments, the free energy of unfolding (DeltaG(D-N)) of alpha-lactalbumin (alpha-LA) as a function of net charge. These same data also yield the hydrodynamic radius, R(H), and net charge measured by CE, Z(CE), of the folded and denatured proteins. Alpha-LA unfolds to a compact denatured state under mildly alkaline conditions; a small increase in R(H) (11%, 2 A) coincides with a large increase in Z(CE) (71%, -4 charge units), relative to the folded state. The increase in Z(CE), in turn, predicts a large pH dependence of free energy of unfolding (-22 kJ/mol per unit increase in pH), due to differences in proton binding in the folded and denatured states. The free energy of unfolding correlates with the square of net charge of the members of the charge ladder. The differential dependence of DeltaG(D-N) on net charge for holo-alpha-LA, (partial differential) DeltaG(D-N)/(partial differential)Z = -0.14Z kJ/mol per unit of charge. This dependence of DeltaG(D-N) on net charge is a result of a net electrostatic repulsion among charge groups on the protein. These results, together with data from pH titrations, show that both the effects of electrostatic repulsion and differences in proton binding in the folded and denatured states can play an important role in the pH dependence of this protein; the relative magnitude of these effects varies with pH. The combination of charge ladders and CE is a rapid and efficient tool that measures the contributions of electrostatics to the energetics of protein folding, and the size and charge of proteins as they unfold. All this information is obtained from a single set of electrophoresis experiments.     

Application of the response surface methodology for modeling demulsification of crude oil emulsion using a demulsifier

The main objective of this research is to determine the capability of four surface-active compounds namely poly(ethylene glycol) distearate, N,N-dimethyldodecylamine N–oxide solution, polyoxyethylene (10) tridecyl ether, and polyethylene glycol sorbitan monooleate as demulsifier agents in breaking water-in-crude oil emulsion through the bottle test method. The influence of temperature, concentration, water content, and pH on demulsification efficiency of the studied demulsifiers was investigated via the response surface methodology (RSM) and the central composite design method (CCD) was applied to design the experiments. The optimum values of input variables to obtain the maximum water separation efficiency were determined based on the developed model by analyze of variance (ANOVA). The R-squared values demonstrate that the developed models could appropriately predict the experimental results of all demulsifier agents.     

Active vibration control using optimized modified acceleration feedback with Adaptive Line Enhancer for frequency tracking

Modified acceleration feedback (MAF) control, an active vibration control method that uses collocated piezoelectric actuators and accelerometer is developed and its gains optimized using an optimal controller. The control system consists of two main parts: (1) frequency adaptation that uses Adaptive Line Enhancer (ALE) and (2) an optimized controller. Frequency adaptation method tracks the frequency of vibrations using ALE. The obtained frequency is then fed to MAF compensators. This provides a unique feature for MAF, by extending its domain of capabilities from controlling a certain mode of vibrations to any excited mode. The optimized MAF controller can provide optimal sets of gains for a wide range of frequencies, based on the characteristics of the system. The experimental results show that the frequency tracking method works quite well and fast enough to be used in a real-time controller. ALE parameters are numerically and experimentally investigated and tuned for optimized frequency tracking. The results also indicate that the MAF can provide significant vibration reduction using the optimized controller. The control power varies for vibration suppression at different resonance frequencies; however, it is always optimized.     

Insights into the current status of privileged N-heterocycles as antileishmanial agents

Molecular Diversity - Leishmania, one of the most important neglected tropical diseases, is endemic in several regions of the world and hence regarded as a serious threat to public health. Major...     

Enhancing endothelial differentiation of human mesenchymal stem cells by culture on a nanofibrous polycaprolactone/(poly-glycerol sebacate)/gelatin scaffold

Cardiovascular diseases have always been one of the main causes of death worldwide and eventually one of the major medical concerns. Tissue engineering is promising strategies of treating cardiovascular, which can be an effective approach with the design of appropriate scaffold. In this study, to develop engineering basement membrane for endothelial differentiation with good cell attachment, we produced polycaprolactone (PCL)/poly (glycerol sebacate) (PGS)/gelatin nanofibrous scaffold via electrospinning. Attenuated total reflectance-Fourier transform infrared and the proton nuclear magnetic resonance results confirmed the chemical structure of synthesized PGS. Scanning electron microscope images of the electrospun scaffold revealed that the nanofibers are smooth, continues and uniform. Moreover, due to the presence of hydrophilic functional groups in the scaffold, the contact angle is in the appropriate range for cell adhesion especially endothelial cells. The elastic modulus and ultimate tensile stress of electrospun scaffold were calculated 1.32 ± 0.27 MPa and 1.23 ± 0.18 MPa respectively. Quantitative polymerase chain reaction was performed for evaluation of endothelial differentiation of mesenchymal stem cells cultured on standard plate and fibrous scaffold under chemical stimulation with growth factor. Specific endothelial gene expression results postulated that our modified scaffold could support and significantly promote endothelial differentiation of MSCs.