Engineering and Kinetic Stabilization of the Therapeutic Enzyme Anabeana variabilis Phenylalanine Ammonia Lyase

Anabeana variabilis phenylalanine ammonia lyase has just recently been discovered and introduced in clinical trials of phenylketonuria enzyme replacement therapy for its outstanding kinetic properties. In the present study, kinetic stabilization of this therapeutically important enzyme has been explored by introduction of a disulfide bond into the structure. Site-directed mutagenesis was performed with quick-change PCR method. Recombinant wild-type and mutated enzymes were expressed in Escherichia coli, and his-tagged proteins were affinity purified. Formation of disulfide bond was confirmed by Ellman’s method, and then chemical unfolding, kinetic behavior, and thermal inactivation of mutated enzyme were compared with the wild type. Based on our results, the Q292C mutation resulted in a significant improvement in kinetic stability and resistance against chemical unfolding of the enzyme while kinetic parameters and pH profile of enzyme activity were remained unaffected. The results of the present study provided an insight towards designing phenylalanine ammonia lyases with higher stability.     

Rapid determination of ultra-trace amounts of acrylamide contaminant in water samples using dispersive liquid–liquid microextraction coupled to gas chromatography-electron capture detector

The objective of the present study was to develop and validate a rapid, highly sensitive, and reliable extraction method to determine acrylamide in water samples. The method was based on the derivatisation of the acrylamide in the presence of KBr, HBr and saturated Br₂ solution into 2,3-dibromopropionamide and dispersive liquid–liquid microextraction (DLLME) followed by gas chromatography–electron capture detection (GC–ECD) of the analyte. Different parameters that affect the DLLME process such as types and volumes of disperser solvent, ionic strength of aqueous solution and extraction time were investigated and optimised. Under optimal conditions, excellent linearity was obtained between concentration of acrylamide and the response of ECD with correlation of determination (R²) of 0.9999. The precision of the method, which was determined by calculating the relative standard deviations (RSD) of the at least three replicate measurements, was 3.6%. The method presented in this study is sensitive enough for the determination of acrylamide in different water samples with the limit of detection (LOD) value of 1?ng?L^?1. The mean percentage recoveries exceeded 91% for all of spiking levels in the real water samples. The results obtained from DLLME method are validated by EPA method 8032A.     

Ionic liquid-based dispersive liquid–liquid microextraction for the separation and preconcentration of lead in water samples prior to FAAS determination without chelating agent

In the present study, an environment-friendly sample preparation method termed ionic liquid-based dispersive liquid–liquid microextraction combined with flame atomic absorption spectrometry has been developed for the determination of Pb(II) ion in water samples prior to flame atomic absorption spectrometry determination. In this method, ionic liquid was used as an extraction solvent instead of the organic solvent used in the conventional dispersive liquid–liquid microextraction (DLLME) assay, and there is no need for a chelating agent. Several variables that may affect extraction efficiencies, including pH, the volume of ionic liquid, the type and volume of disperser solvent, salt addition, and the time for centrifugation and extraction were studied and optimised. Under the optimised conditions, the calibration curve exhibited linearity over the range of 20.0–1000.0 μg L^?1. The enrichment factor and the limit of detection based on 3S_b/m were 35.0 and 5.9 μg L^?1, respectively. Seven replicate determination of a solution containing of 100.0 μg L^?1 Pb(II) ions gave a relative standard deviation of ±2.1%. Finally, the feasibility of the proposed method for Pb(II) determination was assessed by the analysis of certi?ed reference material and various water samples and the satisfactory results were obtained.     

Vinylphosphonium Salt Mediated One-Pot Stereoselective Synthesis of Dialkyl 3,3-Diacetyl-3 a -hydroxy-8-oxo-2,3,3 a ,8-tetrahydrocyclopenta-[ a ]indene-1,2-dicarboxylates

Protonation of the highly reactive 1:1 intermediates produced in the reaction between triphenylphosphine (or tributylphosphine) and dialkyl acetylenedicarboxylates by acetylacetone leads to sterically congested phosphorus ylides, which undergo an intermolecular Wittig reaction with ninhydrin and concomitant annulation in CH 2 Cl 2 at room temperature to produce dialkyl 3,3-diacetyl-3 a -hydroxy-8-oxo-2,3,3 a ,8-tetrahydrocyclopenta[ a ]indene-1,2-dicarboxylates in fairly high yields.     

A Mild and Efficient Method for Chemoselective Silylation of Alcohols Using Hexamethyldisilazane in the Presence of Silica Chloride

Reaction of alcohols with hexamethyldisilazane in the presence of silica chloride provides efficiently the corresponding trimethylsilyl ethers. This system discriminates absolutely amines and thiols from alcohols.     

An Efficient Synthesis of New 3,4-Dihydropyrimidin- 2(1H)-ones Incorporating a Phenyl Moiety at C-5 and C-6 Catalyzed by TMSCl and Co(OAc)2.4H2O

New 3,4-dihydropyrimidin-2-ones having a phenyl moiety at C-5 and C-6 have been prepared by a microwave-assisted Biginelli–like reaction by a three-component, one-pot condensation of an aromatic aldehyde, deoxybenzoin and urea, or thiourea using TMSCl and Co(OAc) ₂ .4H ₂ O as an efficient Lewis acid catalyst.     

Synthesis and evaluation of novel arylisoxazoles linked to tacrine moiety: in vitro and in vivo biological activities against Alzheimer’s disease

Molecular Diversity - Alzheimer’s disease (AD) is now ranked as the third leading cause of death after heart disease and cancer. There is no definite cure for AD due to the multi-factorial...     

Transient analysis of nonlinear Euler–Bernoulli micro-beam with thermoelastic damping,via nonlinear normal modes

In this paper an Euler–Bernoulli model has been used for vibration analysis of micro-beams with large transverse deflection. Thermoelastic damping is considered to be the dominant damping mechanism and introduced as imaginary stiffness into the equation of motion by evaluating temperature profile as a function of lateral displacement. The obtained equation of motion is analyzed in the case of pure single mode motion by two methods; nonlinear normal mode theory and the Galerkin procedure. In contrast with the Galerkin procedure, nonlinear normal mode analysis introduces a nonconventional nonlinear damping term in modal oscillator which results in strong damping in case of large amplitude vibrations. Evaluated modal oscillators are solved using harmonic balance method and tackling damping terms introduced as an imaginary stiffness is discussed. It has been shown also that nonlinear modal analysis of micro-beam with thermoelastic damping predicts parameters such as inverse quality factor, and frequency shift, to have an extrema point at certain amplitude during transient response due to the mentioned nonlinear damping term; and the effect of system?s characteristics on this critical amplitude has also been discussed.     

Investigation of the π–π stacking interactions without direct electrostatic effects of substituents: the aromatic∥aromatic and aromatic∥anti-aromatic complexes

Stability of the π–π stacking interactions in the ben∥substituted-ben and ben∥substituted-COT complexes was studied using the computational quantum chemistry methods (where ben and COT are benzene and cyclooctatetraene, ∥ denotes π–π stacking interaction, substituted-ben and substituted-COT are benzene and cyclooctatetraene which substituted with four ethynyl-X groups, respectively, and X = OH, CH₃, H, F, CF₃, CN and NO₂). In these complexes electron-withdrawing substituents lead to larger binding energies and electron-donating ones lead to weaker interactions compared to X = H. There are meaningful correlations between the Hammett constants and binding energies. The atoms in molecules (AIM) analysis shows that formation of these complexes is accompanied by increase in the electron charge densities at the ring critical points of the substituted-ben and substituted-COT rings which leads to increase/decrease of the π–π stacking interactions in the ben∥substituted-ben/ben∥substituted-COT complexes. The charge transfer occurs from benzene to substituted-ben in the ben∥substituted-ben complexes and from substituted-COT to benzene (with the exception of X = CN) in the ben∥substituted-COT ones. Nuclear magnetic resonance calculations demonstrate that interactions of the more aromatic substituted-ben/less anti-aromatic substituted-COT rings with benzene in the ben∥substituted-ben/ben∥substituted-COT complexes can be helpful to enhance strength of the π–π stacking interactions. Thus, regardless of ring size, the π–π stacking interaction is an aromatic–aromatic interaction and π electron cloud properties of interacting rings affect on the strength of this interaction.     

A novel approach for considering layout problem in cellular manufacturing systems with alternative processing routings and subcontracting approach

In this paper a new integrated approach is presented for designing cellular manufacturing system and its inter- and intra-cell layouts. Various production factors such as part demands, alternative processing routings, operation sequences, processing times, capacity of machines, etc. are incorporated in the problem in order to extend its applicability. To increase the accuracy of the inter- and intra-cell layout design, the material handling cost is calculated in terms of the actual position of machines within the cells and regarding the dimensions of the machines and aisle distances. Also, a subcontracting approach is proposed to determine the production volume of parts within the CF and layout design process regarding the production, material handling and outsourcing costs and under demand and machine capacity constraints. To the best of our knowledge, this is the first study that addresses all these design features simultaneously. As the proposed problem is NP-hard, an efficient GA is employed to solve it. Finally, numerical examples adopted from the literature are used to verify the proposed approach.