Fluorescence of a Histidine-Modified Enhanced Green Fluorescent Protein (EGFP) Effectively Quenched by Copper(II) Ions

Two histidines were introduced by site-directed mutagenesis into the structure of Enhanced Green Fluorescent Protein, replacing the serine at position 202 and the glutamine at position 204 for increasing the sensitivity of the protein towards different metal ions by creating possible metal binding sites near the chromophore group. There is no appreciable difference between the absorbance and fluorescence spectra of the two proteins (wild type and the double-histidine mutant) indicating that the mutation does not change the environment of the fluorophore. Fluorescence quenching was measured at different pH (6.5–8) and temperatures (20–45 °C) varying the concentration of metal ions. Under optimal conditions (pH?=?7.5, 20 °C) the mutant’s K_d is 16 nM, it binds copper more than 200fold stronger than the wild type EGFP.     

Amino Acid Functionalized Superparamagnetic Nanoparticles Inhibit Lysozyme Amyloid Fibrillization

Nanoparticles have great potential to be used in various biomedical applications, including therapy or diagnosis of amyloid-related diseases. The physical and chemical properties of iron oxide superparamagnetic nanoparticles (MNPs) functionalized with different amino acids (AAs), namely, with lysine (Lys), glycine (Gly), or tryptophan (Trp), have been characterized. The cytotoxicity of nanoparticles and their effect on amyloid fibrillization of lysozymes in vitro was also verified. The AA-MNPs under study are nontoxic to human SHSY5Y neuroblastoma cells. Moreover, the AA-MNPs were able to significantly inhibit lysozyme amyloid fibrillization and destroy amyloid fibrils. Kinetic studies revealed that the presence of AA-MNPs affected lysozyme fibrillization, namely, the lag phase and steady-state phase of the growth curves. The most effective activities were observed for Trp-MNPs, which revealed the importance of aromatic rings in the structure of AAs used as coating agents. The obtained results indicate the possible application of these AA-MNPs in the treatment of amyloid diseases associated with lysozyme or other amyloidogenic proteins.     

Ring transformations of β-lactam-condensed 1,3-benzothiazines to isoquinoline and thiazole derivatives by sulfur extrusion and addition sequences

The ring transformations of dichloro-β-lactam-fused 2-aryl-1,3-benzothiazines with sodium methoxide were investigated. With 2 equiv of base, the dichloro-β-lactam derivatives underwent rearrangement and dihydro-1,4-benzothiazepines, 3,4-substituted isoquinolines and substituted thiazole disulfides were isolated. A possible reaction mechanism is proposed for the simultaneous formation of the novel products. The formation of isoquinoline and thiazole derivatives can be explained by sulfur extrusion and addition sequences.     

Multifield computational fluid dynamics model of particulate flow in curved circular tubes

Limitations of mass transfer resulting from non-optimized fluid mechanics can severely affect the performance of synthetic membrane filtration systems. To improve membrane efficiency, modern applications of this technology have extensively used curved membrane ducts that take advantage of Dean vortices (i.e., curvature-induced secondary flows) to minimize membrane fouling. This paper is concerned with a complete three-dimensional analysis of single-phase and two-phase particle/liquid flows around a curved membrane tube. The proposed multidimensional model was implemented in an advanced (next-generation) multiphase computational fluid dynamics (CFD) solver, NPHASE. The results of simulations have been validated against experimental data and compared against other findings available in the literature. The consistency and accuracy of the present approach have been demonstrated. The novel aspects of this work include: the demonstration that azimuthal vortices may bifurcate at Dean numbers lower than previously anticipated, the use of vorticity magnitude as a measure of vortex strength, and the explanation of the role that Dean vortices play to mitigate the effect of gravity on particle settling. The overall results have direct relevance to synthetic membrane fouling during filtration of particle suspensions.     

A new method for measuring the complex shear modulus of a viscoelastic material coupled with an elastic material

Summary In the mechanical-dynamic characterization of viscoelastic materials as a function of temperature, considerable difficulties are encountered, due to the change of joint, to the strong variation of the modulus of elasticity and the increase ofQ ^–1.This paper deals with a theoretical and experimental method for the determination of the shear modulusG₂ and of the internal lossQ ₂ ^–1 of a viscoelastic material by measurements at torsional vibration of a composite test-piece. Experimental measurements were carried out on composite test-pieces by gluing of polystyrene and of pinchbeck. The results obtained are in good agreement with the values found by other methods.     

Achiral bent-core azo compounds: observation of photoinduced effects in an antiferroelectric tilted smectic mesophase

We report the synthesis and physical properties of four novel bent-core compounds, containing an azo linkage and alkyl terminal chains. It is found that, except for the lowest homologue, all the materials are liquid crystalline, exhibiting columnar or tilted polar smectic phases. In one of the compounds a reversible photoinduced variation of the spontaneous polarization of the antiferroelectric state was observed.     

Enhanced In Vitro Refolding of Soluble Human Glucocorticoid-Induced TNF Receptor-Related Ligand

The glucocorticoid-induced tumor necrosis factor receptor (GITR) is a member of the tumor necrosis factor receptor superfamily. Attachment of GITR to its ligand (GITRL) regulates diverse biological functions, including cell proliferation, differentiation, and survival. In this study, the extracellular region of human GITRL (hGITRL) was cloned, expressed, and purified. The coding sequence of the extracellular region of hGITRL was isolated from human brain cDNA and inserted in pET20b vector. The hGITRL was expressed in Escherichia coli BL21 (DE3) Star at 37 and 25 °C. The majority of the protein was found in inclusion bodies. We identified three important factors for efficient refolding of hGITRL: a ratio of GSH/GSSG, pH, and addition of polyethylene glycol. The renaturated protein was purified by Ni-NTA chromatography. The overall yield of the expression and refolding was higher than 50 mg/l E. coli culture grown at 37 °C. Size exclusion chromatography showed that hGITRL exists as mixture of various multimeric forms in solution. We tested the association of recombinant hGITRL with THP-1 and U937 cell lines and its activity to promote extracellular signal-regulated protein kinase phosphorylation. The results showed that the recombinant protein was biologically active.