Studies on the adsorption of cell impurities from plasmid-containing lysates to phenyl boronic acid chromatographic beads

Plasmid DNA (pDNA) is purified directly from alkaline lysis-derived Escherichia coli (E. coli) lysates by phenyl boronate (PB) chromatography. The method explores the ability of PB ligands to bind covalently, but reversibly, to cis-diol-containing impurities like RNA and lipopolysaccharides (LPS), leaving pDNA in solution. In spite of this specificity, cis-diol free species like proteins and genomic DNA (gDNA) are also removed. This is a major advantage since the process is designed to keep the target pDNA from binding. The focus of this paper is on the study of the secondary interactions between the impurities (RNA, gDNA, proteins, LPS) in a pDNA-containing lysate and 3-amino PB controlled pore glass (CPG) matrices. Runs were designed to evaluate the role of adsorption buffer composition, feed type (pH, salt content), CPG matrix and sample pretreatment (RNase A, isopropanol precipitation). Water was chosen as the adsorption buffer over MgCl(2) solutions since it maximised pDNA yield (96.2±4.9%) and protein removal (61.3±3.0%), while providing for a substantial removal of RNA (65.5±3.5%) and gDNA (44.7±14.1%). Although the use of pH 3.5 maximised removal of impurities (~75%), the best compromise between plasmid yield (~96%) and RNA clearance (~60-70%) was obtained for a pH of 5.2. Plasmid yield was maximal (>96%) when the concentration of acetate and potassium ions in the incoming lysate feed were 1.7 M and 1.0 M, respectively. The pre-treatment of lysates with RNase A deteriorated the performance since the resulting oligoribonucleotides lack the cis-diol group at their 3' termini. Overall, the results support the idea that charge transfer interactions between the boron atom at acidic pH and electron donor groups in the aromatic bases of nucleic acids and side residues of proteins are responsible for the non-specific removal of gDNA, RNA and proteins.     

Differentiation of Isomeric Hydroxypyridine <Emphasis Type="Italic">N</Emphasis>-Oxides Using Metal Complexation and Electrospray Ionization Mass Spectrometry

Differentiation between two isomers of hydroxypyridine N-oxide according to the metal cation adducts generated by electrospray ionization (ESI) was investigated for different metal cations, namely Mg (II), Al (III), Ca (II), Sc (III), Fe (III), Co (II), Ni (II), Cu (II), Zn (II), Ga (III), besides the diatomic cation VO(IV). Protonated molecules of the isomeric hydroxypyridine N-oxides as well as the singly/doubly charged adducts formed from neutral or deprotonated ligands and a doubly/triply charged cation were produced in the gas phase using ESI, recording mass spectra with different metal ions for each isomer. While complex formation was successful for 2-hydroxypyridine N-oxide with trivalent ions, in the case of 3-hydroxypyridine N-oxide, only peaks related to the protonated molecule were present. On the other hand, divalent cations formed specific species for each isomer, giving characteristic spectra in every case. Hence, differentiation was possible irrespective of the metal cation utilized. In addition, quantum chemical calculations at the B3LYP/6-31 + G(d,p) level of theory were performed in order to gain insight into the different complexation of calcium(II) with the isomers of hydroxypyridine N-oxide. The relative stability in the gas phase of the neutral complexes of calcium made up of two ligands, as well as the singly charged and doubly charged complexes, was investigated. The results of these calculations improved the understanding of the differences observed in the mass spectra obtained for each isomer.     

A general criterion to determine the number of change-points

A general criterion is proposed to determine the number K of the change-points in a parametric nonlinear multi-response model. Schwarz criterion is a particular case. The change-points depend on regressor values and not on instant of measure. We prove that the proposed estimator for K is consistent. Simulation results, using Monte Carlo technique, for nonlinear models which have numerous applications, support the relevance of the theory.     

Prediction of self-diffusion coefficient and shear viscosity of water and its binary mixtures with methanol and ethanol by molecular simulation

Density, self-diffusion coefficient, and shear viscosity of pure liquid water are predicted for temperatures between 280 and 373 K by molecular dynamics simulation and the Green-Kubo method. Four different rigid nonpolarizable water models are assessed: SPC, SPC/E, TIP4P, and TIP4P/2005. The pressure dependence of the self-diffusion coefficient and the shear viscosity for pure liquid water is also calculated and the anomalous behavior of these properties is qualitatively well predicted. Furthermore, transport properties as well as excess volume and excess enthalpy of aqueous binary mixtures containing methanol or ethanol, based on the SPC/E and TIP4P/2005 water models, are calculated. Under the tested conditions, the TIP4P/2005 model gives the best quantitative and qualitative agreement with experiments for the regarded transport properties. The deviations from experimental data are of 5% to 15% for pure liquid water and 5% to 20% for the water + alcohol mixtures. Moreover, the center of mass power spectrum of water as well as the investigated mixtures are analyzed and the hydrogen-bonding structure is discussed for different states.     

On the correlation between thermal analysis results and corrosion behaviour of some metallic religious artefacts

The aim of this study was to investigate the effects of immersion time on the electrochemical corrosion behaviour of three brass alloys (CuZn) coming from religious artefacts in simulated acid rain at 25 °C, utilising the electrochemical impedance spectroscopy (EIS). The main parameters of the corrosion process were established. On the other hand, the alloys were also analysed by means DSC and TG/DTA before and after immersion in the corrosive environment. Finally, the obtained results were compared in order to correlate them with each other and with the corrosion process.     

TiO2-based photocatalysts impregnated with metallo-porphyrins employed for degradation of 4-nitrophenol in aqueous solutions: role of metal and macrocycle

Photodegradation of organic compounds in water solutions by means of economically advantageous and environment-friendly processes is a topic of growing interest. In recent years a great attention has been devoted to TiO₂-based photocatalysts for the oxidative degradation of various organic pollutants. In this context, we have prepared new photo-catalytic polycrystalline TiO₂ systems impregnated with sensitizers, i.e., copper, iron or manganese porphyrins, and investigated their photoactivity for 4-nitrophenol oxidation compared with that of bare TiO₂. A significant improvement of the photoreactivity was observed in the case of TiO₂ impregnated with copper porphyrin, while only a slight beneficial effect was observed in the case of iron porphyrin. In contrast, the presence of manganese porphyrin appeared to be detrimental.     

Adhesion and bonding of Pt/Ni and Pt/Co overlayers: Density functional calculations

The electronic and energetic properties of bimetallic surfaces Pt/Ni(111) and Pt/Co(111) are examined using the FP-LAPW (Full-PotentialLinearized Augmented Plane Wave) method by means of spin-polarized and non-polarized calculations. We present both the results of the shifts in the d-band centers when one metal (Pt) is pseudomorfically deposited on another with smaller lattice constant (Ni, Co) and those corresponding to the surface and adhesion energies. The surface is modeled by a seven layer slab separated in z direction by a vacuum region of six substrate layers. The results obtained for pure Ni, Co and Pt surfaces are presented in order to compare with experimental and theoretical data reported in the literature     

Some insight into characterizations of minimally nonideal matrices

Lehman (Polyhedral combinatorics 1 of DIMACS series in discrete math. and theoretical computer science, pp 101–105, 1990) described some conditions regular minimally nonideal (mni) matrices must satisfy. Although, there are few results on sufficient conditions for mni matrices. In most of these results, the covering polyhedron must have a unique fractional extreme point. This condition corresponds to ask the matrix to be the blocker of a near-ideal matrix, defined by the authors in a previous work (2006). In this paper we prove that, having the blocker of a near-ideal matrix, only a few very easy conditions have to be checked in order to decide if the matrix is regular mni. In doing so, we define the class of quasi mni matrices, containing regular mni matrices, and we find a generalization on the number of integer extreme points adjacent to the fractional extreme point in the covering polyhedron. We also give a relationship between the covering and stability number of regular mni matrices which allows to prove when a regular mni matrix can be a proper minor of a quasi mni. Partially supported by CONICET Grant PIP 2807/2000 (Argentina) and by CNPq/PROSUL Grant 490333/2004-4 (Brasil).