Engineering deoxysugar biosynthetic pathways from antibiotic-producing microorganisms. A tool to produce novel glycosylated bioactive compounds

A plasmid (pLN2) was generated in which genes involved in the biosynthesis of L-oleandrose in the oleandomycin producer Streptomyces antibioticus ATCC11891 were cloned. pLN2 was used to direct the biosynthesis of different deoxysugars by exchanging and/or adding genes from other antibiotic biosynthetic clusters. Transfer of the synthesized deoxysugars to the tetracenomycin C aglycon, 8-demethyl-tetracenomycin C, through the use of the "sugar flexible" glycosyltransferase ElmGT, validated the system. Several pLN2 derivatives were constructed by replacement of the oleU 4-ketoreductase gene by different 4-ketoreductase genes. Some of them, such as EryBIV and UrdR, reduced the keto group of the 4-keto intermediates, generating L-olivosyl and D-olivosyl derivatives, respectively. The system was also used to generate an L-rhamnosyl derivative (through a two-gene deletion) and an L-rhodinosyl derivative (through a combination of a gene replacement and a gene addition).     

An experimental approach probing the conformational transitions and energy landscape of antibodies: a glimmer of hope for reviving lost therapeutic candidates using ionic liquid

Understanding protein folding in different environmental conditions is fundamentally important for predicting protein structures and developing innovative antibody formulations. While the thermodynamics and kinetics of folding and unfolding have been extensively studied by computational methods, experimental methods for determining antibody conformational transition pathways are lacking. Motivated to fill this gap, we prepared a series of unique formulations containing a high concentration of a chimeric immunoglobin G4 (IgG4) antibody with different excipients in the presence and absence of the ionic liquid (IL) choline dihydrogen phosphate. We determined the effects of different excipients and IL on protein thermal and structural stability by performing variable temperature circular dichroism and bio-layer interferometry analyses. To further rationalise the observations of conformational changes with temperature, we carried out molecular dynamics simulations on a single antibody binding fragment from IgG4 in the different formulations, at low and high temperatures. We developed a methodology to study the conformational transitions and associated thermodynamics of biomolecules, and we showed IL-induced conformational transitions. We showed that the increased propensity for conformational change was driven by preferential binding of the dihydrogen phosphate anion to the antibody fragment. Finally, we found that a formulation containing IL with sugar, amino acids and surfactant is a promising candidate for stabilising proteins against conformational destabilisation and aggregation. We hope that ultimately, we can help in the quest to understand the molecular basis of the stability of antibodies and protein misfolding phenomena and offer new candidate formulations with the potential to revive lost therapeutic candidates.

Probing the energy landscape and thermodynamics of biomolecules for drug design.     

Electrochemical and spectroelectrochemical properties of nitroxyl radical species in PTMA,an organic radical polymer. Influence of the microstructure

In this work, an electrochemical and ESR-spectrochemical study of the oxidation properties for nitroxide radicals contained in the structure of poly(4-methacryloyloxy-2,2,6,6-tetramethylpiperidin-N-oxyl) (PTMA) in acetonitrile solution was performed. Results revealed that this polymer contains two types of nitroxide radical species, the first associated to a multielectronic and reversible electron transfer process. Remaining radical species are irreversibly oxidized at more positive potential values. Proportions obtained for the respective amount of each oxidized species, 78% and 22% respectively, are similar to the values expected due to the difference in syndiotactic:heterotactic triads in the polymer and reported values for the experimental charge/discharge efficiencies of batteries constructed with this material. These effects also influence electron transfer kinetics during the oxidation of each type of radical species in the polymer. These findings suggest that the detailed understanding of the oxidation processes of different microstructures in radical polymers is useful to analyze performance based in these materials.     

Internal correction of hafnium oxide spectral interferences and mass bias in the determination of platinum in environmental samples using isotope dilution analysis

A method has been developed for the accurate determination of platinum by isotope dilution analysis, using enriched ¹⁹⁴Pt, in environmental samples containing comparatively high levels of hafnium without any chemical separation. The method is based on the computation of the contribution of hafnium oxide as an independent factor in the observed isotope pattern of platinum in the spiked sample. Under these conditions, the ratio of molar fractions between natural abundance and isotopically enriched platinum was independent of the amount of hafnium present in the sample. Additionally, mass bias was corrected by an internal procedure in which the regression variance was minimised. This was possible as the mass bias factor for hafnium oxide was very close to that of platinum. The final procedure required the measurement of three platinum isotope ratios (192/194, 195/194 and 196/194) to calculate the concentration of platinum in the sample. The methodology has been validated using the reference material “BCR-723 road dust” and has been applied to different environmental matrices (road dust, air particles, bulk wet deposition and epiphytic lichens) collected in the Aspe Valley (Pyrenees Mountains). A full uncertainty budget, using Kragten’s spreadsheet method, showed that the total uncertainty was limited only by the uncertainty in the measured isotope ratios and not by the uncertainties of the isotopic composition of platinum and hafnium. Figure Simultaneous correction of hafnium oxide spectral interferences and mass bias in the determination of platinum in environmental samples using isotope dilution analysis Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Sharp nonremovability examples for Hölder continuous quasiregular mappings in the plane

Let α ∈ 2 (0, 1), K ≥ 1, and \(d = 2\frac{{1 + \alpha K}}{{1 + K}}\). Given a compact set E ? ?, it is known that if \(\mathcal{H}^d (E) = 0\), then E is removable for α-Hölder continuous K-quasiregular mappings in the plane. The sharpness of the index d is shown with the construction, for any t > d, of a set E of Hausdorff dimension dim(E) = t which is not removable. In this paper, we improve this result and construct compact nonremovable sets E such that \(0 < \mathcal{H}^d (E) < \infty \). For the proof, we give a precise planar K-quasiconformal mapping whose Hölder exponent is strictly bigger than \(\frac{1}{K}\) and which exhibits extremal distortion properties.     

Formal Hopf algebra theory I: Hopf modules for pseudomonoids

In this article we develop some of the basic constructions of the theory of Hopf algebras in the context of autonomous pseudomonoids in monoidal bicategories. We concentrate on the notion of Hopf modules. We study the existence and the internalisation of this notion, called the Hopf module construction. Our main result is the equivalence between the existence of a left dualization for A (i.e., A is left autonomous) and the validity of an analogue of the structure theorem of Hopf modules. In this case a Hopf module construction for A always exists. We recover from the general theory developed here results on coquasi-Hopf algebras.     

Nearest southeast submatrix that makes multiple a prescribed eigenvalue. Part 1

Given four complex matrices A,B,C and D, where A∈C^n×n and D∈C^m×m, and given a complex number z₀: What is the (spectral norm) distance from D to the set of matrices X∈C^m×m such that z₀ is a multiple eigenvalue of the matrix

     

Analysis of chlorophenols,bisphenol-A, 4-tert-octylphenol and 4-nonylphenols in soil by means of ultrasonic solvent extraction and stir bar sorptive extraction with in situ derivatisation

A novel method based on ultrasonic solvent extraction and stir bar sorptive extraction for the analysis of phenolic organic pollutants including chlorophenols, bisphenol-A, 4-tert-octylphenol and 4-nonylphenol in soil samples was developed. The different parameters that affect both the extraction of analytes from the soil samples, such as solvent selection, extraction time, and the partitioning from the solvent/water mix to poly(dimethylsiloxane) (PDMS) were studied. The final selected conditions consisted of the extraction of 1 g of soil with 15 mL methanol by sonication for 30 min. The methanol extract was mixed with 85 mL of Milli-Q water and extracted by means of stir bar sorptive extraction with in situ derivatisation. The stir bars were analyzed by thermal desorption–gas chromatography–mass spectrometry (TD–GC–MS). The effects of the matrix on the recovery of the various pollutants under the developed method were studied using two soils with very different physicochemical properties. Method sensitivity, linearity, repeatability, and reproducibility were also studied. Validation and accuracy of the method were conducted by analyzing two commercial certified reference materials and by comparing the analysis of real samples with the proposed method and a classical method using pressurized solvent extraction (PSE)–GC–MS. The main advantage of this method is a substantial solvent reduction. For the extraction of only 1 g of solid sample allowing limits of detection ranging from 0.2 to 1.7 μg kg⁻¹ dw. Repeatability and reproducibility variation were lower than 20% for all investigated compounds. Results of the certified reference materials and the real samples verify the high accuracy of this method.     

New universal,portable and cryogenic sampler for time weighted average monitoring of H2S,NH3, benzene,toluene, ethylbenzene,xylenes and dimethylethylamine

A new cryogenic integrative air sampler (patent application number 08/00669), able to overcome many of the limitations in current volatile organic compounds and odour sampling methodologies is presented. The sample is spontaneously collected in a universal way at 15 mL/min, selectively dried (reaching up to 95% of moisture removal) and stored under cryogenic conditions. The sampler performance was tested under time weighted average (TWA) conditions, sampling 100 L of air over 5 days for determination of NH₃, H₂S, and benzene, toluene, ethylbenzene and xylenes (BTEX) in the ppm_v range. Recovery was 100% (statistically) for all compounds, with a concentration factor of 5.5. Furthermore, an in-field evaluation was done by monitoring the TWA inmission levels of BTEX and dimethylethylamine (ppb_v range) in an urban area with the developed technology and comparing the results with those monitored with a commercial graphitised charcoal diffusive sampler. The results obtained showed a good statistical agreement between the two techniques.     

Magnetic field effects on the copolymerization of water‐soluble and ionic monomers

The effect of magnetic field (MF) on the radical copolymerization of a series of water‐soluble and ionic monomers is presented including acrylamide (AM), acrylic acid (AA), its ionized form acrylate (A^?), and diallyldimethylammonium chloride (DADMAC). The following combinations have been studied: AM/AA, AM/A^?, AM/DADMAC, and AA/DADMAC. In addition to the MF, strong electrostatic interactions are present for the majority of monomer combinations and conditions. Although the monomer consumption rate (R_p) increased up to 65% applying a MF of 0.1 Tesla, the composition of the resulting copolymers was not affected under such conditions. Despite this increase of R_p by MF, the electrostatic repulsion between ionic monomers and charged growing radicals dominates R_p and governs the copolymer composition with and without MF. The order of the experimentally obtained reactivity ratios reflects the extent of electrostatic interaction: r_AM/AA (1.41) < r (3.10) < r_AA/DADMAC (4.25) < r_AM/DADMAC (6.95) and r_AA/AM (2.20) > r_DADMAC/AA (0.25) > r (0.17) > r_DADMAC/AM (0.03). Overall, weak MF offers to reduce the production time without modifying the product composition. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 373–383, 2009