Characterization of the alnumycin gene cluster reveals unusual gene products for pyran ring formation and dioxan biosynthesis

Alnumycin is closely related to the benzoisochromanequinone (BIQ) polyketides such as actinorhodin. Exceptional structural features include differences in aglycone tailoring that result in the unique alnumycin chromophore and the existence of an unusual 4-hydroxymethyl-5-hydroxy-1,3-dioxan moiety. Cloning and sequencing of the alnumycin gene cluster from Streptomyces sp. CM020 revealed expected biosynthesis genes for polyketide assembly, but several genes encoding subsequent tailoring enzymes were highly atypical. Heterologous expression studies confirmed that all of the genes required for alnumycin biosynthesis resided within the sequenced clone. Inactivation of genes aln4 and aln5 showed that the mechanism of pyran ring formation differs from actinorhodin and granaticin pathways. Further inactivation studies identified two genes, alnA and alnB, involved in the synthesis and attachment of the dioxan moiety, and resulted in the production of the polyketide prealnumycin.     

Thermodynamic assessment of the Cu‐Mg‐Si system in its copper‐rich region

A thermodynamic assessment of the ternary Cu‐Mg‐Si system was made in its copper‐rich region in relation with the development of a thermodynamic database for Cu‐Mg alloys. The adjustable parameters of the binary end‐systems (Cu‐Mg, Cu‐Si and Mg‐Si) were taken from the literature and those of the Cu‐Mg‐Si system were optimized using experimental thermodynamic and topological data. For the sake of simplicity, the solution phases were described with the substitutional solution model and the intermetallic compounds were treated as plain semi‐stoichiometric phases ((A,B)_pC_q‐type). The assessment developed is applicable for magnesium contents up to 20 wt% (i.e. magnesium mole fractions ≈0.4) and silicon contents up to 16 wt% (i.e. silicon mole fractions ≈0.3). (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

On initial populations of a genetic algorithm for continuous optimization problems

Genetic algorithms are commonly used metaheuristics for global optimization, but there has been very little research done on the generation of their initial population. In this paper, we look for an answer to the question whether the initial population plays a role in the performance of genetic algorithms and if so, how it should be generated. We show with a simple example that initial populations may have an effect on the best objective function value found for several generations. Traditionally, initial populations are generated using pseudo random numbers, but there are many alternative ways. We study the properties of different point generators using four main criteria: the uniform coverage and the genetic diversity of the points as well as the speed and the usability of the generator. We use the point generators to generate initial populations for a genetic algorithm and study what effects the uniform coverage and the genetic diversity have on the convergence and on the final objective function values. For our tests, we have selected one pseudo and one quasi random sequence generator and two spatial point processes: simple sequential inhibition process and nonaligned systematic sampling. In numerical experiments, we solve a set of 52 continuous test functions from 16 different function families, and analyze and discuss the results.     

Heuristic for a new multiobjective scheduling problem

We consider a telecommunication problem in which the objective is to schedule data transmission to be as fast and as cheap as possible. The main characteristic and restriction in solving this multiobjective optimization problem is the very limited computational capacity available. We describe a simple but efficient local search heuristic to solve this problem and provide some encouraging numerical test results. They demonstrate that we can develop a computationally inexpensive heuristic without sacrificing too much in the solution quality.     

LC‐MS analysis of estradiol in human serum and endometrial tissue: Comparison of electrospray ionization,atmospheric pressure chemical ionization and atmospheric pressure photoionization

Accurate measurement of estradiol (E2) is important in clinical diagnostics and research. High sensitivity methods are critical for specimens with E2 concentrations at low picomolar levels, such as serum of men, postmenopausal women and children. Achieving the required assay performance with LC–MS is challenging due to the non‐polar structure and low proton affinity of E2. Previous studies suggest that ionization has a major role for the performance of E2 measurement, but comparisons of different ionization techniques for the analysis of clinical samples are not available. In this study, female serum and endometrium tissue samples were used to compare electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) in both polarities. APPI was found to have the most potential for E2 analysis, with a quantification limit of 1 fmol on‐column. APCI and ESI could be employed in negative polarity, although being slightly less sensitive than APPI. In the presence of biological background, ESI was found to be highly susceptible to ion suppression, while APCI and APPI were largely unaffected by the sample matrix. Irrespective of the ionization technique, background interferences were observed when using the multiple reaction monitoring transitions commonly employed for E2 (m/z 271 > 159; m/z 255 > 145). These unidentified interferences were most severe in serum samples, varied in intensity between ionization techniques and required efficient chromatographic separation in order to achieve specificity for E2. Copyright © 2013 John Wiley & Sons, Ltd.     

Correlating Solution- and Solid-State Structures of Conformationally Flexible Resorcinarenes: Significance of a Sulfonyl Group in Intramolecular Self-Inclusion

The synthesis of tetramethoxyresorcinarene podands bearing p-toluene arms connected by -SO₃- ( 1 ) and -CH₂O- ( 2 ) linkers is presented herein. In the solid state, the resorcinarene podand 1 forms an intramolecular self-inclusion complex with the pendant p-toluene group of a podand arm, whereas the resorcinarene podand 2 does not show self-inclusion. The conformations of the flexible resorcinarene podands in solution were investigated by variable-temperature experiments using 1D and 2D NMR spectroscopic techniques as well as by computational methods, including a conformational search and subsequent DFT optimisation of representative structures. The ¹H NMR spectra of 1 and 2 at room temperature show a single set of proton signals that are in agreement with C_4v symmetry. At low temperatures, the molecules exist as a mixture of boat conformations featuring slow exchange on the NMR timescale. Energy barriers (ΔG^≠₂₉₈) of 55.5 and 52.0 kJ mol⁻¹ were calculated for the boat-to-boat exchange of 1 and 2 , respectively. The results of the ROESY experiments performed at 193 K and computational modelling suggest that in solution the resorcinarene podand 1 adopts a similar conformation to that present in its crystal structure, whereas podand 2 populates a more versatile range of conformations in solution.     

On generalized trade-off directions in nonconvex multiobjective optimization

Trade-off information related to Pareto optimal solutions is important in multiobjective optimization problems with conflicting objectives. Recently, the concept of trade-off directions has been introduced for convex problems. These trade-offs are characterized with the help of tangent cones. Generalized trade-off directions for nonconvex problems can be defined by replacing convex tangent cones with nonconvex contingent cones. Here we study how the convex concepts and results can be generalized into a nonconvex case. Giving up convexity naturally means that we need local instead of global analysis. Received: December 2000 / Accepted: October 2001?Published online February 14, 2002     

Analytical strategies for improving the robustness and reproducibility of bioluminescent microbial bioreporters

Whole-cell bioluminescent (BL) bioreporter technology is a useful analytical tool for developing biosensors for environmental toxicology and preclinical studies. However, when applied to real samples, several methodological problems prevent it from being widely used. Here, we propose a methodological approach for improving its analytical performance with complex matrix. We developed bioluminescent Escherichia coli and Saccharomyces cerevisiae bioreporters for copper ion detection. In the same cell, we introduced two firefly luciferases requiring the same luciferin substrate emitting at different wavelengths. The expression of one was copper ion specific. The other, constitutively expressed, was used as a cell viability internal control. Engineered BL cells were characterized using the noninvasive gravitational field-flow fractionation (GrFFF) technique. Homogeneous cell population was isolated. Cells were then immobilized in a polymeric matrix improving cell responsiveness. The bioassay was performed in 384-well black polystyrene microtiter plates directly on the sample. After 2 h of incubation at 37 °C and the addition of the luciferin, we measured the emitted light. These dual-color bioreporters showed more robustness and a wider dynamic range than bioassays based on the same strains with a single reporter gene and that uses a separate cell strain as BL control. The internal correction allowed to accurately evaluate the copper content even in simulated toxic samples, where reduced cell viability was observed. Homogenous cells isolated by GrFFF showed improvement in method reproducibility, particularly for yeast cells. The applicability of these bioreporters to real samples was demonstrated in tap water and wastewater treatment plant effluent samples spiked with copper and other metal ions.