Polyketide Synthase-Mediated O-Methyloxime Formation in the Biosynthesis of the Oximidine Anticancer Agents

Bacterial trans-acyltransferase polyketide synthases (trans-AT PKSs) are modular megaenzymes that employ unusual catalytic domains to assemble diverse bioactive natural products. One such PKS is responsible for the biosynthesis of the oximidine anticancer agents, oxime-substituted benzolactone enamides that inhibit vacuolar H⁺-ATPases. Here, we describe the identification of the oximidine gene cluster in Pseudomonas baetica and the characterization of four novel oximidine variants, including a structurally simpler intermediate that retains potent anticancer activity. Using a combination of in vivo, in vitro and computational approaches, we experimentally elucidate the oximidine biosynthetic pathway and reveal an unprecedented mechanism for O-methyloxime formation. We show that this process involves a specialized monooxygenase and methyltransferase domain and provide insight into their activity, mechanism and specificity. Our findings expand the catalytic capabilities of trans-AT PKSs and identify potential strategies for the production of novel oximidine analogues.     

Gas‐phase copper and silver complexes with phosphorothioate and phosphorodithioate pesticides investigated using electrospray ionization mass spectrometry

Efforts to improve agricultural productivity have led to a growing dependency on organophosphorus pesticides. Phosphorothioate and phosphorodithioate pesticides are organophosphorus pesticide subclasses with widespread application for the control of insects feeding on vegetables and fruits. However, even low doses of these pesticides can cause neurological problems in humans; thus, their determination and monitoring in agricultural foodstuffs is important for human health. Phosphorothioate and phosphorodithioate pesticides may be poorly ionized during electrospray, adversely affecting limits of detection. These pesticides can form complexes with Cu²⁺ and Ag⁺, however, potentially improving ionization. In the present work, we used electrospray ionization/mass spectrometry (ESI/MS) to study fenitrothion, parathion, diazinon, and malathion coordination complexes with silver and copper ions. Stable 1 : 1 and 1 : 2 metal/pesticide complexes were detected. Mass spectra acquired from pesticide solutions containing Ag⁺ or Cu²⁺ showed a significant increase in signal‐to‐background ratio over those acquired from solutions containing only the pesticides, with Ag⁺ improving detection more effectively than Cu²⁺. Addition of Ag⁺ to a pesticide solution improved the limit of detection by ten times. The relative affinity of each pesticide for Ag⁺ was related to complex stability, following the order diazinon > malathion > fenitrothion > parathion. The formation of Ag⁺–pesticide complexes can significantly improve the detection of phosphorothioate and phosphorodithioate pesticides using ESI/MS. The technique could potentially be used in reactive desorption electrospray ionization/mass spectrometry to detect phosphorothioate and phosphorodithioate pesticides on fruit and vegetable skins. Copyright © 2015 John Wiley & Sons, Ltd.     

Development and validation of an LC–MS/MS method for the quantification of artificial sweeteners in human matrices

Artificial sweeteners are widely used as substitutes for sugar. The sweeteners are generally considered safe, however their whereabouts during pregnancy and lactation and the effect on child development are poorly explored. There is a need for new tools to measure these substances during pregnancy and lactation. Here, we describe the development and validation of a sensitive liquid chromatography–tandem mass spectrometry method for the simultaneous quantification of acesulfame, cyclamate, saccharin and sucralose in human plasma, umbilical cord blood, amniotic fluid and breast milk. The samples were prepared by protein precipitation and separated on a Luna Omega Polar C₁₈ column (2.1 × 50 mm, 1.6 μm). Electrospray ionization in negative mode and multiple reaction monitoring were used to monitor the ion transitions. The validated concentration ranges were from 1 to 500 ng/ml (10–500 ng/ml for sucralose). Interassay precisions were all ≤15% and the accuracies were within ±15%. Stability, linearity, dilution integrity, carryover and recovery were also examined and satisfied the validation criteria. Finally, this analytical method was successfully applied on spiked samples of plasma, umbilical cord blood, amniotic fluid and breast milk, proving its suitability for use in clinical studies on artificial sweeteners, including during pregnancy and lactation.     

Conceptual DFT properties‐based 3D QSAR: Analysis of inhibitors of the nicotine metabolizing CYP2A6 enzyme

Structure‐activity relationships of 46 P450 2A6 inhibitors were analyzed using the 3D‐QSAR methodology. The analysis was carried out to confront the use of traditional steric and electrostatic fields with that of a number of fields reflecting conceptual DFT properties: electron density, HOMO, LUMO, and Fukui f⁻ function as 3D fields. The most predictive models were obtained by combining the information of the electron density with the Fukui f⁻ function (r² = 0.82, q² = 0.72), yielding a statistically significant and predictive model. The generated model was able to predict the inhibition potencies of an external test set of five chemicals. The result of the analysis indicates that conceptual DFT‐based molecular fields can be useful as 3D QSAR molecular interaction fields. © 2008 Wiley Periodicals, Inc. J Comput Chem 2009     

Outcome of laryngeal and velopharyngeal biofeedback treatment in children and young adults: A pilot study

A relatively new management strategy for the treatment of voice disorders is the use of laryngeal (LB) and velopharyngeal biofeedback (VB). The main purpose of the present pilot study is to document the outcome of vocal and velopharyngeal performances after a well-defined LB and VB treatment. Four subjects were studied pretreatment (1 week before LB or VB treatment) and posttreatment (1 week after the LB or VB treatment). To measure and compare the effect of LB and VB, objective and subjective assessment techniques were used. Perceptual voice assessment included a perceptual rating of the voice using the GRBAS scale. Furthermore, the vocal quality in this population is modeled by means of the Dysphonia Severity Index. For the objective assessment of nasal resonance, the Nasometer and the Glatzel test were used. A perceptual evaluation of speech, the Gutzmann test, and the tests from Bzoch were used as subjective assessment techniques. Both patients selected for LB and VB treatment showed improvement of their performances. The resulting improvement, as measured by means of an objective approach, is in agreement with the perceived (auditory) improvement of voice and resonance. The use of LB and VB treatment in patients, especially in some subjects who are not responding to traditional voice or velopharyngeal therapy, must be encouraged.     

Radiation damage in Si photodiodes by high-temperature irradiation

Results are presented of a detailed study of the effects of high-temperature 4-MeV neutron irradiation on the performance degradation of Si pin photodiodes together with the radiation-induced defects, observed by deep level transient spectroscopy. The degradation of the device performance and the introduction rate of the lattice defects decrease with increasing sample temperature during irradiation. For a 250°C irradiation, the reduction of the reverse current is only 20% of the starting value. This result suggests that the creation and recovery of the radiation damage proceeds simultaneously at high temperatures. Carbon-related complex as hole capture levels is also mainly responsible for the device degradation for high-temperature neutron irradiation.