Discovery of Tyrosine Kinase Inhibitors by Docking into an Inactive Kinase Conformation Generated by Molecular Dynamics

Several small molecules that bind to the inactive DFG‐out conformation of tyrosine kinases (called type II inhibitors) have shown a good selectivity profile over other kinase targets. To obtain a set of DFG‐out structures, we performed an explicit solvent molecular dynamics (MD) simulation of the complex of the catalytic domain of a tyrosine kinase receptor, ephrin type‐A receptor 3 (EphA3), and a manually docked type II inhibitor. Automatic docking of four previously reported type II inhibitors was used to select a single snapshot from the MD trajectory for virtual screening. High‐throughput docking of a pharmacophore‐tailored library of 175 000 molecules resulted in about 4 million poses, which were further filtered by van der Waals efficiency and ranked according to a force‐field‐based energy function. Notably, around 20 % of the compounds with predicted binding energy smaller than ?10 kcal mol^?1 are known type II inhibitors. Moreover, a series of 5‐(piperazine‐1‐yl)isoquinoline derivatives was identified as a novel class of low‐micromolar inhibitors of EphA3 and unphosphorylated Abelson tyrosine kinase (Abl1). The in silico predicted binding mode of the new inhibitors suggested a similar affinity to the gatekeeper mutant T315I of Abl1, which was verified in vitro by using a competition binding assay. Additional evidence for the type II binding mode was obtained by two 300 ns MD simulations of the complex between N‐(3‐chloro‐4‐(difluoromethoxy)phenyl)‐2‐(4‐(8‐nitroisoquinolin‐5‐yl)piperazin‐1‐yl)acetamide and EphA3.     

Myocardial blood flow quantification from MRI by deconvolution using an exponential approximation basis

We have evaluated the use of deconvolution using an exponential approximation basis for the quantification of myocardial blood flow from perfusion cardiovascular magnetic resonance. Our experiments, based on simulated signal intensity curves, phantom acquisitions, and clinical image data, indicate that exponential deconvolution allows for accurate quantification of myocardial blood flow. Together with automated respiratory motion correction myocardial contour delineation, the exponential deconvolution enables efficient and reproducible quantification of myocardial blood flow in clinical routine.     

Residues of the quinone outside inhibitor fungicide trifloxystrobin after postharvest dip treatments to control Penicillium spp. on citrus fruit

The effectiveness of postharvest dip treatment with trifloxystrobin (TFX) or imazalil (IMZ) was compared for controlling green and blue mold (caused by Penicillium digitatum and Penicillium italicum, respectively) of citrus fruit. Residues retained by fruit were determined as a function of treatment time, dip temperature, and storage conditions. Trials on 'Avana apireno' mandarin oranges artificially inoculated with P. digitatum or P. italicum revealed that treatments with 200 to 600 mg/liter active ingredient TFX at 20 degrees C were less effective than 100 mg/liter TFX at 500C for controlling P. digitatum but equally effective for controlling P. italicum. IMZ treatments with 200 mg/liter IMZ at 20 degrees C or 25 mg/liter IMZ at 50 degrees C resulted in more than 98% reduction of P. digitatum and ca. 93% reduction of P. italicum compared with untreated fruit. Total suppression of pathogens was achieved when higher IMZ doses were applied. Studies on artificially wounded lemons, oranges, clementines, and mandarins revealed that treatment with 100 mg/liter TFX at 50 degrees C effectively controlled decay development (mainly due to P. digitatum) after 7 days of storage at 20 degrees C. These results were confirmed on nonwounded oranges of cv. Tarocco and on grapefruits of cvs. Marsh Seedless and Star Ruby during 3 weeks of simulated quarantine at 1 degrees C, storage (5 weeks at 8 degrees C for oranges and 8 weeks at 11degrees C for grapefruits), and an additional 1 week of simulated marketing conditions at 20 degrees C. IMZ at 50 degrees C was highly effective for controlling decay during storage and the simulated marketing period. TFX treatment at 50 degrees C was as effective as IMZ for controlling decay in most samples. After treatment with 100 mg/liter TFX at 20 degrees C, fungicide residues in 'Tarocco' oranges doubled from 0.15 mg/kg to 0.30 mg/kg when dip time increased from 0.5 to 3 min, whereas when treatments were performed at 50 degrees C TFX residues were not related to dipping time. Residues of TFX were significantly correlated with dip temperature. A 3-min dip treatment at 50 degrees C resulted in a deposition of TFX that was approximately twofold higher than that obtained when treatments were carried out at 20 degrees C.     

Simple Phosphate Buffer Extraction for the Determination of Fumonisins in Masa,Maize, and Derived Products

An accurate determination of fumonisins in masa can be troublesome because of difficulties often experienced in obtaining satisfactory recoveries. In this study, a new extraction method, effective both for masa and for maize and derived products, has been developed. The method extracts fumonisins using a 0.4 M phosphate buffer (PB) at pH 7.5, without any organic solvent; the purification of the extract is performed by an immunoaffinity column and the quantification by HPLC–MS/MS. The average recovery percentages were 95.5 ± 1.9% and 96.7 ± 2.1% for fumonisin B₁ and B₂, respectively. The limit of detection and of quantification for both fumonisins were 10 and 30 μg kg⁻¹, respectively. For masa and tortillas, PB pH 7.5 gave significantly higher results when compared with AOAC and other published extraction methods; for maize flour, PB gave generally higher results, even if the difference with the other extraction methods was not always significant.     

Cow's milk allergens identification by two-dimensional immunoblotting and mass spectrometry

Cow's milk allergy (CMA) has become a common disease in early childhood, its prevalence ranging from 1.6% to 2.8% among children younger than 2 years of age. The role of different cow's milk protein (CMP) in the pathogenesis of CMA is still controversial. Even if the proteins most frequently and most intensively recognized by immunoglobulin E (IgE) seem to be the most abundant in milk (caseins and beta-lactoglobulin), with an although great variability all milk proteins appear to be potential allergens, even those that are present in trace amounts (i.e., lactoferrin, IgG, and BSA). In this work proteomics techniques have been applied for CMP allergens analysis. Allergens have been identified by immunoblotting following resolution of CMP components by two-dimensional electrophoresis. Sera from 20 milk-allergic subjects, as proven by oral provocation test, CAP-RAST and skin prick test, have been used for cow's milk major allergen identification. Cow's milk proteins and their isoforms were identified by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry. In our group of patients, the prevalence of CMP allergens, i.e., the total number of subjects sensitized to CMP divided by the total number of the subjects enrolled in the study, was: 55% alpha(s1)-casein, 90% alpha(s2)-casein, 15% beta-casein, 50% kappa-casein, 45% beta-lactoglobulin, 45% BSA, 95% IgG-heavy chain, 50% lactoferrin, and 0% alpha-lactalbumin.