A novel high-resolution chipCE assay for rapid detection of EGFR gene mutations and amplifications in lung cancer therapy by a combination of fragment analysis, denaturing CE and MLPA

There is a growing interest in evaluating molecular markers as predictors of response to new generation of targeted cancer therapies. One of such areas is biological therapy targeting epidermal growth factor receptor gene (EGFR) in lung cancer. The testing of tumor tissue is focused on specific EGFR mutations and EGFR gene amplification, since tumors exhibiting positivity of either of the two marker types are highly sensitive towards the treatment. Although traditional methods of DNA sequencing and fluorescence in situ hybridization are still in use for the detection of EGFR mutations and gene amplification, respectively, there is a need for new dedicated techniques with the primary emphasis on simplicity, sensitivity, speed and cost effectiveness. The main purpose of this work was to integrate diverse assays for both EGFR tests onto a single platform to eliminate the need for different instruments and separate processing. We demonstrate a chip capillary electrophoresis (chipCE) application for EGFR mutation detection by a combination of fragment analysis and denaturing CE along with multiplex ligation-dependent probe amplification (MLPA) for evaluation of EGFR amplification. All separations are carried out in denaturing sieving polymer on a modified Bioanalyzer 2100 chipCE instrument running at temperatures of up to 65°C. The main strength of the resulting high-resolution chipCE application is in its simplicity, speed of analysis and minimal amount of sample required for complete testing of EGFR status. Such an approach could potentially fit medium throughput laboratories providing molecular pathology services for clinical oncologists with fast turnaround times and limited consumption of tissue material.     

Fast profiling of anthocyanins in wine by desorption nano-electrospray ionization mass spectrometry

Desorption electrospray ionization (DESI) mass spectrometry appears to be a useful technique applicable in different areas (e.g. analysis of pharmaceuticals, identification of biologically active compounds in tissues, imaging mass spectrometry). Its modification termed desorption nano-electrospray (nano-DESI) was tested for analysis of anthocyanins. Acidifying of samples and acidic spray liquid (methanol:water = 75:25 with 0.2% HCOOH) were essential for obtaining good quality spectra. Profiles of main anthocyanins in wine samples, two vintages (2005 and 2007) of three cultivars (Alibernet, Neronet and Rubinet), were successfully acquired. They were in agreement with results of LC/MS experiments (anthocyanins isolated by solid phase extraction were separated by μ-HPLC with gradient elution and detected by ESI-MS). Nano-DESI-MS data also allowed to determine ratio of two cultivars (Neronet and Rubinet) in their mixture and to detect coloring of wine by tenturier or elderberry extract. Detection of main anthocyanins in slices of wine grape, chokeberries and elderberries or in a wine stain on cotton fabric is also presented.     

Modulation of Living Cell Behavior with Ultra‐Low Fouling Polymer Brush Interfaces

Ultra‐low fouling and functionalizable coatings represent emerging surface platforms for various analytical and biomedical applications such as those involving examination of cellular interactions in their native environments. Ultra‐low fouling surface platforms as advanced interfaces enabling modulation of behavior of living cells via tuning surface physicochemical properties are presented and studied. The state‐of‐art ultra‐low fouling surface‐grafted polymer brushes of zwitterionic poly(carboxybetaine acrylamide), nonionic poly(N‐(2‐hydroxypropyl)methacrylamide), and random copolymers of carboxybetaine methacrylamide (CBMAA) and HPMAA [p(CBMAA‐co‐HPMAA)] with tunable molar contents of CBMAA and HPMAA are employed. Using a model Huh7 cell line, a systematic study of surface wettability, swelling, and charge effects on the cell growth, shape, and cytoskeleton distribution is performed. This study reveals that ultra‐low fouling interfaces with a high content of zwitterionic moieties (>65 mol%) modulate cell behavior in a distinctly different way compared to coatings with a high content of nonionic HPMAA. These differences are attributed mostly to the surface hydration capabilities. The results demonstrate a high potential of carboxybetaine‐rich ultra‐low fouling surfaces with high hydration capabilities and minimum background signal interferences to create next‐generation bioresponsive interfaces for advanced studies of living objects.     

Sorption separation of Eu and As from single-component systems by Fe-modified biochar: kinetic and equilibrium study

The utilization of carbonaceous materials in separation processes of radionuclides, heavy metals and metalloids represents a burning issue in environmental and waste management. The main objective of this study was to characterize the effect of chemical modification of corncob-derived biochar by Fe-impregnations on sorption efficiency of Eu and As as a model compounds of cationic lanthanides and anionic metalloids. The biochar sample produced in slow pyrolysis process at 500 °C before (BC) and after (IBC) impregnation process was characterized by elemental, FTIR, SEM-EDX analysis to confirm the effectiveness of Fe-impregnation process. The basic physico-chemical properties showed differences in surface area and pH values of BC- and IBC-derived sorbents. Sorption processes of Eu and As by BC and IBC were characterized as a time- and initial concentration of sorbate-dependent processes. The sorption equilibrium was reached for both sorbates in 24 h of contact time. Batch equilibrium experiments revealed the increased maximum sorption capacities (Q _max) of IBC for As about more than 20 times (Q _max BC 0.11 and Q _max IBC 2.26 mg g^?1). Our study confirmed negligible effect of Fe-impregnation on sorption capacity of biochar for Eu (Q _max BC 0.89 and Q _max IBC 0.98 mg g^?1). The iron-impregnation of biochar-derived sorbents can be utilized as a valuable treatment method to produce stable and more effective sorption materials for various xenobiotics separation from liquid wastes and aqueous solutions.     

Systematic modulation of a bichromic cyclometalated ruthenium(II) scaffold bearing a redox-active triphenylamine constituent

The syntheses and physicochemical properties of nine bis-tridentate ruthenium(II) complexes containing one cyclometalating ligand furnished with terminal triphenylamine (TPA) substituents are reported. The structure of each complex conforms to a molecular scaffold formulated as [Ru(II)(TPA-2,5-thiophene-pbpy)(Me(3)tctpy)] (pbpy = 6-phenyl-2,2'-bipyridine; Me(3)tctpy = trimethyl-4,4',4'-tricarboxylate-2,2':6',2'-terpyridine), where various electron-donating groups (EDGs) and electron-withdrawing groups (EWGs) are installed about the TPA unit and the anionic ring of the pbpy ligand. It is found that the redox chemistry of the Ru center and the TPA unit can be independently modulated by (i) placing EWGs (e.g., -CF(3)) or EDGs (e.g., -OMe) on the anionic ring of the pbpy ligand (substituted sites denoted as R(2) or R(3)) and/or (ii) installing electron-donating substituents (e.g., -H, -Me, -OMe) para to the amine of the TPA group (i.e., R(1)). The first oxidation potential is localized to the TPA unit when, for example, EDGs are placed at R(1) with EWGs at R(2) (e.g., the TPA(?+)/TPA(0) and Ru(III)/Ru(II) redox couples appear at +0.98 and +1.27 V vs NHE, respectively, when R(1) = -OMe and R(2) = -CF(3)). This situation is reversed when R(3) = EDG and R(1) = -H: TPA-based and metal-centered oxidation waves occur at +1.20 and +1.11 V vs NHE, respectively. The UV-vis spectrum for each complex is broad (e.g., absorption bands are extended from the UV region to beyond 800 nm in all cases) and intense (e.g., ε ～ 10(4) M(-1)·cm(-1)) because of the overlapping intraligand charge-transfer and metal-to-ligand charge-transfer transitions. The information derived from this study offers guiding principles for modulating the physicochemical properties of bichromic cyclometalated ruthenium(II) complexes.     

C-2/c-3 annulation and C-2 alkylation of indoles with 2-alkoxycyclopropanoate esters

The annulation reaction between various indoles and 2-alkoxycyclopropanoate esters is reported. Both high efficiency and complete stereochemical control were observed in some cases with this annulation process. A single stereocenter on the cyclopropane controls the diastereoselective formation of up to four new stereocenters. A different reaction course was observed with 3-substituted indole substrates, and an intervening C-3 to C-2-migration process arose that gives synthetically useful C-2 alkylation indole products.     

Achiral and chiral high-performance liquid chromatographic determination of flubendazole and its metabolites in biomatrices using UV photodiode-array and mass spectrometric detection

Flubendazole, methyl ester of [5-(4-fluorobenzoyl)-1H-benzimidazol-2-yl]carbamic acid, belongs to the group of benzimidazole anthelmintics, which are widely used in veterinary and human medicine. The phase I flubendazole biotransformation includes a hydrolysis of the carbamoyl methyl moiety accompanied by a decarboxylation (hydrolysed flubendazole) and a carbonyl reduction of flubendazole (reduced flubendazole). Flubendazole is a prochiral drug, hence a racemic mixture is formed during non-stereoselective reductions at the carbonyl group. Two bioanalytical HPLC methods were developed and validated for the determination of flubendazole and its metabolites in pig and pheasant hepatic microsomal and cytosolic fractions. Analytes were extracted from biomatrices into tert-butylmethyl ether. The first, achiral method employed a 250 mm x 4 mm column with octylsilyl silica gel (5 microm) and an isocratic mobile phase acetonitrile-0.025 M KH(2)PO(4) buffer pH 3 (28:72, v/v). Albendazole was used as an internal standard. The whole analysis lasted 27 min at a flow rate of 1 ml/min. The second, chiral HPLC method, was performed on a Chiralcel OD-R 250 mm x 4.6 mm column with a mobile phase acetonitrile-1 M NaClO(4) (4:6, v/v). This method enabled the separation of both reduced flubendazole enantiomers. The enantiomer excess was evaluated. The column effluent was monitored using a photodiode-array detector (scan or single wavelength at lambda=246 nm). Each of the analytes under study had characteristic UV spectrum, in addition, their chemical structures were confirmed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) experiments. Stereospecificity in the enzymatic carbonyl reduction of flubendazole was observed. While synthetic racemic mixture of reduced flubendazole was separated to equimolar amounts of both enantiomers, practically only one enantiomer was detected in the extracts from all incubates.     

Bulk derivatization and direct injection of human cerebrospinal fluid for trace‐level quantification of endogenous estrogens using trap‐and‐elute liquid chromatography with tandem mass spectrometry

Although there are existing methods for determining estrogen in human bodily fluids including blood plasma and serum, very little information is available regarding estrogen levels in human cerebrospinal fluid (CSF), which is critical to assess in studies of neuroprotective functions and diffusion of neuroprotective estrogens across the blood–brain barrier. To address this problem, a liquid chromatography with tandem mass spectrometry method for the simultaneous quantification of four endogenous estrogens (estrone, 17α‐estradiol, 17β‐estradiol, and estriol) in human CSF was developed. An aliquot (300 μL) of human CSF was bulk derivatized using dansyl chloride in the sample and 10 μL was directly injected onto a restricted‐access media trap column for protein removal. No off‐line sample extraction or cleanup was needed. The limits of detection of estrone, 17α‐estradiol, 17β‐estradiol, and estriol were 17, 28, 13, and 30 pg/mL, respectively, which is in the parts‐per‐trillion regime. The method was then applied to human CSF collected from ischemic trauma patients. Endogenous estrogens were detected and quantified, demonstrating the effectiveness of this method.     

Completion of Semi-uniform Spaces

The category of all Hausdorff complete t-semi-uniform spaces is shown to be epireflective in the category of all Hausdorff t-semi-uniform spaces but the reflection arrows need not be embeddings since there is no nontrivial epireflective subcategory of the category of all Hausdorff t-semi-uniform spaces in which all reflection arrows are embeddings (t-semi-uniform spaces are those semi-uniform spaces inducing a topology). On the other hand for every t-semi-uniform space X there exist a minimal and a maximal completion containing X as a dense subspace. The second one is an almost reflection in complete spaces, i.e., every uniformly continuous mapping on X to a complete semi-uniform space can be extended (as a uniformly continuous map) onto the completion.