Coupling of enzymatic and immunoassay steps to detect E. coli: a new, highly sensitive tandem technique for the analysis of low levels of bacteria

A tandem technique for the detection of very low levels E. coli within about 2 h is demonstrated. The technique couples the widely employed microbial enzymatic detection methods with an immunoassay step. The bacterial marker enzyme, E. coli β-D-galactosidase, was used in conjunction with synthetic enzyme substrates to produce products that could be measured with a highly sensitive enzyme-labelled immunosorbent assay (ELISA). The commercially available 4-methylumbelliferyl-β-D-galactoside and a newly prepared substrate, 4-methylcoumarin-3-propionate-7-O-β-D-galactoside, were used with an ELISA for 7-hydroxy-4-methylcoumarin to demonstrate the detection of low levels of E. coli. The 2 h test indicates that a few viable bacteria cells could be detected by the tandem procedure. The end point of the test is an ELISA with colorimetric measurement step. The novel approach retains the essential features of the microbial enzymatic detection procedures and provides a highly sensitive detection system that can be used for rapid screening or quantification of viable microbial cells in water samples. The tandem test is generic for commonly employed glycosidases and other marker enzymes for which 4-methylumbillerone substrates are available.     

Diisopropyl tartrate (E)-γ-(dimethylphenylsilyl)allylboronate, a chiral allylic alcohol β-carbanion equivalent for the enantioselective synthesis of 2-butene-1,4-diols from aldehydes

An enantioselective synthesis of 4-substituted (E)-2-buten-1,4-diols is described. The method involves the reaction of aldehydes with the chiral PhMe₂Si- substituted allylboronate3 followed by epoxidation (dimethyl dioxirane) and acid catalyzed Petersen rearrangement of the intermediate epoxysilanols.     

Binding of the Zn2+ ion to ferric uptake regulation protein from E. coli and the competition with Fe2+ binding: a molecular modeling study of the effect on DNA binding and conformational changes of Fur

The three dimensional structure of Ferric uptake regulation protein dimer from E. coli, determined by molecular modeling, was docked on a DNA fragment (iron box) and Zn²⁺ ions were added in two steps. The first step involved the binding of one Zn²⁺ ion to what is known as the zinc site which consists of the residues Cys 92, Cys 95, Asp 137, Asp141, Arg139, Glu 140, His 145 and His 143 with an average metal-Nitrogen distance of 2.5 Å and metal-oxygen distance of 3.1–3.2 Å. The second Zn²⁺ ion is bound to the iron activating site formed from the residues Ile 50, His 71, Asn 72, Gly 97, Asp 105 and Ala 109. The binding of the second Zn²⁺ ion strengthened the binding of the first ion as indicated by the shortening of the zinc-residue distances. Fe²⁺, when added to the complex consisting of 2Zn²⁺/Fur dimer/DNA, replaced the Zn²⁺ ion in the zinc site and when a second Fe²⁺ was added, it replaced the second zinc ion in the iron activating site. The binding of both zinc and iron ions induced a similar change in Fur conformations, but shifted residues closer to DNA in a different manner. This is discussed along with a possible role for the Zn²⁺ ion in the Fur dimer binding of DNA in its repressor activity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

An LC–MS/MS assay for the quantitative determination of 2‐pyridyl acetic acid,a major metabolite and key surrogate for betahistine,using low‐volume human K2EDTA plasma

Betahistine is widely used for the treatment of vertigo. Owing to first‐pass metabolism, 2‐pyridyl acetic acid (2PAA, major metabolite of betahistine) was considered as surrogate for quantitation. A specific and sensitive LC–MS/MS method was developed and validated for quantitation of 2PAA using turbo‐ion spray in a positive ion mode. A solid‐phase extraction was employed for the extraction of 2PAA and 2PAA d₆ (IS) from human plasma. Chromatographic separation of analytes was achieved using an ACE CN, 5 μm (50 × 4.6 mm) column with a gradient mobile phase comprising acetonitrile–methanol (90:10% v /v) and 0.7% v/v formic acid in 0.5 mm ammonium trifluoroacetate in purified water (100% v/v). The retention times of 1.15 and 1.17 min for 2PAA and internal standard, respectively, were achieved. Quantitation of 2PAA and internal standard was achieved by monitoring multiple reaction monitoring transition pairs (m /z 138.1 to m /z 92.0 and m /z 142.1 to m /z 96.1, respectively). The developed method was validated for various parameters. The calibration curves of 2PAA showed linearity from 5.0 to 1500 ng/mL, with a lower limit of quantitation of 5.0 ng/mL. The bias and precision for inter‐ and intra‐batch assays were <10%. The developed method was used to support clinical sample analysis.     

Structures and stabilities of group 13 adducts [(NHC)(EX3)] and [(NHC)2(E2X(n))] (E=B to In; X=H, Cl; n=4, 2, 0; NHC=N-heterocyclic carbene) and the search for hydrogen storage systems: a theoretical study

Quantum chemical calculations using density functional theory at the BP86/TZVPP level and ab initio calculations at the SCS-MP2/TZVPP level have been carried out for the group 13 complexes [(NHC)(EX(3))] and [(NHC)(2)(E(2)X(n))] (E=B to In; X=H, Cl; n=4, 2, 0; NHC=N-heterocyclic carbene). The monodentate Lewis acids EX(3) and the bidentate Lewis acids E(2) X(n) bind N-heterocyclic carbenes rather strongly in donor-acceptor complexes [(NHC)(EX(3))] and [(NHC)(2)(E(2)X(n))]. The equilibrium structures of the bidentate complexes depend on the electronic reference state of E(2)X(n), which may vary for different atoms E and X. All complexes [(NHC)(2)(E(2)X(4))] possess C(s) symmetry in which the NHC ligands bind in a trans conformation to the group 13 atoms E. The complexes [(NHC)(2)(E(2)H(2))] with E=B, Al, Ga have also C(s) symmetry with a trans arrangement of the NHC ligands and a planar CE(H)E(H)C moiety that has a E=E π bond. In contrast, the indium complex [(NHC)(2)(In(2) H(2))] has C(i) symmetry with pyramidal-coordinated In atoms in which the hydrogen atoms are twisted above and below the CInInC plane. The latter C(i) form is calculated for all chloride systems [(NHC)(2)(E(2)Cl(2))], but the boron complex [(NHC)(2)(B(2)Cl(2))] deviates only slightly from C(s) symmetry. The B(2) fragment in the linear coordinated complex [(NHC)(2)(B(2))] has a highly excited (3)(1)Σ(g)(-) reference state, which gives an effective B≡B triple bond with a very short interatomic distance. The heavier homologues [(NHC)(2)(E(2))] (E=Al to In) exhibit a anti-periplanar arrangement of the NHC ligands in which the E(2) fragments have a (1)(1) Δ(g) reference state and an E=E double bond. The calculated energies suggest that the dihydrogen release from the complexes [(NHC)(EH(3))] and [(NHC)(2)(E(2)H(n))] becomes energetically more favourable when atom E becomes heavier. The indium complexes should therefore be the best candidates of the investigated series for hydrogen-storage systems that could potentially deliver dihydrogen at close to ambient temperature. The hydrogenation reaction of the dimeric magnesium(I) compound [LMgMgL] (L=β-diketiminate) with [(NHC)(EH(3))] becomes increasingly exothermic with the trend B相似文献   

New In-Situ Sampling and Analysis of the Production of CeO<Subscript>2</Subscript> Powders from Liquid Precursors in a rf Thermal Plasma Reactor. Part 2: Analysis of CeO<Subscript>2</Subscript> Powders,Fuel Addition,and Image Analysis

A novel reactor design, sampling probe and wet collection system were used to investigate the combined effects of plasma operating parameters and particle collection mechanisms on the synthesis of CeO₂ particles from liquid precursors. The sampling of particles in-flight and the collection of particles at several reactor regions were used to provide experimental evidence of particle size at different reactor locations at various plasma operating conditions, i.e., power and plasma gas flow rates. This information provided a picture of how CeO₂ particles were formed and how these particles were collected in various locations. The effect of adding water-soluble fuels (alanine and glycine) to the original cerium nitrate solutions was also investigated. Fuel addition decreased the temperature of CeO₂ formation by acting as a local heat source as a result of fuel auto-ignition. Photographs of the particles in-flight were taken using a fast speed CCD camera.     

Studies on the syntheses of polymetalloxanes and their properties as a precursor for amorphous oxide. IV. Properties of SiO2–TiO2 oxide fibers from polytitanosiloxane

From polytitanosiloxanes (PTS), SiO₂–TiO₂ oxide fibers with fairly good tensile strength were prepared, and their mechanical and thermal properties were investigated. The precursor fibers PTS-0.5 and PTS-1.0 were obtained by dry spinning of a highly viscous PTS solution which were formed as the reaction mixture of silicic acid (SA) with bis(2,4-pentanedionato)titanium diisopropoxide (PTP) in the molar ratios (SA/PTP) of 0.5 and 1.0. The precursor fibers PTS-0.5 were too brittle to measure their tensile strength, whereas PTS-1.0 and the heat-treated fibers were found to have tensile strength of 130 (precursor), 540 (500°C), and 450 (900°C) MPa, respectively. Heat-treatment of the fibers PTS-1.0 at above 1000°C forms anatase and rutile of titanium dioxide. The crystallization is resulted from the unreacted PTP which is not incorporated into the polymer network.     

Determination of vibrational parameters of methanol from matrix-isolation infrared spectroscopy and ab initio calculations. Part 2 – Theoretical treatment including a perturbative approach of the resonances within the methyl group

Ab initio calculations of the potential energy surface of methanol have been developed for the determination of vibrational parameters and their comparison with vibrational data reported in the first part of this work. The strong resonances between the methyl bending and stretching modes, giving rise to polyads of levels P_n in the ranges 3000–2800 (P₂), 4500–4250 (P₃) and 6000–5600 cm⁻¹ (P₄), have been treated by solving for each polyad two Hamiltonian matrices containing off-diagonal terms including both Fermi and Darling-Dennison anharmonic contributions. These terms were calculated from the ab initio determination of the potential energy surface developed up to the quartic terms using the Möller–Plesset 2 method. The choice of the basis set was made to minimize the problem of divergence of the Darling-Dennison constants. Their determination requires however the omission of the terms in which the difference between the harmonic frequencies of the symmetrical methyl stretching and the sum of the two A′ bendings (ω₃–ω₄–ω₅) appears in the denominator. Then, by adjustment of the diagonal elements of the Hamiltonian matrices, it becomes possible to propose a realistic assignment of the matrix spectra.     

Validation of a screening method for the simultaneous identification of fat-soluble and water-soluble vitamins (A,E, B<Subscript>1</Subscript>, B<Subscript>2</Subscript> and B<Subscript>6</Subscript>) in an aqueous micellar medium of hexadecyltrimethylammonium chloride

Simultaneous determination of the fat-soluble vitamins A and E and the water-soluble vitamins B₁, B₂ and B₆ has been carried using a screening method from fluorescence contour graphs. These graphs show different colour zones in relation to the fluorescence intensity measured for the pair of excitation/emission wavelengths. The identification of the corresponding excitation/emission wavelength zones allows the detection of different vitamins in an aqueous medium regardless of the fat or water solubility of each vitamin, owing to the presence of a surfactant which forms micelles in water at the used concentration (over the critical micelle concentration). The micelles dissolve very water insoluble compounds, such as fat-soluble vitamins, inside the aggregates. This approach avoids the use of organic solvents in determining these vitamins and offers the possibility of analysing fat- and water-soluble vitamins simultaneously. The method has been validated in terms of detection limit, cut-off limit, sensitivity, number of false positives, number of false negatives and uncertainty range. The detection limit is about g L^–1. The screening method was applied to different samples such as pharmaceuticals, juices and isotonic drinks.     

Proteomic analysis of cellular soluble proteins from human bronchial smooth muscle cells by combining nondenaturing micro 2DE and quantitative LC‐MS/MS. 2. Similarity search between protein maps for the analysis of protein complexes

Human bronchial smooth muscle cell soluble proteins were analyzed by a combined method of nondenaturing micro 2DE, grid gel‐cutting, and quantitative LC‐MS/MS and a native protein map was prepared for each of the identified 4323 proteins [1]. A method to evaluate the degree of similarity between the protein maps was developed since we expected the proteins comprising a protein complex would be separated together under nondenaturing conditions. The following procedure was employed using Excel macros; (i) maps that have three or more squares with protein quantity data were selected (2328 maps), (ii) within each map, the quantity values of the squares were normalized setting the highest value to be 1.0, (iii) in comparing a map with another map, the smaller normalized quantity in two corresponding squares was taken and summed throughout the map to give an “overlap score,” (iv) each map was compared against all the 2328 maps and the largest overlap score, obtained when a map was compared with itself, was set to be 1.0 thus providing 2328 “overlap factors,” (v) step (iv) was repeated for all maps providing 2328 × 2328 matrix of overlap factors. From the matrix, protein pairs that showed overlap factors above 0.65 from both protein sides were selected (431 protein pairs). Each protein pair was searched in a database (UniProtKB) on complex formation and 301 protein pairs, which comprise 35 protein complexes, were found to be documented. These results demonstrated that native protein maps and their similarity search would enable simultaneous analysis of multiple protein complexes in cells.     

(−)-Quinic acid: a versatile precursor for the synthesis of analogues of 2-crotonyloxymethyl-(4R,5R,6R)-4,5,6-trihydroxycyclohex-2-enone (COTC) which possess anti-tumour properties

Syntheses of three novel analogues of the Streptomyces metabolite COTC are described, using the versatile chiral pool starting material, (−)-quinic acid. The results of bioassays of the target compounds against two lung cancer cell lines, A549 and H460, are presented.     

Triumfettosterol Id and Triumfettosaponin,a New (Fatty Acyl)‐Substituted Steroid and a Triterpenoid ‘Dimer’ Bis(β‐D‐glucopyranosyl) Ester from the Leaves of Wild Triumfetta cordifolia A. Rich. (Tiliaceae)

Two new triterpenoid derivatives were isolated from the leaves of wild Triumfetta cordifolia A. Rich . and identified to be a (fatty acyl)‐substituted steroid 1 and a triterpenoid saponin ‘dimer’ 2 , named (3β)‐stigmasta‐5,22‐diene‐3,29‐diol 3‐propanoate 29‐triacontanoate and (2α,3β,19α)‐2,3,19‐trimethoxyurs‐12‐ene‐24,28‐dioic acid 24‐[(2α,3β)‐24,28‐bis(β‐D ‐glucopyranosyloxy)‐2‐hydroxy‐24,28‐dioxours‐12‐en‐3‐yl] ester, respectively. These compounds were obtained together with a mixture of known sterols (stigmasterol/β‐sitosterol=(3β,22E)‐stigmasta‐5,22‐dien‐3‐ol/(3β)‐stigmast‐5‐en‐3‐ol) and trans‐tiliroside ( 3 ). The structures 1 and 2 were determined on the basis of NMR data (¹H‐, ¹³C‐, and 2D‐NMR analyses) and mass spectrometry and confirmed by chemical transformations. The antimicrobial activities of trans‐tiliroside ( 3 ) against eight bacterial and two fungal strains were evaluated. This compound showed weak activities on some bacterial strains.     

ChemInform Abstract: Lewis Acidities and Hydride,Fluoride, and X‐ Affinities of the BH3‐nXn Compounds for (X: F,Cl, Br,I, NH2, OH,and SH) from Coupled Cluster Theory.

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