Evaluating the catalytic contribution from the oxyanion hole in ketosteroid isomerase

Prior site-directed mutagenesis studies in bacterial ketosteroid isomerase (KSI) reported that substitution of both oxyanion hole hydrogen bond donors gives a 10(5)- to 10(8)-fold rate reduction, suggesting that the oxyanion hole may provide the major contribution to KSI catalysis. But these seemingly conservative mutations replaced the oxyanion hole hydrogen bond donors with hydrophobic side chains that could lead to suboptimal solvation of the incipient oxyanion in the mutants, thereby potentially exaggerating the apparent energetic benefit of the hydrogen bonds relative to water-mediated hydrogen bonds in solution. We determined the functional and structural consequences of substituting the oxyanion hole hydrogen bond donors and several residues surrounding the oxyanion hole with smaller residues in an attempt to create a local site that would provide interactions more analogous to those in aqueous solution. These more drastic mutations created an active-site cavity estimated to be ~650 ?(3) and sufficient for occupancy by 15-17 water molecules and led to a rate decrease of only ~10(3)-fold for KSI from two different species, a much smaller effect than that observed from more traditional conservative mutations. The results underscore the strong context dependence of hydrogen bond energetics and suggest that the oxyanion hole provides an important, but moderate, catalytic contribution relative to the interactions in the corresponding solution reaction.     

Electrochemical Analysis of Acrylamide Using Screen‐Printed Carboxylated Single‐Walled Carbon Nanotube Electrodes

Acrylamide (AA) was electrochemically detected and quantified by means of its voltammetric response on carboxylic modified Single‐Walled Carbon Nanotube Screen Printed Electrodes (COOH‐SWCNT‐SPEs). The electroreduction signal of AA was proportional to AA concentration at low values (below 300 µM) and the observed sensitivity was explained in terms of AA adsorption on the COOH‐SWCNT‐SPEs that was demonstrated using the electrochemical response of [Fe(CN)₆]^3? and [Fe(CN)₆]^4? and Raman spectroscopy experiments. In order to test the suggested analytic approach (LOD of 0.03 µM, LOQ of 0.04 µM), detection and quantification of AA in fried potatoes was carried out using the proposed electrochemical method and HPLC. Both techniques showed similar contents of AA.     

Synthesis,characterization and thermal analysis of ursolic acid solid forms

This study performed a solid‐state characterization of ursolic acid (UA) crystalline forms, a poorly water‐soluble triterpene with anticancer activity. Two new polymorphs (form I, II), two new solvates (propanol and isopropanol solvates), and a known ethanol solvate were determined and elucidated using a combination of multi‐techniques, including X‐ray single crystal and powder diffraction, Fourier transform infrared spectroscopy (FT‐IR), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). A colorless single crystal of UA was grown from a propanol solution, and its crystalline structure was determined through X‐ray single crystal diffraction. It was determined that the propanol solvate was crystallized in the orthorhombic space group P2₁2₁2₁ with unit‐cell parameters a = 7.17200 (8) Å, b = 12.24100 (16) Å, c = 33.8950 (4) Å and Z = 4. The ethanol solvate and propanol solvate were isomorphous crystals. The results of the thermal analysis demonstrate that form I is a meta‐stable form, while form II is a stable form that is monotropically related.     

Isoprene–Styrene Chain Shuttling Copolymerization Mediated by a Lanthanide Half‐Sandwich Complex and a Lanthanidocene: Straightforward Access to a New Type of Thermoplastic Elastomers

A lanthanide half‐sandwich complex and a ansa lanthanidocene have been assessed for isoprene–styrene chain shuttling copolymerization with n‐butylethylmagnesium (BEM). In the presence of 1 equiv BEM, a fully amorphous multiblock microstructure of soft and hard segments is achieved. The microstructure consists of poly(isoprene‐co‐styrene) blocks, with hard blocks rich in styrene and soft blocks rich in isoprene. The composition of the blocks and the resulting glass transition temperatures (T_g) can be easily modified by changing the feed and/or the relative amount of the catalysts, highlighting a new class of thermoplastic elastomers (TPEs) with tunable transition temperatures. The materials self‐organize into nanostructures in the solid state.     

Characterisation of complex amphiphilic cyclodextrin mixtures by high-performance liquid chromatography and mass spectrometry

It is established that amphiphilic beta-cyclodextrins chemically modified with alkyl chains on the secondary face exhibit self-organisation properties yielding stable nanospheres or nanoparticles. The ability of these promising colloidal drug carriers to encapsulate drugs being partly related to the internal structure of nanosystems, precise characterisation methods are required to control their synthesis procedure. The present work describes the development of complementary analytical methods based on reversed-phase high-performance liquid chromatography (RPLC) coupled to evaporative light-scattering detection (ELSD) and electrospray ionisation-mass spectrometry (ESI-MS) to characterize various beta-cyclodextrins enzymatically transesterified by vinyl-acyl fatty esters (the number of carbon atom in the acyl chain varying from 4 to 12). LC-ELSD has been used in a preliminary step to optimize the separation on a monolithic octadecylsiloxane-bonded silica stationary phase. A complex fingerprint was achieved for each mixture, revealing the presence of isomers unnoticed by the sole spectrometric (NMR and MS) techniques.     

An interlaboratory comparison on whole water samples

The European Water Framework Directive 2000/60/EC requires monitoring of organic priority pollutants in so-called whole water samples, i.e. in aqueous non-filtered samples that contain natural colloidal and suspended particulate matter. Colloids and suspended particles in the liquid phase constitute a challenge for sample homogeneity and stability. Within the joint research project ENV08 “Traceable measurements for monitoring critical pollutants under the European Water Framework Directive 2000/60/EC”, whole water test materials were developed by spiking defined amounts of aqueous slurries of ultra-finely milled contaminated soil or sediment and aqueous solutions of humic acid into a natural mineral water matrix. This paper presents the results of an European-wide interlaboratory comparison (ILC) using this type of test materials. Target analytes were tributyltin, polybrominated diphenyl ethers and polycyclic aromatic hydrocarbons in the ng/L concentration range. Results of the ILC indicate that the produced materials are sufficiently homogeneous and stable to serve as samples for, e.g. proficiency testing or method validation. To our knowledge, this is the first time that ready-to-use water materials with a defined amount of suspended particulate and colloidal matter have been applied as test samples in an interlaboratory exercise. These samples meet the requirements of the European Water Framework Directive. Previous proficiency testing schemes mainly employed filtered water samples fortified with a spike of the target analyte in a water-miscible organic solvent.     

Direct patterning of molecularly imprinted microdot arrays for sensors and biochips

We have used fountain pen microlithography to deposit arrays of molecularly imprinted polymer microdots on flat substrates. We visualize analyte binding to the dots by fluorescence microscopy with the aid of fluorescein as a model analyte. Elution and readsorption of the analyte to the MIP dots were possible if the porosity of the dots was improved by a sacrificial polymeric porogen. The imprinting effect was confirmed by using compounds structurally related to fluorescein. In addition, we show with another MIP specific to 2,4-D that, apart from the direct measurement of the binding of fluorescent compounds, a competitive immunoassay-type format can also be used to transduce the binding. We believe that this technique has a strong potential for the fabrication of biomimetic microchips and other types of integrated biosensors.     

Electroactivity of Superoxide Anion in Aqueous Phosphate Buffers Analyzed with Platinized Microelectrodes

The reactivity of platinized ultramicroelectrodes (Pt-black UMEs) towards superoxide anion O₂^.−, an unstable Reactive Oxygen Species (ROS), and its relatives, H₂O₂ and O₂, was studied. Voltammetric studies in PBS demonstrate that Pt-black UMEs provide: i) a well-resolved reversible redox signature for O₂^.− detected in both alkaline and physiological buffers (pH 12 and 7.4); ii) irreversible oxidation and reduction waves for H₂O₂ at pH 7.4. The oxygen reduction reaction (ORR) at Pt-black surfaces solely yields H₂O₂ (2 electrons/2 H⁺) at physiological pH. Consequently, Pt-black UMEs allow to sense different ROS including superoxide anion for future biomedical or physico-chemical investigations.     

Triterpenoids with neurotrophic activity from Ganoderma lucidum

A new triterpenoid, 4,4,14α-trimethyl-5α-chol-7,9(11)-dien-3-oxo-24-oic acid (1), together with seven known triterpenoids, were isolated from the dried fruiting bodies of Ganoderma lucidum. Their structures were elucidated by extensive spectroscopic analyses. Bioassay results revealed that compounds 1 and methyl ganoderic acid B (5) had nerve growth factor-like neuronal survival-promoting effects, whereas compounds 1, and 4-7 showed brain-derived neurotrophic factor-like neuronal survival-promoting activities.     

Application of the iPLEX™ Gold SNP genotyping method for the analysis of Amerindian ancient DNA samples: benefits for ancient population studies

Important developments in the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technique have generated new perspectives regarding SNP genotyping, which are particularly promising for ancient population-based studies. The main aim of the present study was to investigate the application of a MALDI-TOF MS-based SNP genotyping technique, called iPLEX(?) Gold, to analyze Amerindian ancient DNA samples. The first objective was to test the sensitivity of the method, which is recommended for DNA quantities between 10 and 5?ng, for ancient biological samples containing DNA molecules that were degraded and present in minute quantities. The second objective was to detail the advantages of this technique for studies on ancient populations. Two multiplexes were designed, allowing the major Amerindian mitochondrial and Y haplogroups to be determined simultaneously. This analysis has never been described before. Results demonstrated the reliability and accuracy of the method; data were obtained for both mitochondrial and nuclear DNA using picogram (pg) quantities of nucleic acid. This technique has the advantages of both MS and minisequencing techniques; thus, it should be included in the protocols for future ancient DNA studies.