Technical considerations for the use of 15N-DNA stable-isotope probing for functional microbial activity in soils

Stable-isotope DNA probing is a culture-independent technique that may provide a link between function and phylogeny of active microorganisms. The technique has been used in association with 13C substrates while here we evaluate feasibility and limitations of 15N-DNA stable-isotope probing (SIP) using labelled and unlabelled pure microbial cultures or soil extracts. Our results showed that (15)N-DNA probing is feasible for cultures as well as soil samples. Limitations of 15N-DNA-SIP are (a) the need for relatively large quantities of DNA to visualise bands (although molecular resolution is much higher) and (b) 15N-DNA enrichment needed to ideally be >50 at%; however, this requirement can be lowered to approx. 40 atom% 15N with pure cultures using a modified CsCl centrifugation method (140K g for 69 h). These advances in 15N-DNA-SIP methodology open new opportunities to trace active microbial populations utilising specific N substrates in situ.     

A novel approach for the homogenization of cellulose to use micro‐amounts for stable isotope analyses

Climate reconstructions using stable isotopes from tree‐rings are steadily increasing. The investigations concentrate mostly on cellulose due to its high stability. In recent years the available amount of cellulose has steadily decreased, mainly because micro‐structures of plant material have had to be analyzed. Today, the amounts of cellulose being studied are frequently in the milligram and often in the microgram range. Consequently, homogeneity problems with regard to the stable isotopes of carbon and oxygen from cellulose have occurred and these have called for new methods in the preparation of cellulose for reliable isotope analyses. Three different methods were tested for preparing isotopically homogenous cellulose, namely mechanical grinding, freezing by liquid nitrogen with subsequent milling and ultrasonic breaking of cellulose fibres. The best precision of isotope data was achieved by freeze‐milling and ultrasonic breaking. However, equipment for freeze‐milling is expensive and the procedure is labour‐intensive. Mechanical grinding resulted in a rather high loss of material and it is also labour‐intensive. The use of ultrasound for breaking cellulose fibres proved to be the best method in terms of rapidity of sample throughput, avoidance of sample loss, precision of isotope results, ease of handling, and cost. Copyright © 2009 John Wiley & Sons, Ltd.     

RABA (Reductive Alkylation By Acetone): A novel stable isotope labeling approach for quantitative proteomics

Quantitative proteomics is challenging and various stable isotope based approaches have been developed to meet the challenge. Hereby we describe a simple, efficient, reliable, and inexpensive method named reductive alkylation by acetone (RABA) to introduce stable isotopes to peptides for quantitative analysis. The RABA method leads to alkylation of N-terminal and lysine amino groups with isopropyl moiety. Using unlabeled (d₀) and deuterium labeled (d₆) acetone, a 6 Da mass split is introduced to each isopropyl modification between the light and heavy isotope labeled peptides, which is ideally suited for quantitative analysis. The reaction specificity, stoichiometry, labeling efficiency, and linear range of the RABA method have been thoroughly evaluated in this study using standard peptides, tryptic digest of proteins, as well as human cell lysate. Reliable quantitative results have been consistently obtained in all experiments. We also applied the RABA method to quantitative analysis of proteins in spinal cords of transgenic mouse models of amyotrophic lateral sclerosis. Highly homologous proteins (transgenic human SOD1 and endogenous mouse SOD1) were distinguished and quantified using the method developed in this study. In addition, the quantitative results using the RABA approach were independently validated by Western blot.     

Semiclassical approach to model quantum fluids using the statistical associating fluid theory for systems with potentials of variable range

Thermodynamic properties of quantum fluids are described using an extended version of the statistical associating fluid theory for potentials of variable range (SAFT-VR) that takes into account quantum corrections to the Helmholtz free energy A, based on the Wentzel-Kramers-Brillouin approximation. We present the theoretical background of this approach (SAFT-VRQ), considering two different cases depending on the continuous or discontinuous nature of the particles pair interaction. For the case of continuous potentials, we demonstrate that the standard Wigner-Kirkwood theory for quantum fluids can be derived from the de Broglie-Bohm formalism for quantum mechanics that can be incorporated within the Barker and Henderson perturbation theory for liquids in a straightforward way. When the particles interact via a discontinuous pair potential, the SAFT-VR method can be combined with the perturbation theory developed by Singh and Sinha [J. Chem. Phys. 67, 3645 (1977); and ibid. 68, 562 (1978)]. We present an analytical expression for the first-order quantum perturbation term for a square-well potential, and the theory is applied to model thermodynamic properties of hydrogen, deuterium, neon, and helium-4. Vapor-liquid equilibrium, liquid and vapor densities, isochoric and isobaric heat capacities, Joule-Thomson coefficients and inversion curves are predicted accurately with respect to experimental data. We find that quantum corrections are important for the global behavior of properties of these fluids and not only for the low-temperature regime. Predictions obtained for hydrogen compare very favorably with respect to cubic equations of state.     

Merging the potential of microbial genetics with biological and chemical diversity: an even brighter future for marine natural product drug discovery

Marine invertebrates and a growing number of marine bacteria are the sources of novel, bioactive secondary metabolites. Structurally, many of these compounds appear to be biosynthesized by polyketide synthases (PKS) and/or nonribosomal peptide synthetases (NRPS) that have also been found in terrestrial microbes. This review highlights scientific advances from 1999-2003 in the emerging field of molecular genetics of polyketide and nonribosomal peptide natural products isolated from marine organisms. The implications of this research towards the development of marine secondary metabolites as a sustainable source of new drugs are discussed.     

Correlated geminal wave function for molecules: an efficient resonating valence bond approach

We show that a simple correlated wave function, obtained by applying a Jastrow correlation term to an antisymmetrized geminal power, based upon singlet pairs between electrons, is particularly suited for describing the electronic structure of molecules, yielding a large amount of the correlation energy. The remarkable feature of this approach is that, in principle, several resonating valence bonds can be dealt simultaneously with a single determinant, at a computational cost growing with the number of electrons similar to more conventional methods, such as Hartree-Fock or density functional theory. Moreover we describe an extension of the stochastic reconfiguration method, which was recently introduced for the energy minimization of simple atomic wave functions. Within this extension the atomic positions can be considered as further variational parameters, which can be optimized together with the remaining ones. The method is applied to several molecules from Li(2) to benzene by obtaining total energies, bond lengths and binding energies comparable with much more demanding multiconfiguration schemes.     

Pd/Cu-catalyzed facile approach to stilbenes: A novel diversity of TosMIC as an aryl source

Pd/Cu-catalyzed Heck type cross-coupling reaction of p-toluenesulfonylmethyl isocyanide (TosMIC) with various styrenes to access stilbenes in DMSO solvent under mild conditions is developed. This efficient and simple approach employs TosMIC as an aryl source for the first time and demonstrates the novel diversity of TosMIC.     

Configurationally stable,enantioenriched organometallic nucleophiles in stereospecific Pd-catalyzed cross-coupling reactions: an alternative approach to asymmetric synthesis

Several research groups have recently developed methods to employ configurationally stable, enantioenriched organometallic nucleophiles in stereospecific Pd-catalyzed cross-coupling reactions. By establishing the absolute configuration of a chiral alkyltin or alkylboron nucleophile prior to its use in cross-coupling reactions, new stereogenic centers may be rapidly and reliably generated with preservation of the known initial stereochemistry. While this area of research is still in its infancy, such stereospecific cross-coupling reactions may emerge as simple, general methods to access diverse, optically active products from common enantioenriched organometallic building blocks. This minireview highlights recent progress towards the development of general, stereospecific Pd-catalyzed cross-coupling reactions using configurationally stable organometallic nucleophiles.     

Hydroaminomethylation with novel rhodium-carbene complexes: an efficient catalytic approach to pharmaceuticals

Starting from [{Rh(cod)Cl}(2)] and 1,3-dimesitylimidazole-2-ylidenes the novel [RhCl(cod)(carbene)] complexes 1-5 have been synthesized, characterized, and tested in the hydroaminomethylation of aromatic olefins. The influence of different ligands and reaction parameters on the catalytic activity was investigated in detail applying 1,1-diphenylethylene and piperidine as a model system. The scope and limitations of the novel catalysts is shown in the preparation of 16 biologically active 1-amino-3,3-diarylpropenes. In general, high chemo- and regioselectivity as well as good yields of the desired products were achieved.     

Reaction of nonstabilized azomethine ylides with Mannich bases: an approach to 3-acylpyrrolidines

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Bioluminescence-based fibre-optic sensor with entrapped co-reactant: an approach for designing a self-contained biosensor

The possibility of designing a self-contained fibre-optic biosensor, i.e., that can be used without renewing reagents in the probe, was investigated. A probe specific for NADH involves immobilized bioluminescent enzymes which require the presence of two co-reactants [a flavin substrate (FMN) and an aldehyde] to catalyse light emission. The FMN was non-covalently immobilized in a synthetic film and was internally released in the vicinity of the bound enzymes, at the sensing tip of the bioprobe. Release of FMN was achieved from the two different matrices tested: a collagen film and a poly(vinyl alcohol) (PVA) network. Continuous-flow assays of NADH could be performed over a linear dynamic range from 10 pmol to 1 nmol. A PVA matrix appears to be a promising support for designing a self-contained biosensor, as 30–35 reliable measurements (R.S.D.=5%) could be achieved without a decrease in the sensor signal, compared with only 10–15 assays with a collagen film.     

QSAR with experimental and predictive distributions: an information theoretic approach for assessing model quality

We propose that quantitative structure–activity relationship (QSAR) predictions should be explicitly represented as predictive (probability) distributions. If both predictions and experimental measurements are treated as probability distributions, the quality of a set of predictive distributions output by a model can be assessed with Kullback–Leibler (KL) divergence: a widely used information theoretic measure of the distance between two probability distributions. We have assessed a range of different machine learning algorithms and error estimation methods for producing predictive distributions with an analysis against three of AstraZeneca’s global DMPK datasets. Using the KL-divergence framework, we have identified a few combinations of algorithms that produce accurate and valid compound-specific predictive distributions. These methods use reliability indices to assign predictive distributions to the predictions output by QSAR models so that reliable predictions have tight distributions and vice versa. Finally we show how valid predictive distributions can be used to estimate the probability that a test compound has properties that hit single- or multi- objective target profiles.     

Innovative method for determination of 19 polycyclic aromatic hydrocarbons in food and oil samples using gas chromatography coupled to tandem mass spectrometry based on an isotope dilution approach

An efficient and selective analytical method for the determination and the quantification of 19 polycyclic aromatic hydrocarbons (PAHs) in food and oil has been developed. This method includes the monitoring of 15 PAHs stated as a priority by the EU in their 2005/108 recommendation. The samples were extracted according to a selective extraction step using pressurized liquid extraction followed by a purification with polystyrene-divinylbenzene SPE. Identification and quantification were performed using GC-MS/MS, with an isotope dilution approach using (13)C-labelled PAHs. The novel combination of selective extraction followed by purification provides highly purified analytes combined to a fast and automated method. The advantages of GC-MS/MS as compared to other detection methods are tremendous in terms of sensitivity, selectivity and interpretation facilities. Limits of detection varied between 0.008 and 0.15 microg kg(-1), limits of quantification between 0.025 and 0.915 microg kg(-1) for PAHs in food. The calibration curves showed a good linearity for all PAHs (R(2)>0.99) and precision and recovery were fit for purpose. Trueness of the method was carried out using the US National Institute of Standards and Technology SRM 2977 reference material.     

A structure-based design approach for the identification of novel inhibitors: application to an alanine racemase

We report a new structure-based strategy for the identification of novel inhibitors. This approach has been applied to Bacillus stearothermophilus alanine racemase (AlaR), an enzyme implicated in the biosynthesis of the bacterial cell wall. The enzyme catalyzes the racemization of l- and d-alanine using pyridoxal 5-phosphate (PLP) as a cofactor. The restriction of AlaR to bacteria and some fungi and the absolute requirement for d-alanine in peptidoglycan biosynthesis make alanine racemase a suitable target for drug design. Unfortunately, known inhibitors of alanine racemase are not specific and inhibit the activity of other PLP-dependent enzymes, leading to neurological and other side effects.This article describes the development of a receptor-based pharmacophore model for AlaR, taking into account receptor flexibility (i.e. a `dynamic' pharmacophore model). In order to accomplish this, molecular dynamics (MD) simulations were performed on the full AlaR dimer from Bacillus stearothermophilus (PDB entry, 1sft) with a d-alanine molecule in one active site and the non-covalent inhibitor, propionate, in the second active site of this homodimer. The basic strategy followed in this study was to utilize conformations of the protein obtained during MD simulations to generate a dynamic pharmacophore model using the property mapping capability of the LigBuilder program. Compounds from the Available Chemicals Directory that fit the pharmacophore model were identified and have been submitted for experimental testing.The approach described here can be used as a valuable tool for the design of novel inhibitors of other biomolecular targets.     

Structure-LCST relationships for end-functionalized water-soluble polymers: an "accelerated" approach to phase behaviour studies

A novel "high throughput" technique for LCST measurement was developed which is able to identify the effect of subtle changes in end group composition on the aqueous phase behaviour of water-soluble poly(2-(dimethylamino)ethyl methacrylate).     

An approach to the synthesis of tricholomalide A: an effective means for achieving homo-Robinson annulation

A procedure has been developed for the construction of 7,5-fused ring systems through the ring expansion of silyl enol ethers. This method has been applied to the synthesis of an intermediate en route to the natural product, tricholomalide A.     

Protein analysis with large volume sample stacking with an electrosmotic flow pump: a potential approach for proteomics

Large volume sample stacking using electrosmotic flow pump (LVSEP) in capillary electrophoresis is a useful tool for on-line concentration of low-abundant protein samples for proteomics research. While the stacking of only small anions with high pK_a values has been reported at low pH buffers (pH<4), protein samples (trypsin inhibitor and α-lactalbumin) with high pI values (pI>4) were stacked successfully in our system using LVSEP. Initially, the whole column of the SIL, or LPA-coated capillary was filled with protein samples (1 μg/ml), then a high voltage was applied without polarity switching with high pH buffer (pH 8.5). An enhancement factor of more than 100 times was achieved compared to the conventional pressure injection method.