Summary Recent research has disclosed a tight connection between obesity, metabolic gut microbial activities and host health. Obtaining a complete understanding of this relationship remains a major goal. Here, we conducted a comparative metagenomic and metaproteomic investigation of gut microbial communities in faecal samples taken from an obese and a lean adolescent. By analysing the diversity of 16S rDNA amplicons (10% operational phylogenetic units being common), 22 Mbp of consensus metagenome sequences (∼ 70% common) and the expression profiles of 613 distinct proteins (82% common), we found that in the obese gut, the total microbiota was more abundant on the phylum Firmicutes (94.6%) as compared with Bacteroidetes (3.2%), although the metabolically active microbiota clearly behaves in a more homogeneous manner with both contributing equally. The lean gut showed a remarkable shift towards Bacteroidetes (18.9% total 16S rDNA), which become the most active fraction (81% proteins). Although the two gut communities maintained largely similar gene repertoires and functional profiles, improved pili‐ and flagella‐mediated host colonization and improved capacity for both complementary aerobic and anaerobic de novo B12 synthesis, 1,2‐propanediol catabolism (most likely participating in de novo B12 synthesis) and butyrate production were observed in the obese gut, whereas bacteria from lean gut seem to be more engaged in vitamin B6 synthesis. Furthermore, this study provides functional evidence that variable combinations of species from different phyla could ‘presumptively’ fulfil overlapping and/or complementary functional roles required by the host, a scenario where minor bacterial taxa seem to be significant active contributors.

Silica particles containing surface-bound free radical initiators have been used as supports for the grafting of thin films of molecularly imprinted polymers (MIPs). This technique offers a means of fine-tuning the layer thickness for improved kinetic properties or enhanced capacity in chromatographic or sensor applications. Thus prepared MIPs imprinted with L-phenylalanine anilide, have been characterized by FT-IR spectroscopy, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), elemental analysis, fluorescence microscopy, and scanning electron microscopy (SEM), providing evidence concerning the reproducibility in each step and the quantity and quality of the grafted films. The chromatographic properties of the materials have been investigated with respect to the average layer thickness of the polymer on the surface, the solvent, the support pore diameter, the cross-linker concentration, and the composition of the mobile phase. For the porous particles, the column efficiency depended strongly on the amount of grafted polymer. Thus, polymers grafted as thin films (ca. 0.8 nm as average film thickness) on silica with 10 nm average pore diameter showed the highest column efficiency giving plate numbers (N) for the imprinted enantiomer of ca. 700 m -1 and for the antipode ca. 24 000 m -1 . This resulted in baseline resolutions on a 33 mm long column in less than 5min. On the other hand the sample load capacity and separation factor increased up to 7.0 nm layer thickness and dropped again for larger amounts of grafted polymer. A support with an average pore diameter of 100 nm and a 3.8 nm layer thickness showed a far higher saturation capacity than values previously determined for the conventional monolithic materials.

A technique for the synthesis of molecularly imprinted polymers (MIPs) in small scale (∼55 mg) coupled with direct in situ processing and batch rebinding evaluation is reported. The primary assessment is based on quantification by HPLC or UV absorbance measurement of the amount of template released from the polymer in a given solvent. This method allows a rapid screening of the parameters of importance to reach a desired level of binding affinity capacity and selectivity for a given target molecule. This was demonstrated for the triazine herbicide terbutylazine, where an initial screening was performed for the type of functional monomer used in the MIP preparation. Thus among the six functional monomers tested, methyl methacrylate, 4-vinylpyridine, and N-vinyl-α-pyrrolidone led to rapid and quantitative extraction whereas methacrylic acid and (trifluoromethyl)acrylic acid led to polymers that retained the template the most. After having established useful functional monomers, a secondary screening for selectivity was performed. In this, nonimprinted blank polymers were prepared and a normal batch rebinding evaluation was performed. The polymer showing the highest selectivity was the one prepared using methacrylic acid as functional monomer. This polymer was shown to strongly retain chlorotriazines including atrazine when a normal-scale batch of the polymer was evaluated in chromatography.

Two molecularly imprinted polymers were synthesized using either dichloromethane or toluene as the porogen and terbuthylazine as the template and were used as solid-phase extraction cartridges for the enrichment of six chlorotriazines (deisopropylatrazine, deethylatrazine, simazine, atrazine, propazine, and terbuthylazine) in natural water and sediment samples. The extracted samples were analyzed by liquid chromatography/diode array detection (LC/DAD). Several washing solvents, as well as different volumes, were tested for their ability to remove the matrix components nonspecifically adsorbed on the sorbents. This cleanup step was shown to be of prime importance to the successful extraction of the pesticides from the aqueous samples. The optimal analytical conditions were obtained when the MIP imprinted using dichloromethane was the sorbent, 2 mL of dichloromethane was used in the washing step, and the preconcentrated analytes were eluted with 8 mL of methanol. The recoveries were higher than 80% for all the chlorotriazines except for propazine (53%) when 50- or 100-mL groundwater samples, spiked at 1 microg/L level, were analyzed. The limits of detection varied from 0.05 to 0.2 microg/L when preconcentrating a 100-mL groundwater sample. Natural sediment samples from the Ebre Delta area (Tarragona, Spain) containing atrazine and deethylatrazine were Soxhlet extracted and analyzed by the methodology developed in this work. No significant interferences from the sample matrix were noticed, thus indicating good selectivity of the MIP sorbents used.

SummaryIn parallel to a long lasting search for universal multi-purpose sorbents, the area ofsohd phase extraction (SPE) is recently experiencing a rapid development of new lypes of tailor-made class specific or compound-specific sorbents which are designed to respond to the increasing demand for selectivily and efficiency in sample clean-up prior to quantification. An important issue here is the enrichment and clean-up of complex samples, such as environmental waters, sediments, biofluids and foodstuffs prior to detection .This because the analyte is often present in low concentration in a complex mixture of similarcompounds and therefore needs to be isolated and enriched in order to be detected by standard analytical techniques.The fact that molecularly imprinted polymers (MIPs) can in principle be prepared against any low molecular weight target molecule, can endure harsh working conditions and work in organic solvents makes them extremely attractive as class or compou nd-specific sorbents.This review critically discusses the application of the technology of molecular imprinting to solid phase extraction with examples of the different approaches (off-hne, on-line, coupled column), the different sorbent formats (cartridge, membrane, solid phase microextraction (SPME)) and the different protocols (selective adsorption, selective desorption)taken from the literature.The issues involved in the development of a new MISPE protocol (choice of template, functional monomer, crosshnker, evaluation of the phase and validation of the extraction protocol) and the problems associated with the bleeding of non-extracted template are also discussed.

A novel highly selective sample cleanup procedure based on the use of molecularly imprinted polymers (MIPs) as solid-phase extraction materials has been evaluated with respect to its applicability and routine use in environmental analysis. The method comprises the combination of a restricted access material (RAM) and a MIP allowing a selective sample preparation to be achieved in the online mode. This combination is called the size-selective sample separation and solvent switch (six-SPE). The RAM column combines size exclusion and adsorption chromatography, reducing the concentration of matrix molecules by a cutoff of 15 kDa. The MIP column selectively retains the triazine analytes whereas the residual matrix is not retained and separated completely. Thus, the automated RAM-MIP is capable of excluding all matrix and nontarget compounds. The cleaned and enriched extract is subsequently eluted to an HPLC column and analyzed by LC-MS. A complete on-line analysis cycle including multidimensional solid-phase extraction, separation, and detection takes less than 15 min. Terbuthylazine, atrazine, propazine, simazine, ametryn, prometryn, irgarol, and also the metabolites deethylatrazine and deisopropylatrazine can be determined without any matrix interferences, e.g., by humic acids. The whole setup is fully automated and may be continuously operated. Nonspecific interactions with the polymer are below 1% in all cases. The accuracy of the LC-MIP-LC-MS system was controlled using a certified reference material (Aquacheck). The applicability of the method to the cleanup of real samples was demonstrated by injection of contaminated river water samples. The stability of different polymers was tested by consecutive injections, and it was shown that the performance of the materials did not vary even after more than 300 enrichment and desorption cycles.

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