Functional Characterization of the Solute Carrier LAT-1 (SLC7A5/SLC2A3) in Human Brain Capillary Endothelial Cells with Rapid UPLC-MS/MS Quantification of Intracellular Isotopically Labelled L-Leucine

The solute carrier L-type amino acid transporter 1 (LAT-1/SLC7A5) is a viable target for drug delivery to the central nervous system (CNS) and tumors due to its high abundance at the blood–brain barrier and in tumor tissue. LAT-1 is only localized on the cell surface as a heterodimer with CD98, which is not required for transporter function. To support future CNS drug-delivery development based on LAT-1 targeting, we established an ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) assay for stable isotopically labeled leucine ([¹³C₆, ¹⁵N]-L-leucine), with a dynamic range of 0.1–1000 ng/mL that can be applied for the functional testing of LAT-1 activity when combined with specific inhibitors and, consequently, the LAT-1 inhibition capacity of new compounds. The assay was established in a 96-well format, facilitating high-throughput experiments, and, hence, can support the screening for novel inhibitors. Applicable recommendations of the US Food and Drug Administration and European Medicines Agency for bioanalytical method validation were followed to validate the assay. The assay was applied to investigate the IC₅₀ of two well-known LAT-1 inhibitors on hCMEC/D3 cells: the highly specific LAT-1 inhibitor JPH203, which was also used to demonstrate LAT-1 specific uptake, and the general system L inhibitor BCH. In addition, the [¹³C₆, ¹⁵N]-L-leucine uptake was determined on two human brain capillary endothelial cell lines (NKIM-6 and hCMEC/D3), which were characterized for their expressional differences of LAT-1 at the protein and mRNA level and the surface amount of CD98. The IC₅₀ values of the inhibitors were in concordance with previously reported values. Furthermore, the [¹³C₆, ¹⁵N]-L-leucine uptake was significantly higher in hCMEC/D3 cells compared to NKIM-6 cells, which correlated with higher expression of LAT-1 and a higher surface amount of CD98. Therefore, the UPLC-MS/MS quantification of ([¹³C₆, ¹⁵N]-L-leucine is a feasible strategy for the functional characterization of LAT-1 activity in cells or tissue.     

Lactic acid bacteria in marinades used for modified atmosphere packaged broiler chicken meat products

Lactic acid bacteria (LAB) in some marinades commonly used in Finland for modified atmosphere packaged poultry meat products were enumerated and identified to determine whether the marinades contained LAB species that cause meat spoilage. The concentrations of LAB in 51 marinade samples ranged from less than 100 to 8.0 x 10(5) CFU/ml. Seventeen of the samples produced LAB growth only after enrichment, and in five samples no growth was detected either by direct culturing or enrichment. Eighty-eight randomly selected isolates, 51 from the enumerated plates and 37 from enriched samples, were identified using a database of 16S and 23S rRNA gene HindIII restriction fragment length polymorphism patterns of over 300 type and references LAB strains as operational taxonomic units in numerical analyses. The predominating LAB in the enumerated samples was Lactobacillus plantarum (25 of 51 isolates). Eleven isolates were identified as Lactobacillus paracasei subsp. paracasei, and nine were Lactobacillus parabuchneri. None of these species are considered specific spoilage LAB in marinated modified atmosphere packaged poultry meat products nor have they been reported to dominate in unspoiled late-shelf-life products. These results indicate that even though marinades may contain high numbers of LAB, they are not necessarily sources of specific meat spoilage LAB. Therefore, risks associated with meat quality are not predicted by quantitative enumeration of LAB in marinades.     

Pipecolic Acid Hydroxylases: A Monophyletic Clade among cis‐Selective Bacterial Proline Hydroxylases that Discriminates l‐Proline

Proline hydroxylases are iron(II)/2‐oxoglutarate‐dependent enzymes that hydroxylate l ‐proline and derivatives, such as l pipecolic acid, which is the six‐membered‐ring homologue of l ‐proline. It has been established that there is a distinct group of conserved bacterial enzymes that hydroxylate l ‐pipecolic acid and trans‐3‐ and trans‐4‐methyl‐l ‐proline, but virtually no l ‐proline. This allows the organism to produce hydroxyproline congeners without hydroxylation of the physiologically omnipresent l ‐proline. In vitro conversions showed that the substrate spectrum of the pipecolic acid hydroxylases GetF (from a Streptomyces sp.; producer of the tetrapeptide antibiotic GE81112) and PiFa (from Frankia alni) overlaps that of proline hydroxylases, except for the nonacceptance of l ‐proline and smaller homologues. Distinct and conserved residues were determined for both types of enzymes. However, site‐directed mutagenesis in GetF did not yield variants that accepted l ‐proline; this suggested a complex interaction of several residues around the active site, which resulted in delicate changes in substrate specificity. This is supported by substrate docking in a homology model of GetF, which revealed an altered orientation for l ‐proline relative to that of preferred substrates.     

Optimized protocol for the biocompatibility testing of compression stockings and similar products with close skin contact in vitro

Biocompatibility of six different compression stockings and cytotoxic effects were determined using HaCaT keratinocytes, L929 mouse fibroblasts, primary adult and juvenile keratinocytes Cells were quantified using a luminometric ATP assay and the photometric BCA test. Cytotoxic effects were determined by LDH release. An area-based extraction ratio of 1.25 cm²:mL could be shown to be superior to the weight-based extraction of test material. Extraction medium should be an acidic sweat solution as this helps to recreate in vivo conditions. Monolayer cultures of HaCaT keratinocytes or L929 mouse fibroblasts should be used for testing. Primary adult keratinocytes or primary juvenile keratinocytes can also be used. For the latter, testing under DMEM with FCS is recommended to achieve comparable results. It was found that the compression stockings tested exhibited no negative influence on cell viability in vitro and no direct cytotoxic effects measured as release of LDH. Hence, good biocompatibility could be asserted.     

Synthesis,characterization and side chains crystallization of comb-like poly(<Emphasis Type="Italic">p</Emphasis>-<Emphasis Type="Italic">n</Emphasis>-alkylstyrene)s

A series of comb like poly(p-n-alkylstyrene)s with linear alkyl groups, containing even numbers of carbon atoms from 12 up to 22 were synthesized by radical polymerization and characterized by FTIR, ¹H NMR, TGA, TVA DSC, and WAXS. All polymers were obtained in good yields and were soluble in organic solvents as chloroform, dichloromethane or tetrahydrofuran and insoluble in methanol; they are stable up to temperatures near 300 °C. On the other hand, the alkylic side chains of all series are able to crystallize in a paraffinic phase in which the melting temperature and enthalpy increase with the methylene number of the n-alkyl chain.     

Gemeinsame österreichisch-ungarische Sedimentforschung im Rahmen des EFRE-Projektes SEDDON

The multiple uses of the Danube River for navigation, hydropower production and as a drinking water supply influence its ecological quality. Flow restrictions due to regulation as well as impacts on the sediment regime such as retention in the catchment area and interruption of the sediment continuum result in changes of the hydromorphological structures and can produce major problems in the context of sediment management. In light of these facts, cross-border investigations of sediment transport at the Danube River are called for. As there are no uniform research approaches shared by Austria and Hungary, the EU-funded project SEDDON (Sediment Research and Management at the Danube River) aims to compare different monitoring and modeling techniques used to determine sediment transport, and to develop mutual solutions. Further, the laboratory equipment currently used for hydraulic model experiments is evaluated and a research channel with a free flowing discharge of up to 10 m³/s without pumping will be constructed, allowing large-scale joint laboratory tests concerning sediment to be performed.     

The user model-based summarize and refine approach improves information presentation in spoken dialog systems

A common task for spoken dialog systems (SDS) is to help users select a suitable option (e.g., flight, hotel, and restaurant) from the set of options available. As the number of options increases, the system must have strategies for generating summaries that enable the user to browse the option space efficiently and successfully. In the user-model based summarize and refine approach (UMSR, Demberg and Moore, 2006), options are clustered to maximize utility with respect to a user model, and linguistic devices such as discourse cues and adverbials are used to highlight the trade-offs among the presented items. In a Wizard-of-Oz experiment, we show that the UMSR approach leads to improvements in task success, efficiency, and user satisfaction compared to an approach that clusters the available options to maximize coverage of the domain (Polifroni et al., 2003). In both a laboratory experiment and a web-based experimental paradigm employing the Amazon Mechanical Turk platform, we show that the discourse cues in UMSR summaries help users compare different options and choose between options, even though they do not improve verbatim recall. This effect was observed for both written and spoken stimuli.     

Test-taking behaviors in a neurocognitive assessment: Associations with school-age outcomes in a Finnish longitudinal follow-up.

Test-taking behaviors (i.e., task focus, maintenance of attention, and cooperation) affect children's cognitive test performance, and, thus, it is critical to take test-taking behavior into account when drawing conclusions and making recommendations. Prior studies have evaluated test-taking behaviors at the end of the assessment; the present study focused on the fluctuation of cooperation and attention during a neuropsychological assessment. We examined the attention and cooperation of 5-year-old children in a test-taking situation; the associations between these aspects of their test-taking behavior and the children's concurrent neurocognitive test performance, IQ, and parent-rated behavior; and the associations with their IQ, behavioral outcomes, and academic achievement at 8 years of age. The data (for 76 boys and 63 girls) were drawn from the Jyv?skyl? Longitudinal Study of Dyslexia (Lyytinen et al., 2001, 2004). All the children were Caucasian and spoke Finnish as their native language. As a whole, the 5-year-old children showed high cooperation and attention, but a slight decrement in test-taking behavior toward the end of the session was rather common. Three subgroups of children with different levels of cooperation and attention were identified. Children in the subgroup with nonoptimal attention and cooperation showed decreasing neurocognitive test performance toward the end of the assessment session. They also showed more inattentive behavior 3 years later. The findings imply that the examiner's observations of waning attention and cooperation during the assessment session are highly relevant, as these provide stable and clinically meaningful information about the child's behavioral tendencies. (PsycINFO Database Record (c) 2011 APA, all rights reserved)     

Potential use of chemical cues for colony-mate recognition in the big brown bat,Eptesicus fuscus

Bats should benefit from recognition of their roost-mates when colonies form stable social units that persist over time. We used Y-maze experiments and gas chromatography–olfactometry (GC-O) to evaluate whether female big brown bats Eptesicus fuscus (Chiroptera: Vespertilionidae) use chemical cues to distinguish among conspecifics. In dual-choice Y-maze experiments, females chose the scent of another female from their own roost over a conspecific female from a different roost in a majority of trials. Analysis of total body odors using GC-O suggests that individuals from a given colony may share a more common odor signature with roost-mates than with non-roost-mate conspecifics. Using four principle components derived from 15 odor variables, discriminant function analysis correctly assigned most individuals to the correct colony.