Use of a solvent-free dry matrix coating for quantitative matrix-assisted laser desorption ionization imaging of 4-bromophenyl-1,4-diazabicyclo(3.2.2)nonane-4-carboxylate in rat brain and quantitative analysis of the drug from laser microdissected tissue regions

A dry matrix application for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) was used to profile the distribution of 4-bromophenyl-1,4-diazabicyclo(3.2.2)nonane-4-carboxylate, monohydrochloride (BDNC, SSR180711) in rat brain tissue sections. Matrix application involved applying layers of finely ground dry alpha-cyano-4-hydroxycinnamic acid (CHCA) to the surface of tissue sections thaw mounted onto MALDI targets. It was not possible to detect the drug when applying matrix in a standard aqueous-organic solvent solution. The drug was detected at higher concentrations in specific regions of the brain, particularly the white matter of the cerebellum. Pseudomultiple reaction monitoring imaging was used to validate that the observed distribution was the target compound. The semiquantitative data obtained from signal intensities in the imaging was confirmed by laser microdissection of specific regions of the brain directed by the imaging, followed by hydrophilic interaction chromatography in combination with a quantitative high-resolution mass spectrometry method. This study illustrates that a dry matrix coating is a valuable and complementary matrix application method for analysis of small polar drugs and metabolites that can be used for semiquantitative analysis.

Rapid aquaporin translocation regulates cellular water flow:the mechanism of hypotonicity-induced sub-cellular localization of the aquaporin 1 water channel

The control of cellular water flow is mediated by the aquaporin (AQP) family of membrane proteins. The family's structural features and the mechanism of selective water passage through the AQP pore are established, but there remains a gap in our knowledge of how water transport is regulated. Two broad possibilities exist. One is controlling the passage of water through the AQP pore, but this has only been observed as a phenomenon in some plant and microbial AQPs. An alternative is controlling the number of AQPs in the cell membrane. Here we describe a novel pathway in mammalian cells whereby a hypotonic stimulus directly induces intracellular calcium elevations, through transient receptor potential channels, that trigger AQP1 translocation. This translocation, which has a direct role in cell volume regulation, occurs within 30s and is dependent on calmodulin activation and phosphorylation of AQP1 at two threonine residues by protein kinase C. This direct mechanism provides a rationale for the changes in water transport that are required in response to constantly-changing local cellular water availability. Moreover, since calcium is a pluripotent and ubiquitous second messenger in biological systems, the discovery of its role in the regulation of AQP translocation has ramifications for diverse physiological and pathophysiological processes, as well as providing an explanation for the rapid regulation of water flow that is necessary for cell homeostasis.

Structural characterization of CD81-Claudin-1 hepatitis C virus receptor complexes

Tetraspanins are thought to exert their biological function(s) by co-ordinating the lateral movement and trafficking of associated molecules into tetraspanin-enriched microdomains. A second four-TM (transmembrane) domain protein family, the Claudin superfamily, is the major structural component of cellular TJs (tight junctions). Although the Claudin family displays low sequence homology and appears to be evolutionarily distinct from the tetraspanins, CD81 and Claudin-1 are critical molecules defining HCV (hepatitis C virus) entry; we recently demonstrated that CD81-Claudin-1 complexes have an essential role in this process. To understand the molecular basis of CD81-Claudin-1 complex formation, we produced and purified milligram quantities of full-length CD81 and Claudin-1, alone and in complex, in both detergent and lipid contexts. Structural characterization of these purified proteins will allow us to define the mechanism(s) underlying virus-cell interactions and aid the design of therapeutic agents targeting early steps in the viral life cycle.

Reversible addition–fragmentation chain transfer polymerization of 2-chloro-1,3-butadiene

Controlled polymerization of 2-chloro-1,3-butadiene using reversible addition–fragmentation chain transfer (RAFT) polymerization has been demonstrated for the first time. 2-Chloro-1,3-butadiene, more commonly known as chloroprene, has significant industrial relevance as a crosslinked rubber, with uses ranging from adhesives to integral automotive components. However, problems surrounding the inherent toxicity of the lifecycle of the thiourea-vulcanized rubber have led to the need for control over the synthesis of poly(2-chloro-1,3-butadiene). To this end, four chain transfer agents in two different solvents have been trialed and the kinetics are discussed. 2-Cyano-2-propylbenzodithioate (CPD) is shown to polymerize 2-chloro-1,3-butadiene in THF, using AIBN as an initiator, with complete control over the target molecular weight, producing polymers with low polydispersities (Mw/Mn < 1.25 in all cases).

Cortical mechanisms of attention in time:neural correlates of the Lag-1-sparing phenomenon

If humans monitor streams of rapidly presented (approximately 100-ms intervals) visual stimuli, which are typically specific single letters of the alphabet, for two targets (T1 and T2), they often miss T2 if it follows T1 within an interval of 200-500 ms. If T2 follows T1 directly (within 100 ms; described as occurring at 'Lag 1'), however, performance is often excellent: the so-called 'Lag-1 sparing' phenomenon. Lag-1 sparing might result from the integration of the two targets into the same 'event representation', which fits with the observation that sparing is often accompanied by a loss of T1-T2 order information. Alternatively, this might point to competition between the two targets (implying a trade-off between performance on T1 and T2) and Lag-1 sparing might solely emerge from conditional data analysis (i.e. T2 performance given T1 correct). We investigated the neural correlates of Lag-1 sparing by carrying out magnetoencephalography (MEG) recordings during an attentional blink (AB) task, by presenting two targets with a temporal lag of either 1 or 2 and, in the case of Lag 2, with a nontarget or a blank intervening between T1 and T2. In contrast to Lag 2, where two distinct neural responses were observed, at Lag 1 the two targets produced one common neural response in the left temporo-parieto-frontal (TPF) area but not in the right TPF or prefrontal areas. We discuss the implications of this result with respect to competition and integration hypotheses, and with respect to the different functional roles of the cortical areas considered. We suggest that more than one target can be identified in parallel in left TPF, at least in the absence of intervening nontarget information (i.e. masks), yet identified targets are processed and consolidated as two separate events by other cortical areas (right TPF and PFC, respectively).

The extracellular surface of the GLP-1 receptor is a molecular trigger for biased agonism

Ligand-directed signal bias offers opportunities for sculpting molecular events, with the promise of better, safer therapeutics. Critical to the exploitation of signal bias is an understanding of the molecular events coupling ligand binding to intracellular signaling. Activation of class B G protein-coupled receptors is driven by interaction of the peptide N terminus with the receptor core. To understand how this drives signaling, we have used advanced analytical methods that enable separation of effects on pathway-specific signaling from those that modify agonist affinity and mapped the functional consequence of receptor modification onto three-dimensional models of a receptor-ligand complex. This yields molecular insights into the initiation of receptor activation and the mechanistic basis for biased agonism. Our data reveal that peptide agonists can engage different elements of the receptor extracellular face to achieve effector coupling and biased signaling providing a foundation for rational design of biased agonists.

Impact assessment of increasing the 20% Greenhouse gas reduction target of the EU for Hungary (executive summary)

This study has the objective to analyse the impacts on the Hungarian economy of a higher EU GHG (greenhouse gas) reduction undertaking for 2020, namely increasing the GHG reduction target to 20% and to 30% relative to 1990. In order to achieve this objective, we quantify the costs/benefits of these increased undertakings for the various sectors of the Hungarian economy.

Genome-wide association of multiple complex traits in outbred mice by ultra low-coverage sequencing

We thank the High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics and the Wellcome Trust Sanger Institute for the generation of the sequencing data. This work was funded by Wellcome Trust grant 090532/Z/09/Z (J.F.). Primary phenotyping of the mice was supported by the Mary Lyon Centre and Mammalian Genetics Unit (Medical Research Council, UK Hub grant G0900747 91070 and Medical Research Council, UK grant MC U142684172). D.A.B acknowledges support from NIH R01AR056280. The sleep work was supported by the state of Vaud (Switzerland) and the Swiss National Science Foundation (SNF 14694 and 136201 to P.F.). The ECG work was supported by the Netherlands CardioVascular Research Initiative (Dutch Heart Foundation, Dutch Federation of University Medical Centres, the Netherlands Organization for Health Research and Development, and the Royal Netherlands Academy of Sciences) PREDICT project, InterUniversity Cardiology Institute of the Netherlands (ICIN; 061.02; C.A.R., C.R.B). Na Cai is supported by the Agency of Science, Technology and Research (A*STAR) Graduate Academy. The authors wish to acknowledge excellent technical assistance from: Ayako Kurioka, Leo Swadling, Catherine de Lara, James Ussher, Rachel Townsend, Sima Lionikaite, Ausra S. Lionikiene, Rianne Wolswinkel and Inge van der Made. We would like to thank Thomas M Keane and Anthony G Doran for their help in annotating variants and adding the FVB/NJ strain to the Mouse Genomes Project.

Sex steroid feedback regulation of pituitary gonadotropins during early secondary oocyte growth in coho salmon (Oncorhynchus kisutch) – a potential target of endocrine disruption.

Thesis (Ph.D.)--University of Washington, 2016-06

Low spatial frequency filtering modulates early brain processing of affective complex pictures

Recent research on affective processing has suggested that low spatial frequency information of fearful faces provide rapid emotional cues to the amygdala, whereas high spatial frequencies convey fine-grained information to the fusiform gyrus, regardless of emotional expression. In the present experiment, we examined the effects of low (LSF, <15 cycles/image width) and high spatial frequency filtering (HSF, >25 cycles/image width) on brain processing of complex pictures depicting pleasant, unpleasant, and neutral scenes. Event-related potentials (ERP), percentage of recognized stimuli and response times were recorded in 19 healthy volunteers. Behavioral results indicated faster reaction times in response to unpleasant LSF than to unpleasant HSF pictures. Unpleasant LSF pictures and pleasant unfiltered pictures also elicited significant enhancements of P1 amplitudes at occipital electrodes as compared to neutral LSF and unfiltered pictures, respectively; whereas no significant effects of affective modulation were found for HSF pictures. Moreover, mean ERP amplitudes in the time between 200 and 500ms post-stimulus were significantly greater for affective (pleasant and unpleasant) than for neutral unfiltered pictures; whereas no significant affective modulation was found for HSF or LSF pictures at those latencies. The fact that affective LSF pictures elicited an enhancement of brain responses at early, but not at later latencies, suggests the existence of a rapid and preattentive neural mechanism for the processing of motivationally relevant stimuli, which could be driven by LSF cues. Our findings confirm thus previous results showing differences on brain processing of affective LSF and HSF faces, and extend these results to more complex and social affective pictures.

Photochromism of the complex between 40-(2-hydroxyethoxy)-7-hydroxyflavylium and b-cyclodextrin, studied by 1H NMR, UVeVis, continuous irradiation and circular dichroism

1st International Caparica Conference on Chromogenic and Emissive Materials

Hypertrophic Adenoid Is A Major Infection Site Of Human Bocavirus 1.

Human bocavirus 1 (HBoV1) is associated with respiratory infections worldwide, mainly in children. Similar to other parvoviruses, it is believed that HBoV1 can persist for long periods of time in humans, probably through maintaining concatemers of the virus single-stranded DNA genome in the nuclei of infected cells. Recently, HBoV-1 was detected in high rates in adenoid and palatine tonsils samples from patients with chronic adenotonsillar diseases, but nothing is known about the virus replication levels in those tissues. A 3-year prospective hospital-based study was conducted to detect and quantify HBoV1 DNA and mRNAs in samples of the adenoids (AD), palatine tonsils (PT), nasopharyngeal secretions (NPS), and peripheral blood (PB) from patients undergoing tonsillectomy for tonsillar hypertrophy or recurrent tonsillitis. HBoV1 was detected in 25.3% of the AD samples, while the rates of detection in the PT, NPS, and PB samples were 7.2%, 10.5%, and 1.7%, respectively. The viral loads were higher in AD samples, and 27.3% of the patients with HBoV had mRNA detectable in this tissue. High viral loads and detectable mRNA in the AD were associated with HBoV1 detection in the other sample sites. The adenoids are an important site of HBoV1 replication and persistence in children with tonsillar hypertrophy. The adenoids contain high HBoV1 loads and are frequently positive for HBoV mRNA, and this is associated with the detection of HBoV1 in secretions.