T cell cytokines and the risk of blood stream infection in extremely low birth weight infants

Cytokines mediate the host immune response to infectious micro-organisms. The objective of this study was to determine whether immune regulatory interleukins (IL-4, IL-5, IL-6, and IL-10) and inflammatory cytokines (Interferon-γ [INF-γ], tumor necrosis factor-β [TNF-β], IL-2, and IL-17) are associated with an increased risk of developing blood stream bacterial/fungal infection (BSI) in extremely low birth weight (ELBW) infants. ELBW infants from 17 NICHD Neonatal Research Network centers without early onset sepsis were studied. Cytokines were measured from blood on days 1, 3, 7, 14, and 21 after birth. 996 ELBW infants contributed a minimum of 4080 unique measurements for each cytokine during the five sampling periods. Infants with BSI had lower levels of the inflammatory cytokines IL-17 (p=0.01), and higher levels of the regulatory cytokines, IL-6 (p=0.01) and IL-10 (p<0.001). Higher levels of regulatory cytokines relative to pro-inflammatory cytokines were associated with increased risk of BSI even after adjusting for confounding variables. In ELBW infants, the ratio of immune regulatory cytokines to inflammatory cytokines was associated with development of BSI. Altered maturation of regulatory and inflammatory cytokines may increase the risk of serious infection in this population.     

The karyopherin Kap95 and the C-termini of Rfa1, Rfa2, and Rfa3 are necessary for efficient nuclear import of functional RPA complex proteins in Saccharomyces cerevisiae

Nuclear protein import in eukaryotic cells is mediated by karyopherin proteins, which bind to specific nuclear localization signals on substrate proteins and transport them across the nuclear envelope and into the nucleus. Replication protein A (RPA) is a nuclear protein comprised of three subunits (termed Rfa1, Rfa2, and Rfa3 in Saccharomyces cerevisiae) that binds single-stranded DNA and is essential for DNA replication, recombination, and repair. RPA associates with two different karyopherins in yeast, Kap95, and Msn5/Kap142. However, it is unclear which of these karyopherins is responsible for RPA nuclear import. We have generated GFP fusion proteins with each of the RPA subunits and demonstrate that these Rfa-GFP chimeras are functional in yeast cells. The intracellular localization of the RPA proteins in live cells is similar in wild-type and msn5Δ deletion strains but becomes primarily cytoplasmic in cells lacking functional Kap95. Truncating the C-terminus of any of the RPA subunits results in mislocalization of the proteins to the cytoplasm and a loss of protein-protein interactions between the subunits. Our data indicate that Kap95 is likely the primary karyopherin responsible for RPA nuclear import in yeast and that the C-terminal regions of Rfa1, Rfa2, and Rfa3 are essential for efficient nucleocytoplasmic transport of each RPA subunit.     

Study of 'Redhaven' peach and its white-fleshed mutant suggests a key role of CCD4 carotenoid dioxygenase in carotenoid and norisoprenoid volatile metabolism

Background  

Carotenoids are plant metabolites which are not only essential in photosynthesis but also important quality factors in determining the pigmentation and aroma of flowers and fruits. To investigate the regulation of carotenoid metabolism, as related to norisoprenoids and other volatile compounds in peach (Prunus persica L. Batsch.), and the role of carotenoid dioxygenases in determining differences in flesh color phenotype and volatile composition, the expression patterns of relevant carotenoid genes and metabolites were studied during fruit development along with volatile compound content. Two contrasted cultivars, the yellow-fleshed 'Redhaven' (RH) and its white-fleshed mutant 'Redhaven Bianca' (RHB) were examined.     

All four zebrafish Wnt7 genes are expressed during early brain development

Wnt-signalling is involved in a number of biological processes in the course of embryonic development, cell fate determination, proliferation, stem cell maintenance and oncogenesis. Wnt ligands are secreted glycoproteins and the number of Wnt isoforms varies between five in nematodes and 27 in fish. The highly conserved group of Wnt7 genes has been found to signal via at least three Wnt-signalling pathways dependent on the developmental context. These ligands have been identified as important regulators in a number of processes ranging from formation of bones, lungs, kidneys, reproductive organs and placenta to vasculogenesis and synaptogenesis in the brain. The importance of Wnt7 function is underscored by their implication in disease syndromes in man. Unlike the single Wnt7a and Wnt7b mammalian genes we find that the zebrafish genome contains two paralogues genes for each Wnt7 ligand. Here, we compare these four Wnt7 genes evolutionarily and analyse their expression during the first two days of embryonic development. We find Wnt7 genes mainly expressed in a number of CNS structures at developmental stages at which patterning and neural specification takes place. The timely and spatially overlapping as well as complementary gene expression suggests diverse as well as redundant involvements during brain development.     

Development and evaluation of a liquid chromatography tandem mass spectrometry method for simultaneous determination of salivary melatonin, cortisol and testosterone

Circadian disruption can have several possible health consequences, but is not well studied. In order to measure circadian disruption, in relation to shift or night work, we developed a simple and sensitive method for the simultaneous determination of melatonin, cortisol and testosterone in human saliva. We used liquid-liquid extraction (LLE) followed by liquid chromatography coupled to electrospray tandem mass spectrometry (LC-ESI-MS/MS) recorded in positive ion mode. Saliva samples were collected by spitting directly into tubes and 250 μL were used for analysis. The limits of detection were 4.1 pmol/L, 0.27 nmol/L and 10.8 pmol/L for melatonin, cortisol, and testosterone, respectively. The developed method was sensitive enough to measure circadian rhythms of all 3 hormones in a pilot study among four healthy volunteers. It can therefor be used to study the impact of night work and working in artificial light on the workers circadian rhythms. To our knowledge this is the first LC-ESI-MS/MS method for simultaneous determination of salivary melatonin, cortisol and testosterone.     

Investigating a macromolecular complex: the toolkit of methods

Structural biologists studying macromolecular complexes spend considerable effort doing strictly "non-structural" work: investigating the physiological relevance and biochemical properties of a complex, preparing homogeneous samples for structural analysis, and experimentally validating structure-based hypotheses regarding function or mechanism. Familiarity with the diverse perspectives and techniques available for studying complexes helps in the critical assessment of non-structural data, expedites the pre-structural characterization of a complex and facilitates the investigation of function. Here we survey the approaches and techniques used to study macromolecular complexes from various viewpoints, including genetics, cell and molecular biology, biochemistry/biophysics, structural biology, and systems biology/bioinformatics. The aim of this overview is to heighten awareness of the diversity of perspectives and experimental tools available for investigating complexes and of their usefulness for the structural biologist.