Influenza A H3N2 subtype virus NS1 protein targets into the nucleus and binds primarily via its C-terminal NLS2/NoLS to nucleolin and fibrillarin

ABSTRACT: BACKGROUND: Influenza A virus non-structural protein 1 (NS1) is a virulence factor, which is targeted into the cell cytoplasm, nucleus and nucleolus. NS1 is a multi-functional protein that inhibits host cell pre-mRNA processing and counteracts host cell antiviral responses. Previously, we have shown that the NS1 protein of the H3N2 subtype influenza viruses possesses a C-terminal nuclear localization signal (NLS) that also functions as a nucleolar localization signal (NoLS) and targets the protein into the nucleolus. RESULTS: Here, we show that the NS1 protein of the human H3N2 virus subtype interacts in vitro primarily via its C-terminal NLS2/NoLS and to a minor extent via its N-terminal NLS1 with the nucleolar proteins, nucleolin and fibrillarin. Using chimeric green fluorescence protein (GFP)-NS1 fusion constructs, we show that the nucleolar retention of the NS1 protein is determined by its C-terminal NLS2/NoLS in vivo. Confocal laser microscopy analysis shows that the NS1 protein colocalizes with nucleolin in nucleoplasm and nucleolus and with B23 and fibrillarin in the nucleolus of influenza A/Udorn/72 virus-infected A549 cells. Since some viral proteins contain NoLSs, it is likely that viruses have evolved specific nucleolar functions. CONCLUSION: NS1 protein of the human H3N2 virus interacts primarily via the C-terminal NLS2/NoLS and to a minor extent via the N-terminal NLS1 with the main nucleolar proteins, nucleolin, B23 and fibrillarin.     

Phylogeographic origin and genetic diversity of dunlin <Emphasis Type="Italic">Calidris alpina</Emphasis> in Svalbard

We investigated the genetic structure of the presumably small (10–100 pairs) and isolated dunlin (Calidris alpina) population breeding in Svalbard, and compared it with similar data recently published from several dunlin populations in the western Palearctic and East Greenland. Using mitochondrial and nuclear DNA markers, as well as data on bill lengths, we sought to infer the phylogeographic origin of Svalbard dunlins and assess their within-population level of genetic diversity. Only dunlins with haplotypes of the European mtDNA clade (EUR) were found in Svalbard, indicating a close resemblance to dunlin populations in East Greenland and Iceland. Microsatellite data for Svalbard dunlins, as well as their short bills, also supported a western origin. The Svalbard population did not show signs of inbreeding or reduced levels of genetic diversity compared to other investigated populations, which suggests that the population was recently founded or is currently subject to considerable gene flow.     

Metabolomics as a tool to evaluate exercise-induced improvements in insulin sensitivity

Exercise affects substrate utilisation and insulin sensitivity, which in turn improve blood glucose and lipid levels in subjects with type 2 diabetes (T2D). However, making long-lasting lifestyle-changes might be more realistic if the results were easier to record. Screening for biomarkers reflecting metabolic fitness could thus serve as a tool for maintained motivation. The aim of this study was to test the possibility that metabolomics can be used to identify individuals with improved insulin sensitivity as a result of increased physical activity. Healthy and diabetic subjects were investigated before and after 3 months of exercise to determine various metabolic parameters. Insulin sensitivity was determined by hyperinsulinemic euglycemic clamps and found to be improved in the diabetic men. Plasma was collected during the clamp and analyzed through GC/TOFMS. Healthy subjects could be distinguished from diabetics by means of low molecular-weight compounds (LMC) in plasma independently of gender or exercise, and exercise induced differences in LMC patterns both for healthy and T2D subjects. Forty-four significant metabolites were found to explain differences between LMC patterns obtained from trained and non-trained diabetics. Among these compounds, 17 could be annotated and 5 classified. Inositol-1-phosphate showed the highest correlation to insulin sensitivity in diabetic men, whereas an as yet unknown fatty acid correlated best with insulin sensitivity in women. Both metabolites were better correlated to insulin sensitivity than glucose. Finally, the finding that inostitol-1-phosphate negatively correlates with insulin sensitivity in diabetic men, was validated using samples obtained from a similar training study on diabetic men. Jeanette Kuhl and Thomas Moritz contributed equally to this study.     

Geographical segregation in Dunlin Calidris alpina populations wintering along the East Atlantic migratory flyway – evidence from mitochondrial DNA analysis

Dunlin Calidris alpina is one of the most abundant shorebirds using coastal habitats in the East Atlantic migratory flyway, that links arctic breeding locations (Greenland to Siberia) with wintering grounds (West Europe to West Africa). Differential migration and winter segregation between populations have been indicated by morphometrics and ringing recoveries. Here, we analyse the potential of genetic markers (mitochondrial DNA – mtDNA) to validate and enhance such findings. We compared mtDNA haplotypes frequencies at different wintering sites (from north-west Europe to West Africa). All birds from West Africa had western (European) haplotypes, while the eastern (Siberian) haplotypes were only present in European winter samples, reaching higher frequencies further north in Europe. Compilation of published results from migrating birds also confirmed these differences, with the sole presence of European haplotypes in Iberia and West Africa and increasingly higher frequencies of Siberian haplotypes from south-west to north-west Europe. Comparison with published haplotype frequencies of breeding populations shows that birds from Greenland, Iceland, and North Europe were predominant in wintering grounds in West Africa, while populations wintering in West Europe originated from more eastern breeding grounds (e.g. North Russia). These results show that genetic markers can be used to enhance the integrative monitoring of wintering and breeding populations, by providing biogeographical evidence that validate the winter segregation of breeding populations.     

Increased sensitivity to bleomycin in upper aerodigestive tract mucosa of head and neck squamous cell carcinoma patients

Previous studies on lymphocytes have suggested that patients with head and neck squamous cell carcinoma (HNSCC) have an increased susceptibility for chromosomal damage induced by bleomycin, a known radiomimetic mutagen. However, it has so far not been possible to study whether this genetic instability is present also in the epithelial component of the upper aerodigestive tract mucosa, the tissue from which HNSCC originates. In the present study, we have successfully cultured epithelial cells and fibroblasts isolated from non-neoplastic mucosa samples of 30 HNSCC patients and 56 controls. All cell cultures were exposed to bleomycin and chromosome instability was assessed by analysis of chromosome breakage in cells harvested after 2h of exposure and subsequent removal of bleomycin. Furthermore, the status of the fragile histidine triad gene (FHIT) in chromosome band 3p14.2 was studied by fluorescence in situ hybridization (FISH) in epithelial cells that had been cultured after removal of bleomycin. Chromosomal damage, in the form of chromosomal breaks and gaps, was seen in all cell cultures harvested 2h after exposure to bleomycin. In epithelial cells, the frequency of chromosome breakage was significantly higher among HNSCC patients than among controls [mean breaks per cell (b/c) 1.02 vs. 0.77, p=0.02]. When subdivided according to smoking status, age, and sex, a significantly higher frequency of chromosome breakage was still found in HNSCC patients (smokers, p=0.01, age相似文献   

Geographical variation in the timing of breeding and moult in dunlin Calidris alpina on the Palearctic tundra

Studies of how organisms are adapted to regional climatic conditions are valuable when predicting the effects of global climatic changes on biota. Here we report on the geographical variation in timing of breeding and moult of an Arctic breeding wader, the dunlin (Calidris alpina). The Palearctic study sites range latitudinally between 68 and 76°N and longitudinally between 46 and 179°E, and encompass a variety of local climates. The sites were visited in sequence from west to east within 1 year, and therefore the data are not affected by confounding interannual variations. The estimated breeding start ranged from 5 to 25 June across populations. Birds at more southern sites were found to breed earlier than those at more northern breeding sites. Within populations, the breeding start for first clutches spanned a period of 8 days and, when including replacement clutches, 3-4 weeks. No dunlin west of the Taimyr Peninsula were found moulting while incubating at the nest, whereas all dunlin on Taimyr Peninsula and eastwards were in active wing moult while incubating or rearing chicks. The onset of moult in these populations ranged from 23 to 27 June. The consequences of geographical variation of breeding conditions for variation in the annual cycle of this species are discussed.     

Methods of Immobilization of Proteins to Polymethylmethacrylate

An extracorporeal hemoperfusion chamber for insertion into the peripheral circulatory system has previously been described. In 4 to 6 hours perfusion its aim is to exert a subtractive, selective effect on the blood and to stimulate immunocompetent cells. The chamber is made from polymethylmethacrylate (PMMA). Two general methods for immobilization are described here. The first is based on hydrazinolysis of the PMMA surface. Biologically active substances can be attached through diacylhydrazine bridges, amine bonds and amide groups to the PMMA surface. In the second method carboxyl groups are generated by hydrolysis of the PMMA methyl ester groups. The biologically active substance is immobilized using diamine or dialdehyde coupling agents. Methods are provided to attach the substances to the PMMA either via their carboxyl or via their amino groups.