Inhibitors of alphavirus entry and replication identified with a stable Chikungunya replicon cell line and virus-based assays

Chikungunya virus (CHIKV), an alphavirus, has recently caused epidemic outbreaks and is therefore considered a re-emerging pathogen for which no effective treatment is available. In this study, a CHIKV replicon containing the virus replicase proteins together with puromycin acetyltransferase, EGFP and Renilla luciferase marker genes was constructed. The replicon was transfected into BHK cells to yield a stable cell line. A non-cytopathic phenotype was achieved by a Pro718 to Gly substitution and a five amino acid insertion within non-structural protein 2 (nsP2), obtained through selection for stable growth. Characterization of the replicon cell line by Northern blotting analysis revealed reduced levels of viral RNA synthesis. The CHIKV replicon cell line was validated for antiviral screening in 96-well format and used for a focused screen of 356 compounds (natural compounds and clinically approved drugs). The 5,7-dihydroxyflavones apigenin, chrysin, naringenin and silybin were found to suppress activities of EGFP and Rluc marker genes expressed by the CHIKV replicon. In a concomitant screen against Semliki Forest virus (SFV), their anti-alphaviral activity was confirmed and several additional inhibitors of SFV with IC₅₀ values between 0.4 and 24 µM were identified. Chlorpromazine and five other compounds with a 10H-phenothiazinyl structure were shown to inhibit SFV entry using a novel entry assay based on a temperature-sensitive SFV mutant. These compounds also reduced SFV and Sindbis virus-induced cytopathic effect and inhibited SFV virion production in virus yield experiments. Finally, antiviral effects of selected compounds were confirmed using infectious CHIKV. In summary, the presented approach for discovering alphaviral inhibitors enabled us to identify potential lead structures for the development of alphavirus entry and replication phase inhibitors as well as demonstrated the usefulness of CHIKV replicon and SFV as biosafe surrogate models for anti-CHIKV screening.     

Trimeric form of intracellular ATP synthase subunit β of Aggregatibacter actinomycetemcomitans binds human interleukin-1β

Bacterial biofilms resist host defenses and antibiotics partly because of their decreased metabolism. Some bacteria use proinflammatory cytokines, such as interleukin (IL)-1β, as cues to promote biofilm formation and to alter virulence. Although one potential bacterial IL-1β receptor has been identified, current knowledge of the bacterial IL-1β sensing mechanism is limited. In chronic biofilm infection, periodontitis, Aggregatibacter actinomycetemcomitans requires tight adherence (tad)-locus to form biofilms, and tissue destroying active lesions contain more IL-1β than inactive ones. The effect of IL-1β on the metabolic activity of A. actinomycetemcomitans biofilm was tested using alamarBlue™. The binding of IL-1β to A. actinomycetemcomitans cells was investigated using transmission electron microscopy and flow cytometry. To identify the proteins which interacted with IL-1β, different protein fractions from A. actinomycetemcomitans were run in native-PAGE and blotted using biotinylated IL-1β and avidin-HRP, and identified using mass spectroscopy. We show that although IL-1β slightly increases the biofilm formation of A. actinomycetemcomitans, it reduces the metabolic activity of the biofilm. A similar reduction was observed with all tad-locus mutants except the secretin mutant, although all tested mutant strains as well as wild type strains bound IL-1β. Our results suggest that IL-1β might be transported into the A. actinomycetemcomitans cells, and the trimeric form of intracellular ATP synthase subunit β interacted with IL-1β, possibly explaining the decreased metabolic activity. Because ATP synthase is highly conserved, it might universally enhance biofilm resistance to host defense by binding IL-1β during inflammation.     

Production and characterization of a secreted, C-terminally processed tyrosinase from the filamentous fungus Trichoderma reesei

A homology search of the genome database of the filamentous fungus Trichoderma reesei identified a new T. reesei tyrosinase gene tyr2, encoding a protein with a putative signal sequence. The gene was overexpressed in the native host under the strong cbh1 promoter, and the tyrosinase enzyme was secreted into the culture supernatant. This is the first report on a secreted fungal tyrosinase. Expression of TYR2 in T. reesei resulted in good yields, corresponding to approximately 0.3 and 1 g.L(-1) tyrosinase in shake flask cultures and laboratory-scale batch fermentation, respectively. T. reesei TYR2 was purified with a three-step purification procedure, consisting of desalting by gel filtration, cation exchange chromatography and size exclusion chromatography. The purified TYR2 protein had a significantly lower molecular mass (43.2 kDa) than that calculated from the putative amino acid sequence (61.151 kDa). According to N-terminal and C-terminal structural analyses by fragmentation, chromatography, MS and peptide sequencing, the mature protein is processed from the C-terminus by a cleavage of a peptide fragment of about 20 kDa. The T. reesei TYR2 polypeptide chain was found to be glycosylated at its only potential N-glycosylation site, with a glycan consisting of two N-acetylglucosamines and five mannoses. Also, low amounts of shorter glycan forms were detected at this site. T. reesei TYR2 showed the highest activity and stability within a neutral and alkaline pH range, having an optimum at pH 9. T. reesei tyrosinase retained its activity well at 30 degrees C, whereas at higher temperatures the enzyme started to lose its activity relatively quickly. T. reesei TYR2 was active on both l-tyrosine and l-dopa, and it showed broad substrate specificity.     

Relations of APOE promoter polymorphisms to LDL cholesterol and markers of subclinical atherosclerosis in young adults

The common apolipoprotein E (apoE) gene (APOE) epsilon2/epsilon3/epsilon4 polymorphism explains part of serum lipid variation, and polymorphisms in the APOE promoter region have been proposed to participate in the regulation of serum lipid levels within the most common APOE epsilon3/epsilon3 genotype group. We determined APOE -219G/T and +113G/C promoter genotypes and estimated APOE haplotypes in 525 participants of the Cardiovascular Risk in Young Finns Study. We studied the associations of the APOE promoter polymorphisms and their haplotypes with cross-sectional and longitudinal serum lipid and apolipoprotein concentrations as well as with flow-mediated dilatation (FMD), carotid artery compliance (CAC), and intima-media thickness (IMT) within the APOE epsilon3/epsilon3 carriers. We found no significant association between the APOE promoter genotypes and serum lipids [low density lipoprotein-cholesterol (LDL-C), HDL-C, and triglycerides], apolipoproteins (apoA-I and apoB), or brachial artery FMD, CAC, or carotid IMT in either men or women. In longitudinal analyses in males, the carriers of heterozygous genotypes (-219G/T or +113G/C) and, furthermore, carriers of the -219T/+113C/epsilon3 haplotype had significantly higher LDL-C and total cholesterol concentrations throughout the 21 year follow-up period compared with homozygous G allele carriers or noncarriers of the -219T/+113C/epsilon3 haplotype. Such associations were not found in females. In summary, the APOE promoter polymorphisms -219G/T and +113G/C as well as their haplotype are associated with longitudinal changes in LDL-C and total cholesterol concentrations in young Finnish males but do not seem to be major determinants for FMD, CAC, or carotid IMT in males or females.     

Absence of endogenous phospholipid transfer protein impairs ABCA1-dependent efflux of cholesterol from macrophage foam cells

In vitro experiments have demonstrated that exogenous phospholipid transfer protein (PLTP), i.e. purified PLTP added to macrophage cultures, influences ABCA1-mediated cholesterol efflux from macrophages to HDL. To investigate whether PLTP produced by the macrophages (i.e., endogenous PLTP) is also part of this process, we used peritoneal macrophages derived from PLTP-knockout (KO) and wild-type (WT) mice. The macrophages were transformed to foam cells by cholesterol loading, and this resulted in the upregulation of ABCA1. Such macrophage foam cells from PLTP-KO mice released less cholesterol to lipid-free apolipoprotein A-I (apoA-I) and to HDL than did the corresponding WT foam cells. Also, when plasma from either WT or PLTP-KO mice was used as an acceptor, cholesterol efflux from PLTP-KO foam cells was less efficient than that from WT foam cells. After cAMP treatment, which upregulated the expression of ABCA1, cholesterol efflux from PLTP-KO foam cells to apoA-I increased markedly and reached a level similar to that observed in cAMP-treated WT foam cells, restoring the decreased cholesterol efflux associated with PLTP deficiency. These results indicate that endogenous PLTP produced by macrophages contributes to the optimal function of the ABCA1-mediated cholesterol efflux-promoting machinery in these cells. Whether macrophage PLTP acts at the plasma membrane or intracellularly or shuttles between these compartments needs further study.     

Grosmannia and Leptographium spp. associated with conifer-infesting bark beetles in Finland and Russia, including Leptographium taigense sp. nov.

Species of Grosmannia with Leptographium anamorphs include important forest pathogens and agents of blue stain in timber. They are commonly found in association with forest pests, such as bark beetles. During a survey of ophiostomatoid fungi in eastern parts of Finland and neighboring Russia, species belonging to the genus Grosmannia were isolated from 12 different bark beetle species infesting Picea abies and Pinus sylvestris, the most economically important conifers in the region. Identification of these fungi was based on morphology, DNA sequence comparisons for three gene regions and phylogenetic analyses. A total of ten taxa were identified. These belonged to six different species complexes in Grosmannia. The phylogenetic analyses provided an opportunity to redefine the G. galeiformis-, L. procerum-, L. lundbergii-, G. piceiperda-, G. olivacea- and G. penicillata-complexes, and to consider the species emerging from the survey within the context of these complexes. The species included G. galeiformis, G. olivacea, L. chlamydatum, L. lundbergii, L. truncatum and a novel taxon, described here as L. taigense sp. nov. In addition, species closely related to G. cucullata, G. olivaceapini comb. nov., G. piceiperda and L. procerum were isolated but their identity could not be resolved. The overall results indicate that the diversity of Grosmannia species in the boreal forests remains poorly understood and that further studies are needed to clarify the status of several species or species complexes.     

Selection on laying date is connected to breeding density in the pied flycatcher

Timing of reproduction and clutch size are important determinants of breeding success, especially in seasonal environments. Several recent bird population studies have shown changes in breeding time and in natural selection on it. These changes have often been linked with climate change, but few studies have investigated how the traits or natural selection are actually connected with climatic factors. Furthermore, the effect of population density on selection has been rarely considered, despite the potential importance of density in demographic processes. We studied variation in natural selection on laying date and on clutch size in relation to measures of spring phenology and population density in a long-term study of pied flycatchers in SW Finland. The phenological stage of the environment at mean egg-laying did not affect the direction of selection on either laying date or on clutch size. There was, however, stronger selection for earlier laying date when the breeding density of the population was high, suggesting that early breeding is not necessarily beneficial as such, but that its importance is emphasized when high population density increases competition. In addition, early breeding was favoured when the pre-breeding period was cool, which may indicate an increased advantage for the fittest individuals in harsher conditions. In the middle of the twentieth century, there was selection for large clutch size, which subsequently ceased, along with an overall decrease in recruit production. Our results indicate that attention should be paid to demographic factors such as breeding density when studying natural selection and temporal changes in it.     

High-throughput karyotyping of human pluripotent stem cells

Genomic integrity of human pluripotent stem cell (hPSC) lines requires routine monitoring. We report here that novel karyotyping assay, utilizing bead-bound bacterial artificial chromosome probes, provides a fast and easy tool for detection of chromosomal abnormalities in hPSC lines. The analysis can be performed from low amounts of DNA isolated from whole cell pools with simple data analysis interface. The method enables routine screening of stem cell lines in a cost-efficient high-throughput manner.