Evaluation of the influenza A replicon for transient expression of recombinant proteins in mammalian cells

Recombinant protein expression in mammalian cells has become a very important technique over the last twenty years. It is mainly used for production of complex proteins for biopharmaceutical applications. Transient recombinant protein expression is a possible strategy to produce high quality material for preclinical trials within days. Viral replicon based expression systems have been established over the years and are ideal for transient protein expression. In this study we describe the evaluation of an influenza A replicon for the expression of recombinant proteins. We investigated transfection and expression levels in HEK-293 cells with EGFP and firefly luciferase as reporter proteins. Furthermore, we studied the influence of different influenza non-coding regions and temperature optima for protein expression as well. Additionally, we exploited the viral replication machinery for the expression of an antiviral protein, the human monoclonal anti-HIV-gp41 antibody 3D6. Finally we could demonstrate that the expression of a single secreted protein, an antibody light chain, by the influenza replicon, resulted in fivefold higher expression levels compared to the usually used CMV promoter based expression. We emphasize that the influenza A replicon system is feasible for high level expression of complex proteins in mammalian cells.     

Timing is everything: international variations in historical sexual partnership concurrency and HIV prevalence

Background

Higher prevalence of concurrent partnerships is one hypothesis for the severity of the HIV epidemic in the countries of Southern Africa. But measures of the prevalence of concurrency alone do not adequately capture the impact concurrency will have on transmission dynamics. The importance of overlap duration and coital exposure are examined here.

Methodology/Principal Findings

We conducted a comparison of data from three studies of sexual behavior carried out in the early 1990s in Uganda, Thailand and the US. Using cumulative concurrency measures, the three countries appeared somewhat similar. Over 50% of both Thai and Ugandan men reported a concurrency within the last three partnerships and over 20% reported a concurrency in the last year, the corresponding rates among US men were nearly 20% for Blacks and Hispanics, and about 10% for other racial/ethnic groups. Concurrency measures that were more sensitive to overlap duration, however, showed large differences. The point prevalence of concurrency on the day of interview was over 10% among Ugandan men compared to 1% for Thai men. Ugandan concurrencies were much longer duration – a median of about two years – than either the Thai (1 day) or US concurrencies (4–9 months across all groups), and involved 5–10 times more coital risk exposure with the less frequent partner. In the US, Blacks and Hispanics reported higher prevalence, longer duration and greater coital exposure than Whites, but were lower than Ugandans on nearly every measure. Together, the differences in the prevalence, duration and coital exposure of concurrent partnerships observed align with the HIV prevalence differentials seen in these populations at the time the data were collected.

Conclusions/Significance

There were substantial variations in the patterns of concurrent partnerships within and between populations. More long-term overlapping partnerships, with regular coital exposure, were found in populations with greater HIV epidemic severity.     

Plasma Levels of Inter-α Inhibitor Proteins in Children with Acute Dengue Virus Infection

Background

Inter-α inhibitor proteins (IaIp) belong to a family of protease inhibitors that are involved in the haemostatic and the vascular system. Dengue viruses (DENV) infections are characterized by coagulopathy and increased vascular permeability. In this study we measured the concentration of IaIp during DENV infections and evaluated its potential as a biomarker.

Methods and Findings

Concentrations of IaIp were measured in patients with acute DENV infections using a quantitative, competitive enzyme linked immunoassay. Concentrations of IaIp measured in pediatric patients suffering from severe DENV infections were significantly lower than in healthy controls.

Conclusions

This is the first report to demonstrate changes in concentration of IaIp during viral infections. The data also highlight the potential of IaIp as a biological marker for severity of DENV infections.     

Linking ecology, behaviour and conservation: does habitat saturation change the mating system of bearded vultures?

The social organization of a population is the consequence of the decisions made by individuals to maximize their fitness, so differences in social systems may arise from differences in ecological conditions. Here, we show how a long-lived species that used to breed monogamously, and at low densities, can change its mating system in response to habitat saturation. We found that a significant proportion of unpaired birds become potential breeders by entering high-quality territories, or by forming polyandrous trios as a strategy to increase their individual performance. However, productivity of territories was reduced when those occupied by breeding pairs changed to trios, suggesting that the third individual was costly. The decision of some individuals to enter into breeding trios as subordinates also had clear negative consequences to population demography. This unusual mating behaviour is thus compromising the conservation effort directed to this endangered species; management to encourage floaters to settle in other suitable but unoccupied areas may be beneficial.     

Novel biocatalysts for white biotechnology

White Biotechnology uses microorganisms and enzymes to manufacture a large variety of chemical products. Therefore, the demand for new and useful biocatalysts is steadily and rapidly increasing. We have developed methods for the isolation of new enzyme genes, constructed novel expression systems, and optimized existing enzymes for biotechnological applications by methods of directed evolution. Furthermore, we have isolated and characterized biocatalysts relevant for the preparation of enantiopure compounds.     

Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman Syndrome

Adipose triglyceride lipase (ATGL) was recently identified as an important triacylglycerol (TG) hydrolase promoting the catabolism of stored fat in adipose and nonadipose tissues. We now demonstrate that efficient ATGL enzyme activity requires activation by CGI-58. Mutations in the human CGI-58 gene are associated with Chanarin-Dorfman Syndrome (CDS), a rare genetic disease where TG accumulates excessively in multiple tissues. CGI-58 interacts with ATGL, stimulating its TG hydrolase activity up to 20-fold. Alleles of CGI-58 carrying point mutations associated with CDS fail to activate ATGL. Moreover, CGI-58/ATGL coexpression attenuates lipid accumulation in COS-7 cells. Antisense RNA-mediated reduction of CGI-58 expression in 3T3-L1 adipocytes inhibits TG mobilization. Finally, expression of functional CGI-58 in CDS fibroblasts restores lipolysis and reverses the abnormal TG accumulation typical for CDS. These data establish an important biochemical function for CGI-58 in the lipolytic degradation of fat, implicating this lipolysis activator in the pathogenesis of CDS.     

The chemical composition of throughfall beneath oak, birch and pine canopies in Northwest Germany

The chemical composition of rainwater is altered upon its passage through tree canopies. In order to investigate how rainwater chemistry is affected by canopy-dependent processes in characteristic forest types of Northwest German sandy lowland regions – oak–birch-forests, Betula pubescens Ehrh. swamp forests, and stands of Pinus sylvestris L. – comparative studies on the chemical composition of throughfall were carried out at seven forest sites, situated in close proximity within a nature reserve. Additionally, rainwater was sampled at three heathland sites for analysis of open-field precipitation and at three sites along an oak–birch-forest edge. Throughfall concentrations of most of the parameters analysed were significantly higher than open-field concentrations, especially with regard to electric conductivity, NH₄-N, K⁺, and KMnO₄-index. Ion concentrations in throughfall were the lowest in a 10-year-old stand of Betula pendula Roth. and Pinus sylvestris and in a Betula pubescens swamp forest and were highest beneath a stand of Pinus sylvestris. Except for Na⁺, Cl⁻, and NO₃⁻, ion concentrations in both throughfall and open-field precipitation increased during the growing season (May–October). In throughfall, Ca²⁺, Mg²⁺, K⁺, and Mn²⁺ were strongly correlated. Enrichment ratios between throughfall and open-field deposition varied among sites and elements and were the highest for K^‰+, Mg^2‰+, and Mn^2‰+. Estimates of canopy leaching indicated high leaching rates of K^‰+ and Mn^2‰+ and moderate leaching of Mg^2‰+. The contribution of foliar leaching to throughfall deposition was higher at the deciduous than at the coniferous stands.