PASylated interferon α efficiently suppresses hepatitis B virus and induces anti-HBs seroconversion in HBV-transgenic mice.

Interferon α (IFNα) so far is the only therapeutic option for chronic hepatitis B virus (HBV) infection that can lead to virus clearance. Unfortunately, its application is limited by side effects and response rates are low. The aim of this study was to generate a novel long-acting IFNα with the help of PASylation technology that adds a polypeptide comprising Proline, Alanine and Serine (PAS) to increase plasma half-life. Following evaluation of four selected recombinant murine IFNα (mIFNα) subtypes in cell culture, the most active subtype, mIFNα11, was fused with a 600 amino acid PAS chain. The activity of PAS-mIFNα was assessed by interferon bioassay and further evaluated for induction of interferon-stimulated genes (ISG) and antiviral efficacy in cell culture as well as in HBV-transgenic mice. PAS-mIFNα induced expression of ISG comparable to unmodified mIFNα and, likewise, evoked dose-dependent reduction of HBV replication in vitro. In vivo, PAS-mIFNα led to pronounced suppression of HBV replication without detectable liver damage whereas conventional mIFNα treatment only had a modest antiviral effect. Importantly, all PAS-mIFNα treated mice showed an anti-HBs antibody response, lost HBsAg and achieved seroconversion after three weeks. PASylated IFNα showed a profoundly increased antiviral effect in vivo compared to the non-modified version without toxicity, providing proof-of-concept that an improved IFNα can achieve higher rates of HBV antiviral and immune control.

Foam-stabilizing properties of the yeast protein PAU5 and evaluation of factors that can influence its concentration in must and wine.

The absence of the yeast protein seripauperin 5 (PAU5) from Saccharomyces cerevisiae has been suggested as a biomarker for the occurrence of gushing in sparkling wine as samples lacking PAU5 were found to be more susceptible to gushing. In this study, further characterization of PAU5 regarding its foam-stabilizing properties was performed to elucidate whether PAU5 has foam-stabilizing properties and therefore, to elucidate a direct influence on the gushing potential of sparkling wines. PAU5 was successfully purified from non-gushing sparkling wine using reversed-phase high-performance liquid chromatography (RP-HPLC). Pure protein was added to grape juice as a model system for grape must prior to foam stability testing. The results revealed that the protein PAU5 has foam-stabilizing properties. Furthermore, the influence of heat and sulfur treatment in the presence of Botrytis cinerea was analyzed with regard to the amount of PAU5 produced by S. cerevisiae fermented in grape juice. Fermentation experiments using two different S. cerevisiae strains were performed, and the concentration of PAU5 in the samples was compared by RP-HPLC analysis. Unlike sulfur treatment, heat treatment prevented the protein degradation induced by B. cinerea and resulted in even higher amounts of PAU5 compared to the juice fermented with yeast without a previous botrytization. The two different yeast strains applied secreted PAU5 into the surrounding medium in different amounts. In further experiments, the fining process of the wine with bentonite was examined for its potential to remove PAU5 from the wine. RP-HPLC of wines processed with different fining agents revealed that bentonite treatment affected PAU5 concentrations in the final product. The extent of PAU5 removal depended on the type of bentonite applied and on the time of addition during the production process.

Comparative protein profile analysis of wines made from Botrytis cinerea infected and healthy grapes reveals a novel biomarker for gushing in sparkling wine.

Fungal infection of grapes with the plant pathogenic fungus Botrytis (B.) cinerea was shown to cause a degradation of proteins in the resulting wine. Moreover, it influences the foaming properties of the wine. The aim of this study was to compare the protein composition in B. cinerea infected and healthy grapes and of wines produced from such grapes as well as to analyze whether the resulting changes in the protein profiles can be related the occurrence of gushing in sparkling wine. SDS-PAGE and reversed phase HPLC were applied to analyze the protein composition of healthy and botrytized grapes and of wines made from botrytized and healthy grapes. B. cinerea infection led to a general decrease of protein content in infected grapes and wines suggesting proteolytic activity of this fungus. Especially the concentration of a protein with a protein band at ~17kDa underwent a significant decrease in wine made from infected grapes. This protein was identified as Seripauperin 5 (PAU5) from Saccharomyces cerevisiae. A degradation of PAU5 and other proteins and the occurrence of a laccase from B. cinerea were observed in a gushing sparkling wine. Screening of sparkling wines showed that samples lacking PAU5 had a high probability for the occurrence of gushing. We suggest that the absence of protein PAU5 might be a useful biomarker for the occurrence of gushing in sparkling wine.

A novel preparation technique of red (sparkling) wine for protein analysis.

Despite their low concentration, proteins can influence several key enological parameters such as foam stability or haze formation in (sparkling) wine. Most studies focus on white (sparkling) wine since the higher content of phenolic compounds in red wines impairs proteomic research. The aim of the study was the development of a method for the preparation of red (sparkling) wine proteins for proteomic analysis. Three methods of sample preparation were assessed on silver stained SDS-PAGE gels and with MALDI-TOF MS. Our new method was highly suitable for the preparation of proteins for the aforementioned applications. The results showed a substantial increase in signal intensity with a simultaneous decrease in background noise. The preparation protocol consists of (i) dialysis and freeze drying of the sample, (ii) removal of phenolic compounds by water-saturated phenol and (iii) protein precipitation by addition of ammonium acetate. Employment of this method followed by SDS-PAGE analysis allowed for silver stained gels with diminished background or streaking and clearly resolved protein bands. Analysis of spectra obtained from samples prepared according to the proposed protocol showed increased intensity and signal-to-noise ratio in MALDI-TOF MS. Furthermore it was demonstrated that this method can be applied to various kinds of grape products.