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Contiene además supplementary material.-- This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited., We have developed a wine fermentation procedure that takes advantage of the metabolic features of a previously characterized Metschnikowia pulcherrima strain in order to reduce ethanol production. It involves the use of M. pulcherrima/Saccharomyces cerevisiae mixed cultures, controlled oxygenation conditions during the first 48 h of fermentation, and anaerobic conditions thereafter. The influence of different oxygenation regimes and initial inoculum composition on yeast physiology and final ethanol content was studied. The impact of oxygenation on yeast physiology goes beyond the first aerated step and influences yields and survival rates during the anaerobic stage. The activity of M. pulcherrima in mixed oxygenated cultures resulted in a clear reduction in ethanol yield, as compared to S. cerevisiae. Despite relatively low initial cell numbers, S. cerevisiae always predominated in mixed cultures by the end of the fermentation process. Strain replacement was faster under low oxygenation levels. M. pulcherrima confers an additional advantage in terms of dissolved oxygen, which drops to zero after a few hours of culture, even under highly aerated conditions, and this holds true for mixed cultures. Alcohol reduction values about 3.7 % (v/v) were obtained for mixed cultures under high aeration, but they were associated to unacceptable volatile acidity levels. In contrast, under optimized conditions, only 0.35 g/L acetic acid was produced, for an alcohol reduction of 2.2 % (v/v), and almost null dissolved oxygen during the process., Authors would like to thank Cristina Juez Ojeda andMiguel Ángel Fernández Recio for excellent technical assistance, the YeSVitE consortium (EU project, 7FP-IRSES-GA no. 612441) for helpful discussions, and Laura López Ocaña (CECT) for help with yeast species confirmation. This work was supported by grants MINECO AGL2012-32064 and INIA RM2012-00007-00-00 from the Spanish Government. M.Q. was the recipient of a CSIC training contract, JAEDoc, co-funded by the European Social Fund of the EU.

This is an open access article under the CC BY-NC-ND license., Respiration of sugars by non-Saccharomyces yeasts has been recently proposed for lowering alcohol levels in wine. Development of industrial fermentation processes based on such an approach requires, amongst other steps, the identification of yeast strains which are able to grow and respire under the relatively harsh conditions found in grape must. This work describes the characterization of a collection of non-Saccharomyces yeast strains in order to identify candidate yeast strains for this specific application. It involved the estimation of respiratory quotient (RQ) values under aerated conditions, at low pH and high sugar concentrations, calculation of yields of ethanol and other relevant metabolites, and characterization of growth responses to the main stress factors found during the first stages of alcoholic fermentation. Physiological features of some strains of Metschnikowia pulcherrima or two species of Kluyveromyces, suggest they are suitable for lowering ethanol yields by respiration. The unsuitability of Saccharomyces cerevisiae strains for this purpose was not due to ethanol yields (under aerated conditions they are low enough for a significant reduction in final ethanol content), but to the high acetic acid yields under these growth conditions. According to results from controlled aeration fermentations with one strain of M. pulcherrima, design of an aeration regime allowing for lowering ethanol yields though preserving grape must components from excessive oxidation, would be conceivable., This work was funded by the AGL2012-32064 and RM2012-00007-00-00 projects from the Spanish Government. M.Q. was the recipient of a CSIC training JAE-Doc contract, co-funded by the European Social Fund of the EU.

A method has been developed for measurement in vitro of the simultaneous activities alcohol acetyltransferase (AATase) and ester hydrolase (EHase) in yeast extracts taking into account the possibility of interaction between the two opposite activities or the rapid inactivation of the AATase activity. A mathematical model, including as parameters a first order kinetic constant corresponding to the EHase activity and the inactivation constant of AATase, is proposed for the evaluation of AATase activity. To determine ester concentrations, the Headspace-SPME-GC technique has been used. The method has been successfully applied to three yeast strains belonging to the species Pichia anomala, Pichia heedii and Saccharomyces cerevisiae and the corresponding parameters for AATase and EHase have been calculated.

Replication-deficient lentiviral vectors (LV) have been shown to enable the stable genetic modification of multiple cell types in vivo. We demonstrate here that vascular and hepatic delivery of a third-generation HIV-derived lentiviral vector encoding human Factor IX (LV-hFIX) produced potentially therapeutic serum levels of hFIX protein with no vector-mediated local or systemic toxicity of adult mice. Portal vein administration produced the highest serum levels of hFIX and demonstrated proportionally higher levels of gene transfer to the liver with up to 4% of hepatocytes expressing hFIX. Vascular delivery of a lentiviral vector encoding GFP resulted in genetic modification of up to 12% of liver cells. Cell proliferation was not required for hepatocyte transduction with either vector. Serum hFIX levels reached 4% of normal levels following vascular LV-mediated hFIX gene transfer and remained stable for months following vector administration.

Summary β-D-glucosidase activity in 53 yeast strains belonging to the genera Candida, Dekkera, Hanseniaspora, Metschnikowia, Pichia, Rhodotorula, Schizosaccharomyces and Zygosaccharomyces, mainly isolated from grapes and wines, has been detected on the basis of its hydrolytic activity on 4-methylumbelliferyl-β-D-glucoside (MUG) and ρ-nitrophenyl-β-D-glucoside (pNPG). Only yeast species belonging to the genera Dekkera, Rhodotorula and Schizosaccharomyces did not produce the enzyme. The nine yeast strains demonstrating greatest β-D-glucosidase activity on pNPG at pH 3.5 in the presence of 10% (v/v) ethanol belong to the genera Candida, Hanseniaspora and Pichia and exhibited mainly cell wall bound activity. Anthocyanin-β-D-glucosidase activity was checked using an anthocyanin extract by following loss of absorbance at 520 nm. Whilst the nine yeast strains studied degraded the anthocyanin extract, H’spora osmophila 11207 and P. anomala 10590 were selected as the best producers of β-D-glucosidase with the lowest decolorizing activity.

A double coupling bioreactor system was used to fast screen yeast strains for the production of acetate esters. Eleven yeast strains were used belonging to the genera Candida, Hanseniaspora, Metschnikowia, Pichia, Schizosaccharomyces and Zygosacharomyces, mainly isolated from grapes and wine, and two wine Saccharomyces cerevisiae strains. The acetate ester forming activities of yeast strains belonging to the genera Hanseniaspora (Hanseniaspora guilliermondii and H. uvarum) and Pichia (Pichia anomala) showed different substrate specificities and were able to produce ethyl acetate, geranyl acetate, isoamyl acetate and 2-phenylethyl acetate. The influence of aeration culture conditions on the formation of acetate esters by non-Saccharomyces wine yeast and S. cerevisiae was examined by growing the yeasts on synthetic microbiological medium. S. cerevisiae produced low levels of acetate esters when the cells were cultured under highly aeration conditions, while, under the same conditions, H. guilliermondii 11104 and P. anomala 10590 were found to be strong producers of 2-phenylethyl acetate and isoamyl acetate, respectively.

Cancer of the bladder is a good target for oncolytic virotherapy because the bladder has several unique features that allow for facile instillation of virus with minimal systemic exposure. CG8840 is a conditionally replication competent oncolytic adenovirus (OAV) designed to preferentially replicate and kill uroplakin II-positive bladder tumors. CG8840 replicates in and kills bladder transitional carcinoma cells (TCC) in vitro, with highly attenuated cytotoxicity in other cell types and human primary cells. In vivo, CG8840 elicits reproducible and dose-dependent anti-tumor effects in subcutaneous human xenograft tumor models in nude mice. We have developed an orthotopic bladder tumor model in nude mice to closely mimic the location and behavior of bladder tumors in humans. Employing the human bladder transitional cell carcinoma cell line SW780 we established tumors on the luminal surface of the nude mice bladder. In order to visualize the tumor growth and anti-tumor effect following virus infection, the TCC cell line SW780 was stably transduced to express the luciferase gene product. The bladder lumen was initially seeded with 1 × 106 SW780-luc tumor cells in a volume of 100 μl after which the mouth of the urethra was closed with a purse string for 1hr to allow for tumor cell attachment to the bladder wall. Following IP injection of luciferin, the orthotopic tumors were visualized in vivo by luminescence imaging. Measurement of the photon counts emitted from the tumors following luciferin injection allowed us to monitor the growth of the orthotopic tumors. Within 2 weeks tumor size was large enough to initiate efficacy studies. Immuno-histochemical analyses of the orthotopic tumor-bearing bladder with human anti-cytokeratin staining confirmed the human origin of the tumors. Histologically the SW780-Luc orthotopic tumors closely resembled superficial bladder tumors in humans. The need for pretreatment with dodecyl-β-D-maltoside to enhance infectivity of bladder epithelium with adenovirus was demonstrated earlier employing a beta galactosidase-expressing non-replicative adenovirus (Ramesh et al., 2003). For efficient infection of orthotopic tumors with adenovirus similar pretreatment was found to be essential. Following intravesical instillations of the uroplakin-II positive bladder tumor-specific OAV CG8840 we studied the anti-tumor efficacy of the virus. Three doses of CG8840 (1 × 1010 vp/treatment weekly for 3 consecutive weeks) were efficacious in reducing the tumor burden. Similar antitumor activity was not observed following intravesical instillation of a non-bladder tumor specific OAV. Six weeks after the initiation of OAV treatment, immuno-histochemical examination of the bladder showed complete absence of tumor cells in CG8840-treated mice. This simplified technique consistently produced a high incidence of orthotopic tumors in mice. This will be a useful model to conduct intravesical anti-tumor efficacy studies of OAV or other viruses both as single agents and in combination with other therapeutic agents.