Effects of Sulforaphane-Induced Cell Death upon Repeated Passage of Either P-Glycoprotein-Negative or P-Glycoprotein-Positive L1210 Cell Variants.

The expression of the membrane ABCB1 transporter in neoplastic cells is one of the most common causes of reduced sensitivity to chemotherapy. In our previous study, we investigated the effect of a single culture of ABCB1-negative (S) and ABCB1-positive variants of L1210 cells (R and T) in the presence of sulforaphane (SFN). We demonstrated that SFN induces the onset of autophagy more markedly in S cells than in R or T cells. In the current study, we focused on the effect of the repeated culture of S, R and T cells in SFN-containing media. The repeated cultures increased the onset of autophagy compared to the simple culture, mainly in S cells and to a lesser extent in R and T cells, as indicated by changes in the cellular content of 16 and 18 kDa fragments of LC3B protein or changes in the specific staining of cells with monodansylcadaverine. We conclude that SFN affects ABCB1-negative S cells more than ABCB1-positive R and T cells during repeated culturing. Changes in cell sensitivity to SFN appear to be related to the expression of genes for cell-cycle checkpoints, such as cyclins and cyclin-dependent kinases.

Potential of non-Saccharomyces yeast to produce non-alcoholic beer.

Recently, non-Saccharomyces yeast have become very popular in wine and beer fermentation. Their interesting abilities introduce novel aromatic profiles to the fermented product. In this study, screening of eight non-Saccharomyces yeast (Starmerella bombicola, Lindnera saturnus, Lindnera jadinii, Zygosaccharomyces rouxii, Torulaspora delbrueckii, Pichia kluyveri, Candida pulcherrima, and Saccharomycodes ludwigii) revealed their potential in non-alcoholic beer production. Conditions for non-alcoholic beer production were optimised for all strains tested (except T. delbrueckii) with the best results obtained at temperature 10 to 15 °C for maximum of 10 days. Starmerella bombicola, an important industrial producer of biosurfactants, was used for beer production for the first time and was able to produce non-alcoholic beer even at 20°C after 10 days of fermentation. Aromatic profile of the beer fermented with S. bombicola was neutral with no negative impact on organoleptic properties of the beer. The most interesting organoleptic properties were evaluated in beers fermented with L. jadinii and L. saturnus, which produced banana-flavoured beers with low alcohol content. This work confirmed the suitability of mentioned yeast to produce non-alcoholic beers and could serve as a steppingstone for further investigation.

Identification of a Novel Two-Peptide Lantibiotic from Vagococcus fluvialis.

Infections caused by multiresistant pathogens have become a major problem in both human and veterinary medicine. Due to the declining efficacy of many antibiotics, new antimicrobials are needed. Promising alternatives or additions to antibiotics are bacteriocins, antimicrobial peptides of bacterial origin with activity against many pathogens, including antibiotic-resistant strains. From a sample of fermented maize, we isolated a Vagococcus fluvialis strain producing a bacteriocin with antimicrobial activity against multiresistant Enterococcus faecium. Whole-genome sequencing revealed the genes for a novel two-peptide lantibiotic. The production of the lantibiotic by the isolate was confirmed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, which revealed distinct peaks at 4,009.4 m/z and 3,181.7 m/z in separate fractions from reversed-phase chromatography. The combination of the two peptides resulted in a 1,200-fold increase in potency, confirming the two-peptide nature of the bacteriocin, named vagococcin T. The bacteriocin was demonstrated to kill sensitive cells by the formation of pores in the cell membrane, and its inhibition spectrum covers most Gram-positive bacteria, including multiresistant pathogens. To our knowledge, this is the first bacteriocin characterized from Vagococcus. IMPORTANCE Enterococci are common commensals in the intestines of humans and animals, but in recent years, they have been identified as one of the major causes of hospital-acquired infections due to their ability to quickly acquire virulence and antibiotic resistance determinants. Many hospital isolates are multiresistant, thereby making current therapeutic options critically limited. Novel antimicrobials or alternative therapeutic approaches are needed to overcome this global problem. Bacteriocins, natural ribosomally synthesized peptides produced by bacteria to eliminate other bacterial species living in a competitive environment, provide such an alternative. In this work, we purified and characterized a novel two-peptide lantibiotic produced by Vagococcus fluvialis LMGT 4216 isolated from fermented maize. The novel lantibiotic showed a broad spectrum of inhibition of Gram-positive strains, including vancomycin-resistant Enterococcus faecium, demonstrating its therapeutic potential.

Improved Production of Recombinant Myrosinase in Pichia pastoris.

The effect of the deletion of a 57 bp native signal sequence, which transports the nascent protein through the endoplasmic reticulum membrane in plants, on improved AtTGG1 plant myrosinase production in Pichia pastoris was studied. Myrosinase was extracellularly produced in a 3-liter laboratory fermenter using α-mating factor as the secretion signal. After the deletion of the native signal sequence, both the specific productivity (164.8 U/L/h) and volumetric activity (27 U/mL) increased more than 40-fold compared to the expression of myrosinase containing its native signal sequence in combination with α-mating factor. The deletion of the native signal sequence resulted in slight changes in myrosinase properties: the optimum pH shifted from 6.5 to 7.0 and the maximal activating concentration of ascorbic acid increased from 1 mM to 1.5 mM. Kinetic parameters toward sinigrin were determined: 0.249 mM (Km) and 435.7 U/mg (Vmax). These results could be applied to the expression of other plant enzymes.

Optimisation of Recombinant Myrosinase Production in Pichia pastoris.

Myrosinase is a plant defence enzyme catalysing the hydrolysis of glucosinolates, a group of plant secondary metabolites, to a range of volatile compounds. One of the products, isothiocyanates, proved to have neuroprotective and chemo-preventive properties, making myrosinase a pharmaceutically interesting enzyme. In this work, extracellular expression of TGG1 myrosinase from Arabidopsis thaliana in the Pichia pastoris KM71H (MutS) strain was upscaled to a 3 L laboratory fermenter for the first time. Fermentation conditions (temperature and pH) were optimised, which resulted in a threefold increase in myrosinase productivity compared to unoptimised fermentation conditions. Dry cell weight increased 1.5-fold, reaching 100.5 g/L without additional glycerol feeding. Overall, a specific productivity of 4.1 U/Lmedium/h was achieved, which was 102.5-fold higher compared to flask cultivations.