The mRNA cap-binding protein Cbc1 is required for high and timely expression of genes by promoting the accumulation of gene-specific activators at promoters.

The highly conserved Saccharomyces cerevisiae cap-binding protein Cbc1/Sto1 binds mRNA co-transcriptionally and acts as a key coordinator of mRNA fate. Recently, Cbc1 has also been implicated in transcription elongation and pre-initiation complex (PIC) formation. Previously, we described Cbc1 to be required for cell growth under osmotic stress and to mediate osmostress-induced translation reprogramming. Here, we observe delayed global transcription kinetics in cbc1Δ during osmotic stress that correlates with delayed recruitment of TBP and RNA polymerase II to osmo-induced promoters. Interestingly, we detect an interaction between Cbc1 and the MAPK Hog1, which controls most gene expression changes during osmostress, and observe that deletion of CBC1 delays the accumulation of the activator complex Hot1-Hog1 at osmostress promoters. Additionally, CBC1 deletion specifically reduces transcription rates of highly transcribed genes under non-stress conditions, such as ribosomal protein (RP) genes, while having low impact on transcription of weakly expressed genes. For RP genes, we show that recruitment of the specific activator Rap1, and subsequently TBP, to promoters is Cbc1-dependent. Altogether, our results indicate that binding of Cbc1 to the capped mRNAs is necessary for the accumulation of specific activators as well as PIC components at the promoters of genes whose expression requires high and rapid transcription.

Detection and identification of xerophilic fungi in Belgian chocolate confectionery factories.

Chocolate confectionery fillings are generally regarded as microbiologically stable. The stability of these fillings is largely due to the general practice of adding either alcohol or preservatives. Consumer demands are now stimulating producers to move away from adding alcohol or other preservatives to their confectionery fillings and instead to search for innovative formulations. Such changes in composition can influence the shelf life of the product and may lead to spoilage by xerophilic fungi. The aim of this study was to test whether the production environment of Belgian chocolate confectionery factories and common ingredients of chocolate confectioneries could be potential sources of contamination with xerophilic fungal species. In the factory environment, the general and strictly xerophilic fungal spore load was determined using an RCS Air Sampler device in combination with DG18 and MY50G medium, respectively. Four basic ingredients of chocolate confectionery fillings were also examined for fungal spore levels using a direct plating technique. Detected fungi were identified to species level by a combination of morphological characterization and sequence analysis. Results indicated a general fungal spore load in the range of 50-250 colony forming units per cubic meter of air (CFU/m(3) air) and a more strict xerophilic spore load below 50 CFU/m(3) air. These results indicate rather low levels of fungal spores present in the factory environment. The most prevalent fungi in the factory environment were identified as Penicillium spp., particularly Penicillium brevicompactum. Examination of the basic ingredients of confectionery fillings revealed nuts to be the most likely potential source of direct contamination. In nuts, the most prevalent fungal species identified were Eurotium, particularly Eurotium repens.

Effects of ascorbic acid on patulin in aqueous solution and in cloudy apple juice.

Degradation of the mycotoxin patulin (PAT) and the generation of (less toxic) breakdown products, such as (E/Z)-ascladiol (ASC-E/Z) and desoxypatulinic acid (D-PAT), can occur due to chemical, physical and biological treatments. Our study focused on the chemical degradation of PAT in the presence of ascorbic acid (AA) both for pure PAT standard in acidified aqueous solution and for PAT-contaminated cloudy apple juice (CAJ) (obtained via addition of apple mash produced from apples inoculated with Penicillium expansum). Within this framework, different concentrations of AA were evaluated, as well as the presence/absence of oxygen and different storage temperatures. In order to do so, an in-house methodology allowing a good separation of PAT from its reaction and breakdown products was optimized first. The highest PAT reduction (60%) in CAJ with an initial PAT concentration of 100 µg/kg and 0.25% (w/v) AA was achieved after 6 days of incubation at 22 °C in the presence of oxygen. It was also found that the treatment by AA resulted in the generation of degradation products less toxic than PAT (such as (E/Z)-ASC). In conclusion, AA used to improve numerous product quality aspects (e.g. colour (less browning), nutritional value, etc.) and considered as a safe food additive (Food and Drug Administration (FDA) (1999)) has an effect on PAT degradation. It was shown that such degradation generated less toxic compounds in the presence of oxygen. In view of consumers' safety, fortification of apple juice (and possibly apple-based products) with AA could be helpful within an integrated system to ensure products with low levels of patulin. The optimum conditions for such an approach within a legal and practical point of view need to be further explored.

Restriction analysis of an amplified rodA gene fragment to distinguish Aspergillus fumigatus var. ellipticus from Aspergillus fumigatus var. fumigatus.

A previous multidisciplinary study indicated that gliotoxin-producing Aspergillus fumigatus Fresen. isolates from silage commodities mostly belonged to its variant A. fumigatus var. ellipticus Raper & Fennell. Sequence analysis revealed the presence of a single nucleotide polymorphism at five positions in a fragment of the rodA gene (coding for a hydrophobin rodletA protein) between Aspergillus fumigatus var. fumigatus and Aspergillus fumigatus var. ellipticus. A method was developed to distinguish these two types of isolates based on restriction analysis of this rodA gene fragment using the HinfI restriction enzyme. In addition, in silico analysis of 113 rodA gene fragments retrieved from GenBank was performed and confirmed the suitability of this method. In conclusion, the method developed in this study allows easy distinction between A. fumigatus var. fumigatus and its variant ellipticus. In combination with the earlier developed PCR-restriction fragment length polymorphism method of Staab et al. (2009, J Clin Microbiol 47: 2079), this method is part of a sequencing-independent identification scheme that allows for rapid distinction between similar species/variants within Aspergillus section Fumigati, specifically A. fumigatus, A. fumigatus var. ellipticus, Aspergillus lentulus Balajee & K.A. Marr, Neosartorya pseudofischeri S.W. Peterson and Neosartorya udagawae Y. Horie, Miyaji & Nishim.

Yeast mRNA cap-binding protein Cbc1/Sto1 is necessary for the rapid reprogramming of translation after hyperosmotic shock.

In response to osmotic stress, global translation is inhibited, but the mRNAs encoding stress-protective proteins are selectively translated to allow cell survival. To date, the mechanisms and factors involved in the specific translation of osmostress-responsive genes in Saccharomyces cerevisiae are unknown. We find that the mRNA cap-binding protein Cbc1 is important for yeast survival under osmotic stress. Our results provide new evidence supporting a role of Cbc1 in translation initiation. Cbc1 associates with polysomes, while the deletion of the CBC1 gene causes hypersensitivity to the translation inhibitor cycloheximide and yields synthetic "sickness" in cells with limiting amounts of translation initiator factor eIF4E. In cbc1Δ mutants, translation drops sharply under osmotic stress, the subsequent reinitiation of translation is retarded, and "processing bodies" containing untranslating mRNAs remain for long periods. Furthermore, osmostress-responsive mRNAs are transcriptionally induced after osmotic stress in cbc1Δ cells, but their rapid association with polysomes is delayed. However, in cells containing a thermosensitive eIF4E allele, their inability to grow at 37ºC is suppressed by hyperosmosis, and Cbc1 relocalizes from nucleus to cytoplasm. These data support a model in which eIF4E-translation could be stress-sensitive, while Cbc1-mediated translation is necessary for the rapid translation of osmostress-protective proteins under osmotic stress.