Primary Epstein-Barr virus infection with neurological complications.

Several case studies have reported on neurological complications caused by a primary Epstein-Barr virus (EBV) infection. We aimed to investigate the viral loads and the clinical and inflammatory characteristics of this disease entity. We evaluated all 84 cases in which the EBV polymerase chain reaction test (PCR) was requested on cerebrospinal fluid (CSF) for the period 2003-2008. Fourteen patients with proven neuroborreliosis served as the control group. Nine patients were diagnosed with a primary EBV infection and neurological symptoms (median age 36 y; 4 male). Viral DNA copies in CSF were detected by PCR in 7 of 9 patients. The presenting symptoms were meningeal signs, epileptic insults, polyradiculomyelitis, polyradiculitis, and/or sudden cognitive disorders. All EBV cases had a pleocytosis with significantly increased mononuclear leukocytes as compared to the neuroborreliosis group (median 99% interquartile range (96-100%) versus 90% (86-97%). In cases with a primary EBV infection, viral loads ranged from 43 to 3202 copies/ml in CSF and from 61 to 15,595 copies/ml in serum. Seventy-eight percent of the cases had a positive PCR on CSF. This study provides criteria for diagnosing neurological disease during primary EBV infection. Primary EBV infections in immune competent persons can cause a broad range of neurological symptoms, with lymphocytic and monocytic inflammation both in blood and CSF.

Environmental microarray analyses of Antarctic soil microbial communities.

Antarctic ecosystems are fascinating in their limited trophic complexity, with decomposition and nutrient cycling functions being dominated by microbial activities. Not only are Antarctic habitats exposed to extreme environmental conditions, the Antarctic Peninsula is also experiencing unequalled effects of global warming. Owing to their uniqueness and the potential impact of global warming on these pristine systems, there is considerable interest in determining the structure and function of microbial communities in the Antarctic. We therefore utilized a recently designed 16S rRNA gene microarray, the PhyloChip, which targets 8741 bacterial and archaeal taxa, to interrogate microbial communities inhabiting densely vegetated and bare fell-field soils along a latitudinal gradient ranging from 51 degrees S (Falkland Islands) to 72 degrees S (Coal Nunatak). Results indicated a clear decrease in diversity with increasing latitude, with the two southernmost sites harboring the most distinct Bacterial and Archaeal communities. The microarray approach proved more sensitive in detecting the breadth of microbial diversity than polymerase chain reaction-based bacterial 16S rRNA gene libraries of modest size ( approximately 190 clones per library). Furthermore, the relative signal intensities summed for phyla and families on the PhyloChip were significantly correlated with the relative occurrence of these taxa in clone libraries. PhyloChip data were also compared with functional gene microarray data obtained earlier, highlighting numerous significant relationships and providing evidence for a strong link between community composition and functional gene distribution in Antarctic soils. Integration of these PhyloChip data with other complementary methods provides an unprecedented understanding of the microbial diversity and community structure of terrestrial Antarctic habitats.

Rapid identification of target genes for 3-methyl-1-butanol production in Saccharomyces cerevisiae.

Extracellular conditions determine the taste of fermented foods by affecting metabolite formation by the micro-organisms involved. To identify targets for improvement of metabolite formation in food fermentation processes, automated high-throughput screening and cDNA microarray approaches were applied. Saccharomyces cerevisiae was cultivated in 96-well microtiter plates, and the effects of salt concentration and pH on the growth and synthesis of the fusel alcohol-flavoured substance, 3-methyl-1-butanol, was evaluated. Optimal fermentation conditions for 3-methyl-1-butanol concentration were found at pH 3.0 and 0% NaCl. To identify genes encoding enzymes with major influence on product formation, a genome-wide gene expression analysis was carried out with S. cerevisiae cells grown at pH 3.0 (optimal for 3-methyl-1-butanol formation) and pH 5.0 (yeast cultivated under standard conditions). A subset of 747 genes was significantly induced or repressed when the pH was changed from pH 5.0 to 3.0. Expression of seven genes related to the 3-methyl-1-butanol pathway, i.e. LAT1, PDX1, THI3, ALD4, ILV3, ILV5 and LEU4, strongly changed in response to this switch in pH of the growth medium. In addition, genes involved in NAD metabolism, i.e. BNA2, BNA3, BNA4 and BNA6, or those involved in the TCA cycle and glutamate metabolism, i.e. MEU1, CIT1, CIT2, KDG1 and KDG2, displayed significant changes in expression. The results indicate that this is a rapid and valuable approach for identification of interesting target genes for improvement of yeast strains used in industrial processes.

Bat2p is essential in Saccharomyces cerevisiae for fusel alcohol production on the non-fermentable carbon source ethanol.

Branched-chain amino acids (BCAAs) are key substrates in the formation of fusel alcohols, important flavour components in fermented foods. The first step in the catabolic BCAA degradation is a transaminase step, catalyzed by a branched-chain amino acid transaminase (BCAAT). Saccharomyces cerevisiae possesses a mitochondrial and a cytosolic BCAAT, Bat1p and Bat2p, respectively. In order to study the impact of the BCAATs on fusel alcohol production derived from the BCAA metabolism, S. cerevisiae BCAAT-deletion mutants were constructed. The BCAA l-leucine was exogenously supplied during cultivations with mutants of S. cerevisiae. BAT1 deletion is not essential for fusel alcohol production, neither under glucose nor under ethanol growth conditions. The 3-methyl-1-butanol production rate of bat1Delta-cells on ethanol was decreased in comparison with that of wild-type cells, but the cells were still able to produce 3-methyl-1-butanol. However, drastic effects in fusel alcohol production were obtained in cells lacking BAT2. Although the constructed bat2Delta-single deletion strain and the bat1Deltabat2Delta-double deletion strain were still able to produce 3-methyl-1-butanol when grown on glucose, they were incapable of producing any 3-methyl-1-butanol when ethanol was the sole carbon source available. In the circumstances used, gene expression analysis revealed a strong upregulation of BAT2 gene activity in the wild type, when cells grew on ethanol as carbon source. Apparently, the carbon metabolism is able to influence the expression of BCAATs and interferes with the nitrogen metabolism. Furthermore, analysis of gene expression profiles shows that the expression of genes coding for other transaminases present in S. cerevisiae was influenced by the deletion of one or both BCAATs. Several transaminases were upregulated when a BCAAT was deleted. Strikingly, none of the known transaminases was significantly upregulated when BAT2 was deleted. Therefore we conclude that the expression of BAT2 is essential for 3-methyl-1-butanol formation on the non-fermentable carbon source, ethanol.

Identification of salt-induced genes of Zygosaccharomyces rouxii by using Saccharomyces cerevisiae GeneFilters.

Yeast GeneFilters containing all Saccharomyces cerevisiae open reading frame (ORF) sequences were used to elucidate gene activity in the osmotolerant yeast Zygosaccharomyces rouxii. Labelled cDNA derived from Z. rouxii was targeted to spotted S. cerevisiae ORFs. Approximately 90-100% homology of Z. rouxii genes with those of S. cerevisiae was required for definitive identification of the cDNAs hybridised to GeneFilter. Hybridised labelled cDNAs were visualised as small spots on the microarray, providing simultaneous information on homologous genes present in Z. rouxii and on their level of gene activity. Cross-hybridisation of the GeneFilters displayed 155 as yet unidentified genes of Z. rouxii hybridising to S. cerevisiae ORFs. From those 155 genes, the activity of 86 genes was influenced as a result of NaCl stress. In comparison with S. cerevisiae 24% of Z. rouxii genes revealed a different transcription behaviour following NaCl stress. All of these genes had no previously defined function in osmotic-stress response in Z. rouxii. Therefore, cross-hybridisation of GeneFilters proves to be an appropriate and straightforward method for screening transcripts in Z. rouxii, which provides an extension of the knowledge of genes present in a yeast genus other than S. cerevisiae.

Functional analysis of structural genes for NAD(+)-dependent formate dehydrogenase in Saccharomyces cerevisiae.

Co-consumption of formate by aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae CEN.PK 113-7D led to an increased biomass yield relative to cultures grown on glucose as the sole carbon and energy substrate. In this respect, this strain differed from two previously investigated S. cerevisiae strains, in which formate oxidation did not lead to an increased biomass yield on glucose. Enzyme assays confirmed the presence of a formate-inducible, cytosolic and NAD(+)-dependent formate dehydrogenase. To investigate whether this enzyme activity was entirely encoded by the previously reported FDH1 gene, an fdh1Delta null mutant was constructed. This mutant strain still contained formate dehydrogenase activity and remained capable of co-consumption of formate. The formate dehydrogenase activity in the mutant was demonstrated to be encoded by a second structural gene for formate dehydrogenase (FDH2) in S. cerevisiae CEN.PK 113-7D. FDH2 was highly homologous to FDH1 and consisted of a fusion of two open reading frames (ORFs) (YPL275w and YPL276w) reported in the S. cerevisiae genome databases. Sequence analysis confirmed that, in the database genetic background, the presence of two single-nucleotide differences led to two truncated ORFs rather than the full-length FDH2 gene present in strain CEN.PK 113-7D. In the latter strain background an fdh1Deltafdh2Delta double mutant lacked formate dehydrogenase activity and was unable to co-consume formate. Absence of formate dehydrogenase activity did not affect growth on glucose as sole carbon source, but led to a reduced biomass yield on glucose-formate mixtures. These findings are consistent with a role of formate dehydrogenase in the detoxification of exogenous formate.