Metabolite profiling for analysis of yeast stress response during very high gravity ethanol fermentations

A laboratory strain and an industrial strain of Saccharomyces cerevisiae were grown at high substrate concentration, so-called very high gravity (VHG) fermentation. Simultaneous saccharification and fermentation (SSF) was applied in a batch process using 280 g/L maltodextrin as carbon source. It was shown that known ethanol and osmotic stress responses such as decreased growth rate, lower viability, higher energy consumption, and intracellular trehalose accumulation occur in VHG SSF for both strains when compared with standard laboratory medium (20 g/L glucose). The laboratory strain was the most affected. GC-MS metabolite profiling was applied for assessing the yeast stress response influence on cellular metabolism. It was found that metabolite profiles originating from different strains and/or fermentation conditions were unique and could be distinguished with the help of multivariate data analysis. Several differences in the metabolic responses to stressing conditions were revealed, particularly the increased energy consumption of stressed cells was also reflected in increased intracellular concentrations of pyruvate and related metabolites.     

The population structure of African cultivated rice oryza glaberrima (Steud.): evidence for elevated levels of linkage disequilibrium caused by admixture with O. sativa and ecological adaptation

Genome-wide linkage disequilibrium (LD) was investigated for 198 accessions of Oryza glaberrima using 93 nuclear microsatellite markers. Significantly elevated levels of LD were detected, even among distantly located markers. Free recombination among loci at the population genetic level was shown (1) by a lack of decay in LD among markers on the same chromosome and (2) by a strictly increasing composite likelihood function for the recombination parameter. This suggested that the elevation in LD was due not to physical linkage but to other factors, such as population structure. A Bayesian clustering analysis confirmed this hypothesis, indicating that the sample of O. glaberrima in this study was subdivided into at least five cryptic subpopulations. Two of these subpopulations clustered with control samples of O. sativa, subspecies indica and japonica, indicating that some O. glaberrima accessions represent admixtures. The remaining three O. glaberrima subpopulations were significantly associated with specific combinations of phenotypic traits-possibly reflecting ecological adaptation to different growing environments.     

Evidence for genetic control of adult weight plasticity in the snail Helix aspersa

Phenotypic plasticity and canalization are important topics in quantitative genetics and evolution. Both concepts are related to environmental sensitivity. The latter can be modeled using a model with genetically structured environmental variance. This work reports the results of a genetic analysis of adult weight in the snail Helix aspersa. Several models of heterogeneous variance are fitted using a Bayesian, MCMC approach. Exploratory analyses using posterior predictive model checking and model comparisons based on the deviance information criterion favor a model postulating a genetically structured heterogeneous environmental variance. Our analysis provides a strong indication of a positive genetic correlation between additive genetic values affecting the mean and those affecting environmental variation of adult body weight. The possibility of manipulating environmental variance by selection is illustrated numerically using estimates of parameters derived from the snail data set.     

Rapid characterization of N‐linked glycans from secreted and gel‐purified monoclonal antibodies using MALDI‐ToF mass spectrometry

Recombinant monoclonal antibodies (MAbs) are increasingly being used for therapeutic use and correct glycosylation of these MAbs is essential for their correct function. Glycosylation profiles are host cell‐ and antibody class‐dependent and can change over culture time and environmental conditions. Therefore, rapid monitoring of glycan addition/status is of great importance for process validity. We describe two workflows of generally applicability for glycan profiling of purified and gel‐purified MAbs produced in NS0 and CHO cells, in which small‐scale antibody purification and buffer exchange is combined with PNGase F glycan cleavage and graphite HyperCarb desalting. MALDI‐ToF mass spectrometry is used for sensitive detection of glycan forms, with the ability to confirm glycan structures by selective ion fragmentation. Both workflows are rapid, technically simple and amenable to automation, and use in multi‐well formats. Biotechnol. Bioeng. 2010;107: 902–908. © 2010 Wiley Periodicals, Inc.     

Dimerization of ADAR2 is mediated by the double-stranded RNA binding domain

Members of the family of adenosine deaminases acting on RNA (ADARs) can catalyze the hydrolytic deamination of adenosine to inosine and thereby change the sequence of specific mRNAs with highly double-stranded structures. The ADARs all contain one or more repeats of the double-stranded RNA binding motif (DRBM). By both in vitro and in vivo assays, we show that the DRBMs of rat ADAR2 are necessary and sufficient for dimerization of the enzyme. Bioluminescence resonance energy transfer (BRET) demonstrates that ADAR2 also exists as dimers in living mammalian cells and that mutation of DRBM1 lowers the dimerization affinity while mutation of DRBM2 does not. Nonetheless, the editing efficiency of the GluR2 Q/R site depends on a functional DRBM2. The ADAR2 DRBMs thus serve differential roles in RNA dimerization and GluR2 Q/R editing, and we propose a model for RNA editing that incorporates the new findings.     

Recovery and Characterization of a 30.7-kDa Protein from Bacillus licheniformis Associated with Inhibitory Activity Against Methicillin-Resistant Staphylococcus aureus, Vancomycin-Resistant Enterococci, and Listeria monocytogenes

Of 131 bacterial isolates from seaweed, a culture of Bacillus licheniformis produced a novel protein with antibacterial activity against methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, and Listeria monocytogenes. The antibacterial activity was maximal in cultures prepared in Columbia broth containing pieces of synthetic polyurethane sponge and shaken at 210 to 230 rpm. Antibacterial activity was not found in cultures grown statically or with different speeds of rotary shaking. Reduced activity was apparent in supernatants prepared from marine 2216E broth and tryptone soya broth with or without 1% (wt/vol) sodium chloride. The antibacterial compound was sensitive to proteinase K, pronase, and trypsin, but was not affected by Tween−20, −40, −60, or −80, or α− or β-amylase. Activity was not adversely affected by heating up to 40°C or treatment at pH 5 to 14. The bioactive compound was determined to be associated with a protein of 30.7 kDa, which had homology to the YbdN protein of B. licheniformis ATCC 14580.     

Disproportionate abundance between ectomycorrhizal root tips and their associated mycelia

Extensive knowledge of various ectomycorrhizal fungal communities has been obtained over the past 10 years based on molecular identification of the fungi colonizing fine roots. In contrast, only limited information exists about the species composition of ectomycorrhizal hyphae in soil. This study compared the ectomycorrhizal external mycelial community with the adjacent root-tip community in a Danish beech forest. Sand-filled in-growth mesh bags were used to trap external mycelia by incubating the mesh bags in the soil for 70 days. The adjacent ectomycorrhizal root-tip communities were recorded at the times of insertion and retrieval of the mesh bags. Ectomycorrhizal fungi were identified by sequencing the internal transcribed spacer region. In total, 20, 31 and 24 ectomycorrhizal species were recorded from the two root-tip harvests and from the mesh bags, respectively. Boletoid species were significantly more frequent as mycelia than as root tips, while russuloid and Cortinarius species appeared to be less dominant as mycelia than as root tips. Tomentella species were equally frequent as root tips and as mycelia. These discrepancies between the root-tip and the mycelial view of the ectomycorrhizal fungal community are discussed within the framework of ectomycorrrhizal exploration types.