Yeast Genome-Wide Expression Analysis Identifies a Strong Ergosterol and Oxidative Stress Response during the Initial Stages of an Industrial Lager Fermentation

Genome-wide expression analysis of an industrial strain of Saccharomyces cerevisiae during the initial stages of an industrial lager fermentation identified a strong response from genes involved in the biosynthesis of ergosterol and oxidative stress protection. The induction of the ERG genes was confirmed by Northern analysis and was found to be complemented by a rapid accumulation of ergosterol over the initial 6-h fermentation period. From a test of the metabolic activity of deletion mutants in the ergosterol biosynthesis pathway, it was found that ergosterol is an important factor in restoring the fermentative capacity of the cell after storage. Additionally, similar ERG10 and TRR1 gene expression patterns over the initial 24-h fermentation period highlighted a possible interaction between ergosterol biosynthesis and the oxidative stress response. Further analysis showed that erg mutants producing altered sterols were highly sensitive to oxidative stress-generating compounds. Here we show that genome-wide expression analysis can be used in the commercial environment and was successful in identifying environmental conditions that are important in industrial yeast fermentation.     

A comparison of blood pressure measurement over a sleeved arm versus a bare arm

Background

The measurement of blood pressure is a common clinical exam with important health consequences. We sought to determine whether the measurement of blood pressure over a sleeved arm varies from that taken on a bare arm.

Methods

We recruited 376 patients between 18 and 85 years of age from a family medicine clinic between September 2004 and November 2006. They all had their blood pressure recorded using the same automatic oscillometric device, with the cuff placed over their bare arms for the first reading. Each patient was then randomly assigned to either the bare-arm group, for which the second blood pressure reading was also taken on a bare arm, or the sleeved-arm group, for which the second reading was taken with the cuff placed over the patient''s sleeve.

Results

The mean age of the 376 participants was 61.6 years (standard deviation 15.0), 61% of the participants were male, 41% had hypertension and 11.7% had diabetes. We found no clinically important differences between the bare-arm group (n = 180) and the sleeved-arm group (n = 196) in age, sex or body mass index. The mean differences between the first and second readings for patients in the bare-arm group were 4.1 mm Hg (95% confidence interval [CI] 2.8 to 5.5) for systolic blood pressure and 0.1 mm Hg (95% CI –0.7 to 0.9) for diastolic blood pressure. The mean differences between the first and second readings for patients in the sleeved-arm group were 3.4 mm Hg (95% CI 2.1 to 4.7) for systolic blood pressure and 0.4 mm Hg (95% CI –0.4 to 1.3) for diastolic blood pressure. The between-group differences in these values was 0.76 mm Hg (95% CI –1.13 to 2.65) for systolic and –0.31 mm Hg (95% CI –1.48 to 0.86) for diastolic blood pressure; neither of these differences was clinically important or statistically significant.

Interpretation

We found that there was no significant difference in blood pressure recorded over a sleeve or on a bare arm. For practical purposes, the decision to measure blood pressure on a bare arm or over a sleeved arm should be left to the judgment of the health care professional taking the blood pressure.The measurement of blood pressure is one of the most common examinations undertaken in family practice and has important health and management consequences for the patient. Accurate assessment of blood pressure, therefore, is very important. Current guidelines outline standards for obtaining accurate and reproducible blood pressure measurements.¹ These standards include guidelines for the sizes of blood pressure cuffs and the position of the patient''s arm, as well as a recommendation that the patient be seated for 5 minutes before his or her blood pressure is taken. In addition, it has been recommended that blood pressure be measured over the patient''s bare arm.¹ However, the current recommended method to determine blood pressure² has several limitations (e.g., high variability of blood pressure at different times of day or if measured only once, loss of proper technique post-training).Three previous studies have assessed whether blood pressure varies significantly when taken over a sleeved arm compared with a bare arm. Details of the literature review are described in Appendix 1, available online at www.cmaj.ca/cgi/content/full/178/5/585/DC2. In one study, involving 36 patients, Holleman et al³ found no significant differences in systolic or diastolic blood pressure readings taken over the sleeved or bare arm of each patient. However, this study was limited because of its small sample and because blood pressure measurements were taken on both arms simultaneously. A study by Kahan et al,⁴ involving 201 patients, compared blood pressure measurements taken over a sleeved arm, a bare arm and below a rolled-up sleeve. They found that the degree of clothing under the sphygmomanometer cuff did not have a clinically important effect on the reading. Although they found no significant difference in the effect of clothing on blood pressure readings, the study was limited by their design of measuring blood pressure below a rolled-up sleeve. The third study, conducted by Liebl et al,⁵ was published while our study was in progress. Their study, which involved 201 patients, compared blood pressure measurements taken over a sleeved arm and a bare arm with both sphygmomanometric and oscillometric devices. They concluded no significant difference in readings between the sleeved and bare-arm groups. In contrast to previous studies, we sought to determine whether the measurement of blood pressure over a sleeved arm varies from that taken over a bare arm through the use of a control group in which patients'' blood pressure was taken only over a bare arm.     

Site-directed, Ligase-Independent Mutagenesis (SLIM) for highly efficient mutagenesis of plasmids greater than 8kb

Modifying the Site-directed, Ligase-Independent Mutagenesis (SLIM) protocol from a single reaction mode to a two-reaction mode enables highly efficient mutagenesis of plasmid constructs that exceed 8kb. This modified approach reduces the complexity of the PCR step and is optimised for generation of heteroduplexes from long PCR products. The two-reaction mode SLIM has 92% efficiency.     

Mitochondrial Iba57p is required for Fe/S cluster formation on aconitase and activation of radical SAM enzymes

A genome-wide screen for Saccharomyces cerevisiae iron-sulfur (Fe/S) cluster assembly mutants identified the gene IBA57. The encoded protein Iba57p is located in the mitochondrial matrix and is essential for mitochondrial DNA maintenance. The growth phenotypes of an iba57Δ mutant and extensive functional studies in vivo and in vitro indicate a specific role for Iba57p in the maturation of mitochondrial aconitase-type and radical SAM Fe/S proteins (biotin and lipoic acid synthases). Maturation of other Fe/S proteins occurred normally in the absence of Iba57p. These observations identify Iba57p as a novel dedicated maturation factor with specificity for a subset of Fe/S proteins. The Iba57p primary sequence is distinct from any known Fe/S assembly factor but is similar to certain tetrahydrofolate-binding enzymes, adding a surprising new function to this protein family. Iba57p physically interacts with the mitochondrial ISC assembly components Isa1p and Isa2p. Since all three proteins are conserved in eukaryotes and bacteria, the specificity of the Iba57/Isa complex may represent a biosynthetic concept that is universally used in nature. In keeping with this idea, the human IBA57 homolog C1orf69 complements the iba57Δ growth defects, demonstrating its conserved function throughout the eukaryotic kingdom.     

Invasions Slow Down or Collapse in the Presence of Reactive Boundaries

Motivated by the propagation of thin bacterial films around planar obstacles, this paper considers the dynamics of travelling wave solutions to the Fisher–KPP equation \(u_t = u(1-u) + u_{xx} + u_{yy}\) in a planar strip \(-\infty< x < \infty \), \(0 \le y \le L\). We examine the propagation of fronts in the presence of a mixed boundary condition (also referred to as a ‘partially absorbing’ or ‘reactive’ boundary) \(u_y = \alpha u\), with \(\alpha >0\), at \(y=0\). The presence of boundary conditions of this kind leads to the development of front solutions that propagate in x but contain transverse structure in y. Motivated by the observation that the speed of propagation in the Fisher–KPP equation is determined (for exponentially decaying initial conditions) by the behaviour at the leading edge, we analyse the linearised Fisher–KPP equation in order to estimate the speed of the stable travelling front, a function of the width L and the imposed boundary conditions. For wide strips the speed estimate based on the linearised equation agrees well with the results of numerical simulations. For narrow channels numerical simulations indicate that the stable front propagates more slowly, and for sufficiently small L or sufficiently large \(\alpha \) the front speed falls to zero and the front collapses. The reason for the collapse is the non-existence, far behind the front, of a stable positive equilibrium solution u(x, y). While existence of these equilibrium states can be demonstrated via phase plane arguments, the investigation of stability is similar to calculations of critical patch sizes carried out in similar ecological models.     

TB tools to tell the tale-molecular genetic methods for mycobacterial research

In spite of the availability of drugs and a vaccine, tuberculosis--one of man's medical nemeses--remains a formidable public health problem, particularly in the developing world. The persistent nature of the tubercle bacillus, with one third of the world's population is estimated to be infected, combined with the emergence of multi drug-resistant strains and the exquisite susceptibility of HIV-positive individuals, has underscored the urgent need for in-depth study of the biology of Mycobacterium tuberculosis address the resurgence of TB. In aiming to understand the mechanisms by which mycobacteria react to their immediate environments, molecular genetic tools have been developed from naturally occurring genetic elements. These include protein expressing genes, and episomal and integrating elements, which have been derived mainly from prokaryotic but also from eukaryotic organisms. Molecular genetic tools that had been established as routine procedures in other prokaryotic genera were thus mimicked. Knowledge of the underlying mechanisms greatly expedited the harnessing of these elements for mycobacteriological research and has brought us to a point where these molecular genetic tools are now employed routinely in laboratories worldwide.     

The role of respiration, reactive oxygen species and oxidative stress in mother cell-specific ageing of yeast strains defective in the RAS signalling pathway

We show that the dominant activated allele of the yeast RAS gene, RAS2(ala18,val19), led to redox imbalance in exponential-phase cells and to excretion of almost all of the cellular glutathione into the medium when the cells reached early-stationary phase. The mitochondria of the mutant stained strongly with dihydrorhodamine 123 (DHR) and the cells displayed a very short mother cell-specific lifespan. Adding 1 mM reduced glutathione (GSH) to the medium partly restored the lifespan. The corresponding RAS2(+) rho-zero strain also displayed a short lifespan, excreted nearly all of its GSH, and stained positively with DHR. Adding 1 mM GSH completely restored the lifespan of the RAS2(+) rho-zero strain to that of the wild-type cells. The double mutant RAS2(ala18,val19) rho-zero cells showed the same lifespan as the RAS2(ala18,val19) cells, and the effect of glutathione in restoring the lifespan was the same, indicating that both mutations shorten lifespan through a similar mechanism. In the RAS2(ala18,val19) mutant strain and its rho-zero derivative we observed for the first time a strong electron spin resonance (ESR) signal characteristic of the superoxide radical anion. The mutant cells were, therefore, producing superoxide in the absence of a complete mitochondrial electron transport chain, pointing to the existence of a possible non-mitochondrial source for ROS generation. Our results indicate that oxidative stress resulting from a disturbance of redox balance can play a major role in mother cell-specific lifespan determination of yeast cells.