Evaluation of Pyrophosphate-Driven Proton Pumps in Saccharomyces cerevisiae under Stress Conditions.

In Saccharomyces cerevisiae, pH homeostasis is reliant on ATP due to the use of proton-translocating ATPase (H+-ATPase) which constitutes a major drain within cellular ATP supply. Here, an exogenous proton-translocating pyrophosphatase (H+-PPase) from Arabidopsis thaliana, which uses inorganic pyrophosphate (PPi) rather than ATP, was evaluated for its effect on reducing the ATP burden. The H+-Ppase was localized to the vacuolar membrane or to the cell membrane, and their impact was studied under acetate stress at a low pH. Biosensors (pHluorin and mQueen-2m) were used to observe changes in intracellular pH (pHi) and ATP levels during growth on either glucose or xylose. A significant improvement of 35% in the growth rate at a pH of 3.7 and 6 g·L-1 acetic acid stress was observed in the vacuolar membrane H+-PPase strain compared to the parent strain. ATP levels were elevated in the same strain during anaerobic glucose and xylose fermentations. During anaerobic xylose fermentations, co-expression of pHluorin and a vacuolar membrane H+-PPase improved the growth characteristics by means of an improved growth rate (11.4%) and elongated logarithmic growth duration. Our study identified a potential method for improving productivity in the use of S. cerevisiae as a cell factory under the harsh conditions present in industry.

Impact of xylose epimerase on sugar assimilation and sensing in recombinant Saccharomyces cerevisiae carrying different xylose-utilization pathways.

BACKGROUND: Over the last decades, many strategies to procure and improve xylose consumption in Saccharomyces cerevisiae have been reported. This includes the introduction of efficient xylose-assimilating enzymes, the improvement of xylose transport, or the alteration of the sugar signaling response. However, different strain backgrounds are often used, making it difficult to determine if the findings are transferrable both to other xylose-consuming strains and to other xylose-assimilation pathways. For example, the influence of anomerization rates between α- and ß-xylopyranose in pathway optimization and sugar sensing is relatively unexplored. RESULTS: In this study, we tested the effect of expressing a xylose epimerase in S. cerevisiae strains carrying different xylose-consuming routes. First, XIs originating from three different species in isogenic S. cerevisiae strains were tested and the XI from Lachnoclostridium phytofermentans was found to give the best performance. The benefit of increasing the anomerization rate of xylose by adding a xylose epimerase to the XI strains was confirmed, as higher biomass formation and faster xylose consumption were obtained. However, the impact of xylose epimerase was XI-dependent, indicating that anomer preference may differ from enzyme to enzyme. The addition of the xylose epimerase in xylose reductase/xylitol dehydrogenase (XR/XDH)-carrying strains gave no improvement in xylose assimilation, suggesting that the XR from Spathaspora passalidarum had no anomer preference, in contrast to other reported XRs. The reduction in accumulated xylitol that was observed when the xylose epimerase was added may be associated with the upregulation of genes encoding endogenous aldose reductases which could be affected by the anomerization rate. Finally, xylose epimerase addition did not affect the sugar signaling, whereas the type of xylose pathway (XI vs. XR/XDH) did. CONCLUSIONS: Although xylose anomer specificity is often overlooked, the addition of xylose epimerase should be considered as a key engineering step, especially when using the best-performing XI enzyme from L. phytofermentans. Additional research into the binding mechanism of xylose is needed to elucidate the enzyme-specific effect and decrease in xylitol accumulation. Finally, the differences in sugar signaling responses between XI and XR/XDH strains indicate that either the redox balance or the growth rate impacts the SNF1/Mig1p sensing pathway.

Using phosphoglucose isomerase-deficient (pgi1Δ) Saccharomyces cerevisiae to map the impact of sugar phosphate levels on D-glucose and D-xylose sensing.

BACKGROUND: Despite decades of engineering efforts, recombinant Saccharomyces cerevisiae are still less efficient at converting D-xylose sugar to ethanol compared to the preferred sugar D-glucose. Using GFP-based biosensors reporting for the three main sugar sensing routes, we recently demonstrated that the sensing response to high concentrations of D-xylose is similar to the response seen on low concentrations of D-glucose. The formation of glycolytic intermediates was hypothesized to be a potential cause of this sensing response. In order to investigate this, glycolysis was disrupted via the deletion of the phosphoglucose isomerase gene (PGI1) while intracellular sugar phosphate levels were monitored using a targeted metabolomic approach. Furthermore, the sugar sensing of the PGI1 deletants was compared to the PGI1-wildtype strains in the presence of various types and combinations of sugars. RESULTS: Metabolomic analysis revealed systemic changes in intracellular sugar phosphate levels after deletion of PGI1, with the expected accumulation of intermediates upstream of the Pgi1p reaction on D-glucose and downstream intermediates on D-xylose. Moreover, the analysis revealed a preferential formation of D-fructose-6-phosphate from D-xylose, as opposed to the accumulation of D-fructose-1,6-bisphosphate that is normally observed when PGI1 deletants are incubated on D-fructose. This may indicate a role of PFK27 in D-xylose sensing and utilization. Overall, the sensing response was different for the PGI1 deletants, and responses to sugars that enter the glycolysis upstream of Pgi1p (D-glucose and D-galactose) were more affected than the response to those entering downstream of the reaction (D-fructose and D-xylose). Furthermore, the simultaneous exposure to sugars that entered upstream and downstream of Pgi1p (D-glucose with D-fructose, or D-glucose with D-xylose) resulted in apparent synergetic activation and deactivation of the Snf3p/Rgt2p and cAMP/PKA pathways, respectively. CONCLUSIONS: Overall, the sensing assays indicated that the previously observed D-xylose response stems from the formation of downstream metabolic intermediates. Furthermore, our results indicate that the metabolic node around Pgi1p and the level of D-fructose-6-phosphate could represent attractive engineering targets for improved D-xylose utilization.

D-Xylose Sensing in Saccharomyces cerevisiae: Insights from D-Glucose Signaling and Native D-Xylose Utilizers.

Extension of the substrate range is among one of the metabolic engineering goals for microorganisms used in biotechnological processes because it enables the use of a wide range of raw materials as substrates. One of the most prominent examples is the engineering of baker's yeast Saccharomyces cerevisiae for the utilization of d-xylose, a five-carbon sugar found in high abundance in lignocellulosic biomass and a key substrate to achieve good process economy in chemical production from renewable and non-edible plant feedstocks. Despite many excellent engineering strategies that have allowed recombinant S. cerevisiae to ferment d-xylose to ethanol at high yields, the consumption rate of d-xylose is still significantly lower than that of its preferred sugar d-glucose. In mixed d-glucose/d-xylose cultivations, d-xylose is only utilized after d-glucose depletion, which leads to prolonged process times and added costs. Due to this limitation, the response on d-xylose in the native sugar signaling pathways has emerged as a promising next-level engineering target. Here we review the current status of the knowledge of the response of S. cerevisiae signaling pathways to d-xylose. To do this, we first summarize the response of the native sensing and signaling pathways in S. cerevisiae to d-glucose (the preferred sugar of the yeast). Using the d-glucose case as a point of reference, we then proceed to discuss the known signaling response to d-xylose in S. cerevisiae and current attempts of improving the response by signaling engineering using native targets and synthetic (non-native) regulatory circuits.

Workplace Violence and Long-term Sickness Absence: Assessment of the Potential Buffering Effect of Social Support in Two Occupational Cohort Studies.

OBJECTIVES: We aimed to determine the effect of workplace violence on long-term sickness absence, and whether social support from supervisors and colleagues buffer this effect. METHODS: Information on workplace violence and social support were derived from the Danish Work Environment Cohort Study in 2000, 2005, and 2010 and the Swedish Longitudinal Occupational Survey of Health in 2006 and 2008. Individual- and joint-effects on register-based long-term sickness absence were determined using logistic regression models for repeated measurements. Cohort-specific estimates were combined in random effect meta-analyses. RESULTS: Workplace violence and low social support were independently associated with a higher risk of long-term sickness absence, and we did not find evidence of an interaction. CONCLUSION: Exposure to workplace violence is a risk factor for long-term sickness absence while social support is associated with a lower risk of long-term sickness absence.

Effects of procedural justice on prospective antidepressant medication prescription: a longitudinal study on Swedish workers.

BACKGROUND: Procedural justice has been linked to several mental health problems, but most studies have used self-reported data. There exist a need to assess the link between procedural justice and health using outcomes that are not only self-reported. The aim of the current study was to examine whether perceived procedural justice at work is prospectively associated with antidepressant medication prescription. METHODS: Data from 4374 participants from the Swedish Longitudinal Survey of Health (SLOSH) were linked to the Swedish National Prescribed Drug register. Based on their perceived procedural justice at two times (2010 and 2012), participants were divided into four groups: stable low, increasing, decreasing and stable high justice perceptions. Using Cox regression, we studied how the course of stability and change in perceived procedural justice affected the rate of prescription of antidepressant medication over the next 2 years. Participants with missing data and those who had been prescribed antidepressant medication in the period leading up to 2012 were excluded in the main analyses to determine incident morbidity. RESULTS: The results showed that after adjustment for sex, age, education, socioeconomic position, marital status, and insecure employment a decrease in perceived procedural justice over time was associated with greater receipt of antidepressants compared to people with stable high perceptions of procedural justice (HR 1.76, 95% CI: 1.16 to 2.68). Being female and having insecure employment were also associated with higher hazards of antidepressant prescription. CONCLUSIONS: These findings strengthen the notion that procedural justice at work influences psychological well-being, as well as provide new insights into how procedural justice perceptions may affect mental health.

SLC10A4 regulates IgE-mediated mast cell degranulation in vitro and mast cell-mediated reactions in vivo.

Mast cells act as sensors in innate immunity and as effector cells in adaptive immune reactions. Here we demonstrate that SLC10A4, also referred to as the vesicular aminergic-associated transporter, VAAT, modifies mast cell degranulation. Strikingly, Slc10a4 -/- bone marrow-derived mast cells (BMMCs) had a significant reduction in the release of granule-associated mediators in response to IgE/antigen-mediated activation, whereas the in vitro development of mast cells, the storage of the granule-associated enzyme mouse mast cell protease 6 (mMCP-6), and the release of prostaglandin D2 and IL-6 were normal. Slc10a4-deficient mice had a strongly reduced passive cutaneous anaphylaxis reaction and a less intense itching behaviour in response to the mast cell degranulator 48/80. Live imaging of the IgE/antigen-mediated activation showed decreased degranulation and that ATP was retained to a higher degree in mast cell granules lacking SLC10A4. Furthermore, ATP was reduced by two thirds in Slc10a4 -/- BMMCs supernatants in response to IgE/antigen. We speculate that SLC10A4 affects the amount of granule-associated ATP upon IgE/antigen-induced mast cell activation, which affect the release of granule-associated mast cell mediators. In summary, SLC10A4 acts as a regulator of degranulation in vitro and of mast cell-related reactions in vivo.

Validation of Online Versions of Tinnitus Questionnaires Translated into Swedish.

Background: Due to the lack of objective measures for assessing tinnitus, its clinical evaluation largely relies on the use of questionnaires and psychoacoustic tests. A global assessment of tinnitus burden would largely benefit from holistic approaches that not only incorporate measures of tinnitus but also take into account associated fears, emotional aspects (stress, anxiety, and depression), and quality of life. In Sweden, only a few instruments are available for assessing tinnitus, and the existing tools lack validation. Therefore, we translated a set of questionnaires into Swedish and evaluated their reliability and validity in a group of tinnitus subjects. Methods: We translated the English versions of the Tinnitus Functional Index (TFI), the Fear of Tinnitus Questionnaire (FTQ), the Tinnitus Catastrophizing Scale (TCS), the Perceived Stress Questionnaire (PSQ-30), and the Tinnitus Sample Case History Questionnaire (TSCHQ) into Swedish. These translations were delivered via the internet with the already existing Swedish versions of the Tinnitus Handicap Inventory (THI), the Hospital Anxiety and Depression Scale (HADS), the Hyperacusis Questionnaire (HQ), and the World Health Organization Quality of Life questionnaire (WHOQoL-BREF). Psychometric properties were evaluated by means of internal consistency [Cronbach's alpha (α)] and test-retest reliability across a 9-week interval [Intraclass Correlation Coefficient (ICC), Cohen's kappa] in order to establish construct as well as clinical validity using a sample of 260 subjects from a population-based cohort. Results: Internal consistency was acceptable for all questionnaires (α > 0.7) with the exception of the "social relationships" subscale of the WHOQoL-BREF. Test-retest reliability was generally acceptable (ICC > 0.70, Cohens kappa > 0.60) for the tinnitus-related questionnaires, except for the TFI "sense of control" subscale and 15 items of the TSCHQ. Spearmen rank correlations showed that almost all questionnaires on tinnitus are significantly related, indicating that these questionnaires measure different aspects of the same construct. The data supported good clinical validity of the tinnitus-related questionnaires. Conclusion: Our results suggest that most Swedish adaptations of the questionnaires are suitable for clinical and research settings and should facilitate the assessment of treatment outcomes using a more holistic approach by including measures of tinnitus fears, emotional burden, and quality of life.