Pyruvate decarboxylase provides growing pollen tubes with a competitive advantage in petunia

Rapid pollen tube growth places unique demands on energy production and biosynthetic capacity. The aim of this work is to understand how primary metabolism meets the demands of such rapid growth. Aerobically grown pollen produce ethanol in large quantities. The ethanolic fermentation pathway consists of two committed enzymes: pyruvate decarboxylase ( PDC) and alcohol dehydrogenase ( ADH). Because adh mutations do not affect male gametophyte function, the obvious question is why pollen synthesize an abundant enzyme if they could do just as well without. Using transposon tagging in Petunia hybrida, we isolated a null mutant in pollen- specific Pdc2. Growth of the mutant pollen tubes through the style is reduced, and the mutant allele shows reduced transmission through the male, when in competition with wild- type pollen. We propose that not ADH but rather PDC is the critical enzyme in a novel, pollen- specific pathway. This pathway serves to bypass pyruvate dehydrogenase enzymes and thereby maintain biosynthetic capacity and energy production under the unique conditions prevailing during pollen - pistil interaction.

The Pyruvate decarboxylase1 gene of Arabidopsis is required during anoxia but not other environmental stresses

Ethanolic fermentation is classically associated with flooding tolerance when plant cells switch from respiration to anaerobic fermentation. However, recent studies have suggested that fermentation also has important functions in the presence of oxygen, mainly in germinating pollen and during abiotic stress. Pyruvate decarboxylase (PDC), which catalyzes the first step in this pathway, is thought to be the main regulatory enzyme. Here, we characterize the PDC gene family in Arabidopsis. PDC is encoded by four closely related genes. By using real-time quantitative polymerase chain reaction, we determined the expression levels of each individual gene in different tissues, under normal growth conditions, and when the plants were subjected to anoxia or other environmental stress conditions. We show that PDC1 is the only gene induced under oxygen limitation among the PDC1 gene family and that a pdc1 null mutant is comprised in anoxia tolerance but not other environmental stresses. We also characterize the expression of the aldehyde dehydrogenase (ALDH) gene family. None of the three genes is induced by anoxia but ALDH2B7 reacts strongly to ABA application and dehydration, suggesting that ALDH may play a role in aerobic detoxification of acetaldehyde. We discuss the possible role of ethanolic fermentation as a robust back-up energy production pathway under adverse conditions when mitochondrial function is disturbed.

Characterization of choline uptake in Trypanosoma brucei and involvement of a mitochondrial carrier in resistance to choline analogs

Choline is an essential nutrient for eukaryotic cells, where it is used as precursor for the synthesis of choline-­containing phospholipids, such as phosphatidylcholine (PC). Our experiments showed – for the first time – that Trypanosoma brucei, the causative agent of human African sleeping sickness, is able to take up choline from the culture medium to use for PC synthesis, indicating that trypanosomes express a transporter for choline at the plasma membrane. Further characterization in procyclic and bloodstream forms revealed that choline uptake is saturable and can be inhibited by HC-3, a known inhibitor of choline uptake in mammalian cells. To obtain additional insights on choline uptake and metabolism, we investigated the effects of choline-analogs that were previously shown to be toxic for T. brucei parasites in culture. Interestingly, we found that all analogs tested effectively inhibited choline uptake into both bloodstream and procyclic form parasites. Subsequently, selected compounds were used to search for possible candidate genes encoding choline transporters in T. brucei, using an RNAi library in bloodstream forms. We identified a protein belonging to the mitochondrial carrier family, previously annotated as TbMCP14, as prime candidate. Down‐regulation of TbMCP14 by RNAi prevented drug-­induced loss of mitochondrial membrane potential and conferred 8­‐fold resistance of T. brucei bloodstream forms to choline analogs. Conversely, over‐expression of the carrier increased parasite susceptibility more than 13-­fold. However, subsequent experiments demonstrated that TbMCP14 was not involved in metabolism of choline. Instead, growth curves in glucose‐depleted medium using RNAi or knock‐out parasites suggested that TbMCP14 is involved in metabolism of amino acids for energy production. Together, our data demonstrate that the identified member of the mitochondrial carrier family is involved in drug uptake into the mitochondrion and has a vital function in energy production in T. brucei.

Beta-alanine supplementation improves jumping power and affects severe-intensity performance in professional alpine skiers.

INTRODUCTION Supplementation with beta-alanine may have positive effects on severe-intensity, intermittent, and isometric strength-endurance performance. These could be advantageous for competitive alpine skiers, whose races last 45 to 150 s, require metabolic power above the aerobic maximum, and involve isometric muscle work. Further, beta-alanine supplementation affects the muscle force-frequency relationship, which could influence explosiveness. We explored the effects of beta-alanine on explosive jump performance, severe exercise energy metabolism, and severe-intensity ski-like performance. METHODS Nine male elite alpine skiers consumed 4.8 g/d beta-alanine or placebo for 5 weeks in a double-blind fashion. Before and after, they performed countermovement jumps (CMJ), a 90-s cycling bout at 110% VO2max (CLT), and a maximal 90-s box jump test (BJ90). RESULTS Beta-alanine improved maximal (+7 ± 3%, d = 0.9) and mean CMJ power (+7 ± 2%, d = 0.7), tended to reduce oxygen deficit (-3 ± 8%, p = .06) and lactate accumulation (-12 ± 31%) and enhance aerobic energy contribution (+1.3 ± 2.9%, p = .07) in the CLT, and improved performance in the last third of BJ90 (+7 ± 4%, p = .02). These effects were not observed with placebo. CONCLUSIONS Beta-alanine supplementation improved explosive and repeated jump performance in elite alpine skiers. Enhanced muscle contractility could possibly explain improved explosive and repeated jump performance. Increased aerobic energy production could possibly help explain repeated jump performance as well.

Does the Use of Mobile Devices (Tablets and Smartphones) Affect Stated Choice Behavior in Web Surveys? An Application to Environmental Valuation

Web surveys are becoming increasingly popular in survey research. Compared with face-to-face, telephone and mail surveys, web surveys may contain a different and new source of measurement error and bias: the type of device that respondents use to answer the survey questions. To the best of our knowledge, this is the first study that tests whether the use of mobile devices affects survey characteristics and stated preferences in a web-based choice experiment. The web survey was carried out in Germany with 3,400 respondents, of which 12 per cent used a mobile device (i.e. tablet or smartphone), and comprised a stated choice experiment on externalities of renewable energy production using wind, solar and biomass. Our main finding is that survey characteristics such as interview length and acquiescence tendency are affected by the device used. In contrast to what might be expected, we find that, compared with respondents using desktop computers and laptops, mobile device users spent more time to answer the survey and are less likely to be prone to acquiescence bias. In the choice experiment, mobile device users tended to be more consistent in their stated choices, and there are differences in willingness to pay between both subsamples.

An Atypical Mitochondrial Carrier That Mediates Drug Action in Trypanosoma brucei

Elucidating the mechanism of action of trypanocidal compounds is an important step in the development of more efficient drugs against Trypanosoma brucei. In a screening approach using an RNAi library in T. brucei bloodstream forms, we identified a member of the mitochondrial carrier family, TbMCP14, as a prime candidate mediating the action of a group of anti-parasitic choline analogs. Depletion of TbMCP14 by inducible RNAi in both bloodstream and procyclic forms increased resistance of parasites towards the compounds by 7-fold and 3-fold, respectively, compared to uninduced cells. In addition, down-regulation of TbMCP14 protected bloodstream form mitochondria from a drug-induced decrease in mitochondrial membrane potential. Conversely, over-expression of the carrier in procyclic forms increased parasite susceptibility more than 13-fold. Metabolomic analyses of parasites over-expressing TbMCP14 showed increased levels of the proline metabolite, pyrroline-5-carboxylate, suggesting a possible involvement of TbMCP14 in energy production. The generation of TbMCP14 knock-out parasites showed that the carrier is not essential for survival of T. brucei bloodstream forms, but reduced parasite proliferation under standard culture conditions. In contrast, depletion of TbMCP14 in procyclic forms resulted in growth arrest, followed by parasite death. The time point at which parasite proliferation stopped was dependent on the major energy source, i.e. glucose versus proline, in the culture medium. Together with our findings that proline-dependent ATP production in crude mitochondria from TbMCP14-depleted trypanosomes was reduced compared to control mitochondria, the study demonstrates that TbMCP14 is involved in energy production in T. brucei. Since TbMCP14 belongs to a trypanosomatid-specific clade of mitochondrial carrier family proteins showing very poor similarity to mitochondrial carriers of mammals, it may represent an interesting target for drug action or targeting.

Does the use of mobile devices (tablets and smartphones) affect survey quality and choice behaviour in web surveys?

Web surveys are becoming increasingly popular in survey research including stated preference surveys. Compared with face-to-face, telephone and mail surveys, web surveys may contain a different and new source of measurement error and bias: the type of device that respondents use to answer the survey questions. This is the first study that tests whether the use of mobile devices, tablets or smartphones, affects survey characteristics and stated preferences in a web-based choice experiment. The web survey on expanding renewable energy production in Germany was carried out with 3182 respondents, of which 12% used a mobile device. Propensity score matching is used to account for selection bias in the use of mobile devices for survey completion. We find that mobile device users spent more time than desktop/laptop users to answer the survey. Yet, desktop/laptop users and mobile device users do not differ in acquiescence tendency as an indicator of extreme response patterns. For mobile device users only, we find a negative correlation between screen size and interview length and a positive correlation between screen size and acquiescence tendency. In the choice experiment data, we do not find significant differences in the tendency to choose the status quo option and scale between both subsamples. However, some of the estimates of implicit prices differ, albeit not in a unidirectional fashion. Model results for mobile device users indicate a U-shaped relationship between error variance and screen size. Together, the results suggest that using mobile devices is not detrimental to survey quality.

Geological and hydrological histories of the Argyre province, Mars

The geologic history of the multi-ringed Argyre impact basin and surroundings has been reconstructed on the basis of geologic mapping and relative-age dating of rock materials and structures. The impact formed a primary basin, rim materials, and a complex basement structural fabric including faults and valleys that are radial and concentric about the primary basin, as well as structurally-controlled local basins. Since its formation, the basin has been a regional catchment for volatiles and sedimentary materials as well as a dominant influence on the flow of surface ice, debris flows, and groundwater through and over its basement structures. The basin is interpreted to have been occupied by lakes, including a possible Mediterranean-sized sea that formed in the aftermath of the Argyre impact event The hypothesized lakes froze and diminished through time, though liquid water may have remained beneath the ice cover and sedimentation may have continued for some time. At its deepest, the main Argyre lake may have taken more than a hundred thousand years to freeze to the bottom even absent any heat source besides the Sun, but with impact-induced hydrothermal heat, geothermal heat flow due to long-lived radioactivities in early martian history, and concentration of solutes in sub-ice brine, liquid water may have persisted beneath thick ice for many millions of years. Existence of an ice-covered sea perhaps was long enough for life to originate and evolve with gradually colder and more hypersaline conditions. The Argyre rock materials, diverse in origin and emplacement mechanisms, have been modified by impact, magmatic, eolian, fluvial, lacustrine, glacial, periglacial, alluvial, colluvial, and tectonic processes. Post-impact adjustment of part of the impact-generated basement structural fabric such as concentric faults is apparent. Distinct basin-stratigraphic units are interpreted to be linked to large-scale geologic activity far from the basin, including growth of the Tharsis magmatic-tectonic complex and the growth into southern middle latitudes of south polar ice sheets. Along with the migration of surface and sub-surface volatiles towards the central part of the primaiy basin, the substantial difference in elevation with respect to the surrounding highlands and Tharsis and the Thaumasia highlands result in the trapping of atmospheric volatiles within the basin in the form of fog and regional or local precipitation, even today. In addition, the impact event caused long-term (millions of years) hydrothermal activity, as well as deep-seated basement structures that have tapped the internal heat of Mars, as conduits, for far greater time, possibly even today. This possibility is raised by the observation of putative open-system pingos and nearby gullies that occur in linear depressions with accompanying systems of faults and fractures. Long-term water and heat energy enrichment, complemented by the interaction of the nutrient-enriched primordial crustal and mantle materials favorable to life excavated to the surface and near-surface environs through the Argyre impact event, has not only resulted in distinct geomorphology, but also makes the Argyre basin a potential site of exceptional astrobiological significance. (C) 2015 Elsevier Inc. All rights reserved.

Anoxia tolerance in tobacco roots: effect of overexpression of pyruvate decarboxylase

Plant survival during flooding relies on ethanolic fermentation for energy production. The available literature indicates that the first enzyme of the ethanolic fermentation pathway, pyruvate decarboxylase (PDC), is expressed at very low levels and is likely to be rate-limiting during oxygen deprivation. The authors expressed high levels of bacterial PDC in tobacco to study the modulation of PDC activity in vivo, and assess its impact on the physiology of ethanolic fermentation and survival under oxygen stress. In contrast to leaves, wild-type normoxic roots contained considerable PDC activity, and overexpression of the bacterial PDC caused only a moderate increase in acetaldehyde and ethanol production under anoxia compared to wild-type roots. No significant lactate production could be measured at any time, making it unlikely that lactate-induced acidification (LDH/PDC pH-stat) triggers the onset of ethanol synthesis. Instead, the authors favour a model in which the flux through the pathway is regulated by substrate availability. The increased ethanolic flux in the transgenics compared to the wild-type did not enhance anoxia tolerance. On the contrary, rapid utilisation of carbohydrate reserves enhanced premature cell death in the transgenics while replenishment of carbohydrates improved survival under anoxia.

LOW REACTIVE OXYGEN SPECIES AND HIGH GLYCOLYSIS IN GLIOBLASTOMA STEM CELLS:MECHANISMS AND THERAPEUTIC IMPLICATIONS

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor with poor prognosis due in part to drug resistance and high incidence of tumor recurrence. The drug resistant and cancer recurrence phenotype may be ascribed to the presence of glioblastoma stem cells (GSCs), which seem to reside in special stem-cell niches in vivo and require special culture conditions including certain growth factors and serum-free medium to maintain their stemness in vitro. Exposure of GSCs to fetal bovine serum (FBS) can cause their differentiation, the underlying mechanism of which remains unknown. Reactive oxygen species (ROS) play an important role in normal stem cell differentiation, but their role in affecting cancer stem cell fate remains unclear. Whether the metabolic characteristics of GSCs are different from other glioblastoma cells and can be targeted are also unknown. In this study, we used several stem-like glioblastoma cell lines derived from clinical tissues by typical neurosphere culture system or orthotopic xenografts, and showed that addition of fetal bovine serum to the medium induced an increase of ROS, leading to aberrant differentiation and decreases of stem cell markers such as CD133. We found that exposure of GSCs to serum induced their differentiation through activation of mitochondrial respiration, leading to an increase in superoxide (O2-) generation and a profound ROS stress response manifested by upregulation of oxidative stress response pathway. This increase in mitochondrial ROS led to a down-regulation of molecules including SOX2, and Olig2, and Notch1 that are important for stem cell function and an upregulation of mitochondrial superoxide dismutase SOD2 that converts O2- to H2O2. Neutralization of ROS by antioxidant N-acetyl-cysteine in the serum-treated GSCs suppressed the increase of superoxide and partially rescued the expression of SOX2, Olig2, and Notch1, and prevented the serum-induced differentiation phenotype. Additionally, GSCs showed high dependence on glycolysis for energy production. The combination of a glycolytic inhibitor 3-BrOP and a chemotherapeutic agent BCNU depleted cellular ATP and inhibited the repair of BCNU-induced DNA damage, achieving strikingly synergistic killing effects in drug resistant GSCs. This study uncovers the metabolic properties of glioblastoma stem cells and suggests that mitochondrial function and cellular redox status may profoundly affect the fates of glioblastoma stem cells via a ROS-mediated mechanism, and that the active glycolytic metabolism in cancer stem cells may provide a biochemical basis for developing novel therapeutic strategies to effectively eliminate GSCs.

Exploring the Impact of Decreased AMPK Pathway Activity on Brain Injury Outcome

Each year, 150 million people sustain a Traumatic Brain Injury (TBI). TBI results in life-long cognitive impairments for many survivors. One observed pathological alteration following TBI are changes in glucose metabolism. Altered glucose uptake occurs in the periphery as well as in the nervous system, with an acute increase in glucose uptake, followed by a prolonged metabolic suppression. Chronic, persistent suppression of brain glucose uptake occurs in TBI patients experiencing memory loss. Abberant post-injury activation of energy-sensing signaling cascades could result in perturbed cellular metabolism. AMP-activated kinase (AMPK) is a kinase that senses low ATP levels, and promotes efficient cell energy usage. AMPK promotes energy production through increasing glucose uptake via glucose transporter 4 (GLUT4). When AMPK is activated, it phosphorylates Akt Substrate of 160 kDa (AS160), a Rab GTPase activating protein that controls Glut4 translocation. Additionally, AMPK negatively regulates energy-consumption by inhibiting protein synthesis via the mechanistic Target of Rapamycin (mTOR) pathway. Given that metabolic suppression has been observed post-injury, we hypothesized that activity of the AMPK pathway is transiently decreased. As AMPK activation increases energy efficiency of the cell, we proposed that increasing AMPK activity to combat the post-injury energy crisis would improve cognitive outcome. Additionally, we expected that inhibiting AMPK targets would be detrimental. We first investigated the role of an existing state of hyperglycemia on TBI outcome, as hyperglycemia correlates with increased mortality and decreased cognitive outcome in clinical studies. Inducing hyperglycemia had no effect on outcome; however, we discovered that AMPK and AS160 phosphorylation were altered post-injury. We conducted vii work to characterize this period of AMPK suppression and found that AMPK phosphorylation was significantly decreased in the hippocampus and cortex between 24 hours and 3 days post-injury, and phosphorylation of its downstream targets was consistently altered. Based on this period of observed decreased AMPK activity, we administered an AMPK activator post-injury, and this improved cognitive outcome. Finally, to examine whether AMPK-regulated target Glut4 is involved in post-injury glucose metabolism, we applied an inhibitor and found this treatment impaired post-injury cognitive function. This work is significant, as AMPK activation may represent a new TBI therapeutic target.

Characterization of the NOP -1 opsin photoreceptor in the filamentous fungus Neurospora crassa

Light absorption is an important process for energy production and sensory perception in many organisms. In the filamentous fungus, Neurospora crassa, blue-light is an important regulator of both asexual and sexual development, but the identity of the blue-light receptor is unknown. The work presented in this dissertation initiated the characterization of the putative N. crassa opsin photoreceptor, NOP-1. Opsins were thought to exist only in the archaea and mammals until the discovery of nop-1. All opsins have the same conserved structure of seven transmembrane helical domains with a lysine residue in the seventh helix specific for forming a Schiff-base linkage with retinal. The predicted NOP-1 protein sequence is equally similar to archaeal rhodopsins and a newly identified fungal opsin-related protein group (ORPs). ORPs maintain the seven transmembrane helical structure of opsins, but lack the conserved lysine residue for binding retinal. An ORP gene, orp-1 was identified in N. crassa and this work includes the cloning and sequence analysis of this gene. Characterization of NOP-1 function in N. crassa development began with the construction of a Δnop-1 deletion mutant. Extensive phenotypic analysis of Δnop-1 mutants revealed only subtle defects during development primarily under environmental conditions that induce a stress response. NOP-1 was overexpressed in the heterologous system Pichia pastoris, and it was demonstrated that NOP-1 protein bound all-trans retinal to form a green-light absorbing pigment (λmax = 534 nm) with a photochemical reaction cycle similar to archaeal sensory rhodopsins. nop-1 gene expression was monitored during N. crassa development. nop-1 transcript is highly expressed during asexual sporulation (conidiation) and transcript levels are abundant in the later stages of conidial development. nop-1 expression is not regulated by blue-light or elevated temperatures. Potential functions for NOP-1 were discovered through the transcriptional analysis of conidiation-associated genes in Δnop-1 mutants. NOP-1 exhibits antagonistic transcriptional regulation of conidiation-associated genes late in conidial development, by enhancing the carotenogenic gene, al-2 and repressing the conidiation-specific genes, con-10 and con-13. ^

Euphausiid respiration model revamped, link to model results

Euphausiids constitute major biomass component in shelf ecosystems and play a fundamental role in the rapid vertical transport of carbon from the ocean surface to the deeper layers during their daily vertical migration (DVM). DVM depth and migration patterns depend on oceanographic conditions with respect to temperature, light and oxygen availability at depth, factors that are highly dependent on season in most marine regions. Changes in the abiotic conditions also shape Euphausiid metabolism including aerobic and anaerobic energy production. Here we introduce a global krill respiration model which includes the effect of latitude (LAT), the day of the year of interest (DoY), and the number of daylight hours on the day of interest (DLh), in addition to the basal variables that determine ectothermal oxygen consumption (temperature, body mass and depth) in the ANN model (Artificial Neural Networks). The newly implemented parameters link space and time in terms of season and photoperiod to krill respiration. The ANN model showed a better fit (r**2=0.780) when DLh and LAT were included, indicating a decrease in respiration with increasing LAT and decreasing DLh. We therefore propose DLh as a potential variable to consider when building physiological models for both hemispheres. We also tested for seasonality the standard respiration rate of the most common species that were investigated until now in a large range of DLh and DoY with Multiple Linear Regression (MLR) or General Additive model (GAM). GAM successfully integrated DLh (r**2= 0.563) and DoY (r**2= 0.572) effects on respiration rates of the Antarctic krill, Euphausia superba, yielding the minimum metabolic activity in mid-June and the maximum at the end of December. Neither the MLR nor the GAM approach worked for the North Pacific krill Euphausia pacifica, and MLR for the North Atlantic krill Meganyctiphanes norvegica remained inconclusive because of insufficient seasonal data coverage. We strongly encourage comparative respiration measurements of worldwide Euphausiid key species at different seasons to improve accuracy in ecosystem modelling.

Case studies of the effects of a total solar eclipse on weather with varying degrees of cloud cover using a local numerical weather prediction model

The importance of renewable energies for the European electricity market is growing rapidly. This presents transmission grids and the power market in general with new challenges which stem from the higher spatiotemporal variability of power generation. This uncertainty is due to the fact that renewable power production results from weather phenomena, thus making it difficult to plan and control. We present a sensitivity study of a total solar eclipse in central Europe in March. The weather in Germany and Europe was modeled using the German Weather Service's local area models COSMO-DE and COSMO-EU, respectively (http://www.cosmo-model.org/). The simulations were performed with and without considering a solar eclipse for the following 3 situations: 1. An idealized, clear-sky situation for the entire model area (Europe, COSMO-EU) 2. A real weather situation with mostly cloudy skies (Germany, COSMO-DE) 3. A real weather situation with mostly clear skies (Germany, COSMO-DE) The data should help to evaluate the effects of a total solar eclipse on the weather in the planetary boundary layer. The results show that a total solar eclipse has significant effects particularly on the main variables for renewable energy production, such as solar irradiation and temperature near the ground.

Table 6 Generalised properties of Utsira caprocks, based on analysis of cuttings (page 79)

Executive Summary: Carbon dioxide capture and storage (CCS) is one option for mitigating atmospheric emissions of carbon dioxide and thereby contributes in actions for stabilization of atmospheric greenhouse gas concentrations. The Bellona Foundation is striving to achieve wide implementation of carbon dioxide (CO2) capture and storage both in Norway and internationally. Bellona considers CCS as the only viable large scale option to close the gap between energy production and demand in an environmentally sound way, thereby ensuring that climate changes and acidification of the oceans due to increased CO2 concentrations in the atmosphere will be stabilised. ff