Direct methanol fuel cells (DMFCs) have been actively investigated from both fundamental and applied points of view due to their interesting perspectives for the application in the fields of auxiliary power supply and portable power sources. However, research efforts are still needed to solve the drawbacks presently affecting these devices, such as methanol cross-over constraints, high costs of materials, etc. The present paper addresses these issues by using a new FUMATECH proton exchange membrane (fumapem((R)) F-1850), characterized by a nominal equivalent weight of 1800, as the electrolyte, and low electrode noble metal loadings compared to the state of the art. A membrane-electrode assembly (MEA) based on this membrane was investigated in a small area (5 cm(2)) direct methanol fuel cell at 60 degrees C and compared to a benchmark Nafion((R)) 115 membrane in terms of performance and methanol cross-over. The F-1850 membrane showed a lower methanol cross-over rate than Nafion ((R))115, despite a lower thickness of the fumapem((R)) membrane. This allowed to obtain a higher performance (74 vs. 64 mW cm(-2)) for the F-1850 membrane compared to Nafion at ambient pressure. To study the scalability of the DMFC device and related components, the new membrane was integrated in large-area MEAs (100 cm(2)) and investigated in a 5-cell and 10-cell pressurized stack based on a bipolar design. The stack was tested at 75 degrees C, 5 M methanol solution at the anode and pressurized oxygen (2 atm. rel.) at the cathode, giving a normalized power density per cell of about 130 mW cm(-2). The results obtained with the 10-cell stack showed a good agreement with the performance recorded with the 5-cell stack, confirming the scalability of the DMFC device. (C) 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

This paper presents the main results obtained during the European project (FCH-JU) "LoLiPEM - Long-life PEM-FCH & CHP systems". The paper describes significant improvements in the polymer electrolyte by tailored heat treatments for cross-linking of Sulfonated Poly(ether ether ketone) (SPEEK), obtained without any addition of cross-linker species. The reported properties of the ionomers include mechanical properties, gas permeability and ionic conductivity.Innovative gas-diffusion electrodes are fabricated by the electrochemical deposition of Pt catalyst; the fuel cell current voltage characteristics are reported with Nafion and SPEEK-based binder.The fuel cell performances at 80 degrees C of membrane-electrode-assemblies containing a SPEEK membrane with a cross-linking degree of 32% are among the best in the literature compared with the PEMFC using membrane alternative than Nafion. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

Perfluorinated sulphonic acid (PFSA) polymers such as Nafion (R) are widely used for both electrolyte membrane and ionomer in the catalytic layer because of their high proton conductivity (sigma(H)) as well as chemical and thermal stability. When PFSA polymers alternative to Nafion (R) with a different EW are used, it is necessary to optimize the composition of the catalytic ink, considering the different ionomer characteristics. Moreover, fuel cell operating conditions play an important role regarding the catalytic layer optimization. In this work, the influence of ionomer amount in the catalytic layer was evaluated. The electrochemical studies were carried out in a single cell in H-2-O-2 at intermediate temperatures (80 degrees C-130 degrees C) by using a fumapem (R) F-950 membrane as an electrolyte. A hydro-alcoholic dispersion of fumion (R) F was also used as an ionomer in the electrode catalytic structure with an amount ranging from 30 to 60%. Under operating conditions finalised to automotive applications (intermediate temperature, low relative humidity and low pressure of gases) an improvement of performance in terms of maximum power density was obtained by using optimized ionomer content. (C) 2015 Elsevier Ltd. All rights reserved.

Exposure of the yeast Saccharomyces cerevisiae to weak organic acids such as the food preservatives sorbate, benzoate and propionate leads to the pronounced induction of the plasma membrane ATP-binding cassette (ABC) transporter, Pdr12p. This protein mediates efflux of weak acid anions, which is essential for stress adaptation. Recently, we identified War1p as the dedicated transcriptional regulator required for PDR12 stress induction. Here, we report the results from a genetic screen that led to the isolation of two war1 alleles encoding mutant variants, War1-28p and War1-42p, which are unable to support cell growth in the presence of sorbate. DNA sequencing revealed that War1-28 encodes a truncated form of the transcriptional regulator, and War1-42 carries three clustered mutations near the C-terminal activation domain. Although War1-42 is expressed and properly localized in the nucleus, the War1-42p variant fails to bind the weak-acid-response elements in the PDR12 promoter, as shown by in vivo footprinting. Importantly, in contrast with wild-type War1p, War1-42p is also no longer phosphorylated upon weak-acid challenge, demonstrating that phosphorylation of War1p, its activation and DNA binding are tightly linked processes that are essential for adaptation to weak-acid stress.

Weak organic acids such as sorbate are potent fungistatic agents used in food preservation, but their intracellular targets are poorly understood. We thus searched for potential target genes and signaling components in the yeast genome using contemporary genome-wide functional assays as well as DNA microarray profiling. Phenotypic screening of the EUROSCARF collection revealed the existence of numerous sorbate-sensitive strains. Sorbate hypersensitivity was detected in mutants of the shikimate biosynthesis pathway, strains lacking the PDR12 efflux pump or WAR1, a transcription factor mediating stress induction of PDR12. Using DNA microarrays, we also analyzed the genome-wide response to acute sorbate stress, allowing for the identification of more than 100 genes rapidly induced by weak acid stress. Moreover, a novel War1p- and Msn2p/4p-independent regulon that includes HSP30 was identified. Although induction of the majority of sorbate-induced genes required Msn2p/4p, weak acid tolerance was unaffected by a lack of Msn2p/4p. Ectopic expression of PDR12 from the GAL1-10 promoter fully restored sorbate resistance in a strain lacking War1p, demonstrating that PDR12 is the major target of War1p under sorbic acid stress. Interestingly, comparison of microarray data with results from the phenotypic screening revealed that PDR12 remained as the only gene, which is both stress inducible and required for weak acid resistance. Our results suggest that combining functional assays with transcriptome profiling allows for the identification of key components in large datasets such as those generated by global microarray analysis.

Saccharomyces cerevisiae displays very strong induction of a single ATP‐binding cassette (ABC) transporter, Pdr12p, when stressed with certain weak organic acids. This is a plasma membrane pump catalysing active efflux of the organic acid anion from the cell. Pdr12p action probably allows S. cerevisiae to maintain lower intracellular levels of several weak organic acid preservatives than would be expected on the basis of the free equilibration of the acid across the cell membrane. This in turn facilitates growth in the presence of these preservatives and therefore yeast spoilage of food materials. Pdr12p appears to confer resistance to those carboxylic acids that, to a reasonable degree, partition into both the lipid bilayer and aqueous phases. Its gene ( PDR12 ) is strongly induced by sorbate, benzoate and certain other moderately lipophilic carboxylate compounds, but not by organic alcohols or high levels of acetate. PDR12 induction reflects the operation of a previously uncharacterized S. cerevisiae stress response, for which the induction signal is probably a high intracellular pool of the organic acid anion. Copyright © 2003 John Wiley & Sons, Ltd.

The ability of yeasts to grow in the presence of weak organic acid preservatives is an important cause of food spoilage. Many of the determinants of acetate resistance in Saccharomyces cerevisiae differ from the determinants of resistance to the more lipophilic sorbate and benzoate. Interestingly, we show in this study that hypersensitivity to both acetate and sorbate results when the cells have auxotrophic requirements for aromatic amino acids. In tryptophan biosynthetic pathway mutants, this weak acid hypersensitivity is suppressed by supplementing the medium with high levels of tryptophan or, in the case of sorbate sensitivity, by overexpressing the Tat2p high affinity tryptophan permease. Weak acid stress therefore inhibits uptake of aromatic amino acids from the medium. This allows auxotrophic requirements for these amino acids to strongly influence the resistance phenotypes of mutant strains. This property must be taken into consideration when using these phenotypes to attribute functional assignments to genes. We show that the acetate sensitivity phenotype previously ascribed to yeast mutants lacking the Pdr12p and Azr1p plasma membrane transporters is an artefact arising from the use of trp1 mutant strains. These transporters do not confer resistance to high acetate levels and, in prototrophs, their presence is actually detrimental for this resistance.

The Saccharomyces cerevisiae ATP-binding cassette (ABC) transporter Pdr12p effluxes weak acids such as sorbate and benzoate, thus mediating stress adaptation. In this study, we identify a novel transcription factor, War1p, as the regulator of this stress adaptation through transcriptional induction of PDR12. Cells lacking War1p are weak acid hypersensitive, since they fail to induce Pdr12p. The nuclear Zn2Cys6 transcriptional regulator War1p forms homodimers and is rapidly phosphorylated upon sorbate stress. The appearance of phosphorylated War1p isoforms coincides with transcriptional activation of PDR12. Promoter deletion analysis identified a novel cis-acting weak acid response element (WARE) in the PDR12 promoter required for PDR12 induction. War1p recognizes and decorates the WARE both in vitro and in vivo, as demonstrated by band shift assays and in vivo footprinting. Importantly, War1p occupies the WARE in the presence and absence of stress, demonstrating constitutive DNA binding in vivo. Our results suggest that weak acid stress triggers phosphorylation and perhaps activation of War1p. In turn, War1p activation is necessary for the induction of PDR12 through a novel signal transduction event that elicits weak organic acid stress adaptation.

We have previously shown that a S1360F mutation in transmembrane domain 10 (TMD10) of the Pdr5p ABC transporter modulates substrate specificity and simultaneously leads to a loss of FK506 inhibition. In this study, we have constructed and characterized the S1360F/A/T and T1364F/A/S mutations located in the hydrophilic face of the amphipatic Pdr5p TMD10. A T1364F mutation leads to a reduction in Pdr5p‐mediated azole and rhodamine 6G resistance. Like S1360F, the T1364F and T1364A mutants were nearly non‐responsive to FK506 inhibition. Most remarkably, however, the S1360A mutation increases FK506 inhibitor susceptibility, because Pdr5p–S1360A is hypersensitive to FK506 inhibition when compared with either wild‐type Pdr5p or the non‐responsive S1360F variant. Hence, the Pdr5p TMD10 determines both azole substrate specificity and susceptibility to reversal agents. This is the first demonstration of a eukaryotic ABC transporter where a single residue change causes either a loss or a gain in inhibitor susceptibility, depending on the nature of the mutational change. These results have important implications for the design of efficient reversal agents that could be used to overcome multidrug resistance mediated by ABC transporter overexpression.

The nuclear gene encoding the Sit4 protein phosphatase was identified in the budding yeast Kluyveromyces lactis. K. lactis cells carrying a disrupted sit4allele are resistant to oligomycin, antimycin, ketoconazole, and econazole but hypersensitive to paromomycin, sorbic acid, and 4-nitroquinoline-N-oxide (4-NQO). Overexpression ofSIT4 leads to an elevation in resistance to paromomycin and to lesser extent tolerance to sorbic acid, but it has no detectable effect on resistance to 4-NQO. These observations suggest that the Sit4 protein phosphatase has a broad role in modulating multidrug resistance in K. lactis. Expression or activity of a membrane transporter specific for paromomycin and the ABC pumps responsible for 4-NQO and sorbic acid would be positively regulated by Sit4p. In contrast, the function of a Pdr5-type transporter responsible for ketoconazole and econazole extrusion, and probably also for efflux of oligomycin and antimycin, is likely to be negatively regulated by the phosphatase. Drug resistance of sit4 mutants was shown to be mediated by ABC transporters as efflux of the anionic fluorescent dye rhodamine 6G, a substrate for the Pdr5-type pump, is markedly increased in sit4 mutants in an energy-dependent and FK506-sensitive manner.

Yeast cells display an adaptive stress response when exposed to weak organic acids at low pH. This adaptation is important in the spoilage of preserved foods, as it allows growth in the presence of weak acid food preservatives. In Saccharomyces cerevisiae, this stress response leads to strong induction of the Pdr12 ATP-binding cassette (ABC) transporter, which catalyses the active efflux of weak acid anions from the cytosol of adapted cells. S. cerevisiae cells lacking the Cmk1 isoform of Ca2+-calmodulin-dependent protein kinase are intrinsically resistant to weak acid stress, in that they do not need to spend a long adaptive period in lag phase before resuming growth after exposure to this stress. This resistance of the cmk1 mutant is Pdr12 dependent and, unlike with wild-type S. cerevisiae, cmk1 cells are capable of performing Pdr12-specific functions such as energy-dependent cellular extrusion of fluorescein and benzoate. However, they have neither higher PDR12 gene promoter activity nor higher Pdr12 protein levels. The increased Pdr12 activity in cmk1 cells is therefore caused by Cmk1 exerting a negative post-transcriptional influence over the activity of the Pdr12 ABC transporter, a transporter protein that is constitutively expressed in low-pH yeast cultures. This is the first preliminary evidence that shows a protein kinase, either directly or indirectly, regulating the activity of a yeast ABC transporter.

Saccharomyces cerevisiae was the first eukaryotic organism whose complete genome sequence has been determined, uncovering the existence of numerous genes encoding proteins of the ATP-binding cassette (ABC) family. Fungal ABC proteins are implicated in a variety of cellular functions, ranging from clinical drug resistance development, pheromone secretion, mitochondrial function, peroxisome biogenesis, translation elongation, stress response to cellular detoxification. Moreover, some yeast ABC proteins are orthologues of human disease genes, which makes yeast an excellent model system to study the molecular mechanisms of ABC protein-mediated disease. This review provides a comprehensive discussion and update on the function and transcriptional regulation of all known ABC genes from yeasts, including those discovered in fungal pathogens.