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// Katarzyna Niedźwiecka 1 , Mariusz Dyląg 1, * , Daria Augustyniak 2 , Grazyna Majkowska-Skrobek 2 , Magdalena Cal-Bąkowska 1 , Young H. Ko 3 , Peter L. Pedersen 4 , Andre Goffeau 5 , Stanislaw Ulaszewski 1, * 1 Department of Genetics,Institute of Genetics and Microbiology, University of Wroclaw, Wroclaw, Poland 2 Department of Pathogen Biology and Immunology, Institute of Genetics and Microbiology, University of Wroclaw, Wroclaw, Poland 3 KoDiscovery, LLC, UM BioPark, Baltimore, MD, USA 4 Departments of Biological Chemistry and Oncology and Sidney Kimmel Comprehensive Cancer Center (member at large), Johns Hopkins University School of Medicine, Baltimore, MD, USA 5 Institut des Sciences de la Vie, Universite Catholique de Louvain, Louvain-la-Neuve, Belgium * These authors have contributed equally to this work Correspondence to: Mariusz Dyląg, email: mariusz.dylag@uwr.edu.pl Stanislaw Ulaszewski, email: stanislaw.ulaszewski@uwr.edu.pl Keywords: 3-bromopyruvate (3BP), glutathione, buthionine sulfoximine, genes expression, fungi and MM cells Received: January 05, 2016 Accepted: August 13, 2016 Published: August 25, 2016 ABSTRACT In different fungal and algal species, the intracellular concentration of reduced glutathione (GSH) correlates closely with their susceptibility to killing by the small molecule alkylating agent 3-bromopyruvate (3BP). Additionally, in the case of Cryptococcus neoformans cells 3BP exhibits a synergistic effect with buthionine sulfoximine (BSO), a known GSH depletion agent. This effect was observed when 3BP and BSO were used together at concentrations respectively of 4-5 and almost 8 times lower than their Minimal Inhibitory Concentration (MIC). Finally, at different concentrations of 3BP (equal to the half-MIC, MIC and double-MIC in a case of fungi, 1 mM and 2.5 mM for microalgae and 25, 50, 100 μM for human multiple myeloma (MM) cells), a significant decrease in GSH concentration is observed inside microorganisms as well as tumor cells. In contrast to the GSH concentration decrease, the presence of 3BP at concentrations corresponding to sub-MIC values or half maximal inhibitory concentration (IC 50 ) clearly results in increasing the expression of genes encoding enzymes involved in the synthesis of GSH in Cryptococcus neoformans and MM cells. Moreover, as shown for the first time in the MM cell model, the drastic decrease in the ATP level and GSH concentration and the increase in the amount of ROS caused by 3BP ultimately results in cell death.

Cancer cell resistance to chemotherapy is often mediated by overexpression of P-glycoprotein, a plasma membrane ABC (ATP-binding cassette) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. P-glycoprotein (ABCB1, according to the human gene nomenclature committee) consists of two homologous halves each containing a transmembrane domain (TMD) involved in drug binding and efflux, and a cytosolic nucleotide-binding domain (NBD) involved in ATP binding and hydrolysis, with an overall (TMD-NBD)2 domain topology. Homologous ABC multidrug transporters, from the same ABCB family, are found in many species such as Plasmodiumfalciparum and Leishmania spp. protozoa, where they induce resistance to antiparasitic drugs. In yeasts, some ABC transporters involved in resistance to fungicides, such as Saccharomyces cerevisiae Pdr5p and Snq2p, display a different (NBD-TMD)2 domain topology and are classified in another family, ABCG. Much effort has been spent to modulate multidrug resistance in the different species by using specific inhibitors, but generally with little success due to additional cellular targets and/or extrusion of the potential inhibitors. This review shows that due to similarities in function and maybe in three-dimensional organization of the different transporters, common potential modulators have been found. An in vitro 'rational screening' was performed among the large flavonoid family using a four-step procedure: (i) direct binding to purified recombinant cytosolic NBD and/or full-length transporter, (ii) inhibition of ATP hydrolysis and energy-dependent drug interaction with transporter-enriched membranes, (iii) inhibition of cell transporter activity monitored by flow cytometry and (iv) chemosensitization of cell growth. The results indicate that prenylated flavonoids bind with high affinity, and strongly inhibit drug interaction and nucleotide hydrolysis. As such, they constitute promising potential modulators of multidrug resistance.Cancer cell resistance to chemotherapy is often mediated by overexpression of P-glycoprotein, a plasma membrane ABC (ATP-binding cassette) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. P-glycoprotein (ABCB1, according to the human gene nomenclature committee) consists of two homologous halves each containing a transmembrane domain (TMD) involved in drug binding and efflux, and a cytosolic nucleotide-binding domain (NBD) involved in ATP binding and hydrolysis, with an overall (TMD-NBD)2 domain topology. Homologous ABC multidrug transporters, from the same ABCB family, are found in many species such as Plasmodiumfalciparum and Leishmania spp. protozoa, where they induce resistance to antiparasitic drugs. In yeasts, some ABC transporters involved in resistance to fungicides, such as Saccharomyces cerevisiae Pdr5p and Snq2p, display a different (NBD-TMD)2 domain topology and are classified in another family, ABCG. Much effort has been spent to modulate multidrug resistance in the different species by using specific inhibitors, but generally with little success due to additional cellular targets and/or extrusion of the potential inhibitors. This review shows that due to similarities in function and maybe in three-dimensional organization of the different transporters, common potential modulators have been found. An in vitro 'rational screening' was performed among the large flavonoid family using a four-step procedure: (i) direct binding to purified recombinant cytosolic NBD and/or full-length transporter, (ii) inhibition of ATP hydrolysis and energy-dependent drug interaction with transporter-enriched membranes, (iii) inhibition of cell transporter activity monitored by flow cytometry and (iv) chemosensitization of cell growth. The results indicate that prenylated flavonoids bind with high affinity, and strongly inhibit drug interaction and nucleotide hydrolysis. As such, they constitute promising potential modulators of multidrug resistance.

At the beginning of the twenty-first century, 3-bromopyruvate (3BP), a simple alkylating chemical compound was presented to the scientific community as a potent anticancer agent, able to cause rapid toxicity to cancer cells without bystander effects on normal tissues. The altered metabolism of cancers, an essential hallmark for their progression, also became their Achilles heel by facilitating 3BP's selective entry and specific targeting. Treatment with 3BP has been administered in several cancer type models both in vitro and in vivo, either alone or in combination with other anticancer therapeutic approaches. These studies clearly demonstrate 3BP's broad action against multiple cancer types. Clinical trials using 3BP are needed to further support its anticancer efficacy against multiple cancer types thus making it available to more than 30 million patients living with cancer worldwide. This review discusses current knowledge about 3BP related to cancer and discusses also the possibility of its use in future clinical applications as it relates to safety and treatment issues., This work was supported by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569)funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI). João Azevedo-Silva received a fellowship from the Portuguese government from the FCT through FSE (Fundo Social Europeu) and POPH (Programa Operacional Potencial Humano) [grant number SFRH/BD/76038/2011]. Co-author Peter L.Pedersen was supported by NIH grant NCICAl0951 for many years for cancer research that led to a number of the findings described in this review., info:eu-repo/semantics/publishedVersion

Frequently, although not exclusively, multidrug resistance (MDR) results from the action of drug-efflux pumps, which are thought to be able to catalyze the active expulsion of several unrelated cytotoxic compounds out of the cell or their intracellular partitioning. The transporters of the major facilitator superfamily (MFS) presumably involved in MDR belong to the 12-spanner drug:H(+) antiporter DHA1 or to the 14- spanner drug:H(+) antiporter DHA2 families. The expression of most Saccharomyces cerevisiae DHA1 family members was found to confer broad chemoprotection. The evolution of the hemiascomycetous DHA1 proteins, belonging to the Génolevures GL3C007 family, was studied using a combined phylogenetic and gene neighborhood approach. The phylogenetic analysis of 189 DHA1 proteins belonging to the genome of 13 hemiascomycetous species identified 20 clusters. Eleven clusters contained no S. cerevisiae members. The phylogenetic clusters were analyzed by the IONS method developed for Identification of Orthologues by Neighborhood and Similarity. This allowed reconstructing the evolutionary history of most DHA1 members within 10 main gene lineages, spanning the whole hemiascomycetes clade, encompassing an evolutionary history of about 350 million years. In addition, five other more species specific lineages, spanning only two hemiascomycetous species, were identified. It is concluded that 57 out of the 143 members of the DHA1 hemiascomycetous members originated from gene duplication events. In half of these duplicates, the two members belong to different phylogenetic clusters, indicating that at least one of them has sufficiently differentiated to provide potential novel functions to this complex family from which most physiological substrates remain unknown.

The Saccharomyces cerevisiae Yhl035p/Vmr1p is an ABC transporter of the MRP subfamily that is conserved in all post Whole Genome Duplication species. The deletion of the YHL035 gene caused growth sensitivity to several amphiphilic drugs such as cycloheximide, 2,4-dichlorophenoxyacetic acid, 2,4-dinitrophenol as well as to cadmium and other toxic metals. Vmr1p-GFP was located in the vacuolar membrane. The ATP-dependent transport of a DNP-S-glutathione conjugate was reduced in a vesicular fraction from the VMR1 deletant. The energy-dependent efflux of rhodamine 6G was increased by VMR1 deletion. Growth sensitivity to cadmium of the VMR1-deleted strain was more pronounced in glycerol/ethanol than in glucose-grown cells. The VMR1 promoter had higher activity when grown in glycerol/ethanol compared with glucose. In glucose, the VMR1 promoter was activated by the deletion of the glucose-dependent repressor ADR1.

The overexpression of pleiotropic drug resistance (PDR) efflux pumps of the ATP-binding cassette (ABC) transporter superfamily frequently correlates with multidrug resistance. Phylogenetic analysis of 349 full-size (similar to 160 kDa) PDR proteins (Pdrps) from 55 fungal species, including major fungal pathogens, identified nine separate protein clusters (A-G, H1a/H1b and H2). Fungal, plant and human ABCG-family Pdrps possess a nucleotide-binding domain [NBD] and a transmembrane domain [TMD] in a family-defining 'reverse' ABC transporter topology [NBD-TMD] that is duplicated [NBD-TMD](2) in full-size fungal and plant Pdrps. Although full-size Pdrps have similar halves indicating early gene duplication/fusion, they show asymmetry of their NBDs and extracellular loops (ELs). Members of cluster F are most symmetric and may be closely related to the evolutionary ancestor of Pdrps. Unique structural elements are predicted, new PDR-specific motifs identified, and the significance of these and other structural features discussed. (c) 2009 Elsevier Inc. All rights reserved.

Synteny analysis is combined with sequence similarity and motif identification to trace the evolution of the putative monocarboxylate (lactate/pyruvate) transporters Jen1p and the dicarboxylate (succinate/fumarate/malate) transporters Jen2p in Hemiascomycetes yeasts and Euascomycetes fungi. It is concluded that a precursor form of Jen1p, named here preJen1p, arose by the duplication of an ancestral Jen2p, during the speciation of Yarrowia lipolytica, which was transferred into a new syntenic context. The Jen1p transporters differentiated from preJen1p in Kluyveromyces lactis, before the Whole Genome Duplication (WGD), and are conserved as a single copy in the Saccharomyces species. In contrast, the ancestral Jen2p was definitively lost just prior to the WGD and is absent in Saccharomyces.

Background Cholera is caused by Vibrio cholerae , and is transmitted through fecal-oral contact. Infection occurs after the ingestion of the bacteria and is usually asymptomatic. In a minority of cases, it causes acute diarrhea and vomiting, which can lead to potentially fatal severe dehydration, especially in the absence of appropriate medical care. Immunity occurs after infection and typically lasts 6–36 months. Cholera is responsible for outbreaks in many African and Asian developing countries, and caused localised and episodic epidemics in South America until the early 1990s. Haiti, despite its low socioeconomic status and poor sanitation, had never reported cholera before the recent outbreak that started in October 2010, with over 720,000 cases and over 8700 deaths (Case fatality rate: 1.2%) through 8 december 2014. So far, this outbreak has seen 3 epidemic peaks, and it is expected that cholera will remain in Haiti for some time. Methodology/findings To trace the path of the early epidemic and to identify hot spots and potential transmission hubs during peaks, we examined the spatial distribution of cholera patients during the first two peaks in Artibonite, the second-most populous department of Haiti. We extracted the geographic origin of 84,000 patients treated in local health facilities between October 2010 and December 2011 and mapped these addresses to 63 rural communal sections and 9 urban cities. Spatial and cluster analysis showed that during the first peak, cholera spread along the Artibonite River and the main roads, and sub-communal attack rates ranged from 0.1% to 10.7%. During the second peak, remote mountain areas were most affected, although sometimes to very different degrees even in closely neighboring locations. Sub-communal attack rates during the second peak ranged from 0.2% to 13.7%. The relative risks at the sub-communal level during the second phase showed an inverse pattern compared to the first phase. Conclusion/significance These findings demonstrate the value of high-resolution mapping for pinpointing locations most affected by cholera, and in the future could help prioritize the places in need of interventions such as improvement of sanitation and vaccination. The findings also describe spatio-temporal transmission patterns of the epidemic in a cholera-naive country such as Haiti. By identifying transmission hubs, it is possible to target prevention strategies that, over time, could reduce transmission of the disease and eventually eliminate cholera in Haiti.

Leishmaniaspecies cause a spectrum of human diseases in tropical and subtropical regions of the world. We have sequenced the 36 chromosomes of the 32.8-megabase haploid genome ofLeishmania major(Friedlin strain) and predict 911 RNA genes, 39 pseudogenes, and 8272 protein-coding genes, of which 36% can be ascribed a putative function. These include genes involved in host-pathogen interactions, such as proteolytic enzymes, and extensive machinery for synthesis of complex surface glycoconjugates. The organization of protein-coding genes into long, strand-specific, polycistronic clusters and lack of general transcription factors in theL. major, Trypanosoma brucei, andTrypanosoma cruzi(Tritryp) genomes suggest that the mechanisms regulating RNA polymerase II–directed transcription are distinct from those operating in other eukaryotes, although the trypanosomatids appear capable of chromatin remodeling. Abundant RNA-binding proteins are encoded in the Tritryp genomes, consistent with active posttranscriptional regulation of gene expression.

The hydrophobic estradiol-derivative RU49953 inhibits the energy-dependent interaction of yeast multidrug-transporter Pdr5p with its fluorescent drug-substrate rhodamine 6G. The potent inhibition is competitive towards drug binding (Ki=23+/-6 nM), whereas nucleoside-triphosphate hydrolysis is two-orders-of-magnitude less sensitive. RU49953 constitutes the most efficient inhibitor of drug binding to a yeast multidrug ABC exporter reported so far.The hydrophobic estradiol-derivative RU49953 inhibits the energy-dependent interaction of yeast multidrug-transporter Pdr5p with its fluorescent drug-substrate rhodamine 6G. The potent inhibition is competitive towards drug binding (Ki=23+/-6 nM), whereas nucleoside-triphosphate hydrolysis is two-orders-of-magnitude less sensitive. RU49953 constitutes the most efficient inhibitor of drug binding to a yeast multidrug ABC exporter reported so far.

Pdr5p, the major multidrug exporter in Saccharomyces cerevisiae, is a member of the ATP-binding cassette (ABC) superfamily. Pdr5p shares similar mechanisms of substrate recognition and transport with the human MDR1-Pgp, despite an inverted topology of transmembrane and ATP-binding domains. The hexahistidine-tagged Pdr5p multidrug transporter was highly overexpressed in yeast strains where other ABC genes have been deleted. After solubilization and purification, the 160-kDa recombinant Pdr5p has been reconstituted into a lipid bilayer. Controlled detergent removal from Pdr5p-lipid-detergent micelles allowed the production of peculiar square-shaped particles coexisting with liposomes and proteoliposomes. These particles having 11 nm in side were well suited for single particle analysis by electron microscopy. From such analysis, a computed volume has been determined at 25-A resolution, giving insight into the structural organization of Pdr5p. Comparison with the reported structures of different bacterial ABC transporters was consistent with a dimeric organization of Pdr5p in the square particles. Each monomer was composed of three subregions corresponding to a membrane region of about 50 A in height that joins two well separated protruding stalks of about 40 A in height, ending each one with a cytoplasmic nucleotide-binding domain (NBD) lobe of about 50-60 A in diameter. The three-dimensional reconstruction of Pdr5p revealed a close arrangement and a structural asymmetric organization of the two NBDs that appeared oriented perpendicularly within a monomer. The existence of different angular positions of the NBDs, with respect to the stalks, suggest rotational movements during the catalytic cycle.

Ce mémoire à pour but de découvrir plus en profondeur le marché des applications mobiles et plus particulièrement des messageries instantanées. Nous définissons tout d’abord les concepts théoriques importants afin de comprendre le segment des applications mobiles. Ensuite, une fois les bases posées, nous nous concentrons sur une analyse chiffrée du marché des applications mobiles et des Smartphones. Après avoir réalisé cette analyse du marché des applications mobiles, nous tentons de définir la deuxième notion principale apparaissant dans le titre de ce mémoire, à savoir la notion de « Pricing Model ». Grâce à différentes lectures et principalement le livre écrit par Messieurs Alex Osterwalder et Yves Pigneur, s’intitulant « Buisness Model Generation », nous définissons les termes et théories suivants: "Business Model" ; "Cinq forces de Porter" ; "Elasticité-Prix". Une fois ces notions abordées, nous nous concentrons sur la partie pratique de ce papier en tentant de répondre à nos différentes questions de recherches à savoir : « La gratuité des messageries instantanées est-elle vraiment une nécessité au succès de celles-ci ? » et « Si la gratuité est une réelle nécessité pour les messageries instantanées à succès, comment se rémunèrent-elles ? La méthodologie utilisée afin de tenter de répondre à cette question fut : Premièrement, de définir les différents termes principaux présents dans ces questions de recherches à savoir: la notion de messagerie instantanée, les notions de gratuité et de succès ; et deuxièmement, de réaliser une analyse précise du marché des messageries instantanée. Après avoir terminé ces analyses, nous avons introduit notre cas pratique, qu’est l’application PeepOut, jeune messagerie instantanée estudiantine. A l’aide de ce cas pratique, nous avons tenté de savoir si oui ou non, elle serait capable de percer dans le segment des « MIM » (Mobile Instant Messaging) actuellement, en 2015. Master [120] en sciences de gestion, Université catholique de Louvain, 2015

Analysis of the transport functions of individual Candida albicans plasma membrane drug efflux pumps is hampered by the multitude of endogenous transporters. We have stably expressed C. albicans Cdr1p, the major pump implicated in multiple-drug-resistance phenotypes, from the genomic PDR5 locus in a Saccharomyces cerevisiae mutant (AD1-8u − ) from which seven major transporters of the ATP-binding cassette (ABC) family have been deleted. High-level expression of Cdr1p, under the control of the S. cerevisiae PDR5 promoter and driven by S. cerevisiae Pdr1p transcriptional regulator mutation pdr1-3 , was demonstrated by increased levels of mRNA transcription, increased levels of nucleoside triphosphatase activity, and immunodetection in plasma membrane fractions. S. cerevisiae AD1-8u − was hypersensitive to azole antifungals (the MICs at which 80% of cells were inhibited [MIC 80 s] were 0.625 μg/ml for fluconazole, C. albicans Cdr1p was resistant to azoles (MIC 80 s of fluconazole, ketoconazole, and itraconazole, 30, 0.5, and 4 μg/ml, respectively). Drug resistance correlated with energy-dependent drug efflux. AD1002 demonstrated resistance to a variety of structurally unrelated chemicals which are potential drug pump substrates. The controlled overexpression of C. albicans Cdr1p in an S. cerevisiae background deficient in other pumps allows the functional analysis of pumping specificity and mechanisms of a major ABC transporter involved in drug efflux from an important human pathogen.

Multiple or pleiotropic drug resistance often occurs in the yeast Saccharomyces cerevisiae through genetic activation of the Cys(6)-Zn(II) transcription factors Pdr1p and Pdr3p. Hyperactive alleles of these proteins cause overproduction of target genes that include drug efflux pumps, which in turn confer high level drug resistance. Here we provide evidence that both Pdr1p and Pdr3p act to regulate production of an enzyme involved in sphingolipid biosynthesis in S. cerevisiae. The last step in formation of the major sphingolipid in the yeast plasma membrane, mannosyldiinositol phosphorylceramide, is catalyzed by the product of the IPT1 gene, inositol phosphotransferase (Ipt1p). Transcription of the IPT1 gene is responsive to changes in activity of Pdr1p and Pdr3p. A single Pdr1p/Pdr3p response element is present in the IPT1 promoter and is required for regulation by these factors. Loss of IPT1 has complex effects on drug resistance of the resulting strain, consistent with an important role for mannosyldiinositol phosphorylceramide in normal plasma membrane function. Direct assay for lipid contents of cells demonstrates that changes in sphingolipid composition correlate with changes in the activity of Pdr3p. These data suggest that Pdr1p and Pdr3p may act to modulate the lipid composition of membranes in S. cerevisiae through activation of sphingolipid biosynthesis along with other target genes.

Yeast plasma membranes contain a small 55 amino acid hydrophobic polypeptide, Pmp3p, which has high sequence similarity to a novel family of plant polypeptides that are overexpressed under high salt concentration or low temperature treatment. The PMP3 gene is not essential under normal growth conditions. However, its deletion increases the plasma membrane potential and confers sensitivity to cytotoxic cations, such as Na(+) and hygromycin B. Interestingly, the disruption of PMP3 exacerbates the NaCl sensitivity phenotype of a mutant strain lacking the Pmr2p/Enap Na(+)-ATPases and the Nha1p Na(+)/H(+) antiporter, and suppresses the potassium dependency of a strain lacking the K(+) transporters, Trk1p and Trk2p. All these phenotypes could be reversed by the addition of high Ca(2+) concentration to the medium. These genetic interactions indicate that the major effect of the PMP3 deletion is a hyperpolarization of the plasma membrane potential that probably promotes a non-specific influx of monovalent cations. Expression of plant RCI2A in yeast could substitute for the loss of Pmp3p, indicating a common role for Pmp3p and the plant homologue.

Since bilirubin-like pigments are present in the environment as degradation products of heme-containing proteins, yeast could have developed a detoxifying system to transport these compounds into their vacuoles. Vacuoles from Saccharomyces cerevisiae showed an ATP-dependent, saturative transport of unconjugated bilirubin (UCB) that was reduced by 60% and 40% in YCF1 and YLL015w-deleted cells, respectively; the double deletant showed no UCB uptake. Conversely, the transport of bile acids (taurocholate) was comparable in wild and deleted stains. These data identify YCF1 and YLL015w, named BPT1 (Bile Pigment Transporter), as the genes responsible for ATP-dependent UCB transport in yeast. Since YCF1 and YLL015w are rather homologous with multidrug resistant proteins (MRPs), they also suggest the involvement of this class of transporters in the ATP-dependent transport of unconjugated bilirubin. Copyright © 2000 John Wiley & Sons, Ltd.