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Ítem Acceso Abierto Candida infections, causes, targets, and resistance mechanisms: traditional and alternative antifungal agents(Hindawi, 2013) Spampinato, Claudia P.; Leonardi, Darío; https://orcid.org/0000-0003-2292-3570The genus Candida includes about 200 different species, but only a few species are human opportunistic pathogens and cause infections when the host becomes debilitated or immunocompromised.Candida infections can be superficial or invasive. Superficial infections often affect the skin or mucous membranes and can be treated successfully with topical antifungal drugs. However, invasive fungal infections are often life-threatening, probably due to inefficient diagnostic methods and inappropriate initial antifungal therapies. Here, we briefly review our current knowledge of pathogenic species of the genus Candida and yeast infection causes and then focus on current antifungal drugs and resistance mechanisms. An overview of new therapeutic alternatives for the treatment of Candida infections is also provided.Ítem Acceso Abierto Beyond the binding site: In vivo Identification of tbx2, smarca5 and wnt5b as molecular targets of CNBP during embryonic development(Public Library of Science, 2013-05-07) Armas, Pablo; Margarit, Ezequiel; Mouguelar, Valeria Soraya; Allende, Miguel L.; Calcaterra, Nora B.CNBP is a nucleic acid chaperone implicated in vertebrate craniofacial development, as well as in myotonic dystrophy type 2 (DM2) and sporadic inclusion body myositis (sIBM) human muscle diseases. CNBP is highly conserved among vertebrates and has been implicated in transcriptional regulation; however, its DNA binding sites and molecular targets remain elusive. The main goal of this work was to identify CNBP DNA binding sites that might reveal target genes involved in vertebrate embryonic development. To accomplish this, we used a recently described yeast one-hybrid assay to identify DNA sequences bound in vivo by CNBP. Bioinformatic analyses revealed that these sequences are G-enriched and show high frequency of putative G-quadruplex DNA secondary structure. Moreover, an in silico approach enabled us to establish the CNBP DNA-binding site and to predict CNBP putative targets based on gene ontology terms and synexpression with CNBP. The direct interaction between CNBP and candidate genes was proved by EMSA and ChIP assays. Besides, the role of CNBP upon the identified genes was validated in loss-of-function experiments in developing zebrafish. We successfully confirmed that CNBP up-regulates tbx2b and smarca5, and down-regulates wnt5b gene expression. The highly stringent strategy used in this work allowed us to identify new CNBP target genes functionally important in different contexts of vertebrate embryonic development. Furthermore, it represents a novel approach toward understanding the biological function and regulatory networks involving CNBP in the biology of vertebrates.Ítem Acceso Abierto Molecular fingerprints to identify Candida species(Hindawi, 2013-06-17) Spampinato, Claudia P.; Leonardi, DaríoA wide range of molecular techniques have been developed for genotyping Candida species. Among them, multilocus sequence typing (MLST) and microsatellite length polymorphisms (MLP) analysis have recently emerged. MLST relies on DNA sequences of internal regions of various independent housekeeping genes, while MLP identifies microsatellite instability. Both methods generate unambiguous and highly reproducible data. Here, we review the results achieved by using these two techniques and also provide a brief overview of a new method based on high-resolution DNA melting (HRM). This method identifies sequence differences by subtle deviations in sample melting profiles in the presence of saturating fluorescent DNA binding dyesÍtem Acceso Abierto Crystal structure of the FAD-containing ferredoxin-NADP+ reductase from the plant pathogen Xanthomonas axonopodis pv. citri(Hindawi, 2013-08-01) Tondo, María Laura; Hurtado-Guerrero, Ramón; Ceccarelli, Eduardo Augusto; Medina, Milagros; Orellano, Elena G.; Martínez-Júlvez, Marta; https://orcid.org/0000-0002-3122-9401We have solved the structure of ferredoxin-NADP(H) reductase, FPR, from the plant pathogen Xanthomonas axonopodis pv. citri, responsible for citrus canker, at a resolution of 1.5 Å. This structure reveals differences in the mobility of specific loops when compared to other FPRs, probably unrelated to the hydride transfer process, which contributes to explaining the structural and functional divergence between the subclass I FPRs. Interactions of the C-terminus of the enzyme with the phosphoadenosine of the cofactor FAD limit its mobility, thus affecting the entrance of nicotinamide into the active site. This structure opens the possibility of rationally designing drugs against the X. axonopodis pv. citri phytopathogen.Ítem Acceso Abierto Centrosomal AKAP350 modulates the G1/S transition(Landes Bioscience, 2013-10-10) Mattaloni, Stella M.; Ferretti, Anabela Cecilia; Tonucci, Facundo Mauro; Favre, Cristian; Goldenring, James R.; Larocca, María CeciliaAKAP350 (AKAP450/AKAP9/CG-NAP) is an A-kinase anchoring protein, which recruits multiple signaling proteins to the Golgi apparatus and the centrosomes. Several proteins recruited to the centrosomes by this scaffold participatein the regulation of the cell cycle. Previous studies indicated that AKAP350 participates in centrosome duplication. In the present study we specifically assessed the role of AKAP350 in the progression of the cell cycle. Our results showed that interference with AKAP350 expression inhibits G1/S transition, decreasing the initiation of both DNA synthesis and centrosome duplication. We identified an AKAP350 carboxyl-terminal domain (AKAP350CTD), which contained the centrosomal targeting domain of AKAP350 and induced the initiation of DNA synthesis. Nevertheless, AKAP350CTD expression did not induce centrosomal duplication. AKAP350CTD partially delocalized endogenous AKAP350 from the centrosomes, but increased the centrosomal levels of the cyclin-dependent kinase 2 (Cdk2). Accordingly, the expression of this AKAP350 domain increased the endogenous phosphorylation of nucleophosmin by Cdk2, which occurs at the G1 /S transition and is a marker of the centrosomal activity of the cyclin E-Cdk2 complex. Cdk2 recruitment to the centrosomes is a necessary event for the development of the G1/S transition. Altogether, our results indicate that AKAP350 facilitates the initiation of DNA synthesis by scaffolding Cdk2 to the centrosomes, and enabling its specific activity at this organelle. Although this mechanism could also be involved in AKAP350-dependent modulation of centrosomal duplication, it is not sufficient to account for this process.Ítem Acceso Abierto Deciphering the metabolic pathways influencing heat and cold responses during post-harvest physiology of peach fruit(Wiley, 2014-01-21) Lauxmann, Martín Alexander; Borsani, Julia; Osorio, Sonia; Lombardo, Verónica Andrea; Budde, Claudio O.; Bustamante, Claudia Anabel; Monti, Laura Lucía; Andreo, Carlos Santiago; Fernie, Alisdair R.; Drincovich, María Fabiana; Lara, María ValeriaPeaches are highly perishable and deteriorate quickly at ambient temperature. Cold storage is commonly used to prevent fruit decay; however, it affects fruit quality causing physiological disorders collectively termed ‘chilling injury’ (CI). To prevent or ameliorate CI, heat treatment is often applied prior to cold storage. In the present work, metabolic profiling was performed to determine the metabolic dynamics associated with the induction of acquired CI tolerance in response to heat shock. ‘Dixiland’ peach fruits exposed to 39 °C, cold stored, or after a combined treatment of heat and cold, were compared with fruits ripening at 20 °C. Dramatic changes in the levels of compatible solutes such as galactinol and raffinose were observed, while amino acid precursors of the phenylpropanoid pathway were also modified due to the stress treatments, as was the polyamine putrescine. The observed responses towards temperature stress in peaches are composed of both common and specific response mechanisms to heat and cold, but also of more general adaptive responses that confer strategic advantages in adverse conditions such as biotic stresses. The identification of such key metabolites, which prime the fruit to cope with different stress situations, will likely greatly accelerate the design and the improvement of plant breeding programs.Ítem Acceso Abierto Flavoprotein monooxygenases for oxidative biocatalysis: recombinant expression in microbial hosts and applications(Frontiers Media, 2014-02-06) Ceccoli, Romina Denis; Bianchi, Dario A.; Rial, Daniela V.External flavoprotein monooxygenases comprise a group of flavin-dependent oxidoreductases that catalyze the insertion of one atom of molecular oxygen into an organic substrate and the second atom is reduced to water. These enzymes are involved in a great number of metabolic pathways both in prokaryotes and eukaryotes. Flavoprotein monooxygenases have attracted the attention of researchers for several decades and the advent of recombinant DNA technology caused a great progress in the field. These enzymes are subjected to detailed biochemical and structural characterization and some of them are also regarded as appealing oxidative biocatalysts for the production of fine chemicals and valuable intermediates toward active pharmaceutical ingredients due to their high chemo-, stereo-, and regioselectivity. Here, we review the most representative reactions catalyzed both in vivo and in vitro by prototype flavoprotein monooxygenases, highlighting the strategies employed to produce them recombinantly, to enhance the yield of soluble proteins, and to improve cofactor regeneration in order to obtain versatile biocatalysts. Although we describe the most outstanding features of flavoprotein monooxygenases, we mainly focus on enzymes that were cloned, expressed and used for biocatalysis during the last years.Ítem Acceso Abierto Recombinant protein expression in Escherichia coli: advances and challenges(Frontiers Media, 2014-04-17) Rosano, Germán L.; Ceccarelli, Eduardo AugustoEscherichia coli is one of the organisms of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols at hand for the high-level production of heterologous proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies.We review the different approaches for the synthesis of recombinant proteins in E. coli and discuss recent progress in this ever-growing field.Ítem Acceso Abierto Recombinant protein expression in microbial systems(Frontiers, 2014-07-08) Rosano, Germán L.; Ceccarelli, Eduardo AugustoThe emergence of recombinant DNA technology during the early 70's set a revolution in molecular biology. This set of techniques was strengthened even further later on with the introduction of the polymerase chain reaction and allowed scientists to explore and understand essential life processes in an easy and straightforward way. It also marked the birth of the modern biotech industry [...]Ítem Acceso Abierto Characterization of the accessory protein ClpT1 from Arabidopsis thaliana: oligomerization status and interaction with Hsp100 chaperones(BMC, 2014-08-24) Colombo, Clara V.; Ceccarelli, Eduardo Augusto; Rosano, Germán L.Background: The caseinolytic protease (Clp) is crucial for chloroplast biogenesis and proteostasis. The Arabidopsis Clp consists of two heptameric rings (P and R rings) assembled from nine distinct subunits. Hsp100 chaperones (ClpC1/2 and ClpD) are believed to dock to the axial pores of Clp and then transfer unfolded polypeptides destined to degradation. The adaptor proteins ClpT1 and 2 attach to the protease, apparently blocking the chaperone binding sites. This competition was suggested to regulate Clp activity. Also, monomerization of ClpT1 from dimers in the stroma triggers P and R rings association. So, oligomerization status of ClpT1 seems to control the assembly of the Clp protease. Results: In this work, ClpT1 was obtained in a recombinant form and purified. In solution, it mostly consists of monomers while dimers represent a small fraction of the population. Enrichment of the dimer fraction could only be achieved by stabilization with a crosslinker reagent. We demonstrate that ClpT1 specifically interacts with the Hsp100 chaperones ClpC2 and ClpD. In addition, ClpT1 stimulates the ATPase activity of ClpD by more than 50% when both are present in a 1:1 molar ratio. Outside this optimal proportion, the stimulatory effect of ClpT1 on the ATPase activity of ClpD declines. Conclusions: The accessory protein ClpT1 behaves as a monomer in solution. It interacts with the chloroplastic Hsp100 chaperones ClpC2 and ClpD and tightly modulates the ATPase activity of the latter. Our results provide new experimental evidence that may contribute to revise and expand the existing models that were proposed to explain the roles of this poorly understood regulatory protein.Ítem Acceso Abierto Activation of spleen tyrosine kinase (Syk) at fertilization in Rhinella arenarum eggs(University of the Basque Country Press (UBC Press), 2015-07-02) Mouguelar, Valeria Soraya; Coux, GabrielaRecently, we have provided evidence for the involvement of a cytosolic tyrosine–phosphorylatable 70 kDa oocyte protein in Rhinella arenarum (Anura: Bufonidae) fertilization. The aim of the present work was to characterize its phosphorylation, determine the identity of this protein and establish its biological role during the fertilization process. Tyrosine phosphorylation of the 70 kDa protein was not observed in eggs activated with the calcium ionophore A23187. Pretreatment of oocytes with the tyrosine kinase inhibitor genistein effectively blocked the fertilization-dependent phosphorylation of the 70 kDa protein. In order to identify this protein, we examined the presence in amphibian oocytes of non-receptor 70 kDa tyrosine kinase members of the Syk/Zap70 and Tec families by RT-PCR using degenerate primers. We found that R. arenarum oocytes contain the transcripts coding for Syk and Tec kinases. Western blot analysis confirmed the presence of Syk protein in unfertilized oocytes and eggs. Studies using phospho-Syk specific antibodies showed that fertilization rapidly (less than 10 minutes) induces phosphorylation on Syk tyrosine residues (352 and 525/526) that are necessary for the activation of the enzyme. Finally, specific inhibition of Syk with the R406 compound provoked a diminished fertilization score, thereby confirming a functional role of the Syk protein during R. arenarum fertilization. To our knowledge this is the first time that Syk is described as a player in the signaling cascade activated after fertilization.Ítem Acceso Abierto Genome comparison of two Exiguobacterium strains from high altitude andean lakes with different arsenic resistance: identification and 3D modeling of the Acr3 efflux pump(Frontiers, 2015-07-22) Ordoñez, Omar F.; Lanzarotti, Esteban; Kurth, Daniel; Cortez, Néstor; Farías, María E.; Turjanski, Adrian G.Ítem Acceso Abierto G-quadruplexes as novel cis-elements controlling transcription during embryonic development(Oxford University Press, 2016-01-14) David, Aldana P.; Margarit, Ezequiel; Domizi, Pablo; Banchio, Claudia; Armas, Pablo; Calcaterra, Nora B.G-quadruplexes are dynamic structures folded in G-rich single-stranded DNA regions. These structures have been recognized as a potential nucleic acid based mechanism for regulating multiple cellular processes such as replication, transcription and genomic maintenance. So far, their transcriptional role in vivo during vertebrate embryonic development has not yet been addressed. Here, we performed an in silico search to find conserved putative G-quadruplex sequences (PQSs) within proximal promoter regions of human, mouse and zebrafish developmental genes. Among the PQSs able to fold in vitro as G-quadruplex, those present in nog3, col2a1 and fzd5 promoters were selected for further studies. In cellulo studies revealed that the selected G-quadruplexes affected the transcription of luciferase controlled by the SV40 nonrelated promoter. G-quadruplex disruption in vivo by microinjection in zebrafish embryos of either small ligands or DNA oligonucleotides complementary to the selected PQSs resulted in lower transcription of the targeted genes. Moreover, zebrafish embryos and larvae phenotypes caused by the presence of complementary oligonucleotides fully resembled those ones reported for nog3, col2a1 and fzd5 morphants. To our knowledge, this is the first work revealing in vivo the role of conserved G-quadruplexes in the embryonic development, one of the most regulated processes of the vertebrates biology.Ítem Acceso Abierto KatG, the bifunctional catalase of xanthomonas citri subsp. citri, responds to hydrogen peroxide and contributes to epiphytic survival on citrus leaves(Public Library of Science (PLOS), 2016-03-18) Tondo, María Laura; Delprato, María Laura; Kraiselburd, Ivana; Fernández Zenoff, María Verónica; Farías, María Eugenia; Orellano, Elena G.Ítem Acceso Abierto Suppression of reactive oxygen species accumulation in chloroplasts prevents leaf damage but not growth arrest in salt-stressed tobacco plants(Public Library of Science (PLOS), 2016-07-21) Lodeyro, Anabella F.; Giró, Mariana; Poli, Hugo O.; Bettucci, Gabriel; Cortadi, Adriana; Ferri, Alejandro M.; Carrillo, NéstorÍtem Acceso Abierto Cnbp ameliorates Treacher Collins Syndrome craniofacial anomalies through a pathway that involves redox-responsive genes(Springer Nature, 2016-10-06) Porcel de Peralta, Mauro S.; Mouguelar, Valeria Soraya; Sdrigotti, María Antonella; Ishiy, Felipe A. A.; Fanganiello, Roberto D.; Passos Bueno, Maria R.; Coux, Gabriela; Calcaterra, Nora B.Treacher Collins Syndrome (TCS) is a rare congenital disease (1:50 000 live births) characterized by craniofacial defects, including hypoplasia of facial bones, cleft palate and palpebral fissures. Over 90% of the cases are due to mutations in the TCOF1 gene, which codifies the nucleolar protein Treacle. Here we report a novel TCS-like zebrafish model displaying features that fully recapitulate the spectrum of craniofacial abnormalities observed in patients. As it was reported for a Tcof1+/ − mouse model, Treacle depletion in zebrafish caused reduced rRNA transcription, stabilization of Tp53 and increased cell death in the cephalic region. An increase of ROS along with the overexpression of redox-responsive genes was detected; furthermore, treatment with antioxidants ameliorated the phenotypic defects of craniofacial anomalies in TCS-like larvae. On the other hand, Treacle depletion led to a lowering in the abundance of Cnbp, a protein required for proper craniofacial development. Tcof1 knockdown in transgenic zebrafish overexpressing cnbp resulted in barely affected craniofacial cartilage development, reinforcing the notion that Cnbp has a role in the pathogenesis of TCS. The cnbp overexpression rescued the TCS phenotype in a dose-dependent manner by a ROScytoprotective action that prevented the redox-responsive genes’ upregulation but did not normalize the synthesis of rRNAs. Finally, a positive correlation between the expression of CNBP and TCOF1 in mesenchymal cells from both control and TCS subjects was found. Based on this, we suggest CNBP as an additional target for new alternative therapeutic treatments to reduce craniofacial defects not only in TCS but also in other neurocristopathies.Ítem Acceso Abierto Prospecting biotechnologically-relevant monooxygenases from cold sediment metagenomes: an In silico approach(MDPI, 2017-04-09) Musumeci, Matías A.; Lozada, Mariana; Rial, Daniela V.; Mac Cormack, Walter P.; Jansson, Janet K.; Sjöling, Sara; Carroll, JoLynn; Dionisi, HebeÍtem Acceso Abierto Cloning and characterization of the Type I Baeyer–Villiger monooxygenase from Leptospira biflexa(Springer, 2017-04-27) Ceccoli, Romina Denis; Bianchi, Dario A.; Fink, Michael J.; Mihovilovic, Marko D.; Rial, Daniela V.Ítem Acceso Abierto Genome-wide plant responses during the non-host interaction of tobacco with the Hemibiotrophic Bacterium Xanthomonas campestris pv. Vesicatoria(Frontiers Media, 2017-07-04) Pierella Karlusich, Juan J.; Matias D. Zurbriggen, Matias D.; Shahinnia, Fahimeh; Sonnewald, Sophia; Sonnewald, Uwe; Hosseini, Seyed A.; Hajirezaei, Mohammad-Reza; Carrillo, NéstorÍtem Acceso Abierto Unravelling early events in the Taphrina deformans–Prunus persica interaction: an insight into the differential responses in resistant and susceptible genotypes(Wiley, 2017-07-12) Svetaz, Laura Andrea; Bustamante, Claudia Anabel; Goldy, Camila; Rivero, Nery Alberto; Müller, Gabriela Leticia; Valentini, Gabriel Hugo; Fernie, Alisdair R.; Drincovich, María Fabiana; Lara, María Valeria; https://orcid.org/0000-0003-4914-0242; Dr. Bellini, E.: provide P. persica selections DOFI-84.364.089 and DOFI-84.364.060; Dr. Giordani, E.: provide P. persica selections DOFI-84.364.089 and DOFI-84.364.060Leaf peach curl is a devastating disease affecting leaves, flowers and fruits, caused by the dimorphic fungus Taphrina deformans. To gain insight into the mechanisms of fungus pathogenesis and plant responses, leaves of a resistant and two susceptible Prunus persica genotypes were inoculated with blastospores (yeast), and the infection was monitored during 120 h post inoculation (h.p.i.). Fungal dimorphism to the filamentous form and induction of reactive oxygen species (ROS), callose synthesis, cell death and defence compound production were observed independently of the genotype. Fungal load significantly decreased after 120 h.p.i. in the resistant genotype, while the pathogen tended to grow in the susceptible genotypes. Metabolic profiling revealed a biphasic re-programming of plant tissue in susceptible genotypes, with an initial stage co-incident with the yeast form of the fungus and a second when the hypha is developed. Transcriptional analysis of PRs and plant hormone-related genes indicated that pathogenesis-related (PR) proteins are involved in P. persica defence responses against T. deformans and that salicylic acid is induced in the resistant genotype. Conducted experiments allowed the elucidation of common and differential responses in susceptible versus resistant genotypes and thus allow us to construct a picture of early events during T. deformans infection.