Artículos y presentaciones en Congresos
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Acceso Abierto Hydrogen-bonding interactions and compostability of bionanocomposite films prepared from corn starch and nano-fillers with and without added Jamaica flower extract(2018) Gutiérrez, Tomy J.; Toro-Márquez,Luis A.; Merino, Danila; Mendieta, Julieta RenéeBionanocomposite films processed by twin screw extrusion followed by thermo molding were prepared from corn starch (Zea mays) and pH-sensitive nano-clays packaged with Jamaica flower (Hibiscus sabdariffa) extract (JFE). The hydrogen (H)-bonding interactions of the materials obtained were evaluated by ATR/FTIR spectroscopy, and their influence on the physicochemical and surface properties of the materials was analyzed. The degree of biodegradability and compostability of the films was also recorded. This latter was analyzed in terms of the ecotoxicity of the films using the variations in the growth of the primary root of lettuce (Lactuca sativa) seedlings exposed to three concentrations (1, 10 and 100 μg/mL) of the powdered films as a biomarker. The addition of the JFE-containing nano-fillers strengthened the H-bonding interactions with the thermoplastic starch (TPS) matrix, and these interactions were more efficient when there were fewer steric impediments between the JFE and the TPS. Additionally, stronger H-bonding interactions produced more hydrophilic surfaces, with greater surface energy and rougher surface morphology. All the films tested were biodegradable. Our research group had previously encountered high cytotoxicity in one of the evaluated nano-clay systems, and in this study, we confirmed that this same nano-clay system produced a non-compostable material at high concentrations (100 μg/mL), as measured by its effect on lettuce seedlings. This confirms that biodegradable materials are not necessarily compostable. - Artículo
Acceso Abierto Are Oxidative Stress Biomarkers Sensitive to Environmental Concentrations of Chlorpyrifos Exposed to the Freshwater Crab, Zilchiopsis collastinensis (Decapoda; Trichodactylidae)?(2019) Negro, C. L.; Iturburu, F. G.; Mendieta, Julieta Renée; Menone, M. L.; Collins, P.Global trends in pesticide use can increase aquatic pollution and affect resident fisheries. Crabs exposed to organophosphate pesticides, such as chlorpyrifos, may increase production of reactive oxygen species (ROS), affecting the pro-oxidant/antioxidant balance. Zichiopsis collastinensis crabs were exposed to environmentally relevant concentrations of chlorpyrifos (0.1 and 0.5 μg L− 1). Effects on the oxidative stress enzymes catalase, superoxide dismutase, glutathione S-transferases, glutathione reductase, and on thiobarbituric acid reactive substances and hydrogen peroxide concentrations were evaluated at four intervals during 96 h exposures. Exposures caused decreased GST activity and increased H2O2 levels in gills. There were modifications of GST, CAT and SOD activities in the hepatopancreas after 12 h of exposure, and an increase of H2O2 levels at every exposure interval observed. The present study proved that chlorpyrifos lead to oxidative stress in Z. collastinensis. However other enzymatic/non-enzymatic responses should be further investigated in order to be included as part of a battery of biomarkers, together with H2O2 levels, which is a parameter highly recommended to be taken into account. - Artículo
Acceso Abierto An integrated biomarker response study explains more than the sum of the parts: Oxidative stress in the fish Australoheros facetus exposed to imidacloprid(2018) Iturburu, Fernando G.; Bertrand, Lidwina; Mendieta, Julieta Renée; Amé, María V.; Menone, Mirta L.Integrated Biomarkers Response (IBR) index have been developed as a practical and robust tool to assess the susceptibility to pollutants using multiple biomarker responses. Neonicotinoid insecticides are nowadays one of the most sold pesticides worldwide. Nevertheless, imidacloprid (IMI) sub-lethal effects such as oxidative stress (OS) on fishes are scarcely studied. Hence, the aims of this work were: (1) to evaluate exposure- and damage biomarkers related to OS in the freshwater fish Australoheros facetus exposed to IMI and (2) to apply the IBR index to achieve a comprehensive understanding of OS in the fish. The results of the present study showed that all the biomarkers presented different responses in the three monitored tissues: liver, brain and gills. Results for an initial battery of 19 biomarkers were obtained and for the IBR index only those with significant differences have been considered. The biomarkers that had the most important weight on the IBR index were SOD activity in brain and gills, H2O2 concentration in liver, and carbonyl groups concentration in gills in fishes exposed to 100 and 1000 μg L−1 IMI. This index allowed affirming that a short term exposure to environmentally relevant concentrations of IMI (≥10 μg L−1) produces OS in A. facetus. However, a more deep understanding of some biomarkers response is necessary to improve the index and for finally apply it in field studies. - Artículo
Acceso Abierto Strategies for cadmium detoxification in the white shrimp Palaemon argentinus from clean and polluted field locations(2019) Chiodi Boudet, Leila; Mendieta, Julieta Renée; Romero, María Belén; Dolagaratz Carricavur, Arantxa; Polizzi, Paula; Marcovecchio, Jorge E.; Gerpe, MarcelaIn this study, we investigated the metal handling capacity of non-tolerant and tolerant populations of Palaemon argentinus to cadmium (Cd), through evaluating of the main mechanisms of metal detoxification, metallothioneins (MT), and metal-rich granules (MRG), to prove that the presence of MRG in the second population is responsible for that condition. The tolerant population were exposed to 3.06 and 12.26 mg Cd$L 1, while the non-tolerant shrimp were exposed to 3.06 mg Cd$L 1. Each experiment involved the exposure during 3, 7, 10, and 15 days and, the depuration during 7, 14, 21, and 28 days, for which shrimp were transferred to clean water. The range values of MT concentrations for non-tolerant shrimp were: 12.24e23.91 mg g (w.w), while for tolerant shrimp were: 8.75e16.85 mg g (w.w); MRG levels were: 0.12e0.57 mg g (w.w) and 0.3e2.1 mg g (w.w), respectively. The results showed different strategies for Cd detoxification: the induction of MT was the main pathway in the non-tolerant population, while the formation of Cd-MRG was the main mechanism for tolerant shrimp. These differences could be related to environmental history and the health status of each population. - Artículo
Acceso Abierto Characterization of biodegradable/non-compostable films made from cellulose acetate/corn starch blends processed under reactive extrusion conditions(2019) Herniou–Julien, Clémence; Mendieta, Julieta Renée; Gutiérrez, Tomy J.The manufacture of food packaging materials from food hydrocolloids has been widely studied during the last decades and multiple alternatives have been investigated, with research mainly focusing on improving the physicochemical and mechanical properties of the different materials. Processing food hydrocolloids by reactive extrusion (REx) for the development of food packaging has, however, been poorly studied. Four film systems were prepared from corn (Zea mays) thermoplastic starch (TPS) containing either cellulose acetate (C) or chromium octanoate (Cat - a potential food grade catalyst), or a blend of both (C + Cat). Processing was done under REx conditions using a twin-screw extruder. An exhaustive study of the resulting materials was carried out in terms of the structural, physicochemical, thermal, surface, mechanical and compostable properties related to their potential use in food packaging applications. The most hydrophobic material was the C-containing film. However, this physicochemical behavior was different on the film surface, thus suggesting molecular rearrangements within the material. The Cat-containing films were darker than the other materials. The mechanical behavior observed in the Cat-containing films was particularly interesting as it suggests that these film systems could be used as shape memory materials for food packaging applications, as long as the following mechanical conditions are not exceeded: 5.02% strain and 0.43 MPa stress. All the films tested were biodegradable. We confirmed that Cat-containing film systems produced non-compostable materials at high concentrations (1 mg/mL), as measured by its effect on lettuce seedlings. This confirms that biodegradable materials are not necessarily compostable. - Artículo
Acceso Abierto A Rhomboid protease gene deletion affects a novel oligosaccharide N-linked to the S-layer glycoprotein of Haloferax volcanii(2014) Parente, Juliana Elena; Casabuono, Adriana; Ferrari, María Celeste; Paggi, Roberto Alejandro; De Castro, Rosana Esther; Couto, Alicia Susana; Giménez, María InésRhomboid proteases occur in all domains of life; however, their physiological role is not completely understood, and nothing is known of the biology of these enzymes in Archaea. One of the two rhomboid homologs ofHaloferax volcanii(RhoII) is fused to a zinc finger domain. Chromosomal deletion ofrhoIIwas successful, indicating that this gene is not essential for this organism; however, the mutant strain (MIG1) showed reduced motility and increased sensitivity to novobiocin. Membrane preparations of MIG1 were enriched in two glycoproteins, identified as the S-layer glycoprotein and an ABC transporter component. TheH. volcaniiS-layer glycoprotein has been extensively used as a model to study haloarchaeal proteinN-glycosylation. HPLC analysis of oligosaccharides released from the S-layer glycoprotein after PNGase treatment revealed that MIG1 was enriched in species with lower retention times than those derived from the parent strain. Mass spectrometry analysis showed that the wild type glycoprotein released a novel oligosaccharide species corresponding to GlcNAc-GlcNAc(Hex)2-(SQ-Hex)6in contrast to the mutant protein, which contained the shorter form GlcNAc2(Hex)2-SQ-Hex-SQ. A glycoproteomics approach of the wild type glycopeptide fraction revealed Asn-732 peptide fragments linked to the sulfoquinovose-containing oligosaccharide. This work describes a novelN-linked oligosaccharide containing a repeating SQ-Hex unit bound to Asn-732 of theH. volcaniiS-layer glycoprotein, a position that had not been reported as glycosylated. Furthermore, this study provides the first insight on the biological role of rhomboid proteases in Archaea, suggesting a link between protein glycosylation and this protease family. - Parte de libro
Acceso Abierto Hidrolizados proteicos de pescado a partir de residuos de la industria pesquera con potencialidad en Biotecnología(2016) Massa, Agueda E.; Manca, Emilio A.; Mansilla, Andrea Yamila; Mendieta, Julieta Renée; Casalongué, Claudia A.Durante las operaciones pesqueras destinadas al procesamiento de pescados y mariscos para consumo humano, se generan residuos (cabezas, vísceras, piel y espinas) que constituyen más del 40% del peso total de los desembarques pesqueros. Estos subproductos presentan compuestos con importantes propiedades nutricionales, funcionales y bioactivas que pueden ser utilizados en diversos sectores industriales. El objetivo del presente estudio fue elaborar hidrolizados proteicos a partir de residuos pesqueros y evaluar su aplicabilidad en la industria agrícola. La materia prima, que fue obtenida de industrias pesqueras marplatenses, se homogeneizó y se sometió a una hidrólisis enzimática. Finalizado dicho proceso, el hidrolizado proteico fue separado y caracterizado químicamente. La composición química de estos productos incluyó compuestos orgánicos (péptidos aminoácidos libres, ácidos grasos omega-3, vitaminas) y minerales (nitrógeno, fósforo, potasio, calcio, magnesio y otros oligoelementos) altamente nutritivos para las plantas y microorganismos beneficiosos. En este contexto, el desarrollo de hidrolizados proteicos de pescado puede considerarse como una alternativa económicamente viable y ecológicamente sustentable con un alto potencial de aplicación biotecnológica. - Artículo
Embargado Calcium-dependent Protein Kinases are Involved in Potato Signal Transduction.(2008) Blanco, F.A.; Zanetti, M.E.; Daleo, Gustavo RaúlPlant response to pathogens involves an intricate network of signal transduction pathways. Here, potato cell cultures were used to study signal transduction in response to elicitors fromPhytophthora infestans. Pretreatment of cells with Ser/Thr protein kinase inhibitors, EGTA, calmodulin antagonists or a channel blocker abolished the induction of two enzymes involved in defence responses, phenylalanine ammonia-lyase (PAL) and peroxidase. Phosphatase inhibitors caused an increase of these activities in the absence of elicitors. Hyphal cell wall components (HWC) from an incompatible race (HWC 0) produced a rapid and transient increment of histone phosphorylation, whereas induction by HWC from a compatible race (HWC C) was less pronounced and more sustained. As activities were calcium-dependent, a fraction enriched in calcium-dependent protein kinases (CDPKs) was obtained by DEAE chromatography. Fractions from HWC 0- and HWC C-treated cells presented higher kinase activity than that from untreated cells. Moreover, total activity was higher in the incompatible than in the compatible interaction. Activity was calcium-dependent, partially inhibited by calmodulin antagonists and able to phosphorylate syntide-2, a specific substrate of CDPKs. An in-gel kinase assay showed the presence of a band of approximately 50kDa whose activity was higher in HWC 0- than in HWC C-treated cells and was not detected in control extracts. This report presents evidences of the differential activation of CDPKs in response to elicitors from different races ofP. infestans, revealing that these protein kinases participate in the defence response to oomycete. - Artículo
Acceso Abierto Antimicrobial activity of phosphites against different potato pathogens(2010) Lobato, M.C.; Olivieri, F.P.; Daleo, G.R.; Andreu., A.B.Phosphites have low-toxicity on the environment and show high efficacy in controlling oomycete diseases in plants, both by a direct and an indirect mechanism. We have shown that they are also effective in reducing disease symptoms produced byPhytophthora infestans, Fusarium solaniandRhizoctonia solaniwhen applied to potato seed tubers. To gain better insight into the direct mode of action of phosphites on different potato pathogens, and to ascertain chemical determinants in their direct antimicrobial activity, four potato pathogens were assayed with respect to sensitivity toward calcium, potassium and copper phosphites (CaPhi, KPhi and CuPhi, respectively). The influence of acidification and ionic strength changes after Phi addition on the antimicrobial activity, and the fungicidal or fungistatic activity, were evaluated. Results showed that phosphites were able to inhibit growth of all pathogens.Phytophthora infestanswas the most inhibited pathogen by all phosphites, followed byStreptomyces scabies, whileRhizoctonia solaniandFusarium solaniwere less inhibited. CuPhi had the highest antimicrobial activity against the four pathogens analysed, and CaPhi and KPhi showed similar antimicrobial activities. Inhibitions by CuPhi and CaPhi could be partially explained by acidification of the media. However, results obtained with KPhi demonstrated that the phosphite anion has antimicrobial activity itself. The increase in ionic strength after Phi addition was not important in the antimicrobial activity of Phi. The activity of phosphites on germination ofF. solanispores showed to be fungistatic rather than fungicidal. - Artículo
Acceso Abierto Swaposin domain of potato aspartic protease (StAsp-PSI) exerts antimicrobial activity on plant and human pathogens(2010) Muñoz, Fernando F.; Mendieta, Julieta Renée; Pagano, Mariana R.; Paggi, Roberto A.; Daleo, Gustavo Raúl; Guevara, María G.Plant-specific insert domain (PSI) is a region of approximately 100 amino acid residues present in most plant aspartic protease (AP) precursors. PSI is not a true saposin domain; it is the exchange of the N- and C-terminal portions of the saposin like domain. Hence, PSI is called a swaposin domain. Here, we report the cloned, heterologous expression and purification of PSI from StAsp 1 (Solanum tuberosum aspartic protease 1), called StAsp-PSI. Results obtained here show that StAsp-PSI is able to kill spores of two potato pathogens in a dose-dependent manner without any deleterious effect on plant cells. As reported for StAPs (S. tuberosum aspartic proteases), the StAsp-PSI ability to kill microbial pathogens is dependent on the direct interaction of the protein with the microbial cell wall/or membrane, leading to increased permeability and lysis. Additionally, we demonstrated that, like proteins of the SAPLIP family, StAsp-PSI and StAPs are cytotoxic to Gram-negative and Gram-positive bacteria in a dose dependent manner. The amino acid residues conserved in SP_B (pulmonary surfactant protein B) and StAsp-PSI could explain the cytotoxic activity exerted by StAsp-PSI and StAPs against Gram-positive bacteria. These results and data previously reported suggest that the presence of the PSI domain in mature StAPs could be related to their antimicrobial activity - Artículo
Acceso Abierto Cytotoxic effect of potato aspartic proteases (StAPs) on Jurkat T cells(2010) Mendieta, Julieta Renée; Fimognari, Carmela; Daleo, Gustavo Raúl; Hrelia, Patrizia; Guevara, María G.StAPs are potato aspartic proteases with cytotoxic activity against plant pathogens and spermatozoa.StAPs cytotoxic activity is selective, since these proteins do not exert toxic effect on plant cells and erythrocytes. In this work, we investigated the capacity ofStAPs to exert cytotoxicity on human leukaemia cells. Obtained results show thatStAPs induce apoptosis on Jurkat T cells after a short time of incubation in a dose-dependent manner. However, no significative effect on the T lymphocytes viability was observed at allStAPs incubation times and concentrations tested. These results suggest thatStAPs can be conceptually promising leads for cancer therapy. - Artículo
Acceso Abierto BABA effects on the behaviour of potato cultivars infected by Phytophthora infestans and Fusarium solani(2009) Olivieri, F.P; González Altamiranda, E.; Lobato, M.C.; Huarte, M.A.; Daleo, Gustavo Raúl; Guevara, M.G.; Andreu, A.B.Since most plants possess resistance mechanisms which can be induced upon pre-treatment with a variety of chemical compounds, the use of β-aminobutyric acid (BABA) as a defence inducer without reported toxic effect on the environment was studied. The aim of this work was to analyse the effectiveness of BABA to induce resistance againstPhytophthora infestansandFusarium solaniin potato cultivars differing in their level of resistance to late blight. The behaviour of some components of biochemical mechanisms by which BABA increases resistance againstP. infestans, as well as the effect of BABA on the activity of a potential pathogenic factor ofF. solani, were studied. Plants with four applications of BABA throughout the crop cycle produced tubers more resistant toP. infestansandF. solanithan non-treated plants. In addition, tuber slices from treated plants, inoculated withP. infestans,showed an increase in phenol and phytoalexin content. The aspartyl proteaseStAP1 accumulation was also higher in tubers obtained from treated plants and inoculated withP. infestans. This result was observed only in the more resistant potato cv. Pampeana, early after infection. In the potato–F. solaniinteraction, infected tubers coming from BABA-treated plants showed minor fungal proteolytic activity than infected, non-treated ones. For potato cvs Pampeana and Bintje, the BABA treatment improved the yield of harvested tubers. The number of tubers per plant and total weight of harvested tubers was greater for those obtained from treated plants with two early or four applications of BABA. The results show that the BABA treatment increases the resistance of potatoes but the degree of increase depends on the original level of resistance present in each cultivar. - Documento de conferencia
Acceso Abierto Expression of plant specific domain of potato aspartic proteases (StAP-PSI) restricts P. infestans spread in potato leaves.(2015) Frey, María Eugenia; Pepe, Alfonso; Daleo, Gustavo Raúl; Guevara, María GabrielaPlant specific insert (PSI) is a domain present in the precursors and mature atypical plant aspartic proteases (APs). Several plant APs have been associated with the plant mechanism of defence against pathogens. However, only two (StAP1 and StAP3, for Solanum tuberosum APs) of these proteases, contain the PSI domain into the mature form. We have previously reported the cytotoxic activity of the recombinant StAP-PSI towards plant pathogens. However the role of PSI domain of StAPs in the plant mechanism defense is still unknown. The aim of this work was to analyze the effect of transient expression of StAP-PSI in potato leaves infected by P. infestans. Results obtained show that StAP-PSI expression reduces the P. infestans affected area in a 60 % compared with the control ones. Analysis by qPCR shows an increase of the transcript level of the hypersensitive response marker (hsr203J) in potato leaves that express StAP-PSI, independent of the P. infestans infection; however the highest increase of this gene was detected in leaves at 6 h. after infection. Additionally, an increase of the WRKI1 transcript level was detected in potato leaves that express StAP-PSI. Results obtained here indicate that, PSI domain of StAPs could have a direct (as antimicrobial compound) and indirect (as an inductor molecule) role in the plant mechanism to restrict the pathogen spread. - Artículo
Acceso Abierto Isolation and characterization of a Solanum tuberosum subtilisin-like protein with caspase-3 activity (StSBTc-3)(2015) Fernandez, María Belén; Daleo, Gustavo Raúl; GuevaRA, María GabrielaPlant proteases with caspase-like enzymatic activity have been widely studied during the last decade. Previously, we have reported the presence and induction of caspase-3 like activity in the apoplast of potato leaves during Solanum tuberosum- Phytophthora infestans interaction. In this work we have purified and identified a potato extracellular protease with caspase-3 like enzymatic activity from potato leaves infected with P. infestans. Results obtained from the size exclusion chromatography show that the isolated protease is a monomeric enzyme with an estimated molecular weight of 70 kDa approximately. Purified protease was analyzed by MALDI-TOF MS, showing a 100% of sequence identity with the deduced amino acid sequence of a putative subtilisin-like protease from S. tuberosum (Solgenomics protein ID: PGSC0003DMP400018521). For this reason the isolated protease was named as StSBTc-3. This report constitutes the first evidence of isolation and identification of a plant subtilisin-like protease with caspase-3 like enzymatic activity. In order to elucidate the possible function of StSBTc-3 during plant pathogen interaction, we demonstrate that like animal caspase-3, StSBTc-3 is able to produce in vitro cytoplasm shrinkage in plant cells and to induce plant cell death. This result suggest that, StSBTc-3 could exert a caspase executer function during potato- P. infestans interaction, resulting in the restriction of the pathogen spread during plantepathogen interaction. - Artículo
Acceso Abierto Potassium phosphite increases tolerance to UV-B in potato(2015) Oyarburo, Natalia Soledad; Machinandiarena, Milagros; Feldman, Mariana; Daleo, Gustavo Raúl; Andreu, Adriana B.; Olivieri, Florencia PThe use of biocompatible chemical compounds that enhance plant disease resistance through Induced Resistance (IR) is an innovative strategy to improve the yield and quality of crops. Phosphites (Phi), inorganic salts of phosphorous acid, are environment friendly, and have been described to induce disease control. Phi, similar to other plant inductors, are thought to be effective against different types of biotic and abiotic stress, and it is assumed that the underlying signaling pathways probably overlap and interact. The signaling pathways triggered by UV-B radiation, for instance, are known to crosstalk with other signaling routes that respond that biotic stress. In the present work, the effect of potassium phosphite (KPhi) pre-treatment on UV-B stress tolerance was evaluated in potato leaves. Plants were treated with KPhi and, after 3 days, exposed to 2 h/day of UV-B (1.5 Watt m(-2)) for 0, 3 and 6 days. KPhi pre-treatment had a beneficial effect on two photosynthetic parameters, specifically chlorophyll content and expression of the psbA gene. Oxidative stress caused by UV-B was also prevented by KPhi. A decrease in the accumulation of hydrogen peroxide (H2O2) in leaves and an increase in guaiacol peroxidase (POD) and superoxide dismutase (SOD) activities were also observed. In addition, the expression levels of a gene involved in flavonoid synthesis increased in UV-B-stressed plants only when pre-treated with KPhi. Finally, accumulation of glucanases and chitinases was induced by UV-B stress and markedly potentiated by KPhi pre-treatment. Altogether, this is the first report that shows a contribution of KPhi in UV-B stress tolerance in potato plants. - Artículo
Acceso Abierto Fibrin(ogen)olytic and antiplatelet activities of a subtilisin-like protease from Solanum tuberosum (StSBTc-3)(2016) Pepe, Alfonso; Frey, María Eugenia; Muñoz, Fernando; Fernández, María Belén; Pedraza, Anabela; Galbán, Gustavo; García, Diana Noemí; Daleo, Gustavo Raúl; Guevara, María GabrielaPlant serine proteases have been widely used in food science and technology as well as in medicine. In this sense, several plant serine proteases have been proposed as potential anti-coagulants and antiplatelet agents. Previously, we have reported the purification and identification of a plant serine protease from Solanum tuberosum leaves. This potato enzyme, named as StSBTc-3, has a molecular weight of 72 kDa and it was characterized as a subtilisin like protease. In this work we determine and characterize the biochemical and medicinal properties of StSBTc-3. Results obtained show that, like the reported to other plant serine proteases, StSBTc-3 is able to degrade all chains of human fibrinogen and to produces fibrin clot lysis in a dose dependent manner. The enzyme efficiently hydrolyzes b subunit followed by partially hydrolyzed a and g subunits of human fibrinogen. Assays performed to determine StSBTc-3 substrate specificity using oxidized insulin b-chain as substrate, show seven cleavage sites: Asn3-Gln4; Cys7-Gly8; Glu13-Ala14; Leu15-Tyr16; Tyr16-Leu17; Arg22-Gly23 and Phe25-Tyr26, all of them were previously reported for other serine proteases with fibrinogenolytic activity. The maximum StSBTc-3 fibrinogenolytic activity was determined at pH 8.0 and at 37 C. Additionally, we demonstrate that StSBTc- 3 is able to inhibit platelet aggregation and is unable to exert cytotoxic activity on human erythrocytes in vitro at all concentrations assayed. These results suggest that StSBTc-3 could be evaluated as a new agent to be used in the treatment of thromboembolic disorders such as strokes, pulmonary embolism and deep vein thrombosis.