Nuclear translocation and DNA recognition signals colocalized within the bZIP domain of cyclic adenosine 3',5'-monophosphate response element-binding protein CREB.

CREB is a cAMP-responsive nuclear DNA-binding protein that binds to cAMP response elements and stimulates gene transcription upon activation of the cAMP signalling pathway. The protein consists of an amino-terminal transcriptional transactivation domain and a carboxyl-terminal DNA-binding domain (bZIP domain) comprised of a basic region and a leucine zipper involved in DNA recognition and dimerization, respectively. Recently, we discovered a testis-specific transcript of CREB that contains an alternatively spliced exon encoding multiple stop codons. CREB encoded by this transcript is a truncated protein lacking the bZIP domain. We postulated that the antigen detected by CREB antiserum in the cytoplasm of germinal cells is the truncated CREB that must also lack its nuclear translocation signal (NTS). To test this hypothesis we prepared multiple expression plasmids encoding carboxyl-terminal deletions of CREB and transiently expressed them in COS-1 cells. By Western immunoblot analysis as well as immunocytochemistry of transfected cells, we show that CREB proteins truncated to amino acid 286 or shorter are sequestered in the cytoplasm, whereas a CREB of 295 amino acids is translocated into the nucleus. Chimeric CREBs containing a heterologous NTS fused to the first 248 or 261 amino acids of CREB are able to drive the translocation of the protein into the nucleus. Thus, the nine amino acids in the basic region involved in DNA recognition between positions 287 and 295 (RRKKKEYVK) of CREB contain the NTS. Further, mutation of the lysine at position 290 in CREB to an asparagine diminishes nuclear translocation of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Feeding strategies and energy to protein ratio on tambaqui performance and physiology

The objective of this work was to evaluate the effect of feed deprivation and refeeding with diets containing different energy to protein ratios (E/P) on the performance and physiology of juvenile tambaqui (Colossoma macropomum). A 4x2 factorial arrangement with three replicates was used, with four E/P ratios (11.5, 10.5, 9.5, and 8.5 kcal g-1 digestible energy per protein) and two feeding regimens (with and without deprivation), during 60 days. Fish from the food-deprived group were fasted for 14 days and refed from the fifteenth to the sixtieth day, whereas the remaining fish were fed for 60 days. At the end of the experimental period, weight of fish subjected to food deprivation was lower than that of those continuously fed; however, this condition did not influence the physiological parameters analyzed. Tambaqui fed 11.5 kcal g-1 achieved lower final weight than those fed with the other diets, in both regimens. Among the physiological parameters, only plasma protein presented significant increase in fish fed 8.5 kcal g-1, in both feeding regimens, probably due to the higher dietary protein concentration. These results indicate that fish show a partial compensatory growth, and that 10.5 kcal g-1 can be recommended for the diet of juvenile tambaqui.

Molecular characterization of the Carma1-related protein card9

ABSTRACT : Fungal infections have become a major source of diseases in immuncompromised patients, but are quite benign in healthy individuals. As fungi are eukaryotes, and share many biological processes with humans, many antifungal drugs can cause toxicity in the patients. Therefore, the characterization of signaling pathways specific to the anti-fungal immune response is relevant for the better understanding of the disease and the development of new therapeutic approaches. Dectin-1 is the major mammalian pattern recognition receptor for the fungal component zymosan. Dectin-1 is an innate non-Toll-like receptor containing immunoreceptor tyrosine-based activation motifs (ITAMs). Card9, Bc110 and Maltl are proteins that have been shown to play a key role in the Dectin-l-induced signaliñg pathway by controlling Dectin-l-mediated cell activation, cytokine production and innate anti-fungal immunity in mice. Here we investigate the role of the Card9-Bc110-Maltl complex in humans using the monocytic cell line THP-1. We show that Card9 interacts with Bc110 through a CARD-CARD interaction and that interaction of Card9 with Bc110 is required for NF-xB activation. We further demonstrate that Card9 is phosphorylated in its C-terminal part on serine residues. The phosphorylation status of Card9 can influence its ability to active NF-xB, since mutation of the phosphorylation sites increases its ability to activate NF-xB. We find that Card9 is expressed in myeloid derived cells, such as the human monocytic cell lines THP1 and U937, and in human monocyte-enriched PBLs and monocyte-derived DCs. Our findings demonstrate that Card9 is implicated in anti-fungal responses, since silencing of Card9 as well as of Bc110 and Maltl diminishes the capacity of THP1 cells to produce TNF-a in response to zymosan. Interestingly, activation of the NF-xB and MAPK pathway remained normal and levels of TNF-a mRNA produced were also not affected in THP 1 cells silenced for the expression of Card9, Bc110 or Malt1. Using a Malt1 inhibitor, we provide evidence that the proteolytic activity of Malt1 is needed for zymosan-induced TNF-a production in THP 1 cells and bone marrow-derived macrophages of mice, but further experiments are required to confirm these findings and identify the substrate(s) of Malt1. In conclusion, our results reveal an important role for Card9 in the innate immune response of human macrophages to fungi. RÉSUMÉ : Les infections fongiques sont une source majeure de maladie chez les patients immunodéprimés, alors qu'elles sont plutôt bénignes chez les individus sains. Comme les champignons sont des eucaryotes et partagent beaucoup de processus biologiques avec les humains, les médicaments antifongiques peuvent être source de toxicité chez les patients. Il est donc important de mieux caractériser les voies de signalisation intracellulaire des réponses anti-fongiques pour pouvoir développer de nouvelles approches thérapeutiques. La protéine Dectin-1 est le récepteur principal du composé fongique zymosan. Les protéines Card9, Bc110 et Maltl ont été décrites comme jouant un rôle primordial dans les signaux d'activation induits par Dectin-l, en contrôlant l'activité cellulaire, la production de cytokines et la défense anti-fongique dans les souris. Dans cette étude, nous investiguons le rôle du complexe Card9-Bc110-Maltl dans la lignée monocytaire humaine THP1. Nous montrons que Card9 interagit avec Bc110 par une interaction CARD-CARD et que cette interaction est requise pour activer le facteur de transcription NF-xB. Nous observons que Card9 est phosphorylé dans sa partie C-terminale sur des résidus serine et que l'état de phosphorylation de Card9 influence sa capacité à activer NF-xB. En effet, sa capacité à activer NF-xB est augmentée, après mutation des sites de phosphorylation. La génération d'un anticorps spécifique dirigé contre Card9 nous a permis de démontrer que Card9 est exprimé dans des cellules myéloïdes comme les lignées cellulaires monocytiques THP-1 et U-937, ainsi que dans les cellules dendritiques humaines. Nos résultats démontrent que Card9 est impliqué dans la réponse immunitaire antifongique puisque la réduction de l'expression de Card9 ainsi que de Bc110 et de Malt1 diminue la capacité des THP-1 à produire du TNF-a en réponse au zymosan. Par contre, les voies de signalisation NF-xB et MAPK ainsi que les niveaux de mRNA de TNF-a produits en réponse au zymosan ne sont pas affectés dans ces cellules. En utilisant un inhibiteur de Malt1, nous montrons que l'activité protéolytique de Malt1 est nécessaire pour la production de TNF-a induite par le zymosan dans les cellules THP-1 ainsi que dans les macrophages de souris, mais d'autres expériences seront nécessaires pour confirmer cette observation et identifier le(s) substrat(s) de Malt1 responsables de cet effet. En conclusion, nos résultats révèlent un rôle important de la protéine Card9 dans la réponse immunitaire innée antifongique dans les macrophages humains.

Different concentrations of Mg++ ions affect nuclear matrix protein distribution during thermal stabilization of isolated nuclei.

The nuclear matrix, a proteinaceous network believed to be a scaffolding structure determining higher-order organization of chromatin, is usually prepared from intact nuclei by a series of extraction steps. In most cell types investigated the nuclear matrix does not spontaneously resist these treatments but must be stabilized before the application of extracting agents. Incubation of isolated nuclei at 37C or 42C in buffers containing Mg++ has been widely employed as stabilizing agent. We have previously demonstrated that heat treatment induces changes in the distribution of three nuclear scaffold proteins in nuclei prepared in the absence of Mg++ ions. We studied whether different concentrations of Mg++ (2.0-5 mM) affect the spatial distribution of nuclear matrix proteins in nuclei isolated from K562 erythroleukemia cells and stabilized by heat at either 37C or 42C. Five proteins were studied, two of which were RNA metabolism-related proteins (a 105-kD component of splicing complexes and an RNP component), one a 126-kD constituent of a class of nuclear bodies, and two were components of the inner matrix network. The localization of proteins was determined by immunofluorescent staining and confocal scanning laser microscope. Mg++ induced significant changes of antigen distribution even at the lowest concentration employed, and these modifications were enhanced in parallel with increase in the concentration of the divalent cation. The different sensitivity to heat stabilization and Mg++ of these nuclear proteins might reflect a different degree of association with the nuclear scaffold and can be closely related to their functional or structural role.

Grx5 glutaredoxin plays a central role in protection against protein oxidative damage in Saccharomyces cerevisiae

Glutaredoxins are members of a superfamily of thiol disulfide oxidoreductases involved in maintaining the redox state of target proteins. In Saccharomyces cerevisiae, two glutaredoxins (Grx1 and Grx2) containing a cysteine pair at the active site had been characterized as protecting yeast cells against oxidative damage. In this work, another subfamily of yeast glutaredoxins (Grx3, Grx4, and Grx5) that differs from the first in containing a single cysteine residue at the putative active site is described. This trait is also characteristic for a number of glutaredoxins from bacteria to humans, with which the Grx3/4/5 group has extensive homology over two regions. Mutants lacking Grx5 are partially deficient in growth in rich and minimal media and also highly sensitive to oxidative damage caused by menadione and hydrogen peroxide. A significant increase in total protein carbonyl content is constitutively observed in grx5cells, and a number of specific proteins, including transketolase, appear to be highly oxidized in this mutant. The synthetic lethality of the grx5 and grx2 mutations on one hand and ofgrx5 with the grx3 grx4 combination on the other points to a complex functional relationship among yeast glutaredoxins, with Grx5 playing a specially important role in protection against oxidative stress both during ordinary growth conditions and after externally induced damage. Grx5-deficient mutants are also sensitive to osmotic stress, which indicates a relationship between the two types of stress in yeast cells.

Multilevel control of arabidopsis 3-hydroxy-3-methylglutaryl coenzyme A reductase by protein phosphatase 2A

Plants synthesize a myriad of isoprenoid products that are required both for essential constitutive processes and for adaptive responses to the environment. The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes a key regulatory step of the mevalonate pathway for isoprenoid biosynthesis and is modulated by many endogenous and external stimuli. In spite of that, no protein factor interacting with and regulating plant HMGR in vivo has been described so far. Here, we report the identification of two B99 regulatory subunits of protein phosphatase 2A (PP2A), designated B99a and B99b, that interact with HMGR1S and HMGR1L, the major isoforms of Arabidopsis thaliana HMGR. B99a and B99b are Ca2+ binding proteins of the EF-hand type. We show that HMGR transcript, protein, and activity levels are modulated by PP2A in Arabidopsis. When seedlings are transferred to salt-containing medium, B99a and PP2A mediate the decrease and subsequent increase of HMGR activity, which results from a steady rise of HMGR1-encoding transcript levels and an initial sharper reduction of HMGR protein level. In unchallenged plants, PP2A is a posttranslational negative regulator of HMGR activity with the participation of B99b. Our data indicate that PP2A exerts multilevel control on HMGR through the fivemember B99 protein family during normal development and in response to a variety of stress conditions.

The fusion protein SS18-SSX1 employs core Wnt pathway transcription factors to induce a partial Wnt signature in synovial sarcoma.

Expression of the SS18/SYT-SSX fusion protein is believed to underlie the pathogenesis of synovial sarcoma (SS). Recent evidence suggests that deregulation of the Wnt pathway may play an important role in SS but the mechanisms whereby SS18-SSX might affect Wnt signaling remain to be elucidated. Here, we show that SS18/SSX tightly regulates the elevated expression of the key Wnt target AXIN2 in primary SS. SS18-SSX is shown to interact with TCF/LEF, TLE and HDAC but not β-catenin in vivo and to induce Wnt target gene expression by forming a complex containing promoter-bound TCF/LEF and HDAC but lacking β-catenin. Our observations provide a tumor-specific mechanistic basis for Wnt target gene induction in SS that can occur in the absence of Wnt ligand stimulation.

Overexpression of guanylate cyclase activating protein 2 in rod photoreceptors in vivo leads to morphological changes at the synaptic ribbon.

Guanylate cyclase activating proteins are EF-hand containing proteins that confer calcium sensitivity to retinal guanylate cyclase at the outer segment discs of photoreceptor cells. By making the rate of cGMP synthesis dependent on the free intracellular calcium levels set by illumination, GCAPs play a fundamental role in the recovery of the light response and light adaptation. The main isoforms GCAP1 and GCAP2 also localize to the synaptic terminal, where their function is not known. Based on the reported interaction of GCAP2 with Ribeye, the major component of synaptic ribbons, it was proposed that GCAP2 could mediate the synaptic ribbon dynamic changes that happen in response to light. We here present a thorough ultrastructural analysis of rod synaptic terminals in loss-of-function (GCAP1/GCAP2 double knockout) and gain-of-function (transgenic overexpression) mouse models of GCAP2. Rod synaptic ribbons in GCAPs−/− mice did not differ from wildtype ribbons when mice were raised in constant darkness, indicating that GCAPs are not required for ribbon early assembly or maturation. Transgenic overexpression of GCAP2 in rods led to a shortening of synaptic ribbons, and to a higher than normal percentage of club-shaped and spherical ribbon morphologies. Restoration of GCAP2 expression in the GCAPs−/− background (GCAP2 expression in the absence of endogenous GCAP1) had the striking result of shortening ribbon length to a much higher degree than overexpression of GCAP2 in the wildtype background, as well as reducing the thickness of the outer plexiform layer without affecting the number of rod photoreceptor cells. These results indicate that preservation of the GCAP1 to GCAP2 relative levels is relevant for maintaining the integrity of the synaptic terminal. Our demonstration of GCAP2 immunolocalization at synaptic ribbons at the ultrastructural level would support a role of GCAPs at mediating the effect of light on morphological remodeling changes of synaptic ribbons.

Most of the Abundant Protein Fractions of Embryonic Cerebrospinal Fluid are Produced Out of the Brain Anlagen

The microenvironment of the central nervous system is important for neuronal function and development. During the early stages of embryo development the cephalic vesicles are filled by embryonic cerebrospinal fluid, a complex fluid containing different protein fractions, which contributes to the regulation of the survival, proliferation and neurogenesis of neuroectodermal stem cells. The protein content of embryonic cerebrospinal fluid from chick and rat embryos at the start of neurogenesis has already been determined. Most of the identified gene products are thought to be involved in the regulation of developmental processes during embryogenesis. However, due to the crucial roles played by embryonic cerebrospinal fluid during brain development, the embryological origin of the gene products it contains remains an intriguing question. According to the literature most of these products are synthesised in embryonic tissues other than the neuroepithelium. In this study we examined the embryological origin of the most abundant embryonic cerebrospinal fluid protein fractions by means of slot-blot analysis and by using several different embryonic and extraembryonic protein extracts, immunodetected with polyclonal antibodies. This first attempt to elucidate their origin is not based on the proteins identified by proteomic methods, but rather on crude protein fractions detected by SDS-PAGE analysis and to which polyclonal antibodies were specifically generated. Despite some of the limitations of this study, i.e. that one protein fraction may contain more than one gene product, and that a specific gene product may be contained in different protein fractions depending on post-translational modifications, our results show that most of the analysed protein fractions are not produced by the cephalic neuroectoderm but are rather stored in the egg reservoir; furthermore, few are produced by embryo tissues, thus indicating that they must be transported from their production or storage sites to the cephalic cavities, most probably via embryonic serum. These results raise the question as to whether the transfer of proteins from these two embryo compartments is regulated at this early developmental stage.

Polyclonal antibodies to the coat protein of Apple stem grooving virus expressed in Escherichia coli: production and use in immunodiagnosis

The coat protein gene of Apple stem grooving virus (ASGV) was amplified by RT-PCR, cloned, sequenced and subcloned in the expression vector pMal-c2. This plasmid was used to transform Escherichia coli BL21c+ competent cells. The ASGV coat protein (cp) was expressed as a fusion protein containing a fragment of E. coli maltose binding protein (MBP). Bacterial cells were disrupted by sonication and the ASGVcp/MBP fusion protein was purified by amylose resin affinity chromatography. Polyclonal antibodies from rabbits immunized with the fusion protein gave specific reactions to ASGV from infected apple (Malus domestica) cv. Fuji Irradiada and Chenopodium quinoa at dilutions of up to 1:1,000 and 1:2,000, respectively, in plate trapped ELISA. The ASGVcp/MBP fusion protein reacted to a commercial antiserum against ASGV in immunoblotting assay. The IgG against ASGVcp/MBP performed favorably in specificity and sensitivity to the virus. This method represents an additional tool for the efficient ASGV-indexing of apple propagative and mother stock materials, and for use in support of biological and molecular techniques.

Nuclear Matrix in Apoptotic Cell Death and Cell Proliferation. The role of Nuclear Mitotic Apparatus (NuMA) Protein

The nucleus is a membrane enclosed organelle containing most of the genetic information of the cell in the form of chromatin. The nucleus, which can be divided into many sub-organelles such as the nucleoli, the Cajal bodies and the nuclear lamina, is the site for several essential cellular functions such as the DNA replication and its regulation and most of the RNA synthesis and processing. The nucleus is often affected in disease: the size and the shape of the nucleus, the chromatin distribution and the size of the nucleoli have remained the basis for the grading of several cancers. The maintenance of the vertebrate body shape depends on the skeleton. Similarly, in a smaller context, the shape of the cell and the nucleus are mainly regulated by the cytoskeletal and nucleoskeletal elements. The nuclear matrix, which by definition is a detergent, DNase and salt resistant proteinaceous nuclear structure, has been suggested to form the nucleoskeleton responsible for the nuclear integrity. Nuclear mitotic apparatus protein, NuMA, a component of the nuclear matrix, is better known for its mitotic spindle organizing function. NuMA is one of the nuclear matrix proteins suggested to participate in the maintenance of the nuclear integrity during interphase but its interphase function has not been solved to date. This thesis study concentrated on the role of NuMA and the nuclear matrix as structural and functional components of the interphase nucleus. The first two studies clarified the essential role of caspase-3 in the disintegration of the nuclear structures during apoptosis. The second study also showed NuMA and chromatin to co-elute from cells in significant amounts and the apoptotic cleavage of NuMA was clarified to have an important role in the dissociation of NuMA from the chromatin. The third study concentrated on the interphase function of NuMA showing NuMA depletion to result in cell cycle arrest and the cytoplasmic relocalization of NuMA interaction partner GAS41. We suggest that the relocalization of the transcription factor GAS41 may mediate the cell cycle arrest. Thus, this study has given new aspects in the interactions of NuMA, chromatin and the nuclear matrix.

Milk protein polymorphisms in Brazilian Zebu cattle

Five bovine milk protein polymorphisms were studied in Zebuine cattle raised in Brazil, through horizontal electrophoresis on starch gel containing urea and 2-mercaptoethanol, using basic and acidic buffer systems. Allelic frequencies for a-La, b-Lg, aS1-Cn, b-Cn and k-Cn loci were estimated in six Gyr herds (N = 283), six Guzerat herds (N = 205), one Nelore herd (N = 17) and one Sindi herd (N = 22), all from São Paulo or Minas Gerais State, Brazil. Genotypic frequencies observed for each locus and breed studied are in accordance with the assumption of genetic equilibrium, demonstrating absence of high inbreeding levels for the breeds tested. The FST value found indicated significant genetic differentiation among breeds; however, the Gyr and Guzerat herds showed significantly different gene frequencies. Genetic distance estimates among zebuine breeds studied and the Holstein breed, taken as a reference for a taurine breed, showed strong differences between these two racial groups

Thyroid function in post-weaning rats whose dams were fed a low-protein diet during suckling

This study was designed to evaluate the thyroid and pituitary hormone levels in post-weaning rats whose dams were fed a low-protein diet during suckling (21 days). The dams and pups were divided into 2 groups: a control group fed a diet containing 22% protein that supplies the necessary amount of protein for the rat and is the usual content of protein in most commercial rat chow, and a diet group fed a low-protein (8%) diet in which the protein was substituted by an isocaloric amount of starch. After weaning all dams and pups received the 22% protein diet. Two hours before sacrifice of pups aged 21, 30 and 60 days, a tracer dose (0.6 µCi) of 125I was injected (ip) into each animal. Blood and thyroid glands of pups were collected for the determination of serum T4, T3 and TSH and radioiodine uptake. Low protein diet caused a slight decrease in radioiodine uptake at 21 days, and a significant decrease in T3 levels (128 ± 14 vs 74 ± 9 ng/dl, P<0.05), while T4 levels did not change and TSH was increased slightly. At 30 days, T3 and TSH did not change while there was a significant increase in both T4 levels (4.8 ± 0.3 vs 6.1 ± 0.2 µg/dl, P<0.05) and in radioiodine uptake levels (0.34 ± 0.02 vs 0.50 ± 0.03%/mg thyroid, P<0.05). At 60 days serum T3, T4 and TSH levels were normal, but radioiodine uptake was still significantly increased (0.33 ± 0.02 vs 0.41 ± 0.03%/mg thyroid, P<0.05). Thus, it seems that protein malnutrition of the dams during suckling causes hypothyroidism in the pups at 21 days that has a compensatory mechanism increasing thyroid function after refeeding with a 22% protein diet. The radioiodine uptake still remained altered at 60 days, when all the hormonal serum levels returned to the normal values, suggesting a permanent change in the thyroid function

Construction of occluded recombinant baculoviruses containing the full-length cry1Ab and cry1Ac genes from Bacillus thuringiensis

The administration of baculoviruses to insects for bioassay purposes is carried out, in most cases, by contamination of food surfaces with a known amount of occlusion bodies (OBs). Since per os infection is the natural route of infection, occluded recombinant viruses containing crystal protein genes (cry1Ab and cry1Ac) from Bacillus thuringiensis were constructed for comparison with the baculovirus prototype Autographa californica nucleopolyhedrovirus (AcNPV). The transfer vector pAcUW2B was used for construction of occluded recombinant viruses. The transfer vector containing the crystal protein genes was cotransfected with linearized DNA from a non-occluded recombinant virus. The isolation of recombinant viruses was greatly facilitated by the reduction of background "wild type" virus and the increased proportion of recombinant viruses. Since the recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve the pathogenicity of the recombinant viruses when compared with the wild type AcNPV, and in order to compare expression levels of the full-length crystal proteins produced by non-occluded and occluded recombinant viruses the full-length cry1Ab and cry1Ac genes were chosen for construction of occluded recombinant viruses. The recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve its pathogenicity but the size of the larvae infected with the recombinant viruses was significantly smaller than that of larvae infected with the wild type virus.

A role for histone-like protein H1 (H-NS) in the regulation of hemolysin expression by Serratia marcescens

The histone-like protein H1 (H-NS) is an abundant structural component of the bacterial nucleoid and influences many cellular processes including recombination, transcription and transposition. Mutations in the hns gene encoding H-NS are highly pleiotropic, affecting the expression of many unrelated genes. We have studied the role of H-NS on the regulation of hemolysin gene expression in Serratia marcescens. The Escherichia coli hns mutant carrying S. marcescens hemolysin genes on a plasmid constructed by ligation of the 3.2-kb HindIII-SacI fragment of pR02 into pBluescriptIIKS, showed a high level of expression of this hemolytic factor. To determine the osmoregulation of wild-type and hns defective mutants the cells were grown to mid-logarithmic phase in LB medium with 0.06 or 0.3 M NaCl containing ampicillin and kanamycin, whereas to analyze the effect of pH on hemolysin expression, the cells were grown to late-logarithmic phase in LB medium buffered with 0.1 M Tris-HCl, pH 4.5 to 8.0. To assay growth phase-related hemolysin production, bacterial cells were grown in LB medium supplemented with ampicillin and kanamycin. The expression of S. marcescens hemolysin genes in wild-type E. coli and in an hns-defective derivative at different pH and during different growth phases indicated that, in the absence of H-NS, the expression of hemolysin did not vary with pH changes or growth phases. Furthermore, the data suggest that H-NS may play an important role in the regulation of hemolysin expression in S. marcescens and its effect may be due to changes in DNA topology influencing transcription and thus the amount of hemolysin expression. Implications for the mechanism by which H-NS influences gene expression are discussed.