An innate antiviral pathway acting before interferons at epithelial surfaces.

Mucosal surfaces are exposed to environmental substances and represent a major portal of entry for microorganisms. The innate immune system is responsible for early defense against infections and it is believed that the interferons (IFNs) constitute the first line of defense against viruses. Here we identify an innate antiviral pathway that works at epithelial surfaces before the IFNs. The pathway is activated independently of known innate sensors of viral infections through a mechanism dependent on viral O-linked glycans, which induce CXCR3 chemokines and stimulate antiviral activity in a manner dependent on neutrophils. This study therefore identifies a previously unknown layer of antiviral defense that exerts its action on epithelial surfaces before the classical IFN response is operative.

The three-dimensional structure of an H-superfamily conotoxin reveals a granulin fold arising from a common ICK cysteine framework.

Venomous marine cone snails produce peptide toxins (conotoxins) that bind ion channels and receptors with high specificity and therefore are important pharmacological tools. Conotoxins contain conserved cysteine residues that form disulfide bonds that stabilize their structures. To gain structural insight into the large, yet poorly characterized conotoxin H-superfamily, we used NMR and CD spectroscopy along with MS-based analyses to investigate H-Vc7.2 from Conus victoriae, a peptide with a VI/VII cysteine framework. This framework has CysI-CysIV/CysII-CysV/CysIII-CysVI connectivities, which have invariably been associated with the inhibitor cystine knot (ICK) fold. However, the solution structure of recombinantly expressed and purified H-Vc7.2 revealed that although it displays the expected cysteine connectivities, H-Vc7.2 adopts a different fold consisting of two stacked ß-hairpins with opposing ß-strands connected by two parallel disulfide bonds, a structure homologous to the N-terminal region of the human granulin protein. Using structural comparisons, we subsequently identified several toxins and nontoxin proteins with this "mini-granulin" fold. These findings raise fundamental questions concerning sequence-structure relationships within peptides and proteins and the key determinants that specify a given fold.

FAM20C phosphorylation of the RGDSVVYGLR motif in osteopontin inhibits interaction with the αvß3 integrin.

Osteopontin (OPN) is a ubiquitously expressed, multifunctional, and highly phosphorylated protein. OPN contains two neighboring integrin-binding motifs, RGD and SVVYGLR, which mediate interaction with cells. Phosphorylation and proteolytic processing affect the integrin-binding activities of OPN. Here we report that the kinase, FAM20C, phosphorylates Ser146 in the 143 RGDSVVYGLR152 motif of OPN and that Ser146 is phosphorylated in vivo in human and bovine milk. Ser146 is located right next to the RGD motif and close by the regulatory thrombin and plasmin cleavage sites in the OPN sequence. Phosphorylation of Ser146 could potentially affect the proteolytic processing and the integrin-binding activities of OPN. We show that phosphorylation of Ser146 does not affect the susceptibility of OPN for thrombin or plasmin cleavage. However, phosphorylation of Ser146 significantly reduces the RGD-mediated interaction with the αv ß3 integrin in MDA-MB-435 and Moαv cells. This suggests a new mechanism by which specific phosphorylation of OPN can regulate interaction with the αv ß3 integrin and thereby affect OPN-cell interaction.

Hemoglobin polymerization via disulfide bond formation in the hypoxia-tolerant turtle Trachemys scripta: implications for antioxidant defense and O2 transport.

The ability of many reptilian hemoglobins (Hbs) to form high-molecular weight polymers, albeit known for decades, has not been investigated in detail. Given that turtle Hbs often contain a high number of cysteine (Cys), potentially contributing to the red blood cell defense against reactive oxygen species, we have examined whether polymerization of Hb could occur via intermolecular disulfide bonds in red blood cells of freshwater turtle Trachemys scripta, a species that is highly tolerant of hypoxia and oxidative stress. We find that one of the two Hb isoforms of the hemolysate HbA is prone to polymerization in vitro into linear flexible chains of different size that are visible by electron microscopy but not the HbD isoform. Polymerization of purified HbA is favored by hydrogen peroxide, a main cellular reactive oxygen species and a thiol oxidant, and inhibited by thiol reduction and alkylation, indicating that HbA polymerization is due to disulfide bonds. By using mass spectrometry, we identify Cys5 of the αA-subunit of HbA as specifically responsible for forming disulfide bonds between adjacent HbA tetramers. Polymerization of HbA does not affect oxygen affinity, cooperativity, and sensitivity to the allosteric cofactor ATP, indicating that HbA is still fully functional. Polymers also form in T. scripta blood after exposure to anoxia but not normoxia, indicating that they are of physiological relevance. Taken together, these results show that HbA polymers may form during oxidative stress and that Cys5αA of HbA is a key element of the antioxidant capacity of turtle red blood cells.

Pericyte modulation by a functional antibody obtained by a novel single-cell selection strategy.

OBJECTIVE: Pericytes surround the endothelial cells of the microvasculature where they serve as active participants in crucial vascular functions such as angiogenesis, stability, and permeability. However, pericyte loss or dysfunction has been described in a number of pathologies. Targeting pericytes could therefore prove instrumental in the further development of vascular therapeutics. METHODS: To target the pericyte, a proteomic-based approach using antibody phage display was conducted. We present a novel single-cell selection strategy, with a modified selection step to drive the selection of antibodies toward relevant pericyte epitopes. RESULTS: Characterization of the selected antibodies revealed two antibodies with binding specificity for pericytes. The cognate antigen of one of the antibodies was identified as pericyte-expressed fibronectin. This antibody was shown to be a potent inhibitor of pericyte migration and to induce a pro-angiogenic response when included in a pericyte-endothelial cell co-culture angiogenesis assay. CONCLUSIONS: The selection method provides an efficient platform for the selection of functional antibodies which target pericytes. We obtain an antibody that interacts with a fibronectin epitope important for pericyte mobility and functionality. Targeting of this epitope in pathologies where pericytes are implicated could potentially be of therapeutic benefit.

Structural Basis for the Function of Complement Component C4 within the Classical and Lectin Pathways of Complement.

Complement component C4 is a central protein in the classical and lectin pathways within the complement system. During activation of complement, its major fragment C4b becomes covalently attached to the surface of pathogens and altered self-tissue, where it acts as an opsonin marking the surface for removal. Moreover, C4b provides a platform for assembly of the proteolytically active convertases that mediate downstream complement activation by cleavage of C3 and C5. In this article, we present the crystal and solution structures of the 195-kDa C4b. Our results provide the molecular details of the rearrangement accompanying C4 cleavage and suggest intramolecular flexibility of C4b. The conformations of C4b and its paralogue C3b are shown to be remarkably conserved, suggesting that the convertases from the classical and alternative pathways are likely to share their overall architecture and mode of substrate recognition. We propose an overall molecular model for the classical pathway C5 convertase in complex with C5, suggesting that C3b increases the affinity for the substrate by inducing conformational changes in C4b rather than a direct interaction with C5. C4b-specific features revealed by our structural studies are probably involved in the assembly of the classical pathway C3/C5 convertases and C4b binding to regulators.

Redox Regulation of the Superoxide Dismutases SOD3 and SOD2 in the Pulmonary Circulation.

When evaluating the role of redox-regulating signaling in pulmonary vascular diseases, it is intriguing to consider the modulation of key antioxidant enzymes like superoxide dismutase (SOD) because SOD isoforms are regulated by redox reactions, and, in turn, modulate downstream redox sensitive processes. The emerging field of redox biology is built upon understanding the regulation and consequences of tightly controlled and specific reduction-oxidation reactions that are critical for diverse cellular processes including cell signaling. Of relevance, both the site of production of specific reactive oxygen and nitrogen species and the site of the antioxidant defenses are highly compartmentalized within the cell. For example, superoxide is generated during oxidative phosphorylation in the mitochondria as well as by a number of enzymatic sources within the cytosol and at the cell membrane. In the pulmonary circulation, these sources include the mitochondrial electron transport chain, NADPH oxidases (NOX1-4, Duox1,2), nitric oxide synthases, and xanthine oxidase; this important topic has been thoroughly reviewed recently [1]. In parallel with these different cellular sites of superoxide production, the three SOD isoforms are also specifically localized to the cytosol (SOD1), mitochondria (SOD2) or extracellular compartment (SOD3). This chapter focuses on the role of redox mechanisms regulating SOD2 and SOD3, with an emphasis on these processes in the setting of pulmonary hypertension.

Transglutaminase 2-Catalyzed Intramolecular Cross-Linking of Osteopontin.

Osteopontin (OPN) is a multifunctional integrin-binding protein present in several tissues and body fluids. OPN is a substrate for the enzyme transglutaminase 2 (TG2), which catalyzes inter- and intramolecular cross-linking affecting the biological activity of the protein. Polymerization of OPN by intermolecular cross-linking has mostly been studied using relatively high TG2 concentrations, whereas the effect of lower concentrations of TG2 has remained unexplored. Here we show that TG2 at physiologically relevant concentrations predominantly catalyzes the formation of intramolecular cross-links in OPN. By site-directed mutagenesis and mass spectrometry, we demonstrate that Gln(42) and Gln(193) serve as the primary amine acceptor sites for isopeptide bond formation. We find that Gln(42) predominantly is linked to Lys(4) and that Gln(193) participates in a cross-link with Lys(154), Lys(157), or Lys(231). The formation of specific isopeptide bonds was not dependent on OPN phosphorylation, and similar patterns of cross-linking were observed in human and mouse OPN. Furthermore, we find that OPN purified from human urine contains the Lys(154)-Gln(193) isopeptide bond, indicating that intramolecular cross-linking of OPN occurs in vivo. Collectively, these data suggest that specific intramolecular cross-linking in the N- and C-terminal parts of OPN is most likely the dominant step in TG2-catalyzed modification of OPN.

Stanniocalcin-1 Potently Inhibits the Proteolytic Activity of the Metalloproteinase Pregnancy-associated Plasma Protein-A.

Stanniocalcin-1 (STC1) is a disulfide-bound homodimeric glycoprotein, first identified as a hypocalcemic hormone important for maintaining calcium homeostasis in teleost fish. STC1 was later found to be widely expressed in mammals, although it is not believed to function in systemic calcium regulation in these species. Several physiological functions of STC1 have been reported, although many molecular details are still lacking. We here demonstrate that STC1 is an inhibitor of the metzincin metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A), which modulates insulin-like growth factor (IGF) signaling through proteolytic cleavage of IGF-binding proteins (IGFBPs). STC1 potently (Ki = 68 pm) inhibits PAPP-A cleavage of IGFBP-4, and we show in a cell-based assay that STC1 effectively antagonizes PAPP-A-mediated type 1 IGF receptor (IGF1R) phosphorylation. It has recently been found that the homologous STC2 inhibits PAPP-A proteolytic activity, and that this depends on the formation of a covalent complex between the inhibitor and the proteinase, mediated by Cys-120 of STC2. We find that STC1 is unable to bind covalently to PAPP-A, in agreement with the absence of a corresponding cysteine residue. It rather binds to PAPP-A with high affinity (KD = 75 pm). We further demonstrate that both STC1 and STC2 show inhibitory activity toward PAPP-A2, but not selected serine proteinases and metalloproteinases. We therefore conclude that the STCs are proteinase inhibitors, probably restricted in specificity to the pappalysin family of metzincin metalloproteinases. Our data are the first to identify STC1 as a proteinase inhibitor, suggesting a previously unrecognized function of STC1 in the IGF system.

The solution structure of the MANEC-type domain from hepatocyte growth factor activator inhibitor-1 reveals an unexpected PAN/apple domain-type fold.

A decade ago, motif at N-terminus with eight-cysteines (MANEC) was defined as a new protein domain family. This domain is found exclusively at the N-terminus of >400 multi-domain type-1 transmembrane proteins from animals. Despite the large number of MANEC-containing proteins, only one has been characterized at the protein level: hepatocyte growth factor activator inhibitor-1 (HAI-1). HAI-1 is an essential protein, as knockout mice die in utero due to placental defects. HAI-1 is an inhibitor of matriptase, hepsin and hepatocyte growth factor (HGF) activator, all serine proteases with important roles in epithelial development, cell growth and homoeostasis. Dysregulation of these proteases has been causatively implicated in pathological conditions such as skin diseases and cancer. Detailed functional understanding of HAI-1 and other MANEC-containing proteins is hampered by the lack of structural information on MANEC. Although many MANEC sequences exist, sequence-based database searches fail to predict structural homology. In the present paper, we present the NMR solution structure of the MANEC domain from HAI-1, the first three-dimensional (3D) structure from the MANEC domain family. Unexpectedly, MANEC is a new subclass of the PAN/apple domain family, with its own unifying features, such as two additional disulfide bonds, two extended loop regions and additional α-helical elements. As shown for other PAN/apple domain-containing proteins, we propose a similar active role of the MANEC domain in intramolecular and intermolecular interactions. The structure provides a tool for the further elucidation of HAI-1 function as well as a reference for the study of other MANEC-containing proteins.

Fibril Core of Transforming Growth Factor Beta-Induced Protein (TGFBIp) Facilitates Aggregation of Corneal TGFBIp.

Mutations in the transforming growth factor beta-induced (TGFBI) gene result in a group of hereditary diseases of the cornea that are collectively known as TGFBI corneal dystrophies. These mutations translate into amino acid substitutions mainly within the fourth fasciclin 1 domain (FAS1-4) of the transforming growth factor beta-induced protein (TGFBIp) and cause either amyloid or nonamyloid protein aggregates in the anterior and central parts of the cornea, depending on the mutation. The A546T substitution in TGFBIp causes lattice corneal dystrophy (LCD), which manifests as amyloid-type aggregates in the corneal stroma. We previously showed that the A546T substitution renders TGFBIp and the FAS1-4 domain thermodynamically less stable compared with the wild-type (WT) protein, and the mutant FAS1-4 is prone to amyloid formation in vitro. In the present study, we identified the core of A546T FAS1-4 amyloid fibrils. Significantly, we identified the Y571-R588 region of TGFBIp, which we previously found to be enriched in amyloid deposits in LCD patients. We further found that the Y571-R588 peptide seeded fibrillation of A546T FAS1-4, and, more importantly, we demonstrated that native TGFBIp aggregates in the presence of fibrils formed by the core peptide. Collectively, these data suggest an involvement of the Y571-R588 peptide in LCD pathophysiology.

Off-pathway aggregation can inhibit fibrillation at high protein concentrations.

Ribosomal protein S6 fibrillates readily at slightly elevated temperatures and acidic pH. We find that S6 fibrillation is retarded rather than favored when the protein concentration is increased above a threshold concentration of around 3.5mg/mL. We name this threshold concentration C(FR), the concentration at which fibrillation is retarded. Our data are consistent with a model in which this inhibition is due to the formation of an off-pathway oligomeric species with native-like secondary structure. The oligomeric species dominates at high protein concentrations but exists in dynamic equilibrium with the monomer so that seeding with fibrils can overrule oligomer formation and favors fibrillation under C(FR) conditions. Thus, fibrillation competes with formation of off-pathway oligomers, probably due to a monomeric conversion step that is required to commit the protein to the fibrillation pathway. The S6 oligomer is resistant to pepsin digestion. We also report that S6 forms different types of fibrils dependent on protein concentration. Our observations highlight the multitude of conformational states available to proteins under destabilizing conditions.

Murine extracellular superoxide dismutase is converted into the inactive fold by the Ser195Cys mutation.

We have previously shown that human extracellular superoxide dismutase (EC-SOD) exists as two variants with differences in their disulfide bridge patterns: one form is the active enzyme (aEC-SOD), and the other is inactive (iEC-SOD). The availability of both active and inactive folding variants significantly reduces the specific activity of EC-SOD in vivo. Both forms are produced during biosynthesis, but the underlying folding mechanisms remain unclear. To address this issue, we expressed EC-SOD in heterologous systems that do not endogenously express iEC-SOD. Rodents express only aEC-SOD because they lack Cys195 (human EC-SOD sequence numbering), which is essential for the formation of iEC-SOD. However, cultured hamster cells and transgenic mice expressing human EC-SOD were able to produce both human a- and iEC-SOD variants, which led us to hypothesize that the folding was sequence-dependent rather than a property of the expression system. To substantiate this hypothesis, we expressed murine EC-SOD in a human cell line, and as expected, only aEC-SOD was produced. Significantly, when Cys195 was introduced, both murine aEC-SOD and a novel murine iEC-SOD were generated, and the specific activity of the murine EC-SOD was significantly reduced by the mutation. Collectively, these data suggest that Cys195 actuates the formation of iEC-SOD, independent of the expression system or host. In addition, the dual-folding pathway most likely requires biosynthesis factors that are common to both humans and rodents.

Bone Formation in Zebrafish: The Significance of DAF-FM DA Staining for Nitric Oxide Detection.

DAF-FM DA is widely used as a live staining compound to show the presence of nitric oxide (NO) in cells. Applying this stain to live zebrafish embryos is known to indicate early centers of bone formation, but the precise (cellular) location of the signal has hitherto not been revealed. Using sections of zebrafish embryos live-stained with DAF-FM DA, we could confirm that the fluorescent signals were predominantly located in areas of ongoing bone formation. Signals were observed in the bone and tooth matrix, in the notochord sheath, as well as in the bulbus arteriosus. Surprisingly, however, they were exclusively extracellular, even after very short staining times. Von Kossa and Alizarin red S staining to reveal mineral deposits showed that DAF-FM DA stains both the mineralized and non-mineralized bone matrix (osteoid), excluding that DAF-FM DA binds non-specifically to calcified structures. The importance of NO in bone formation by osteoblasts is nevertheless undisputed, as shown by the absence of bone structures after the inhibition of NOS enzymes that catalyze the formation of NO. In conclusion, in zebrafish skeletal biology, DAF-FM DA is appropriate to reveal bone formation in vivo, independent of mineralization of the bone matrix, but it does not demonstrate intracellular NO.

Flexibility of the thrombin-activatable fibrinolysis inhibitor pro-domain enables productive binding of protein substrates.

We have previously reported that thrombin-activatable fibrinolysis inhibitor (TAFI) exhibits intrinsic proteolytic activity toward large peptides. The structural basis for this observation was clarified by the crystal structures of human and bovine TAFI. These structures evinced a significant rotation of the pro-domain away from the catalytic moiety when compared with other pro-carboxypeptidases, thus enabling access of large peptide substrates to the active site cleft. Here, we further investigated the flexible nature of the pro-domain and demonstrated that TAFI forms productive complexes with protein carboxypeptidase inhibitors from potato, leech, and tick (PCI, LCI, and TCI, respectively). We determined the crystal structure of the bovine TAFI-TCI complex, revealing that the pro-domain was completely displaced from the position observed in the TAFI structure. It protruded into the bulk solvent and was disordered, whereas TCI occupied the position previously held by the pro-domain. The authentic nature of the presently studied TAFI-inhibitor complexes was supported by the trimming of the C-terminal residues from the three inhibitors upon complex formation. This finding suggests that the inhibitors interact with the active site of TAFI in a substrate-like manner. Taken together, these data show for the first time that TAFI is able to form a bona fide complex with protein carboxypeptidase inhibitors. This underlines the unusually flexible nature of the pro-domain and implies a possible mechanism for regulation of TAFI intrinsic proteolytic activity in vivo.

Functional amyloid in Pseudomonas.

Amyloids are highly abundant in many microbial biofilms and may play an important role in their architecture. Nevertheless, little is known of the amyloid proteins. We report the discovery of a novel functional amyloid expressed by a Pseudomonas strain of the P. fluorescens group. The amyloid protein was purified and the amyloid-like structure verified. Partial sequencing by MS/MS combined with full genomic sequencing of the Pseudomonas strain identified the gene coding for the major subunit of the amyloid fibril, termed fapC. FapC contains a thrice repeated motif that differs from those previously found in curli fimbrins and prion proteins. The lack of aromatic residues in the repeat shows that aromatic side chains are not needed for efficient amyloid formation. In contrast, glutamine and asparagine residues seem to play a major role in amyloid formation as these are highly conserved in curli, prion proteins and FapC. fapC is conserved in many Pseudomonas strains including the opportunistic pathogen P. aeruginosa and is situated in a conserved operon containing six genes, of which one encodes a fapC homologue. Heterologous expression of the fapA-F operon in Escherichia coli BL21(DE3) resulted in a highly aggregative phenotype, showing that the operon is involved in biofilm formation.

Superoxide dismutase 3 is expressed in bone tissue and required for normal bone homeostasis and mineralization.

Superoxide dismutase 3 (SOD3) is an extracellular protein with the capacity to convert superoxide into hydrogen peroxide, an important secondary messenger in redox regulation. To investigate the utility of zebrafish in functional studies of SOD3 and its relevance for redox regulation, we have characterized the zebrafish orthologues; Sod3a and Sod3b. Our analyses show that both recombinant Sod3a and Sod3b express SOD activity, however, only Sod3b is able to bind heparin. Furthermore, RT-PCR analyses reveal that sod3a and sod3b are expressed in zebrafish embryos and are present primarily in separate organs in adult zebrafish, suggesting distinct functions in vivo. Surprisingly, both RT-PCR and whole mount in situ hybridization showed specific expression of sod3b in skeletal tissue. To further investigate this observation, we compared femoral bone obtained from wild-type and SOD3-/- mice to determine whether a functional difference was apparent in healthy adult mice. Here we report, that bone from SOD3-/- mice is less mineralized and characterized by significant reduction of cortical and trabecular thickness in addition to reduced mechanical strength. These analyses show that SOD3 plays a hitherto unappreciated role in bone development and homeostasis.

Transport of a Peptide from Bovine αs1-Casein across Models of the Intestinal and Blood-Brain Barriers.

The effect of food components on brain growth and development has attracted increasing attention. Milk has been shown to contain peptides that deliver important signals to the brains of neonates and infants. In order to reach the brain, milk peptides have to resist proteolytic degradation in the gastrointestinal tract, cross the gastrointestinal barrier and later cross the highly selective blood-brain barrier (BBB). To investigate this, we purified and characterized endogenous peptides from bovine milk and investigated their apical to basal transport by using human intestinal Caco-2 cells and primary porcine brain endothelial cell monolayer models. Among 192 characterized milk peptides, only the αS1-casein peptide 185PIGSENSEKTTMPLW199, and especially fragments of this peptide processed during the transport, could cross both the intestinal barrier and the BBB cell monolayer models. This peptide was also shown to resist simulated gastrointestinal digestion. This study demonstrates that a milk derived peptide can cross the major biological barriers in vitro and potentially reach the brain, where it may deliver physiological signals.

The dynamic uptake and release of SOD3 from intracellular stores in macrophages modulates the inflammatory response.

Superoxide dismutase 3 (SOD3) is an extracellular enzyme with the capacity to modulate extracellular redox conditions by catalyzing the dismutation of superoxide to hydrogen peroxide. In addition to synthesis and release of this extracellular protein via the secretory pathway, several studies have shown that the protein also localizes to intracellular compartments in neutrophils and macrophages. Here we show that human macrophages release SOD3 from an intracellular compartment within 30 min following LPS stimulation. This release acutely increases the level of SOD3 on the cell surface as well as in the extracellular environment. Generation of the intracellular compartment in macrophages is supported by endocytosis of extracellular SOD3 via the LDL receptor-related protein 1 (LRP1). Using bone marrow-derived macrophages established from wild-type and SOD3-/- mice, we further show that the pro-inflammatory profile established in LPS-stimulated cells is altered in the absence of SOD3, suggesting that the active release of this protein affects the inflammatory response. The internalization and acute release from stimulated macrophages indicates that SOD3 not only functions as a passive antioxidant in the extracellular environment, but also plays an active role in modulating redox signaling to support biological responses.

Grafting phenolics onto milk protein via conjugated polymerization for delivery of multiple functionalities: Synthesis and characterization.

A synthetic scenario for functionalization of ß-lactoglobulin (ßLg) with polymeric units containing caffeic acid (ßLg-polyCA) was developed; and all intermediates and final products were structurally confirmed using nuclear magnetic resonance spectroscopy, matrix assisted laser desorption ionization time-of-flight mass spectrometry, and physico-chemically characterized using differential scanning calorimetry and circular dichroism. The antioxidant properties and emulsion stability of ßLg, ßLg-CA conjugate and ßLg-polyCA based systems containing high percentage of fish oil (50%) were evaluated; and ßLg-polyCA presented the highest antioxidant and free radical-scavenging activity based on DPPH, ABTS and HS scavenging assays (92.4, 87.92 and 67.35% respectively). Thiobarbituric acid (TBARS) test demonstrated that compared to native ßLg, ßLg-polyCA afford up 4-5 fold of inhibition of oxidative rancidity and displayed drastic secondary structure changes. Compared to native ßLg based emulsions, ßLg-polyCA had larger oil droplet sizes, stronger negative zeta potentials (-69.9 mv), narrower size distributions (PDI: 0.22) and less creaming index.