The Relationship between S. aureus and Branched-Chain Amino Acids Content in Composite Cow Milk.

The early diagnosis of mastitis is an essential factor for the prompt detection of the animal for further actions. In fact, if not culled, infected cows must be segregated from the milking herd and milked last, or milked with separate milking units. Besides microbiological analysis, the somatic cell count (SCC) commonly used as predictor of intramammary infection, frequently lead to a misclassification of milk samples. To overcome these limitations, more specific biomarkers are continuously evaluated. The total amino acid content increases significantly in mastitic milk compared to normal milk. S. aureus requires branched-chain amino acids (BCAAs-isoleucine, leucine, and valine) for protein synthesis, branched-chain fatty acids synthesis, and environmental adaptation by responding to their availability via transcriptional regulators. The increase of BCAAs in composite milk has been postulated to be linked to mammary infection by S. aureus. The aim of this work is to demonstrate, by a direct ion-pairing reversed-phase method, based on the use of the evaporative light-scattering detector (IP-RP-HPLC-ELSD), applied to 65 composite cow milk samples, a correlation between the concentration of isoleucine and leucine, and S. aureus load. The correlation coefficient, r, was found to be 0.102 for SCC (p = 0.096), 0.622 for isoleucine (p < 0.0001), 0.586 for leucine (p < 0.0001), 0.013 for valine (p = 0.381), and 0.07 for tyrosine (p = 0.034), standing for a positive correlation between S. aureus and isoleucine and leucine concentration. The link between the content of BCAAs, isoleucine and leucine, and udder infection by S. aureus demonstrated with our study has an important clinical value for the rapid diagnosis of S. aureus mastitis in cows.

Evaluation of (arene)Ru(II) complexes of curcumin as inhibitors of dipeptidyl peptidase IV.

Curcumin, the main component of Curcuma longa, shows an anti-hyperglycemic effect and improved insulin sensitivity. This action may be attributed at least in part to its anti-inflammatory properties and also to its possible interaction with dipeptidyl peptidase-4 (DPPIV), the enzyme that the conversion of glucagon-like peptide-1 (GLP-1), responsible for glucose tolerance into inactive GLP-1. In this work we evaluated the inhibitory activities of a series of different arene-Ru(II)-curcumin complexes on bovine kidney dipeptidyl peptidase-4 (DPPIV). We studied also the interaction of these inhibitors on the enzyme with fluorescence studies displaying the binding poses with molecular docking studies. Specifically organometallic ruthenium(II) complexes of general formula [(η(6)-arene)Ru(curcuminato)Cl], with arene being p-(i)PrC6H4Me (1), C6H6 (2), and C6Me6 (3), were evaluated for their inhibition activity toward the mammalian enzyme. Among them, 2 suppressed DPPIV activities more potently (Ki = 20.2(±0.8) µM) than 1, 3, or free curcumin, and all complexes showed an antioxidant activity as free curcumin. As shown from our docking simulations a putative binding site of the compound 2 was found on subdomains S1 and S2 of DPP-IV, where S1 hydrophobic pocket includes catalytic residues and is the primary determinant of substrate specificity for the enzyme. Collectively, our results demonstrate that the complexation of curcumin with ruthenium(II) could be a promising starting point for the development of curcumin-based DPPIV inhibitors.

Long-lasting neuroprotection and neurological improvement in stroke models with new, potent and brain permeable inhibitors of poly(ADP-ribose) polymerase.

BACKGROUND AND PURPOSES: Thienyl-isoquinolone (TIQ-A) is a relatively potent PARP inhibitor able to reduce post-ischaemic neuronal death in vitro. Here we have studied, in different stroke models in vivo, the neuroprotective properties of DAMTIQ and HYDAMTIQ, two TIQ-A derivatives able to reach the brain and to inhibit PARP-1 and PARP-2. EXPERIMENTAL APPROACH: Studies were carried out in (i) transient (2 h) middle cerebral artery occlusion (tMCAO), (ii) permanent MCAO (pMCAO) and (iii) electrocoagulation of the distal portion of MCA in conjunction with transient (90 min) bilateral carotid occlusion (focal cortical ischaemia). KEY RESULTS: In male rats with tMCAO, HYDAMTIQ (0.1-10 mg·kg(-1)) injected i.p. three times, starting 4 h after MCAO, reduced infarct volumes by up to 70%, reduced the loss of body weight by up to 60% and attenuated the neurological impairment by up to 40%. In age-matched female rats, HYDAMTIQ also reduced brain damage. Protection, however, was less pronounced than in the male rats. In animals with pMCAO, HYDAMTIQ administered 30 min after MCAO reduced infarct volumes by approximately 40%. In animals with focal cortical ischaemia, HYDAMTIQ treatment decreased post-ischaemic accumulation of PAR (the product of PARP activity) and the presence of OX42-positive inflammatory cells in the ischaemic cortex. It also reduced sensorimotor deficits for up to 90 days after MCAO. CONCLUSION AND IMPLICATIONS: Our results show that HYDAMTIQ is a potent PARP inhibitor that conferred robust neuroprotection and long-lasting improvement of post-stroke neurological deficits.

Pharmacological inhibition of poly(ADP-ribose) polymerase (PARP) activity in PARP-1 silenced tumour cells increases chemosensitivity to temozolomide and to a N3-adenine selective methylating agent.

We recently demonstrated that poly(ADP-ribose) polymerase (PARP)-1 is involved in angiogenesis and tumour aggressiveness. In this study we have compared the influence of abrogation of PARP-1 expression by stable gene silencing to that of the pharmacological inhibition of cellular PARP activity using PARP-1/-2 inhibitors on the chemosensitivity of tumour cells to the wide spectrum methylating agent temozolomide (TMZ) and to the N3-adenine selective methylating agent {1-methyl-4-[1-methyl-4-(3-methoxysulfonylpropanamido)pyrrole-2-carboxamido]-pyrrole-2-carboxamido}propane (Me-Lex). Silencing of PARP-1 in melanoma or cervical carcinoma lines enhanced in vitro sensitivity to TMZ and Me- Lex, and induced a higher level of cell accumulation at the G2/M phase of cell cycle with respect to controls. GPI 15427, which inhibits both PARP-1 and PARP-2, increased sensitivity to TMZ and Me-Lex both in PARP-1-proficient and - deficient cells. However, it induced different cell cycle modulations depending on PARP-1 expression, provoking a G2/M arrest only in PARP-1 silenced cells. Treatment of PARP-1 silenced cells with TMZ or Me-Lex resulted in a more extensive phosphorylation of Chk-1 and p53 as compared to PARP-1 proficient cells. The combination of the methylating agents with GPI 15427 increased Chk-1 and p53 phosphorylation both in PARP-1 proficient or deficient cells. When mice challenged with PARP-1 silenced melanoma cells were treated with the TMZ and PARP inhibitor combination there was an additional reduction in tumour growth with respect to treatment with TMZ alone. These results suggest the involvement of PARP-2 or other PARPs, in the repair of DNA damage provoked by methylating agents, highlighting the importance of targeting both PARP-1 and PARP-2 for cancer therapy.

Thymoquinone, a potential therapeutic agent of Nigella sativa, binds to site I of human serum albumin.

Thymoquinone (TQ) is the main constituent of Nigella sativa essential oil which shows promising in vitro and in vivo antineoplastic growth inhibition against various tumor cell lines. Because of the increasing interest to test it in pre-clinical and clinical researches for assessing its health benefits, we here evaluate the interactions between TQ and human serum albumin (HSA), a possible carrier of this drug in vivo. Binding to HSA was studied using different spectroscopic techniques. Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopies suggest that the association between TQ and HSA does not affect the secondary structure of HSA. Using fluorescence spectroscopy, one mole of TQ was found to bind one mole of HSA with a binding constant of 2.39 +/- 0.2 10(4)M(-1). At 25 degrees C (pH 7.4), van't Hoff's enthalpy and entropy that accompany the binding were found to be -10.24 kJ/mol(-1) and 45 J/mol(-1)K(-1) respectively. The thermodynamic analysis of the TQ-HSA complex formation shows that the binding process is enthalpy driven and spontaneous, and that hydrophobic interactions are the predominant intermolecular forces stabilizing the complex. Furthermore, displacement experiments using warfarin and ibuprofen indicate that TQ could bind to site I of HSA, which is also in agreement with the results of the molecular modeling study.

Selective PARP-2 inhibitors increase apoptosis in hippocampal slices but protect cortical cells in models of post-ischaemic brain damage.

BACKGROUND AND PURPOSE: Poly(ADP-ribose) polymerases (PARP)-1 and PARP-2 play complementary tasks in the maintenance of genomic integrity, but their role in cell death or survival processes is rather different. A recently described series of selective PARP-2 inhibitors (UPF-1035, UPF-1069) were used to study the role of PARP-1 and PARP-2 in post-ischaemic brain damage. EXPERIMENTAL APPROACH: We evaluated post-ischaemic brain damage in two different in vitro models: rat organotypic hippocampal slices exposed to oxygen-glucose deprivation (OGD) for 20-30 min, a model characterized by apoptosis-like cell death and mouse mixed cortical cell cultures exposed to 60 min OGD, a model in which cells die with mostly necrosis-like features. KEY RESULTS: In organotypic hippocampal slices, PARP-2 inhibition with UPF-1069 (0.01-1 micromolxL(-1)) caused a concentration-dependent exacerbation (up to 155%) of OGD-induced CA1 pyramidal cell death. Higher concentrations, acting on both PARP-1 and PARP-2, had no effect on OGD injury. In mouse mixed cortical cells exposed to OGD, on the contrary, UPF-1069 (1-10 micromolxL(-1)) significantly reduced post-ischaemic damage. CONCLUSION AND IMPLICATIONS: Selective PARP-2 inhibitors increased post-OGD cell death in a model characterized by loss of neurons through a caspase-dependent, apoptosis-like process (hippocampal slice cultures), but they reduced post-OGD damage and increased cell survival in a model characterized by a necrosis-like process (cortical neurons). UPF-1069 may be a valuable tool to explore the function of PARP-2 in biological systems and to examine the different roles of PARP isoenzymes in the mechanisms of cell death and survival.

In vitro retentive strength of metal superstructures cemented to solid abutments.

AIM: The aim of this study was to evaluate the effect of sandblasting on the retentive strength of metal single crowns luted with a resin cement to Straumann implant/abutment assemblies. METHODS: Fifty 4.1 mm-wide Straumann solid screw implants were mounted in self-polymerizing soft resin. Standard 5.5 mm-high, 8 degree tapered solid abutments were placed on each implant and torqued to 35 Ncm. Fifty metal castings were made using prefabricated burn-out caps. Each implant/abutment assembly and its corresponding metal casting was numbered and they were divided into two groups of 25. In the test group, the external surface of the abutments and the cavosurface of the corresponding metal casting were sandblasted. In the control group, neither the implant abutment nor the metal casting underwent sandblasting. Each metal casting was cemented onto its respective implant/abutment assembly using Panavia 21 (Kuraray Europe GmbH, Dusseldorf, Ger-many) resin cement. Specimens were then subjected to a pull-out test using a universal Instron testing machine. The load required to dislodge each crown was recorded and mean values were calculated for each group. Retention values were analyzed using the ANOVA test. RESULTS: The test group showed a higher mean retention value (83.78 kgf+/-19.61) than the control group (44.03 kgf+/-9.45) and the difference was statistically significant. CONCLUSION: Within the limitations of this in vitro study, the results suggest that sandblasting treatment significantly increases mechanical retention of crowns cemented using a resin cement. It is at the clinician's discretion to evaluate whether additional retention is desired in cementing an implant-supported fixed partial denture.

Modeling of poly(ADP-ribose)polymerase (PARP) inhibitors. Docking of ligands and quantitative structure-activity relationship analysis.

Poly(ADP-ribose)polymerase-1 (PARP-1) is a nuclear enzyme that has recently emerged as an important player in the mechanisms leading to postischemic neuronal death, and PARP inhibitors have been proposed as potential neuroprotective agents. With the aim of clarifying the structural basis responsible for PARP inhibition, we carried out a computational study on 46 inhibitors available through the literature. Our computational approach is composed of three parts. In the first one, representative PARP inhibitors have been docked into the crystallographic structure of the catalytic domain of PARP by using the Autodock 2.4 program. The docking studies thus carried out have provided an alignment scheme that has been instrumental for superimposing all the remaining inhibitors. Upon the basis of this alignment scheme, a quantitative structure-activity relationship (QSAR) analysis has been carried out after electrostatic and steric interaction energies have been computed with the RECEPTOR program. The QSAR analysis yielded a predictive model able to explain much of the variance of the 46-compound data set. The inspection of the QSAR coefficients revealed that the major driving force for potent inhibition is given by the extension of the contact surface between enzyme and inhibitors while electrostatic energy and hydrogen bonding capability play a minor role. Finally, the projection of the QSAR coefficients back onto the X-ray structure of the catalytic domain of PARP provides insights into the role played by specific amino acid residues. This information will be useful to address the design of new selective and potent PARP inhibitors.

Metabotropic glutamate receptors: structure and new subtype-selective ligands.

Metabotropic glutamate receptors (mGluRs) constitute an attractive target for the development of potential neuroprotective agents. Recent advances in the elucidation of the peculiar molecular architecture of mGluRs and in the design and synthesis of subtype selective ligands are discussed.

Synthesis and biological evaluation of 2-(3'-(1H-tetrazol-5-yl) bicyclo[1.1.1]pent-1-yl)glycine (S-TBPG), a novel mGlu1 receptor antagonist.

The design and synthesis of 2-(3'-(1H-tetrazol-5-yl)bicyclo[1.1.1]pent-1-yl)glycine (S-TBPG), a novel mGluR1 antagonist is reported. S-TBPG is characterized by the bioisosteric replacement of the distal carboxy group of 2-(3'-carboxybicyclo [1.1.1]pent-1-yl)glycine (S-CBPG) by a tetrazolyl moiety. Despite a moderate reduction in potency, S-TBPG is a selective mGluR1 antagonist (69 microM), with no activity at other mGluR subtypes. The interesting biological profile of S-TBPG, coupled with its peculiar chemical structure, is discussed in terms of the structure activity relationship (SAR) of mGluR1 antagonists.

Adenosine deaminase: functional implications and different classes of inhibitors.

Adenosine deaminase (ADA) is an enzyme of the purine metabolism which catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine, respectively. This ubiquitous enzyme has been found in a wide variety of microorganisms, plants, and invertebrates. In addition, it is present in all mammalian cells that play a central role in the differentiation and maturation of the lymphoid system. However, despite a number of studies performed to date, the physiological role played by ADA in the different tissues is not clear. Inherited ADA deficiency causes severe combined immunodeficiency disease (ADA-SCID), in which both B-cell and T-cell development is impaired. ADA-SCID has been the first disorder to be treated by gene therapy, using polyethylene glycol-modified bovine ADA (PEG-ADA). Conversely, there are several diseases in which the level of ADA is above normal. A number of ADA inhibitors have been designed and synthesized, classified as ground-state and transition-state inhibitors. They may be used to mimic the genetic deficiency of the enzyme, in lymphoproliferative disorders or immunosuppressive therapy (i.e., in graft rejection), to potentiate the effect of antileukemic or antiviral nucleosides, and, together with adenosine kinase, to reduce breakdown of adenosine in inflammation, hypertension, and ischemic injury.

2-Substituted N-ethylcarboxamidoadenosine derivatives as high-affinity agonists at human A3 adenosine receptors.

A number of 2-substituted 5'-N-ethylcarboxamidoadenosine (NECA) derivatives was investigated for their affinity and selectivity at human A3 adenosine receptors. The compounds were tested in radioligand competition studies and modulation of adenylyl cyclase activity on membranes from CHO cell lines stably transfected with the four human adenosine receptor subtypes. In binding studies the most potent compound, 2-(3-hydroxy-3-phenyl)propyn-1-yl-NECA (PHPNECA), exhibited a subnanomolar affinity for A3 adenosine receptors with a Ki value of 0.4 nM. As opposed to the limited A3 selectivity of PHPNECA, a 100-fold selectivity compared to both A1 and A2A receptors was found for 2-(2-phenyl)ethynyl-NECA (PENECA; Ki 6 nM). The EC50 values for activation of adenylyl cyclase via A2A adenosine receptors were in good agreement with the respective Ki values from binding experiments. In contrast, IC50 values for A1 and A3 receptor-mediated inhibition of adenylyl cyclase were shifted to higher values compared to the respective affinities determined in radioligand competition studies. Similar discrepancies between binding and functional data have been observed for the inhibitory A1 adenosine receptor in previous studies. Therefore, the same A3 selectivity of PENECA compared to A1 receptors was found in binding and adenylyl cyclase inhibition whereas the selectivity compared to A2A receptors that was detected in ligand binding was obscured in the functional assay. The series of compounds presented in this study identifies 2-substitution of the purine system as a promising target for the development of A3-selective high-affinity ligands.

Synthesis and receptor affinity of polysubstituted adenosines.

In a search for potent and selective adenosine agonists it has been found that 2-hexynyladenosine-5'-N-ethyluronamide (HENECA) displays high affinity at rat A2A receptor combined with a good A2A vs A1 selectivity. The finding that HENECA shows good affinity also for A3 receptors prompted us to investigate the effect of various substituents in different positions of this molecule.

Structure-activity relationships of bisphosphate nucleotide derivatives as P2Y1 receptor antagonists and partial agonists.

The P2Y1 receptor is present in the heart, in skeletal and various smooth muscles, and in platelets, where its activation is linked to aggregation. Adenosine 3',5'- and 2',5'-bisphosphates have been identified as selective antagonists at the P2Y1 receptor (Boyer et al. Mol. Pharmacol. 1996, 50, 1323-1329) and have been modified structurally to increase receptor affinity (Camaioni et al. J. Med. Chem. 1998, 41, 183-190). We have extended the structure-activity relationships to a new series of deoxyadenosine bisphosphates with substitutions in the adenine base, ribose moiety, and phosphate groups. The activity of each analogue at P2Y1 receptors was determined by measuring its capacity to stimulate phospholipase C in turkey erythrocyte membranes (agonist effect) and to inhibit phospholipase C stimulation elicited by 10 nM 2-(methylthio)adenosine 5'-diphosphate (antagonist effect). 2'-Deoxyadenosine bisphosphate analogues containing halo, amino, and thioether groups at the 2-position of the adenine ring were more potent P2Y1 receptor antagonists than analogues containing various heteroatom substitutions at the 8-position. An N6-methyl-2-chloro analogue, 6, was a full antagonist and displayed an IC50 of 206 nM. Similarly, N6-methyl-2-alkylthio derivatives 10, 14, and 15 were nearly full antagonists of IC50 < 0.5 microM. On the ribose moiety, 2'-hydroxy, 4'-thio, carbocyclic, and six-membered anhydrohexitol ring modifications have been prepared and resulted in enhanced agonist properties. The 1,5-anhydrohexitol analogue 36 was a pure agonist with an EC50 of 3 microM, i.e., similar in potency to ATP. 5'-Phosphate groups have been modified in the form of triphosphate, methyl phosphate, and cyclic 3',5'-diphosphate derivatives. The carbocyclic analogue had enhanced agonist efficacy, and the 5'-O-phosphonylmethyl modification was tolerated, suggesting that deviations from the nucleotide structure may result in improved utility as pharmacological probes. The N6-methoxy modification eliminated receptor affinity. Pyrimidine nucleoside 3', 5'-bisphosphate derivatives were inactive as agonists or antagonists at P2Y receptor subtypes.

Synthesis of di- and tri-substituted adenosine derivatives and their affinities at human adenosine receptor subtypes.

The synthesis of 2-(hex-1-ynyl)adenosine derivatives substituted at the N6- and/or 5'-position was carried out on the basis that 2-(hex-1-ynyl)adenosine-5'-N-ethyluronamide (HENECA, 2) showed good affinity and different degree of selectivity for rat adenosine receptors. All new compounds were tested in radioligand binding and adenylyl cyclase assays with recently cloned human A1, A2A, A2B, and A3 adenosine receptors.

New substituted 9-alkylpurines as adenosine receptor ligands.

In the present study an investigation of the structure-activity relationships in 9-ethylpurine derivatives, aimed at preparing A1, A2A, A2B, and A3 selective adenosine receptor antagonists, was undertaken. Our synthetic approach was to introduce various substituents (amino, alkoxy and alkynyl groups) into the 2-, 6-, or 8-positions of the purine ring. The starting compounds for each series of derivatives were respectively: 2-iodo-9-ethyladenine (9), obtained from 2-amino-6-chloropurine (5); 9-ethyl-6-iodo-9H-purine (11), 8-bromo-9-ethyl-adenine (3) and 8-bromo-9-ethyl-6-iodo-9H-purine (13), obtained from 9-ethyl-adenine (2). The synthesized compounds were tested in in vitro radioligand binding assays at A1, A2A, and A3 human adenosine receptor subtypes. Due to the lack of a suitable radioligand the affinity of the 9-ethyladenine derivatives at A2B adenosine receptors was determined in adenylyl cyclase experiments. In general, the series of 9-ethylpurine derivatives exhibited a similar pharmacological profile at A1 and A2A receptors whereas some differences were found for the A3 and the A2B subtypes. 8-Bromo-9-ethyladenine (3) showed higher affinity for all receptors in comparison to the parent compound 2, and the highest affinity in the series for the A2A and A2B subtypes (Ki = 0.052 and 0.84 microM, respectively). Analyzing the different substituents, a phenethoxy group in 2-position (10a) gave the highest A2A versus A2B selectivity (near 400-fold), whereas a phenethylamino group in 2- and 6-position (10b and 12b, respectively) improved the affinity at A2B receptors, compared to the parent compound 2. The presence of a hexynyl substituent in 8-position led to a compound with good affinity at the A3 receptor (4d, Ki = 0.62 microM), whereas (ar)alkynyl groups are detrimental for the potency at the A2B subtype. These differences give raise to the hope that further modifications will result in the development of currently unavailable leads with good affinity and selectivity for A2B adenosine receptors.

Competitive and selective antagonism of P2Y1 receptors by N6-methyl 2'-deoxyadenosine 3',5'-bisphosphate.

The antagonist activity of N6-methyl 2'-deoxyadenosine 3',5'-bisphosphate (N6MABP) has been examined at the phospholipase C-coupled P2Y1 receptor of turkey erythrocyte membranes. N6MABP antagonized 2MeSATP-stimulated inositol phosphate hydrolysis with a potency approximately 20 fold greater than the previously studied parent molecule, adenosine 3',5'-bisphosphate. The P2Y1 receptor antagonism observed with N6MABP was competitive as revealed by Schild analysis (pK(B) = 6.99 +/- 0.13). Whereas N6MABP was an antagonist at the human P2Y1 receptor, no antagonist effect of N6MABP was observed at the human P2Y2, human P2Y4 or rat P2Y6 receptors.

Human P2Y1 receptor: molecular modeling and site-directed mutagenesis as tools to identify agonist and antagonist recognition sites.

The molecular basis for recognition by human P2Y1 receptors of the novel, competitive antagonist 2'-deoxy-N6-methyladenosine 3', 5'-bisphosphate (MRS 2179) was probed using site-directed mutagenesis and molecular modeling. The potency of this antagonist was measured in mutant receptors in which key residues in the transmembrane helical domains (TMs) 3, 5, 6, and 7 were replaced by Ala or other amino acids. The capacity of MRS 2179 to block stimulation of phospholipase C promoted by 2-methylthioadenosine 5'-diphosphate (2-MeSADP) was lost in P2Y1 receptors having F226A, K280A, or Q307A mutations, indicating that these residues are critical for the binding of the antagonist molecule. Mutation of the residues His132, Thr222, and Tyr136 had an intermediate effect on the capacity of MRS 2179 to block the P2Y1 receptor. These positions therefore appear to have a modulatory role in recognition of this antagonist. F131A, H277A, T221A, R310K, or S317A mutant receptors exhibited an apparent affinity for MRS 2179 that was similar to that observed with the wild-type receptor. Thus, Phe131, Thr221, His277, and Ser317 are not essential for antagonist recognition. A computer-generated model of the human P2Y1 receptor was built and analyzed to help interpret these results. The model was derived through primary sequence comparison, secondary structure prediction, and three-dimensional homology building, using rhodopsin as a template, and was consistent with data obtained from mutagenesis studies. We have introduced a "cross-docking" procedure to obtain energetically refined 3D structures of the ligand-receptor complexes. Cross-docking simulates the reorganization of the native receptor structure induced by a ligand. A putative nucleotide binding site was localized and used to predict which residues are likely to be in proximity to agonists and antagonists. According to our model TM6 and TM7 are close to the adenine ring, TM3 and TM6 are close to the ribose moiety, and TM3, TM6, and TM7 are near the triphosphate chain.