Top PDF G Protein-Coupled Odorant Receptors: from sequence to structure

G Protein-Coupled Odorant Receptors: from sequence to structure

G Protein-Coupled Odorant Receptors: from sequence to structure

Introduction Odorant molecules are perceived by mammals through extraordinary subtle mechanisms, notably involving odorant receptors (ORs).(1) In human, the family of genes coding for ORs is one of the largest, as it represents more than 2% of our genome. At the protein level, ORs account for more than 4% of our proteome and constitute the largest sub-family of Class-A (or Rhodopsin like) G Protein-Coupled Receptors (GPCR). GPCRs are seven-transmembrane domain (7 TM) proteins that transmit extracellular signals across the plasma membrane. Although structures of some Class-A members have been experimentally solved, no experimental structure is to date available for any OR. For now, molecular modeling appears as the only way to propose atomic-level mechanisms of either ligand selectivity or receptor activation for these proteins on a structural basis. Models can either be made ab-initio or based on sequence homology with respect to known experimental structures.(2; 3) In both cases, sequence alignment between the candidate receptor and the experimentally determined templates is undoubtedly the crucial step.
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Protein Evolution From Sequence To Structure.

Protein Evolution From Sequence To Structure.

In contrast to the first two sections, which attempt to predict the function of a protein, chapter 3 elucidates the structural/functional characteristics of regions of a protein. The conserved complex structure of serpins and their extensive sequence divergence make them good model proteins to study covariation among protein residues. It is expected that the residues at certain sites of the protein will strongly affect evolution of other sites in close three-dimensional proximity. At such sites, residue substitutions which tend to destabilize a particular structure or function are probably corrected or compensated for by other substitutions at neighboring sites. For example, if a substitution causes a reduction of volume in the protein core, which destabilizes the protein, only compensating substitutions in a few adjacent residues would be able to fill the space. The resulting correlation between these sites should be detectable by detailed statistical analyses. In addition to structural and functional correlations, the evolutionary history of the proteins creates correlations between sites.
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Prediction of structure, function, and spectroscopic properties of G-protein-coupled receptors: methods and applications

Prediction of structure, function, and spectroscopic properties of G-protein-coupled receptors: methods and applications

The MembStruk method (Floriano et al., 2000; Vaidehi et al., 2002; Trabanino et al., 2004) was developed by myself, Spencer Hall and Vaidehi, to predict GPCR structure using an approach very different from that of homology modeling. The MembStruk protocol determines the 3D structure of a GPCR beginning from the sequence and using mostly first principles in predicting the structure in various steps. The step which uses some crude structural information is the template building step, where the tilts of the 7Å frog rhodopsin structure (Schertler, 1998) are used to form an initial TM helical bundle. Aside from this, the TM helical extent, the translations of the helices along their axes, their rotations within the bundle, and their bends are determined from first principles. Judging from the success in predicting function (by ligand binding site and affinity determination) and direct structure (compared to crystal structure or mutagenesis studies), the use of this initial template seems to be justified (Freddolino et al., 2004; Kalani et al., 2004). Even so, currently the group is working on determining the tilts using Monte Carlo methods treating each helix as a rigid body with mesoscale forcefield interactions with adjacent helices.
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Emerging roles for G protein coupled receptors in development and activation of macrophages

Emerging roles for G protein coupled receptors in development and activation of macrophages

GPCRs have a common structure of seven transmembrane α-helical segments (H1–H7) joined with three intracellular (I1, I2, and I3) and three extracellular (E1, E2, and E3) loops, an extracellular N-, and an intracellular C-terminus. The crystallization and structural analysis of GPCRs including rhodopsin, β1 adrenergic, β2 adrenergic, A2A adenosine, glucagon, glucagon-like peptide, corticotropin-releasing factor, metabotropic glutamate, and smoothen receptors has provided new insights into the structure, mechanism, and regulation of this class of receptors (11). Based on earlier studies (51, 52) the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification has categorized GPCRs into 5 different families, which are class A (rhodopsin-like receptors), class B (secretin-like receptors), class C (glutamate- like receptors), frizzled, and other 7TM protiens (47). Class A also known as rhodopsin-like receptors is the largest and most diverse group amongst the five families and consists of receptors for light, olfactory, biogenic amines, chemokines, prostanoids, adrenaline, and many others. Class B includes receptors for the parathyroid hormone, calcitonin and the diverse family of gastrointestinal hormones such as glucagon and secretin. Class C GPCRs consists of the GABAB receptor, calcium- sensing receptor and the family of metabotropic glutamate receptors. Class C is relatively small and members generally contain a large extracellular amino terminus thought to be important for ligand capture (47). Despite the general sequence and structural similarities of members within each family (over 25%), individual GPCRs have prominent differences in their extracellular and intracellular loops and these regions are important for ligand binding and interaction with downstream mediators (53). These differences allow individual GPCRs to exhibit unique signaling properties due to different receptor couplings to different G proteins, resultant difference intracellular pathway signaling, different G protein independent pathway activation, as well as complex regulatory processes such as receptor desensitization, internalization, endocytosis, and re-sensitization (53). An updated list of human GPCRs and their ligands has been provided by a committee of the International Union of Pharmacology (47) and a frequently updated webpage can be interrogated for the newest relevant information (https://gpcrdb.org/).
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Current applications of mini G proteins to study the structure and function of G protein coupled receptors

Current applications of mini G proteins to study the structure and function of G protein coupled receptors

members, thus further work is required to complete development of the full mini G protein panel. The relatively high degree of sequence homology shared by the different mini G proteins means that it should be possible to use gene shuffling approaches to pinpoint areas responsible for the poor stability of certain subtypes. This would enable the development of more biologically relevant chimeras in which only minimal regions of the mini G protein need to be substituted to achieve optimal expression and stability [67]. Although a number of different tools are available for characterising receptors in their active state, the fact that mini G proteins can be used off-the-shelf without the need for laborious receptor-specific development makes them an ideal first choice in many applications. Further development and modification of mini G proteins should result in many more novel applications being identified, particularly in relation to GPCR functional studies. Therefore, in the future, mini G proteins will continue to contribute to our understanding of these important receptors, and aid in the development of drugs to treat a wide variety of diseases.
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Fragment-based lead discovery on G-protein-coupled receptors

Fragment-based lead discovery on G-protein-coupled receptors

Protein structures might be available from both experimental and theoretical sources. Experimental structures are usually better suited for virtual screening; however, the flexibility of the protein is often considered using theoretical approaches such as docking to conformational ensembles typically generated by molecular dynamics simulations (15). Comparative modeling represents another option for targets having sequential homologues with known 3D structure. Although homology models were used successfully in a number of cases their usefulness depends strongly on the level of sequence identity and the character of the target (16). The limited availability of experimental GPCR structures made homology models popular for virtual screening applications. On the other hand, however, recent developments in GPCR structural biology resulted in a high number of GPCR structures initiating a significant number of virtual screening studies (18-22). Fast and often parallelized docking algorithms allow the prediction of the binding mode for hundreds of thousands of potential ligands in reasonable time. There are, however, several limitations of these
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Vibrational resonance, allostery, and activation in rhodopsin like G protein coupled receptors

Vibrational resonance, allostery, and activation in rhodopsin like G protein coupled receptors

outlined in Fig. 1. Computed protein evolutionary networks are coupled with a molecular level view of allosteric effect and communication guided by experimentally detected protein associations. Explicitly, we used THz spec- troscopy to directly probe the conformational fluctuations associated with the ensemble dynamics in rhodopsin in addition to the receptor interaction networks that promote allosteric interactions in the receptor 3-D structure. We complement our experimental results with MD simulation as a means of calculating and assigning vibrational modes associated with specific residue interactions and helical associations. We also use principal component analyses (PCA) from the MD simulations as a way of comprehending the nature and role of global dynamics in stabilizing specific signaling pathways in the photo-activated receptor, Meta II. We extract local (LSFs) and global structural fluctuations (GSFs) from the MD trajectories to gain a better understanding of the mechanism in which specific protein interactions form allosteric signaling pathways in rhodopsin and this is weighed by com- puting evolutionary conserved interactions from a sequence alignment of the rhodopsin-like family to determine distinctive allosteric sites that may contribute to the common functionality of the receptor family. Lastly, we use force-distribution analyses (FDA) from the MD trajectories to consider the role of the retinal in both distributing and propagating stress within the interior of the receptor due to the retinal interactions that take place within the ligand-binding pocket and to further comprehend the influence of the retinal dynamics on the global modes of the receptor.
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Structure Prediction of G Protein Coupled Receptors

Structure Prediction of G Protein Coupled Receptors

Even though the sequence similarity between the classes is low, there are structural similarities. Especially important are the similarities on the intracellular side of the GPCRs, since the same G- proteins bind to all GPCR classes. Let us now compare class A to the other classes. The blue color in Fig. 2.8 denotes the contacts common to all classes, and orange denotes contacts specific to class A. Only one contact, 6.51-7.39, is present in all of class A structures (active and inactive), but not in the structures of the other classes. Furthermore, the interactions of TMs 1-5 are more conserved across all classes, but the TM 6 and 7 contacts are very class A specific. It is possible that during the GPCR assembly the helices 1-5 form some intermediate partially folded state before helices 6 and 7 are fully present in the membrane. This might be the reason why the contacts between helices 1-5 are more similar across the classes. Another possibility is that the motion of TM6, which is critical for activation, is different for classes B, C, and F.
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Eukaryotic G protein-coupled receptors as descendants of prokaryotic sodium-translocating rhodopsins

Eukaryotic G protein-coupled receptors as descendants of prokaryotic sodium-translocating rhodopsins

In KR2, the imino group of the Schiff base that connects the retinal with Lys255 plugs the potential Na + -binding site when KR2 is in the ground state [39–41] (Fig. 1e). Functional and structural studies of the Na + translocating rhodopsins [16, 38, 40, 41, 51, 59] indicate that photoi- somerization of the retinal, by twisting the side chain of Lys255, is bound to unplug the Na + -binding site of KR2, cause deprotonation of the Schiff base (as it has been shown in bacteriorhodopsin [60]), and, concurrently, in- duce an outward movement of the 6 th helix (F-helix), opening a cleft that is needed for ion translocation across the hydrophobic part of the protein, as in other microbial rhodopsins [21, 61–65]. The nucleophilic nitrogen atom of the deprotonated Schiff base would thereby provide one more ligand for the Na + ion on its way through KR2. The resulting layers of polar residues along the interacting surfaces of helices 3 and 7 (Fig. 1d) yield a typical cation- conducting structure, which was previously described in ion channels [66]. Binding of the Na + ion by the stretches of polar residues of the 3 d and 7 th helices, which are con- served throughout MRs and GPCRs (see a structure-based sequence alignment in Fig. 1f, Additional file 1: Figure S1 and S4), strongly support a common origin of all these proteins.
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Misclassification of class C G-protein-coupled receptors as a label noise problem

Misclassification of class C G-protein-coupled receptors as a label noise problem

function but, so far, no class C full 3-D structure has yet been discovered and their functional study must mostly rely on primary structure: the amino acid (AA) sequences, publicly available from several databases. There are seven class C subtypes with their corresponding labels. Label noise is unavoidable in this context because sequence labeling is itself model-based and follows a complex many-step procedure that can only guarantee limited success [5]. GPCR classification may use aligned or unaligned versions of sequences. Some methods of sequence alignment-free analysis entail transforming sequences according to the physicochemical properties of their constituent AAs [6].
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Development and Application of Virtual Screening Methods for G Protein-Coupled Receptors

Development and Application of Virtual Screening Methods for G Protein-Coupled Receptors

16 structure models from scratch. Some groups have approached this problem with physics-based approaches, such as molecular dynamics simulations. For instance, one study utilized replica- exchange molecular dynamics simulations on a set of 9 small proteins from the PDB; 31 8 of the 9 structures folded correctly, though the experiment took about 6 months to run. Other strategies include using reduced models with only the peptide backbone and side chain centers of mass in folding simulations. Algorithms such as QUARK 32 and ROSETTA 33 are able to produce reasonable models, but unfortunately, the resolution of the structures are usually not high enough for in-depth analysis. Additionally, typically only small proteins can be folded, due to the high computational costs. Comparative modeling methods, such as MODELLER, 34 operate under the notion that sequence similarity implies structural similarity. 35 Homologous proteins with solved structures are as used as input for modeling, and when they are below 30% sequence identity, the accuracy of the models takes a hit and may end up with an entirely different fold. 36 Therefore, the usage of this methodology is limited in many cases, when a good homologue with a solved structure is not available. In response to this, fold recognition methods aim to overcome this drawback by selecting templates for modeling through fold-level homology. This is made possible by the prediction that that there is a limited number of folds found in nature. 37 Furthermore, it is likely that a correct fold will be selected a majority of the time, as there are already approximately 1,300 folds currently known. Numerous algorithms have been developed in the same vein, such as HHpred, 38 Phyre, 39 and MUSTER. 40
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Oligomerization of G-Protein-Coupled Receptors: Lessons from the Yeast Saccharomyces cerevisiae

Oligomerization of G-Protein-Coupled Receptors: Lessons from the Yeast Saccharomyces cerevisiae

Despite exhibiting striking diversity in primary sequence and biologic function, GPCRs possess the same fundamental ar- chitecture, consisting of seven transmembrane (TM) domains and share common mechanisms of signal transduction (85). GPCRs transduce extracellular signals by coupling to hetero- trimeric guanine nucleotide binding proteins (G proteins) con- sisting of ␣ , ␤ , and ␥ subunits. Activated GPCRs stimulate exchange of GTP for GDP on G ␣ subunits, dissociating G ␣ and G ␤␥ subunits that, in turn, trigger biological responses by binding effector proteins that regulate second messenger pro- duction, protein kinase cascades, cytoskeletal organization, gene transcription, and ion channel activity. GPCRs also signal by G protein-independent mechanisms through recruitment of scaffold proteins such as ␤ -arrestins (reviewed in reference 54). In the budding yeast S. cerevisiae, GPCR signaling regulates two biologic processes: conjugation and nutrient sensing (re- viewed in references 15 and 106). During conjugation, a mat- ing type cells secrete a-factor, a 12-residue farnesylated oli- gopeptide pheromone that binds the G-protein-coupled a-factor receptor (STE3 gene product) expressed only by cells of the ␣ mating type. Conversely, ␣ cells secrete ␣ -factor, an unmodified 13-residue peptide pheromone that binds the G- protein-coupled ␣ -factor receptor (STE2 gene product) ex- pressed only by cells of the a mating type. Although a- and ␣ -factor receptors are unrelated in primary sequence, they
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Active state structures of G protein coupled receptors highlight the similarities and differences in the G protein and arrestin coupling interfaces

Active state structures of G protein coupled receptors highlight the similarities and differences in the G protein and arrestin coupling interfaces

this region could play a wider role in the coupling specificity of both arrestins and G proteins. A unique feature of the opsin–arrestin complex is the central role played by electrostatic interactions [15,41,42]. Two b-arrestins are potentially responsible for the desensitisation of ~800 human GPCRs, so they need to be far more promiscuous than G proteins. Complementation between negatively charged finger loop residues and positively charged regions on the cytoplasmic surface of the receptor may represent a simple mechanism that has evolved to facilitate arrestin binding to a large number of GPCRs with low sequence homology [15,43].
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Regulation of CaV2 calcium channels by G protein coupled receptors

Regulation of CaV2 calcium channels by G protein coupled receptors

Although there is currently no crystal structure for voltage-gated calcium channels that could be used to visualize their interactions with G proteins, site directed mutagenesis, chimeric, and biochemical approaches have been used to elucidate channel structural determi- nants involved in modulation. The first investigations involved chi- meras between Cav2.1 and Cav2.2 channels [140] . These chimeras were expressed in Xenopus oocytes and their sensitivities to G pro- teins assessed via two electrode voltage clamp. These experiments identi fied domain I as a key determinant of G protein inhibition, along with the C-terminus of the channel. Subsequent biochemical studies using in vitro translated G βγ subunits revealed two spatially distinct regions on the I –II linker of CaV2.1 as possible Gβγ targets
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Receptor component protein, an endogenous allosteric modulator of family B G protein coupled receptors

Receptor component protein, an endogenous allosteric modulator of family B G protein coupled receptors

Journal Pre-proof 12 Current pharmacological studies point to a direct interaction with intracellular loops 2 (ICL2) of CLR [8]. For Family B GPCRs, there is a high degree of sequence conservation within this region, and could therefore indicate possible interactions of RCP with other family B receptors. We therefore investigated whether knock down of RCP affects signalling of other family B receptors. We observed impairment of the maximum cAMP production (but not pEC 50 ) in transfected HEK293S cells by CT at the

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Identification of novel arthropod vector G protein coupled receptors

Identification of novel arthropod vector G protein coupled receptors

A multi-step validation process combining database an- notations, similarity searches, domain identification, and structure prediction of the Ensemble* classifier predictions was performed (Figure 4). A total of 1,369 arthropod se- quences, of which 697 belong to the vector species, had positive likelihood scores. Using a threshold value of 0.085, 416 (148 for Ae. aegypti; 148 for An. gambiae; and 120 for Pe. humanus) sequences were predicted as putative vector GPCRs. Of the 416 predicted sequences, 329 were in the original training set, confirming them as known GPCRs, (Figure 4). Eighty-seven predicted sequences were not in the training set and required further validation and confirmation. Of these 87 predicted sequences, 12 se- quences were false positives: either their database annota- tion or identification of domains by ScanPROSITE [56] indicated the sequences as something other than a GPCR. From their respective database annotations, 23 predicted sequences were identified as previously-known GPCRs. Of the remaining 52 sequences, 27 were validated as having GPCR domains by ScanPROSITE but did not have a data- base GPCR annotation and 25 could be neither confirmed by their respective database annotation nor validated by ScanPROSITE as containing a GPCR domain (Additional file 1: Table S1).
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Large-scale production and protein engineering of G protein-coupled receptors for structural studies

Large-scale production and protein engineering of G protein-coupled receptors for structural studies

complementary baculovirus transfer vector carrying a GPCR gene under control of polyhedrin promoter. Virus viability is restored by homologous recombination, so only viruses which carry the GPCR gene are viable ( Kitts and Possee, 1993 ). This approach is used in e.g., BaculoGold (BD Biosciences), BestBac (Expression Systems) and Sapphire (Allele Biotechnology) bac- ulovirus expression systems. The second approach, used in e.g., Bac-to-Bac baculovirus expression system (Invitrogen), is based on site-specific transposition of an expression cassette from a donor vector into the parent baculovirus shuttle vector (bacmid) in Escherichia coli DH10Bac competent cells ( Luckow et al., 1993 ). Insertion of the expression cassette disrupts the lacZ sequence in a bacmid, so the bacterial colonies with the recombi- nant bacmid can be detected by blue/white selection. Insect cells are then transfected with the recombinant bacmid to produce a virus with the gene of interest. Sf 9 or Sf 21 cells are prefer- ably used for either cotransfection or transfection with bacmid, because they allegedly show higher transfection efficiency and virus replication than High Five cells. In all cases, the recombi- nant virus is amplified in successive rounds of infection (usually two) and finally used for protein expression in the selected insect cells. It is essential to quantify virus concentration by one of a few available methods: e.g., plaque assay, end-point dilution assay, in flow cytometry analysis after immunostaining with gp64-PE antibody (Expression Systems) or ligand-biding assays to mea- sure amounts of expressed protein. Multiplicities of infection in a range of 5–10 viral particles per insect cell are usually necessary for efficient protein expression.
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Engineering a minimal G protein to facilitate crystallisation of G protein coupled receptors in their active conformation

Engineering a minimal G protein to facilitate crystallisation of G protein coupled receptors in their active conformation

G protein-coupled receptors (GPCRs) modulate cytoplasmic signalling in response to extracellular stimuli, and are important therapeutic targets in a wide range of diseases. Structure determination of GPCRs in all activation states is important to elucidate the precise mechanism of signal trans- duction and to facilitate optimal drug design. However, due to their inherent instability, crystallisa- tion of GPCRs in complex with cytoplasmic signalling proteins, such as heterotrimeric G proteins and β -arrestins, has proved challenging. Here, we describe the design of a minimal G protein, mini-G s , which is composed solely of the GTPase domain from the adenylate cyclase stimulating
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Tubby is required for trafficking G protein-coupled receptors to neuronal cilia

Tubby is required for trafficking G protein-coupled receptors to neuronal cilia

The tubby-like proteins are defined by a highly con- served carboxyl terminal half of their primary sequence known as the tubby signature domain [1,2]. This family of proteins includes the prototype tubby, and TULP1, 2 and 3, for tubby-like proteins 1, 2 and 3 [3-5]. Other than members of the tubby family, search of sequence databases reveals no significant homology with known proteins or functional motifs. The tubby gene ( Tub ) was originally discovered by way of a spontaneously arisen obesity model in mice, and other members of the family were subsequently identified by homology cloning [3]. Mutations in human TULP1 are a cause of retinitis pig- mentosa [6]. Loss of TULP1 function in mice replicates this rapid photoreceptor degeneration phenotype [7,8]. Prior to photoreceptor degeneration in the mouse retina, pronounced ectopic distribution of rhodopsin is apparent indicating a defect in trafficking across the connecting cilia to reach their normal destination, the outer segments [9]. Loss of TULP3 function in mice leads to neural tube patterning defects and embryonic lethality [10], and the cellular basis can be traced to a failure of Hedgehog signaling due to defective ciliary trafficking [11]. Little is known about Tulp2 , but its Chlamydomonas ortholog was identified as one of strongly induced genes during flagellar regeneration [12] and it was also reported as a can- didate gene for human obesity in linkage analysis [13]. The tubby signature domain binds polyphosphorylated phos- phatidylinositol [14], but their N-terminal domain is much more diverse. In the best characterized example, the TULP3 N-terminal domain binds to the IFT-A complex, which is part of the essential cellular machinery for ciliary transport, through a short conserved motif. In cultured cells, TULP3 facilitates membrane receptor trafficking to primary cilia. Thus it serves as bipartite bridges through their phosphoinositide-binding tubby domain and N-terminal IFT-binding motif, coordinating multiple signaling path- ways including membrane receptor trafficking [15].
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Role of G-Protein Coupled Receptors in Cancer Research and Drug Discovery

Role of G-Protein Coupled Receptors in Cancer Research and Drug Discovery

G-Protein Coupled Receptors are the largest class of cell surface signaling proteins which form the biggest class of drug targets. Their physiological functions and their link with emerging different types of diseases especially development of cancers and metastasis, have been made them significant for drug discovery programs and hiring them as therapeutic drug targets. In this paper we first focus on their structure, classification, mechanism of peptide and non-peptide interactions then discuss their role in drug discovery and recent finding related to their position as novel drug targets in drug discovery. [1,2].The purpose of this review is to redefine the structure-function relationship of the CPCRs as a valuable source for development of novel drugs and provide favorable and tremendous opportunities in prevention and treatment of cancers. Mechanism of their
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