PIPwas said to prejudice the channels in the closed state and relief from inhibition could be received by the activation of PLC. This notion was based on indirect tests where the effects of phosphoinositideswere maybe not directly examined in excised patches. On the other hand, in excised angiogenesis pathway patches it absolutely was unearthed that PtdIns P, its precursor PtdIns P, and other phosphoinositides stimulate TRPV1 and positively charged aminoacids R701 and K701 inside the TRPV1 sequence are accountable for the primary initiating activities of PIPIn another study the debate regarding the position of PIPmay have now been settled. Using HEK293 cells, the authors discovered that after exposing TRPV1 to large capsaicin concentrations, the following Cainflux initiates PLC, which results in the destruction of channel activity is reduced by PtdIns Pand PtdIns P, which, resulting in desensitization. Inhibition of PLC activity led to deficiencies in desensitization. It was also Infectious causes of cancer shown in excised patches that PtdIns P, the precursor of PtdIns P, triggered TRPV1 and inhibited desensitization, and, in addition, that PtdIns Phad an inhibitory influence on the channel, but only at low capsaicin concentrations. This inhibitory effect could only be detected in whole cells and not in excised patches, suggesting that this effect may be indirect. In this study, the authors conclude that the balance between the inhibitory and activating effects of PtdIns Pdepends around the stimulation level ofthe channel, because all through sensitization PLC coupled agonists cause a moderate depletion of PtdIns R, removing its inhibitory effect, however not providing low enough lipid levels to inhibit channel activity. In contrast, high capsaicin concentrations induce a severedepletion of PtdIns Pthat limits channel activity and brings todesensitization, proving TRPV1 regulation by lipids to be somewhat complicated. In this regard, it has been proven that phosphoinositide 3 kinase interacts specifically with TRPV1 and that this complex encourages TRPV1 trafficking to the plasma membrane. That trafficking is noticed in reaction to nerve growth factor, a process that might be responsible for NGF and other relevant professional algesic agents capability to produce Docetaxel ic50 hyperalgesia. Other membrane taken lipids also determine TRPV1. As an example, oleylethanolamide, an organic analogue of the endogenous cannabinoid anandamide, anandamide it self, and some lipoxygenase products and services all regulate TRPV1 function. TRPV1 can also be activated by the metabolic products of lipoxygenases, such as for instance 15 and 5 HETEs and 12 and 15 HPETEs. Recently, omega-3 fatty acids, which exhibit analgesic properties, have been demonstrated to interact directly with TRPV1. These essential fatty acids stimulate TRPV1 in a dependent fashion and improve its responses to extra-cellular protons. Apparently, these lipids competitively inhibit the reactions of vanilloid agonists. there is controversy concerning whether PtdIns Pincreases or reduces the open probability of the station.

Loperamide is often used to treat ritonavir related diarrhea in patients with human immunodeficiency virus. While ritonavir increased plasma AUC of loperamide and its natural compound library metabolite 2, tipranavir containing programs decreased the plasma AUC of loperamide and its CNS active metabolite, N desmethyl loperamide. 2 fold and 1. 4 flip, respectively. However, despite the increased plasma contact with loperamide and its metabolite, there was no clinically applicable change in the respiratory reaction to carbon dioxide or in pupil size between the treatment groups, showing that ritonavir did not enhance the exchange of loperamide in to the CNS. Similarly, Tayrouz et al. Applied loperamide to 12 healthy volunteers with either 600 mg ritonavir or placebo. Although ritonavir increased 2. 7 fold the plasma AUC of loperamide, no main pharmacodynamic results were observed following coadministration of loperamide with either ritonavir or placebo. Thus, it seems that coadministration of loperamide with ritonavir doesn’t pose particular challenges to the individual. We examined the aftereffect of cyclosporine on verapamil plasma and brain levels in 12 healthy volunteers, to quantitatively assess the effect of G gp inhibition Plastid at the human BBB. At pseudo steady-state 2. 8 uM cyclosporine concentration in blood, mental performance to plasma AUC ratio of radioactivity increased by 888-766 without a major change in plasma verapamil k-calorie burning or plasma protein binding. This increase was modest in comparison with the optimum increases reported in non human primates and in rodents. If the grey matter and white matter of the mind were compared, the upsurge in radioactivity distribution was similar. The difference in degree of this DDI at the human versus non human primates or rodents BBB is partly due to variations in the blood levels of the inhibitor, cyclosporine. Certainly, at lower blood concentration of cyclosporine, the size of the verapamil cyclosporine DDI at the rat BBB is smaller. The lower maximum increase in the brain distribution of radioactivity in non Letrozole 112809-51-5 human primates, compared to rodents, is probable explained by species differences in the share of BBB R gp activity to the distribution of verapamil into the brain. Ergo, while there is an exemplary agreement between the interaction noticed at the rat and the human BBB at the reduced cyclosporine blood levels, if the non human primates is representative of people, there might be a divergence between the rat and human as the chemical concentration is elevated and as Pgp inhibition approaches a maximum. Six healthy volunteers were scanned under standard conditions or post administration of quinidine or cyclosporine. Cyclosporine increased 2 fold mental performance uptake of loperamide, but quinidine did not notably influence it. The authors suggested that along with G gp, other mechanisms are involved in preventing loperamide central activity.

Assessment of loperamide temperament in mice revealed that the effect of P gp inhibition on brain uptake of loperamide was blunted by low G gp substrate radioactive metabolites of loperamide. Zosuquidar significantly improved the distribution of nelfinavir to the head, without a change in its CSF to blood concentration ratio. These data suggest that CSF concentration as a surrogate marker for brain drug concentration should be used in combination with caution, specially to determine drug interactions CTEP in the BBB. Likewise, doxorubicin CSF concentrations in four adult rhesus monkeys were below the limit of detection if the drug was administered alone or in combination with intravenous cyclosporine. Kurdziel et al. Employed PET to test the tissue distribution of paclitaxel in the absence and the presence of tariquidar in 3 rhesus monkeys. Despite changes in the distribution of radioactivity into liver, lung, and kidney with tariquidar administration, paclitaxel uptake into the head was really low and appeared unchanged after the administration of the chemical. Although Choo et al have previously demonstrated in rats that P gp at the BBB is more resistant to inhibition by tariquidar than in other areas, when loperamide was used as the substrate, the reason behind this tissue specificity of the relationship is unknown. This finding can also be contrary to the 4. 3 fold increase in paclitaxel brain uptake when it had been company applied Urogenital pelvic malignancy with tariquidar to mice. In contrast to the wealth of information on G gp inhibition, much less is known about the effect of Pgp induction in the BBB. In one of the earlier in the day studies, subjects were treated with morphine or dexamethasone for 5 days. Both compounds MAPK phosphorylation decreased the antinociceptive effect of morphine and enhanced P glycoprotein expression in the mind, in comparison to those observed in animals treated with the vehicle. The researchers postulated that improved brain P gp activity following chronic exposure to morphine or dexamethasone could have caused the lower brain levels of the drug. Chronic exposure of rat brain endothelial cells to other medications, including phenobarbital, phenytoin and carbamazepine may also lead to induction of P gp expression and functionality in vitro and in vivo. Also, HIV protease inhibitors have been shown to up regulate G gp expression in vitro in a mind endothelial cell line. Studies about expression and activity of transcription factors that regulate the BBB expression of G gp and other transporters are conflicting. Bauer and colleagues presented evidence that the nuclear receptor pregnane X receptor exists in rat brain capillaries, where it can potentially mediate DDIs. Upon activation by dexamethasone, PXR regulates the expression of P gp in rat brain capillaries in vitro and in vivo.

We observed early proliferation of home restricted T cell clones in the most limiting dilution cultures, but T cell colonies showing powerful proliferation over PF299804 solubility several weeks all became nonspecific. We suppose that early clonal growth of survivin specific clones may have occurred when minimal numbers of T cells were buffered by large numbers of feeder cells, but HLA A2 limited apoptosis may have hindered their later outgrowth. Because HLA A2 cells couldn’t display the corresponding pMHC ligands, they were immune to HLA A2 restricted fratricide, and numerous HLA A2 allorestricted survivin certain T cell clones could be separated. These results seem to be concordant with previous studies that described HLA A2 limited survivin specific T cells that were disseminated as T cell lines in vitro or detected in peripheral blood samples of cancer patients ex vivo, whereas survivin specific T cell clones were difficult to have. Lately, one survivin specific CTL clone that was isolated from an HLA A2 breast cancer patient acknowledged the same pMHC ligand because the Tg TCR described here. That patientderived CTL clone was shown to recognize all HLA A2 survivin tumefaction cell lines Metastasis in a little screen, with the exception of the FM 86 cell line. The authors surmised that FM 86 cells were not known because of disturbed pMHC ligand appearance, since the tumor cells were found to possess high quantities of survivin mRNA. As demonstrated here, this tumor cell line expresses relatively low levels of surface HLA A2. We included FM 86 cells in our analyses and found that these tumor cells were identified by effector cells transduced with each of the 3 Tg TCRs, nevertheless, killing was less with effector cells expressing TCR A71, the Tg TCR that endowed the PBLs with the best functional avidity. purchase Capecitabine The failure of the patient CTL clone to destroy FM 86 cells would be explained if it’d an operating avidity decidedly lower than that of PBLs revealing TCR A71. Moreover, our Tg TCRs were codonoptimized and modified to express murine frequent regions, which imbued them with great surface expression and strong ability to interact with tumor cells expressing low levels of pMHC ligand. Furthermore, it’s been reported that TCR/CD3 expression or TCR signaling is often disturbed in patientderived T cells, thereby limiting their ability to acknowledge tumor cells. Such alterations could also have disturbed the ability of the patient derived CTLs to recognize FM 86 tumefaction cells showing low pMHC ligand density. Because our survivin certain TCRs were well expressed as transgenic proteins in activated person lymphocytes of HLA A2 healthier donors, we’re able to avoid failures that impinge on appearance, signaling, or function of patient produced CTL clones. The 3 Tg TCRs irritated wide differences in functional avidity in receiver lymphocytes, different by over 4 orders of magnitude in awareness.

The option of the p110 X ray crystal structure granted modelling of A66 in the p110 kinase domain and the likely mechanisms for its selectivity towards this element were determined. Critically in this expected binding function, the ligand forms a connection with Val851 of the inter selective c-Met inhibitor lobe linker region. The backbone amide and carbonyl of Val851 interact with the hydrogen bond donor and acceptor nitrogen atoms set in the main aminothiazole key, consistent with the binding mode noticed for PIK 93 bound to p110. The tertiary butyl thiazole moiety extends from the aminothioazole key in to the lipophilic affinity pocket, although the pyrrolidine carboxamide group extends in the contrary direction towards a region of the binding site wall described by the C terminal lobe which has p110 certain elements, proven to influence ligand binding. In this predicted binding present, the carboxamide amine moiety forms hydrogen bonds with backbone carbonyl group of Ser854 the sidechain carbonyl group of Gln859 and possibly. Somewhat, the unminimized present believed a hydrogen bond interaction between both the carboxamide amide and carbonyl categories of the ligand and those within the Gln859 side chain. These residues Organism were predicted formerly to be engaged in chemical interactions within the p110 active site. We also examined possible binding modes for your A66 R form, and discovered that a cause just like that of the S form was not found, and it failed to form a hydrogen bond interaction with the backbone amide of Val851 too. Inside the top ranked cause, the R pyrrolidine carboxamide amino group was believed to form a hydrogen bond using the Val851 backbone carbonyl. In this direction, the ligands key urea carbonyl was expected to connect to the side chain amino group of Gln859 and also the appreciation pocket Evacetrapib was not filled. Apparently, a few clashes between the protein and ligand were seen with the S form, although more were present for that of the R form. These results, taken together along with the higher Chemscore exercise price, indicate that the A66 S type seems to complement better the p110 active site. In agreement with this, we discover that the A66 R form dropped inhibitory action and didn’t prevent p110 at 10 uM. A66 was docked to the p110 structure with the aminothiazole containing PIK 93 bound and the p110 apo chemical structure, as both these amino-acid side chains have hydrogen bond donor and acceptor groups that’ll interact with the ligands pyrrolidine carboxamide. The top ranked offer predicted for A66 binding to p110 had a similar direction to that predicted with p110, but, the Chemscore fitness value was lower, suggesting a worse fit. An interaction using the p110 Val882 backbone amide was also not believed, although the PIK 93 aminothiazole forms this interaction.

To evaluate whether ABT 869 might inhibit the activation of ERK or AKT pathways downstream of PDGFR and c KIT in EWS cells, we addressed purchase Ivacaftor and A4573 cells with the ligands for PDGFR and c KIT in the existence of the drug or vehicle control and done Western blot analyses with phosphospecific antisera. Our results suggest that ABT 869 therapy prevents activation of p42/p44MAPK and in certain EWS cells, AKT. ABT 869 inhibits the development and growth of EWS cells in vivo To determine if the inhibition of PDGFR and h KIT induced by ABT 869 inhibits cyst growth in vivo, NOD/SCID mice were inoculated subcutaneously with TC71 or A4573 cells. Rats were handled daily by oral gavage with either ABT 869 at 40 mg/kg or even a corn oil vehicle control. The delayed treatment group received ABT 869 at 40 mg/kg/day when the tumors reached an amount of 300 mm3. Previous studies demonstrated that the drug does not affect normal body function. We did not see any signs of physical stress or fat loss during the treatment course with ABT 869 during our studies. Treatment with ABT 869 directly after inoculation resulted in action Lymph node preventing tumefaction development from injected cells. In previous experiments, therapy with the drug after significant cyst burden didn’t lead to increased survival. Thus, this experiment was done to measure the effects of drug in a setting of microscopic disease, prior to the onset of significant metastatic disease. One of the issues with eradicating EWS infection is that there are residual cells that are resistant to chemotherapy, which raise the danger of relapse. Cyst growth was significantly inhibited subsequent delayed treatment of drug at 40 mg/kg/ day. Mathematical mean tumor volumes at 25 days after injection with TC71 cells were 2 and 22-year. 0.02-0.05 of vehicle get a grip on under delayed and immediate treatment, respectively. Similarly, geometric mean sizes applying the A4573 cell line were 23-year and 3. Six months of get a grip on, respectively. By hematoxylin and eosin staining, the histology demonstrated that tumors from rats treated with ABT 869 had increased evidence of necrosis and supplier Bortezomib infection compared to vehicle controls. TUNEL discoloration showed increased apoptosis within the immediate and delayed treatment groups in comparison with the car controls for both cell lines. There have been no differences in the cell cycle account of cells treated with ABT 869 in comparison to vehicle control. Thus, ABT 869 is effective in controlling growth and causing cell death of EWS cells in vivo. ABT 869 inhibits development of cancer cells in a metastatic EWS model To evaluate the potential ramifications of ABT 869 on the metastatic model of Ewing sarcoma, GFP/ Luciferase showing A4573 and TC71 cells were made through lentiviral transduction followed by selecting for GFP. The fixed cells were cultured and injected through the tail vein into female NOD/SCID rats. Six rats were examined per treatment group.

We hypothesized that any compound in a position to inverse the infection signature should be bad for influenza virus replication. Rilmenidine and aminobenzenesulfonamide had only a moderate antiviral effect on one particular virus. Harmol and merbromin were poor inhibitors on most of the worms. Midodrine and brinzolamide were Bortezomib clinical trial poor to moderate inhibitors of all of the examined worms. As expected, ribavirin was a solid inhibitor of tried worms. In light of these results, we conclude that we’ve identified a common trademark whose partial inversion is strong enough to inhibit viral replication. We cannot exclude that some in silico chosen drugs exert a possible direct influence on an exercise or on a process abused by herpes. On the list of seven molecules, three specifically could have this effect: ribavirin and merbromin which could both directly inhibit a purpose, and harmol which could inhibit a path. Harmol is really a beta carboline alkaloid of the medicinal plant, Perganum harmala L.. Several certain effects are described Urogenital pelvic malignancy for harmol except that it puts a psychoactive influence by inhibiting monoamine oxydase, somewhat inhibits platelet aggregation by inhibiting PLCc2 and induces apoptosis in certain cell lines by activating caspase 8. PLCc2 is implicated in the protein kinase C activation path, the game of which is a must for influenza virus entry. Thus its inhibition by harmol might simply be responsible for the antiviral effect shown by this molecule. Similarly, activation of apoptosis could control viral replication. However, three kinds of evidence support our theory the molecules have an antiviral effect by modifying the host cell gene expression. First, the outcomes of our test of infection advantages show that none of the substances except for merbromin had an effect on structure or function before infection. Second, the high confirmation charge of the in silico selected drug panel examine the rational of the selection. natural compound library Last, some compounds that governed the host cell transcription in the same way that influenza virus infection increased viral production. To your knowledge, modulation of the cell gene expression never been described to support the effects of the in silico chosen drug, apart from ribavirin. This antiviral drug with in vitro activity against both DNA and RNA viruses, has several mechanisms of action proposed to guide its antiviral effect the exhaustion of the intracellular GTP share by inhibition of inosine monophosphate dehydrogenase compromises the forming of progeny viral RNA, ii) the inhibition of viral RNA dependent RNA polymerase activity has been shown for hepatitis C and influenza viruses, and iii) it might become a RNA virus mutagen creating error problem. Which mechanisms contribute to its anti flu effect in vivo remains undetermined.

We next determined the results of exposure to 17 DMAG for 8 or 24-hours about the myeloid progenitor mobile line 32D overexpressing both wild-type or mutant TrkA. This suggested a chaperone relationship of TrkA with hsp90 in human leukemia cells that is disrupted by treatment with 17 DMAG. Finally, Aurora A inhibitor we show that treatment of K562 cells with 17 DMAG leads to a dose-dependent increase in apoptosis, which likely arises as a result of the abrogation of chaperone relationship of hsp90 with pro success signaling proteins including h Raf and AKT. 1Treatment with chemical is famous to decrease the organization of the customer proteins with hsp90 with simultaneous increase in binding to hsp70. Treatment with 17 DMAG light emitting diode to a period dependent decrease in binding of TrkA with hsp90 and a reciprocal upsurge in the binding of TrkA to hsp70, as shown in Figure 2A. We next determined the results of 17 DMAG to the connection of TrkA with hsp90 corp chaperone cdc37, that is mixed up in filling of kinase customer meats onto hsp90. Figure 2B shows that, in K562 cells, following treatment with 17 DMAG for an interval as short together hour TrkA binding to cdc37 was paid down, with a further drop in binding of TrkA to cdc37 by two hours. Treatment with 17 DMAG also inhibited the association of hsp90 with the co chaperone p23. We next determined whether inhibition Ribonucleic acid (RNA) of chaperone relationship of hsp90 with TrkA would cause polyubiquitylation of TrkA. Treatment with 17 DMAG enhanced the intracellular levels of polyubiquitylated TrkA within two hours without a decrease in the total TrkA levels. The results of 17 DMAG to the intracellular localization of TrkA was determined by immunofluorescence microscopy. In untreated K562 cells, TrkA was primarily localized to the cell surface membrane. In contrast, following treatment with 0. 25 uM of 17 DMAG, the cell surface expression of TrkA was lowered. Taken together, these results suggest that 17 DMAG treatment inhibits the chaperone organization of TrkA GW0742 with hsp90, accompanied by polyubiquitylation, proteasomal degradation and reduced membrane localization of TrkA. NGF is famous to bind TrkA and triggers downstream signaling involving autophosphorylation of AKT, TrkA and ERK1/2. To look for the ramifications of hsp90 inhibition on NGF induced signaling, 32D/wtTrkA and K562 cells were treated with NGF alone or with the mix of 17 and NGF DMAG. NGF therapy caused fast autophosphorylation of TrkA and improved p AKT and ERK1/2 in both K562 and 32D cells with endogenous and exogenous expression of TrkA, respectively. Co therapy with 17 DMAG inhibited NGF mediated increase in p AKT, p TrkA, and p ERK1/2. The drop in p TrkA and p AKT levels was more pronounced than in p ERK1/2 levels.

The elimination rate constant was estimated by linear regression of the blood or plasma levels within the log linear terminal phase. The residual rodents survived through 72 h and demonstrated c-Met Inhibitor no visible signs of acute poisoning. Observations conducted by blinded observers reported that 12 hours post i. v. dosing of free 17 DMAG at levels above 10 mg/kg, the subjects offered nose bleeding, disorientation, heavy breathing, and slight reduction in response to noise. The animals that acquired 17GAC16Br in the mPEG b PCL micelle system did not display undesireable effects for the first 24 hours at 40 mg/kg serving, but did show nose and mild diarrhoea bleeding 48 hours post administration of the measure. In the pharmacokinetic reports, five animals were dosed at 10 mg/kg of 17GAC16Br in mPEGb PCL micelles for comparison to free 17 DMAG, and at the 200 mg/kg 17GAC16Br formula for comparison to an unique 10 mg/kg dose. In Figure 3, the serum levels of free 17 DMAG and 17GAC16Br concentration versus. time profiles at 10 Cholangiocarcinoma mg/kg differed, with all the micellar formula representing extended blood circulation in the body set alongside the more rapidly expunged free 17 DMAG. It was also seen that 17GAC16Br was quickly changed into 17GAOH subsequent administration, as evidenced by its early presence in serum. This rapid release of the prodrug from micelles at the beginning of the pharmacokinetic profile is almost certainly a direct result prodrug compounds that had not been fully encapsulated within the semi crystalline PCL primary, which quickly diffuses out in to the blood following injection. This is also seen to correlate with a rapid 17GAOH distribution phase and a significantly slower elimination phase following sustained release of prodrugs from micelles more than 48 h. At 200 mg/kg 17GAC16Br, we noticed greater initial contact us concentrations of the micelles in serum in addition to a greater amount of hydrolyzed prodrug due to initial fast release of the drug. Nevertheless at 12 h, the serum levels of the 200 mg/kg micellar dose were much like 10 mg/ kilogram levels but the product was removed from serum at a faster rate compared to 10 mg/kg dose. There clearly was a 1. 8 flip better hepatic clearance of 17GAOH by the liver at 200 mg/kg set alongside the same 10 mg/kg amount. The us hydrolyzed lipophilic prodrug is secured in the micelles, and consequently its rate of removal is compared to the rate of clearance of the micelle in addition to release of lipophilic prodrug elements from the micelles at both levels. Specifically, we realize that at 10 mg/kg, the AUC of 17GAC16Br in micelles is 72 fold higher than free 17 DMAG implemented at the same measure. More over, at 200 mg/kg of 17GAC16Br in micelles, the AUC gets to an extraordinary 2000 fold progress and the volume of distribution decreased 21 fold compared to free 17 DMAG.

The effects of diminished ATF3 expression on tumor development in vivo were first examined in a subcutaneous tumor product using HCT116 cells. More over, in a recent publication, colleagues and Ameri could show that induction of ATF3 in hypoxic conditions, a typical feature noticeable supplier PF299804 in solid tumors, is in addition to the transcription factor HIF 1a. The factors HIF 1a and ATF3 are both caused by hypoxia and other mobile causes, and both transcription factors regulate the expression of multiple genes during tumor progression and metastasis. Significantly, and of high clinical relevance, we could show in the present and in one initial previous research that ATF3 term may be induced in cancer cells by Hsp90 inhibition in vitro and in vivo. Inhibitors to Hsp90 are being investigated in a growing quantity of clinical trials. Ergo, the present study not only provides an appealing new element to the multiple Cellular differentiation mechanisms of Hsp90 inhibition, but also provides reasonable evidence that an induction by inhibition may be positive for therapy of high level colon cancer. Our data suggest that induction of ATF3 may be valuable for improving therapy of colorectal cancer patients when it comes to preventing peritoneal and hepatic metastasis. Furthermore, our study provides evidence that such ATF3 induction is possible by inhibition, which is especially intriguing since Hsp90 inhibitors are promising new agents for targeted treatment of advanced colorectal cancer and other malignancies. Heat-shock protein 90 has a vital role in both the regulation and stabilisation of numerous proteins, including those associated with radioresistance. Inhibition of Hsp90 may contact us therefore provide a strategy for enhancing the radiosensitivity of tumor cells. This study explores the reactions of four tumor cell lines to combined therapy with ionising radiation and two novel inhibitors of NVP AUY922, Hsp90 and NVP BEP800. The practices used included colony and cell counts, phrase of Hsp70, Hsp90, Akt, survivin, cleaved caspase 3, p53, cell cycle progression and related proteins. DNA injury was analysed by histone gH2AX and Comet assays. We found that NVP BEP800 and NVP AUY922 increased radiosensitivity in every examined cell lines. In contrast, only two cell lines showed a heightened rate of apoptosis after drug pre-treatment, as revealed by western blot. In all tested mobile lines, the expression of histone gH2AX, a marker of DNA double strand breaks, after combined drug IR treatment was higher and its decay rate was slower than those after each single treatment modality. Drug IR treatment also triggered impaired cell cycle progression, as indicated by S phase destruction and G2/M charge.