This hypothesis proved for being proper, particularly given that PIC formation possibly takes place within a capsid that is not entirely dissociated, as a result precluding quick accessibility to 100 % free IN . The style of new assays for screening ligands of the IN?vDNA complicated sooner or later led for the identification within the 1st strand transfer inhibitors, L-731, 988 and L-708, 906 on the flip of your century . These compounds compete using the target DNA by binding on the IN?vDNA complex. They recognize a particular webpage close to the catalytic triad, which opens following a adjust in conformation induced by the binding and three? processing of the viral DNA . The initial selective inhibitors of strand transfer to get identified had been ?, ?- diketoacids . This kind of compounds dependant on the ?-ketoenol fragment effectively chelate the Mg2+ cation demanded to the exercise of integrase and their total affinity for that target depends on their surrounding substituent groups .
The prerequisites to get a exact strand transfer inhibitor comprise the presence of the chemical group including the heteroatoms, nitrogen or oxygen, capable of binding two divalent cations plus a hydrophobic aromatic part of the molecule likely to bind and stabilize the IN?vDNA complex, forming selleck chemical PF-01367338 ic50 an lively pharmacophore accountable for the action of all strand transfer inhibitors . Compounds with these properties selectively target and bind to the IN?vDNA complicated, near to the 3′ finish with the donor DNA, therefore inhibiting target DNA binding, resulting in selective inhibition on the strand transfer response with no important impact over the 3′-processing response . They hence act as IN?DNA interfacial inhibitors, and therefore are known as integrase strand transfer inhibitors .
The substitute from the carboxylate group by its tetrazolium bioisostere led to the growth of 5- CITEP and its analog, S-1360. Regardless of the weak exercise of those molecules towards integrase, the framework of the integrase/5-CITEP complicated is determined, making it conceivable to construct a model from the construction within the inhibitor pharmacophore bound towards the lively internet site metal cation . Modifications on the ?, ?-diketoacid a part of the molecule at first led on the substitute of this group by 8-hydroxy quinoline, to improve antiviral exercise and to conquer pharmacological limits, like serum protein binding . Compounds from this family members, like Merck L870, 812 , have potent antiviral action, delivering the proofof- notion for INSTI activity in vivo despite their toxicity in vivo .
The L870, 812 series of compounds was not produced even further, however the dihydroquinoline JTK303/GS9137 derived from quinolone antibiotics was used for additional drug development and is now in the innovative clinical improvement stage, under the identify of elvitegravir .

For this, we taken care of KG-1 and NB4 cells that has a series of doses of SNS-032 or/and perifosine. As demonstrated in Inhibitor 7A, remedy of KG-1 and NB4 cells with SNS- 032 plus perifosine resulted in significantly reduced cell viability than either SNS-032 or perifosine treatment method. The blend index evaluation showed synergistic cytotoxic effects when two medicines had been mixed at relatively increased concentrations. Following, no matter if perifosine enhances the effect of SNS-032 in long-term colony formation assay was also examined. We observed that, underneath the circumstances when SNS-032 or perifosine alone had moderate inhibition result of colony formation of leukemic cell lines the mixture treatment practically fully suppressed the colony-forming ability of those leukemic cells . Related benefits have been also present in main blasts obtained from two patients with AML .
To additional delineate the effect of mixture remedy on development signaling, we examined the effect of SNS-032, perifosine, and combination about the activiation of caspase TWS119 structure pathway, phosphorylation of mTOR and downstream targets, at the same time as expression of phosphor-ERK1/2. As proven in Inhibitor 7D, we discovered that though SNS-032 and perifosine alone had very little effect on caspase three and PRAP, the two together have been highly effective, suggesting that perifosine can enhance SNS-032-induced apoptosis. Several scientific studies have proven that perifosine inhibits activation of Akt in cancer cells . Steady with these reports, perifosine drastically inhibited the degree of phosphorylated Akt in KG-1 and NB4 cells and consequently decreased the degree of phosphorylated mTOR , which represent the activity of mTORC1, but not that of phosphorylated mTOR .
Whereas, phosphorylated mTOR amounts declined in KG-1 and NB4 cells in the lower concentrations of 60 and 80 nM of SNS-032, respectively. Importantly, mixed SNS-032 and perifosine therapy more hints resulted in basically finish elimination of phosphorylated Akt and action of mTORC1. Consequently, furthermore, it appreciably attenuated 4EBP1 phosphorylation at all tested online websites and phosphorylated p70S6K , both of that are direct target of mTORC1. With each other, this combination remedy is very likely to have major benefit to AML sufferers since it can synergistically inhibit action of mTORC1 and Akt in leukemic cells. Discussion CDK inhibitors are gaining results in the clinic as antitumor agents for cancers as well as hematologic malignancies .
SNS-032 is actually a potent CDK inhibitor, which targets CDK2, CDK7, and CDK9, the CDKs that regulate the initiation and elongation of transcription by phosphorylating Ser2 and Ser5 of RNA Pol II, respectively.

1 method to improve responsiveness to EGFR inhibitors may be to simultaneously target several HER members of the family. Afatinib is presently probably the most superior compound in this class. Afatinib is definitely an irreversible EGFR/ HER2 inhibitor, with activity towards wild-type and mutant forms of EGFR . Afatinib was even more potent than gefitinib, erlotinib, and lapatinib in inducing the cell death of NSCLC cell lines, together with these harboring wild-type EGFR, and also the erlotinib-resistant T790M mutation . It had been also found in the present research the molar potency of afatinib against these cells was considerably higher than either gefitinib or erlotinib. HCC827 cells harboring the activating E746_A750 deletion had been really sensitive to afatinib, whereas other NSCLC cell lines had been moderately delicate, which can be in agreement with other reports .
The action towards the resistance mutation T790M and cell lines with downstream resistance mechanisms was, nonetheless, only slightly much better compared to the reversible TKIs. The various EGFR-targeting approaches vary in action mechanisms. TKIs compete with ATP to bind on the EGFR kinase, consequently inhibiting EGFR autophosphorylation and activation Salinomycin of downstream signaling. Anti- EGFR antibodies reduce receptor dimerization and consequently activation . However, none of these agents alone does maximally suppress EGFR signaling or the result of mutant EGFR during the malignant phenotype, as also shown in our experiments. The mixture of cetuximab with the distinctive TKI has already been examined . The in vitro and in vivo effects showed that the combined treatment can augment the potency of EGFR signaling inhibition.
Ramalingam et al. made use of a mixture of cetuximab and gefitinib for individuals with advanced/metastatic lung cancer who had been previously taken care of with platinum-based chemotherapy. It was concluded that dual inhibition is feasible and protected, and might possibly have modest action in advanced/metastatic NSCLC. The combination of afatinib and cetuximab can even conquer resistance resulting from the T790M mutation each preclinically too as clinically . During the current study, the mixed remedy of EGFR siRNA and TKIs or antibody accomplished greater tumor cell development suppression in each of the 5 NSCLC cell lines and increased apoptosis as large as by 100% . The result using the various agents from the different cell lines was additive, not synergistic, as calculated by a mixture index .
Once again, the differential sensitivity from the cell lines on the mixture mimicked their sensitivity to TKI alone: the cell lines that demonstrated essentially the most sensitivity to siRNA had the biggest result from your mixture, as well as the cell lines with downstream TKI-resistance mutations or even the T790M mutation.

The VEGFR2 gene is essential, as knockout mice display embryonic lethality with almost full reduction of endothelial cells and defective vasculogenesis . Constitutive activation of FGFR1 in mice can induce mammary invasive lesions and prostate cancer . There’s considerably interest while in the advancement of membrane permeable molecules that target receptor tyrosine kinases this kind of as VEGFR2 and FGFR and thereby block tumour angiogenesis . Indolinones are 1 this kind of class of ATPstyle mimetics that bind the VEGFR2 tyrosine kinase domain and inhibit enzyme exercise, exemplified through the anti-cancer drug SU11248 and its predecessor compound SU5416 . These compounds are characterized by a 2-oxindole core having a variant side chain on the 3-position. Sutent has become authorized for remedy of renal cell carcinoma and imatinib-resistant gastrointestinal stromal tumours .
A different class of tyrosine kinase inhibitors are anilinophthalazines such as PTK787 . These compounds consist of a phthalazine or quinazoline heterocycle with numerous substitutions. PTK787 has been shown to inhibit development within the microvasculature and a variety of myeloma development and has proven guarantee to the treatment of advanced metastatic colorectal cancer . The mechanism of action explanation of these compounds on VEGFR2 has been effectively characterized in vitro; yet, the specificity of indolinones and anilinophthalazines is unclear because they have already been proven to inhibit a variety of Sort III receptor tyrosine kinases . It will be becoming increasingly clear that inhibition of many pro-angiogenic axes might possibly provide a much better treatment than targeting just one pathway or possibly a single enzymatic step .
On this study, we have now examined the means of these compounds to target both the VEGF-A-VEGFR2 or bFGF-FGFR axes, with consequences for endothelial cell migration, wound healing and tube formation, all important features of angiogenesis. Go 6983 Human umbilical vein endothelial cells had been retrieved from human tissues obtained by neighborhood ethical approval through the Leeds Hospitals NHS Trust and cultured as previously described . Recombinant human VEGF-A was a present from Genentech . Recombinant human EGF, bFGF, VEGFR2 and FGFR1iiic and antibodies towards VEGFR1 and VEGFR2 extracellular domain had been obtained from R&D Systems . Phospho-ERK1/2, phospho-PLCg1 and ERK1/2 antibodies were purchased from Cell Signalling Technology . FGFR1, PLCg1 and PECAM-1 antibodies have been from Santa Cruz Biotechnology .
Antibody to early endosomal antigen-1 was from BD Biosciences and horseradish peroxidase -conjugated secondary antibodies had been from PerBio Sciences . AlexaFluor-conjugated secondary antibodies and Concanavalin A had been from Invitrogen . SU5416 , Sutent and PTK787 were prepared as 10 mM stock solutions in dimethyl sulphoxide . Serial 10-fold dilutions were made in tissue culture medium.

Whilst a high variability amid the lines was observed, docetaxel, paclitaxel and SN-38 showed usually greater efficacy than gemcitabine . Pemetrexed, a multitargeted folate pathway inhibitor, induced hMPM cell toxicity in numerous cell lines, whilst showing really distinctive IC50 , independently in the folate receptor expression amounts . Nonetheless, various insights to the mechanism of action of those medicines permitted the identification of your treatment method sequence to become adopted to maximize the outcomes. A study aimed to identify the optimal schedule for blend treatment of pemetrexed and gemcitabine, showed that the simultaneous exposure of MSTO-211H hMPM cells to each medicines was antagonistic, but a powerful synergism was observed when pemetrexed preceded gemcitabine; the inverted sequence was again antagonistic .
Comparable final results were obtained in a research addressing the optimization of gemcitabine-cisplatin protocols employing cell lines derived from pleural effusions of untreated PF-562271 solubility hMPM individuals . Four-hour pretreatment with gemcitabine followed by 68-hour exposure to cisplatin was uncovered to exert synergistic exercise in the two epithelioid and sarcomatoid hMPM cell lines, inducing a strong S-phase arrest that correlated with accumulation of double-strand breaks . Consequently, it had been proposed that gemcitabine increases cisplatin-induced double-strand breaks by inhibiting DNA adduct fix. EGFR family members TK inhibitors Somewhere around 70% of hMPMs present aberrant expression of EGFR, though in quite a few cases, and in the subset of hMPM cell lines, both EGFR and TGF-a are expressed, suggesting an autocrine regulation of EGFR in hMPM .
In 4 EGFR-expressing cell lines derived from previously untreated individuals with epithelial , sarcomatoid and biphasic Bleomycin hMPMs, gefitinib significantly inhibited EGF-dependent cell signalling including phosphorylation of Akt and ERK1/2 . On top of that, therapy with gefitinib led to a significant dose-dependent reduction of colony formation when hMPM cells have been grown in soft agar. A differential sensitivity among the cell lines was reported with MSTO-211H, H2461 and H2373 displaying higher responsiveness than H2591. Gefitinib induced 89% of growth inhibition in H2373 hMPM cells, exhibiting a dose-dependent arrest on the G1/S and a corresponding boost in p27kip1 amounts . Gefitinib, erlotinib and canertinib , not simply induced apoptosis but additionally inhibited migration and matrix metalloprotease manufacturing in M14K, ZL34 and SPC212 hMPM cells, confirming the likely effectiveness in focusing on numerous components of EGFR relatives in hMPM .
In a further study , gefitinib inhibited EGF-induced proliferation in two hMPM cell lines, derived from pleural effusion or tumour biopsy .

Protection was observed even at concentrations as much as 64 mg/ml MNNG, displaying that in vivo AfAGT is indeed a functional orthologue within the Mgt1 protein. Up-regulation of methyltransferase activities by MNNG We determined methyltransferase action by quantifying the transfer of -methyl groups from – N-methyl-N-nitrosourea-methylated calf thymus DNA to protein: methyl transfer from O6 meG and methylphosphotriesters allowed determination of AGT and MPT activity, repsectively . In original scientific studies, we primary investigated if the putative AfAGT and AfMPT routines had been quantifiable applying this assay procedure. Exercise was quickly detected in cell-free extracts within the S. cerevisiae mgt1 deletion that had been transformed with an expression vector harbouring Afagt but exercise in extracts of Afmpt transformants was near to that of extracts of vector-transfected controls . To verify that the assay was capable of detecting the methylphosphotriester demethylation perform of AfMPT, we ligated its ORF into pMALC2, expressed the fusion protein in E. coli and purified employing amylose affinity chromatography.
The purified AfMPT fusion protein transferred radioactivity small molecule library screening from substrate DNA to an extent that was closely similar to that observed for the amino-terminal area from the Ada MPT protein, that had been expressed and purified under identical disorders . Furthermore, when rAfMPT was mixed with purified recombinant human MGMT, roughly twice as much radioactivity was transferred to protein, but mixtures of rAfMPT with r E. coli MPT were not additive indicating that such as the E. coli protein, rAfMPT acted only on among the methylPT isomers. We also found that pre-incubation on the rAfMPT at 37_C led to a speedy reduction of action whereas at area temperature there was pretty minor loss of activity in excess of forty min . At room temperature, methyl transfer to rAfMPT was complete in about 20 min .
Space temperature incubation was thus applied for assessing transferase exercise in crude extracts of your A. fumigatus strains. No methyltransferase activity was deteckinase in cell-free extracts of any on the uninduced A. fumigatus strains beneath the assay ailments more info here put to use. Yet, action was conveniently detected in extracts of the MNNGinduced wild-type strain, but not in these of your deletants . To investigate the nature with the induced action, under substrate-limiting circumstances, we added recombinant AfMPT or human MGMT towards the extracts before addition of substrate DNA. This resulted in higher amounts of transferase activity only when rAfMPT was additional to your induced wild-type extract , demonstrating the activity noticed was exclusively the O6-meG repair perform, AfAGT. MNNG treatment method consequently substantially up-regulates the expression of AfAGT.
The extent of this induction cannot be accurately calculated simply because action was not quantifiable in the pre-MNNG extract under the situations made use of, but the induced level of activity was calculated to equate to two.7_103 molecules of AfAGT per cell.

Arginine, a semiessential amino acid in people, is essential for some cancers, which include mesothelioma. Argininosuccinate synthetase catalyzes a rate-limiting step from the synthesis of arginine. Downregulation of this enzyme success inside the cell turning into dependent on extracellular sources of arginine, a process known as arginine auxotrophy. Numerous tumors have demonstrated this dependence, such as mesothelioma. In these arginine-dependent cells, the depletion of arginine by pegylated kinds from the enzyme arginine deiminase may possibly demonstrate for being a novel anticancer technique.66,67 Phase I/II trials of this system have shown action in hepatocellular carcinomas and melanomas, two tumor styles that are regarded arginine auxotrophs. Equivalent investigation in malignant pleural mesothelioma is warranted and underneath consideration .
Tumor necrosis factor-alpha has been studied for many years for its antitumor exercise in the range of malignancies; nevertheless, early enthusiasm continues to be dampened by serious toxicities related with its administration. In an work to create a more targeted approach, investigators read what he said have coupled TNF-a with ligand-directed agents. A phase II trial of human TNF-a fused that has a cyclic tumor-homing peptide, asparagine-glycine-arginine, was administered to 57 previously treated patients with mesothelioma on an each 3-week schedule in 43 individuals along with a weekly routine in 14 patients.68 Toxicity was generally chills for the duration of administration and was no greater within the dosedense weekly routine than within the triweekly schedule. The general disorder control price was 46%, and median progression-free survival was 2.eight months using a median total survival of twelve.
1 months, hence warranting the investigators to recommend more investigation of the weekly regimen. TP53 gene mutation could be the most frequent genetic alteration found in human Fisetin cancer. Because the primary discovery of TP53 alterations in 1989, >35 000 mutations are already described in various varieties of tumours . One particular with the biggest contributions to your research of TP53 mutations is presented by molecular epidemiology and its applications. We are going to not examine these epidemiological research in detail, as they are actually the topic of a lot of thorough opinions and are summarized in Supplementary Kinase S1 . These scientific studies show a website link involving publicity to several kinds of carcinogens, certain mutational occasions in the TP53 gene as well as the improvement of distinct cancers.
By far the most striking illustration is the fact that of tandem mutations, specifically induced by ultraviolet radiation, which are only observed in skin cancers. The partnership concerning G->T transversion and lung cancer in smokers or even the mutation of codon 249 observed in aflatoxin B1-induced liver cancers is additionally demonstrative. More just lately, a particular pattern of mutational occasions during the TP53 gene is observed in nephropathy-associated tumours that help the involvement of aristolochic acid, a potent mutagen found in conventional medicines, from the aetiology of this cancer.

Oncogene addition and synthetic lethality: unbiased searchs for novel anti Ras therapies In light in the present lack of success in establishing clinically practical anti Ras medicines, latest scientific studies have taken benefit of KRAS oncogene addiction to look for synthetic lethal partners of mutant KRAS. Making use of RNA interference technologies, huge scale interfering RNA screens are already applied to get a practical and unbiased technique to recognize therapeutic targets for anti Ras inhibition . Perturbation of these genes may well outcome in oncogene specific synthetic lethal genetic interactions that might produce new therapeutic opportunities. These screens are based upon the notion of synthetic lethality, in which two genes are defined as synthetically lethal if mutation of either gene alone is compatible with viability however the simultaneous mutation of the two genes prospects to death . Mutationally activated RAS genes hence signify 1 gene and RNAi mediated ablation in cancer cells in the expression of the 2nd gene will provide the 2nd hit.
Synthetic lethal interactions can involve find out this here genes in the same pathway, genes inside of parallel pathways that cooperate with respect to an very important perform, or genes within distant pathways that become functionally linked as a consequence of the response in the cell to a specific perturbation. Because typical cells lack mutant RAS, genes recognized in this manner ought to in principle be selectively lethal for tumors but not typical cells. In one particular examine which included a restricted RNAi library focusing on 1,011 genes using a target on protein kinases, it was found that cells that were dependent on mutant KRAS genetically interacted with the STK33 serine threonine kinase as a synthetic lethal partner irrespective of your tissue of origin, whereas STK33 was not demanded by KRAS independent cells .
STK33 promotes cancer cell viability in the kinase action dependent method by regulating the suppression of mitochondrial apoptosis mediated by S6K1 induced inactivation Triciribine of the death agonist Poor selectively in mutant KRAS dependent cells. The synthetic lethality functional screen was significant, seeing that there was no alteration in STK33 expression, no mutations, and no transforming exercise of STK33 was detected. Therefore, with the classical analyses of cancer causing genes, STK33 would have not been identified. In a second research that included a genome wide RNAi display, identification of synthetic lethal interaction partners together with the KRAS oncogene was carried out targeting 32,293 one of a kind human transcripts . The genes identified encode a functionally various set of proteins that regulate a number of biological processes, primarily mitotic functions.
One of those genes that was characterized in this study was Polo like kinase 1 , a serine threonine kinase that plays a essential role in mitosis. PLK1 is often a component on the anaphase marketing complicated cyclosome, plus the proteasome that, when inhibited, results in prometaphase accumulation as well as the subsequent death of Ras mutant cells.

Consequently, Sabutoclax downregulated proliferation of both the C4-2 and ARCaPM xenografts, concomitant with greater apoptosis, as evidenced by dramatic tumor size reduction, similar to that observed in Tgfbr2ColTKO transgenic mice. Together, it suggested that antagonism ofMcl-1 and also other antiapoptotic Bcl-2 household proteins by Sabutoclax may be effective to target the growth of the two main and metastatic CRPCs. Sabutoclax Sensitized PC-3 Cells to Docetaxel-Induced Apoptosis In Vitro and In Vivo Mortality from PCa most regularly outcomes through the resistance to chemotherapy with docetaxel. To evaluate the capacity on the panactive Bcl-2 inhibitor Sabutoclax to synergize with docetaxel for your treatment method of human PCa, we implemented the Chou-Talalay procedure for synergy examination .
This model is capable to take into consideration the nonuniform dose-response curves that we had previously observed in single-agent dosing experiments . For these tests, we plated a threelog purchase dose-response curve for each of your compounds individually too as in the selleck chemical compound library cancer ratio blend on the single 96-well plate with every single dose collected in triplicate. At 72 hours immediately after administration in the drug to the plated androgen receptor?deficient PC-3 cells, the relative ATP concentration in just about every properly was measured with respect to an internal DMSO management being a proxy for cell viability. The resultant dose-response curves were utilized to recognize the respective ED50 and ED90 values for every drug individually and in mixture. Furthermore, these curves have been analyzed to calculate the CI worth for every cell line .
Working with the CI model of drug blend, CI = one signifies Pimobendan an additive effect whereas CI < 1 indicates synergy and CI > one signifies antisynergy. Our examination identified strong synergy of Sabutoclax and docetaxel therapy . To find out no matter whether the synergy observed in vitro could possibly be replicated in vivo, we tested the compounds individually and in mixture for his or her capability to suppress growth of PC-3mouse xenograft tumors. We 1st established the dose dependence of Sabutoclax while in the PC-3 xenograft model. The 3 groups of mice had a powerful dose-dependent response and exhibited minimal weight-loss in excess of the course with the examine. From this preliminary examination, we chose a three-time weekly dose of 2 mg/kg Sabutoclax along with a once-weekly dose of 12.5 mg/kg docetaxel for that blend review.
Handle injections not having drug have been administered to the control group and to each on the single-agent groups to simulate strain from your total number of injections administered to the mixture group. Mouse weight and tumor size was tracked in the course of the program within the treatment . A substantial big difference in tumor size was observed amongst the Sabutoclax or docetaxel arms along with the combination arm .

We anticipate that mutation of residues inside these surfaces will alter the free of charge power of protein protein interaction but not the totally free energy of DNA interaction . Third, the designs predict that all AGT molecules within a cooperative unit will have exactly the same orientation with respect to a single DNA strand. In our designs and inside the structure of Daniels et al the N terminal finish from the protein is oriented towards the five? end from the repaired strand11. Polarized binding may perhaps account for your differences in restore efficiency observed for lesions found near the 5? and 3? ends of single stranded substrates25. If binding is polarized with respect to one strand, complexes formed with duplex DNA can have a twofold degeneracy reflecting the truth that there are two probable orientations of the bound protein, when complexes with single stranded DNA will be homogeneous.
We anticipate that this distinction will end result in really distinct patterns of DNA protection when complexes containing single stranded and duplex DNAs are probed with hydroxyl radicals selleck chemical PF-03814735 or other reagents. Lastly, the smaller unwinding angles obtained in our topoisomerase assays along with the small alterations during the CD spectra of duplex DNA when AGT binds27 indicate that the formation of the cooperative AGT DNA complex has tiny impact on base stacking and DNA backbone conformations. Collectively, these success suggest that inside the cell, AGT might interact preferentially with areas of torsionally relaxed duplex DNA. Since the publicity of this kind of DNA within the nucleus is determined by the activities of chromatin remodelling and topoisomerase enzymes47; 48 and seeing that these routines are expected for DNA replication and transcription, we predict the routines of AGT is going to be concentrated near replication forks and in regions of actively transcribed chromatin.
The ability to bind and restore single stranded DNAs additional suggests that AGT may well be energetic in the replication fork itself. Experiments created to test these predictions are underway. The chlorinated marine normal item salinosporamide A , a potent 20S proteasome inhibitor now in phase one human clinical trials to the treatment method of a variety of myeloma and also other informative post cancers, is 500 instances more lively than its deschloro analog salinosporamide B one 5. The biosynthetic origin of SalA?s pharmacologically active chloroethyl side chain from an unknown sugar precursor differs through the analogous ethyl group in SalB, which instead originates from butyrate 6.
Examination in the 41 kilobase sal biosynthetic gene cluster from S. tropica uncovered a polyketide synthase nonribosomal peptide synthetase hybrid pathway involving a brand new chlorination route for SalA production7.