Nonetheless, in the GLV 1h285 handled group, all mice were alive till 91 dpi, indicating a substantial survival advantage imparted by viral BMP four expression. VACV mediated BMP 4 expression significantly delays tumor progression and improves survival in immunocompromised mice The efficacy of GLV 1h285 in tumors initiated by GBM FLuc CSCs was also assessed in the larger tumor burden setting. The tumors had been allowed to increase for seven weeks rather than two weeks and the viruses were inoculated sub sequently. Comparison of the tumor signals right after inocu lation of GLV 1h189 or GLV 1h285 virus exposed a delay in tumor signal peak for GLV 1h285 in contrast to GLV 1h189. Furthermore, a recurrence of tumor signal was observed only for GLV 1h189 inocu lation at 62 dpi onwards, with rapid tumor progression in 80% in the surviving mice.
Interestingly, once the survival data was plotted underneath the tumor signal data, GLV 1h189 inoculated mice commenced to expire around 24 dpi with an increase in tumor VX661 signal. Yet another steep decline in survivability was observed at the level wherever recurrence of tumor signal occurred at 62 dpi. In situation of your GLV 1h285 inoculated group, the tumor signal peak also correlated with animal reduction. On the other hand, it had been drastically less than that on the GLV 1h189 inoculated group, with practically 60% of your mice surviving. On euthanasia or termination within the review, the brains from the animals had been harvested for examination. Brains in the uninfected group animals showed a high degree of necrosis and hematoma, primarily over the correct side of your brain wherever the cells were implanted. Brains in the bulk within the GLV 1h285 inoculated mice showed considerable improvement in gross morphology compared to your uninfected mice. The couple of mice that survived right after GLV 1h189 inoculation also showed only small scarring on the webpage of implantation.
Discussion Practical exercise of oncolytic viruses is regarded as for being proof against mechanisms attributed to make cancer PD173074 resistance against chemotherapeutic agents and radiation modalities that are regarded to reside in CSCs. Yet, there’s a lack of precedence for robust and validated CSC methods to be tested extensively with oncolytic viruses, primarily with oncolytic VACVs. The data presented within this study demonstrates the feasibility of creating a VACV that expresses a stem cell differenti ation agent, BMP four to efficiently target infected and non infected undifferentiated GBM CSCs. The resulting result of a BMP 4 expressing VACV infection triggers an enhanced growth inhibition of GBM stem cells in vitro and substantial tumor regression in mice compared towards the parental, non BMP 4 carrying VACV. BMP 4, a member with the TGF B super relatives of secreted proteins has become proven to get probable applications in treating GBM and colon cancer.

Thanks to their capacity to potently inhibit EGFR, the two afatinib and neratinib are assessed in lung cancer that has become resistant to gefitinib and erlotinib due to the T790M point mutation inside the kinase domain. In the preceding publication by our group, we identified a panel of genes whose expression in response to twelve hours of lapatinib therapy altered in the manner proportionate towards the sensitivity from the cell lines assessed to this agent. Co inertia examination was utilized to assess microarray information from untreated and lapatinib treated BT474 and SKBR3. A panel of 27 genes had been validated working with RT PCR and from this analysis, genes that had a differential expression of two were viewed as vital. This multi variate statistical strategy is implemented to link transcription factor binding web site target predictions and gene expression data in an effort to identify transcription aspects connected with the cellular response to lapatinib.
CIA allowed us to determine commonality concerning the expression from the genes as well as the TFs which can be predicted to target these genes. Working with this gene panel of five, we examined the differential expression of those genes in response to pharmacologically pertinent concentrations inhibitor Topotecan of neratinib, afatinib and traztuzumab to characterise if this panel informed to the sensitivity from the cell designs to lapatinib alone or may additionally be practical in predicting cellular response to other HER2 targetting therapies. Superior prediction from the most likely efficacy of a targeted therapy could have massive implications for enhanced efficacy of can cer treatment, patient individualised optimisation from the offered arsenal of treatment options and, by means of rapid identification of most likely response non response, greatly re ducing the general financial burden of those high priced but at times lifesaving pharmaceuticals.
Materials and approaches Drug preparations Lapatinib tosylate, neratinib, afatinib, dasatinib and gefitinib were all sourced from Sequoia Chemicals Inc. The medication had been selleckchem prepared to 10 mM in DMSO. Traztuzumab was sourced from Roche, Basel, Switzerland and epirubicin was sourced from Pfizer, Ny, NY, USA. 5dFUR, an lively metabolite derivative of capecitabine, was sourced from Sigma, St Louis, MO, USA. As with all the TKI medicines, the 5dFUR was ready in DMSO. Cell culture The cell lines that had been examined have been BT474 and SKBR3, HER2 overexpressing, lapatinib sensitive breast cancer cell lines, and MDAMB453, a HER2 overexpressing but lapatinib insensitive breast cancer cell line. SKBR3 and MDAMB453 breast cancer cell lines have been maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum. BT474 cells were maintained in Dulbeccos Modified Eagles medium supplemented with 10% fetal bovine serum, 2% L glutamine and 1% Sodium Pyruvate. All cell lines had been kept at 37 C in 5% CO2 95% air humidified incubators.

The more observation that silencing of Frzb also success in a lower in these collagens might be explained by lack of chondrogenic dif ferentiation from the latter strategy. We also uncovered that overexpression of FRZB appeared to stimulate chondrogenesis within this model, as proven by increased aggrecan and col2a1 expression. Matured aggrecan monomers inside the cartilage are glycosylated macro molecules through which the glycoconjugates are formed by sulphatation of GAG side chains to the core protein. The quantity of sulphated GAGs in the micro masses, measured by Safranin O staining, was surprisingly decreased in FRZB overexpressing micro masses. Even though the differences we observed had been lim ited, these results might possibly suggest that FRZB overexpres sion within this strategy impairs the maturation of those aggrecan monomers, for instance, by a relative excess in substrate due to the higher expression ranges.
Staining for collagens by Picrosirius Red indicated no leading distinctions in total collagen content in FRZB overex pressing micro masses and controls. The observed spreading from the fibers from your center, however, which was also noted inside the Safranin O staining, selleck suggests that overexpression of FRZB could modify matrix distribu tion, potentially by increasing ATDC5 migration. Each one of these results are in line with earlier observations on FRZB and chondrogenesis. Collagen style III and V can also be uncovered from the bone, co distributed in significantly decrease quantities subsequent to the main collagen element type I collagen. Kind V col lagen expression is regulated by TGFb in osteoblasts throughout osteogenesis. Considering that members on the TGFb pathway are up regulated in our Frzb samples, this could possibly impact expression from the subchondral bone.
Collagen form V is enhanced in some sufferers with brittle bone disease and in individuals with osteogenesis imperfecta, wherever collagen variety V probable interferes with the normal system of mineralization. Related final results Tubastatin A have been noticed for collagen type III, suggesting a purpose for collagen sort III and V in defects in maturation with the bone. The responsive factors for TCF LEF but additionally other transcription components, related to WNT signaling, within the Col3 and Col5 promoters recommend a direct website link with WNT signaling by which FRZB can influence the com position from the cartilage and subchondral bone ECM. Alternatively, thinking of the reasonably mild effects on WNT signaling with the tissue level, our study also leaves open the probability that FRZB has unex pected, far more robust publish transcriptional or epigenomic results in these tissues suggesting new directions for investigation. Reduction of Frzb resulted within a reduce of genes linked to cell cycle progression. Proliferation analysis of ribcage chondrocytes isolated from Frzb mice com pared to those isolated from wild form mice agreed with this particular observation.

High HSP90 expression was connected with large expression of HER2 and ER, sizeable tumors, substantial nuclear grade, and lymph node involvement. Our outcomes demonstrated that up regulation of many isoforms of HSP90 in main breast cancer have been inde pendent poor prognosis components, indicating that HSP90 targeted therapies in blend with cytotoxic che motherapies or other targeted agents, might boost diag nosis and treatment of really aggressive breast cancers. Mainly because HSP90 is usually a critical part of oncogenic sig naling, an rising quantity of candidate HSP90 inhibi tors are already developed and evaluated, each in preclinical models and in clinical trials. Despite the fact that HSP90 inhibitors have exhibited clinical exercise from the deal with ment of breast and other cancers, focusing on HSP90 alone usually ends in cytostatic rather then cytotoxic results on tumors.
In the bulk of individuals, sickness progression occurs following cessation of treatment method with an HSP90 inhibitor. Our effects recommend selleckchem that up regulated HSP90 might not be an independent bad prognosis aspect amongst sufferers with HER2 optimistic breast cancer, as no statistically sizeable correlation was observed among bad survival and high degree expression of any HSP90 isoforms, that’s constant together with the former locating the most common clinical response in individuals with HER2 good breast cancer who obtained HSP90 monotherapy is steady sickness. In contrast, numerous studies employing cell based mostly or several tumor xenograft models of breast cancer have shown a considerable degree of synergy by combining HSP90 inhibitors with therapies targeting HER2. Certainly, in animal xenograft designs, tumors frequently never immediately re grow upon drug withdrawal, and often sizeable tumor regression is often observed.
In clinical selelck kinase inhibitor trials, continual administration in the vast majority of HSP90 inhibitors is properly tolerated by people, with manageable toxicity. At first glance this seems surprising provided the crucial position on the protein in many ordinary cellular processes. yet, the apparent lack of toxicity of HSP90 inhibitors may perhaps be linked to the recent realization that cancer cells are addicted to HSP90 a prime instance of tumor cell non oncogene addiction. This may perhaps provide a sufficiently significant therapeutic window to the safe utilization of HSP90 inhi bitors in cancer. Moreover, there is proof that oncogenic clients can alter the conformation of HSP90. Quite a few inhibitors of your protein have already been formulated that only recognize this activated conformation suggesting an even better therapeutic index. TNBC continues to be regarded as a additional aggressive breast cancer subtype with a larger price of distant recurrence as well as a poorer prognosis.

Tumor volume was determined according to the formula tumor volume shorter diameter2 ? longer diameter two. Sets of mice were sacrificed at eight weeks publish injec tion to examine invasiveness with the major tumor. With the end of these research, mammary tumors with surrounding excess fat pad and tissues have been fixed in 10% neutral buffered for malin for one day. Sections of mammary tumor have been embedded in Tissue Tek O. C. T. compound and 9 ?m thick sections had been stained with hematoxylin and eosin. Photos in the tumors had been photographed by light microscope applying ten? and twenty? objectives. For intratibia injections, parental and shRNA p21 SCP2 cells were injected intramuscularly into the left tibia of two group mice. The mice were monitored weekly for tumor burden. Digital radiography in the hind limbs of all animals was used to watch the development of skeletal lesions at 4, six and eight weeks submit injection in a MX 20 cabinet X ray program.
On Week 8, radiographs of anesthetized mice had been taken and also the osteolytic lesion place was analyzed as previously described. The score of lesion location was measured as 0, no lesions. 1, minor lesions. two, tiny lesions. 3, important lesions with small selleck chemical Bosutinib break of margins. 4, considerable lesions with major break in peripheral lesions. Statistical analyses College students t test was made use of and differences in between groups have been deemed significant at P 0. KX2-391 05. Results p21 expression correlates with bad survival in breast cancer individuals Prior studies have recommended that larger expression of cytoplasmic p21 correlated with bad prognosis in breast carcinomas. To additional take a look at the corre lation of p21 gene expression degree with clinical final result in breast cancer patients, we utilized a recently pub lished gene profiling database of breast cancer individuals to assess p21 gene expression in total survival and distant metastasis zero cost survival outcomes.
We analyzed the prognostic value according to the median, upper and lower quartile expression amounts of p21 in the 20 12 months follow up for OS and DMFS. As shown in Figure 1A C, elevated p21 expression appreciably correlated with poor OS in each median, one. seven. one. 1 to 2. 6. P 0. 012 and upper quartile, but not during the lower quartile. Moreover, larger p21 ranges showed a related pattern in DMFS. Right after twenty years follow up, sufferers who’re cost-free of distant metas tasis showed diminished expression on the p21 gene plus a improved survival rate. Though the prediction didn’t display statistically substantial ends in the median expression, the P value of the p21 upper quartile did attain statistical signifi cance. We also analyzed the partnership of p21 expression and clinical outcomes in each estrogen receptor optimistic and damaging breast cancer sufferers.

Right after the loading, cells have been washed with PBS and suspended in DMEM. Fluorescence measurements had been carried out using an Olympus Fluoview 500 confocal process. Fluo three was enthusiastic by argon laser light at 488 nm and fluorescence was measured at wavelengths of 515 nm. ELISA Assay of DAG DAG concentrations were measured by ELISA, in accordance for the suppliers instruction. This assay employs the quantita tive sandwich enzyme immunoassay approach. Antibody particular for DAG has been pre coated onto a micro plate. Standards and samples are pipetted in to the wells and any DAG existing is bound by the immobilized antibody. After removing any unbound substances, a biotin conjugated antibody unique for DAG is additional to the wells. Following washing, avidin conjugated Horseradish Peroxidase is extra towards the wells. Following a wash to get rid of any unbound avidin enzyme reagent, a substrate resolution is added to your wells and color develops in proportion to the amount of DAG bound in the first phase.
The shade improvement is stopped plus the intensity with the shade is measured. Subcellular Fractionation Subcellular fractionation into cytosol and membrane fractions was performed by using Membrane and Cytosol Protein Extraction Kit, in accordance for the makers instruction. Monolayer cultures had been washed three times with ice cold PBS choice and scraped into cold homogenization buffer containing twenty mM Tris HCl, pop over here 4 mM EDTA, order inhibitor two mM EGTA, 10% glycerol, 10 g ml leupeptin, and 1 mM PMSF. The cells were lysed by means of sonication with 4 15 s intervals and comprehensive lysis was monitored microscopically. The homogenate was ultracentrifuged at 86,000 g for 45 min at 4uC, along with the supernatant was designated as the cytosolic fraction. The pellet was resuspended in HB containing 1% Triton X 100 and incubated on ice for thirty min.
The samples were then centrifuged at 14,000 g for 20 min at 4uC, and the supernatant was designated because the membrane fraction. All samples have been boiled and cleared by centrifugation. Cell Migration Assay Migration action of AGS cells have been detected by transwell system. Just after trypsinization, 5 104cells were seeded in to the upper chamber containing culture medium without FBS. Cell migration towards the bottom side of membrane was induced by medium containing 10% FBS inside the reduce chamber for twelve h at 37uC in the tissue culture incubator. Migrated cells around the bottom side in the membrane had been fixed in 40% paraformaldehyde option for 30 min, stained in Giemsa choice for ten min, after which rinsed in water. The stained cells were subjected to microscopic examination below a light microscope.

We obtained antibodies against MxA from Otto Haller, and antibodies towards Sox3 had been generously presented by Michael Klymkowsky. All secondary reagents for immunoblot, immunocytochemistry, and flow cytom etry analyses were obtained from Jackson Immunoresearch, except the Alexa FluorH 488 conjugated streptavidin. The secreted alkaline phosphatase reporter plasmid driven by an IFN stimulated response component promoter continues to be previously described, plus the pTet On plasmid was obtained from BD Biosciences. The overexpres sion plasmids encoding human IRF 9 and STAT2 were obtained from OriGene, and an overexpression plasmid encoding the human IFNAR2c isoform using a C terminal hemagglutinin epitope tag was generously offered by John Krolewski. IRF 9, STAT2, and IFNAR2 HA cDNAs had been subcloned into pTRE2hyg to realize doxycycline inducible expression. Cloning tactic details are available upon request.
Viruses Fort Morgan virus strain CM4 146 was obtained from the American selleck Kind Culture Assortment and WEEV strain Cba 87 was produced from your complete length WEEV cDNA clone pWE2000 as previously described. All exper iments with infectious WEEV have been carried out under Biosafety Degree 3 problems in accordance with University of Michigan Institutional Biosafety Committee and CDC NIH pointers. All viruses had been propagated in Vero cells and infectious virus titers in tissue culture supernatants had been established on Vero cell monolayers as previously described, the place plaque assay sensitivity was one hundred plaque forming units ml. We contaminated cells with either FMV or WEEV at a multiplicity of infection of 0. 01 for all experiments with BE C neuronal cells.
We harvested tissue culture supernatants for WEEV titers at 36 h post infection, Alisertib as previously published experiments indicated that 24 to 48 hpi was the optimum timeframe to detect maximal differences amongst undifferentiated BE C and differentiated BE C m cells with respect to both virus induced cytotoxicity and virus manufacturing after sort I IFN stimulation. FMV is significantly less virulent than WEEV, which final results in delayed advancement of cytotoxicity in cultured cells at a equivalent inoculum. Preliminary experiments indicated that 72 hpi with an MOI of 0. 01 was the optimum time stage to measure virus manufacturing immediately after style I IFN stimulation and separate FMV induced cytotoxicity concerning undifferentiated BE C and differentiated BE C m cells. To isolate purified virus for hESC derived cell culture experiments, Vero cells have been harvested at 48 hpi, tissue culture supernatants were centrifuged at 1,0006 g for five min to pellet cellular debris, and virions have been precipitated overnight by addition of polyethylene glycol and sodium chloride to 7% and two.

Myt3 suppression in islets modestly, but significantly decreased cellular insulin ranges, but had no result on their potential to secrete insulin following stimulation with glucose, KCl or arginine. To find out how suppression of Myt3 lowers cellular insulin amounts we assessed the result of Myt3 suppression within the expression of selected transcriptional regulators vital in pancreas growth or perform, or genes with effectively established roles in b cell function. Myt3 suppression in ex vivo islets had a substantial result on many transcription variables and cofactors known to regulate b cell perform, which include Hnf1a, Hnf1b, Hnf4a, Insm1, Sox9, Pdx1, and Mafa, which had been all lowered by at the very least one. 6 fold. From the genes involved in b cell perform, Myt3 suppression decreased Abcc8 and Slc30a8 the most, by 1. 54 fold and 1. 67 fold respectively.
Myt3 suppression also impaired Ins1 and Ins2 expression, even though the expression ranges of your other islet selleck chemical JAK Inhibitor hormones had been unaltered. Treating MIN6 cells with siRNAs targeting Myt3 developed related final results for picked genes, particularly for Pdx1 and Mafa. Offered this, and as Pdx1 and Mafa have properly established roles in b cell perform, we attempted to validate their repression selleck inhibitor on the protein degree. Western blot analysis of islets transduced with adenovirus expressing shMyt3 reduced Mafa ranges by 1. 67 fold and Pdx1 levels by one. 48 fold, consistent with our qPCR data. These outcomes suggest that Myt3 has an effect on cellular insulin articles through the regulation of numerous genes together with Ins1, Ins2, Pdx1 and Mafa. Myt3 Regulates b cell Survival Publicity of islets to cytokines both in vitro and in vivo suppresses Myt3 expression suggesting a prospective role for Myt3 in b cell survival.
To test this hypothesis we transduced MIN6 cells with our adenoviruses expressing shRNAs targeting Myt3 or perhaps a scramble sequence and incubated the cells with propidium iodide. Escalating shMyt3 virus concentration drastically increased b cell death over time. Similarly, Myt3 suppression elevated Annexin V optimistic cells by 2 fold, as well as level of cleaved caspase three. To validate these success we performed TUNEL analysis on dispersed islets treated with both the shScramble or shMyt3 virus. Our information demonstrate that apoptosis was enhanced by somewhere around two fold, much like our benefits in MIN6 cells. This was also confirmed in whole islets. As cytokine exposure final results in lowered Myt3 expression, and adenoviral mediated suppression of Myt3 increases apoptosis, we examined the skill of Myt3 in excess of expression to safeguard islets from cytokine mediated cell death. Dispersed islets treated with an adenovirus more than expressing Myt3 had a greater than 2 fold lower in cytokine induced apoptosis, as when compared with islets treated by using a handle adenovirus expressing eGFP, as revealed by TUNEL staining.

Despite the fact that not independently validated, these findings place SMA patients in to the class of large chance candidates who are likely to be impacted quite possibly the most below the situations of OS. On top of that, OS induced enhanced skipping of SMN2 exon 7 raises the basic question regardless of whether any in the methods aimed in the correction of SMN2 exon 7 splicing in SMA will retain its efficacy under the situations of OS. Provided a high degree of sequence conservation in between SMN1 and SMN2, it truly is most likely that a number of the however uncharacterized splicing occasions in OS are typical to the two, SMN1 and SMN2. A definitive response to this question awaits additional experimentation in precise cell varieties that express either SMN1 or SMN2. Right here we report a systematic examination of splice isoforms produced by SMN1 and SMN2 below normal and OS conditions. Our examine also addresses a significant question of neuronal versus non neuronal regulation of different splicing of diverse SMN exons.
A single of the defining reversible STAT inhibitor elements of this examine may be the application of a numerous exon skipping detection assay that captures the relative abundance of all big splice variants of SMN. We also benefit from a different cell variety that we serendipitously identified to lack SMN2 transcripts quite possibly thanks to a partial or complete deletion of SMN2 alleles. Our findings reveal novel splice variants, like these created by an sudden skipping of exon 4 and or many adjacent exons of SMN. We show that OS has an effect on alternative splicing of various exons of SMN1 and SMN2 in the two, neuronal and non neuronal cells. Our outcomes suggest an OS induced collaborative skipping of SMN2 exons 5 and seven. Even more, we show that skipping of SMN2 exon seven beneath OS is dependent upon numerous aspects including promoter sequence, endogenous context along with the strength of ss.
We also show that an antisense oligonucleotide primarily based tactic to appropriate SMN2 exon 7 splicing retains its efficacy BMS599626 beneath the conditions of OS. Success Identification of the Cell Line that Lacks SMN2 To check out the probability that distinctive pathological problems can have an impact on splicing of SMN exon 7, we screened a variety of publically readily available patient cell lines, like batten illness, Parkinsons disorder and Alzheimers disease cell lines. As being a handle, we also employed undifferentiated neuronal SH SY5Y cells. SMN1 SMN2 transcripts had been analyzed by a sensitive radioactive RT PCR employing forward and reverse primers annealing to exon six and exon eight, respectively. Importantly, PCR merchandise contained the SMN2 distinct DdeI restriction webpage inside of exon 8. Thus, following DdeI digestion of PCR goods, samples from cells containing both, SMN1 and SMN2, made 4 bands. The slowest migrating band in the polyacrylamide gel represented the exon 7 included transcripts from SMN1, whereas, three rapid migrating bands originated from SMN2.

The enforced expression of miR 29 members of the family in lung cancer cells can restore ordinary patterns of DNA methylation, induce the re expression of methylation silenced tumor suppressor genes, this kind of as FHIT and WWOX, and inhibit tumorigenicity. GO 0022008. Several genes annotated to this GO phrase are linked with acantha and brain metastases. for example, mutations in activating epidermal development factor receptor had been uncovered in lots of lung cancer sufferers. Human lung cancer features comprehensive alterations of microRNA expression that may deregulate cancer related genes. for example, hsa miR 125a 5p silencing unregulated ROCK1, miR 34b methylation induced c Met overexpression, and miR 200c was silenced by methylation and downregulated TCF8 and E cadherin, which resulted in cancer invasion and deterioration. Demethylation and mutation of genes could also trigger carcinogenesis.
Methylation in the Death associated protein kinase promoter plus the opioid binding protein cell adhesion molecule like gene is observed in each adenocarcinoma and squamous cell carcinoma. GO 0005576. Epithelial Mesenchymal Transition is definitely the major course of action expected for tumor invasion and translocation. Mutations in TIMP3, LAMA B C, TMEFF2, CDH13 and other genes are associated with lung cancer deterioration. IL hop over to this site eight can initiate an airway epithelial signaling pathway, and deregulation of this gene may perhaps lead to tobacco relevant lung cancer. 5 microRNAs are seen for being expressed differently in lung cancer tissues versus the corresponding noncancerous lung tissues. Among these microRNAs, allow 7a can regulate RAS action. Epigenetic activation of human kallikrein 13 enhances the malignancy of lung adenocarcinoma by selling N cadherin expression and laminin degradation. Not long ago, MMP1 was reported for being linked with lung cancer.
The 16071G 2G polymorphism of MMP1 results in transcriptional up regulation. X Xiang et al. reported the secure expression of miR 155 substantially full report minimizes the aggressiveness of tumor cell dissemination by preventing the EMT of tumor cells in vivo. Furthermore, miR 155 straight suppresses the expression within the transcription aspect TCF4, and that is a crucial regulator of EMT. The large frequency genes and microRNAs during the top rated dysfunctional gene sets We calculated the frequency of genes or microRNAs from the prime 300 dysfunctional gene sets. The genes in both mRNA or methylation gene sets with frequency increased than 50 were defined as high frequency genes. Similarly, the high frequency microRNAs were defined as microRNAs that have frequency greater than 50 from the top rated 300 dysfunctional gene sets. The high frequency genes and microRNAs are given in Table S4.