Having said that, it can be also doable that there are other downstream genes differentially regu lated by MiTF WT and MiTF S73A, consequently affecting the cell cycle progression. The short-term G1 arrest mediated by MiTF WT seemed to enhance cell survival after UVC, because the cell death was decreased to about half of that in cells expressing MiTF S73A or management GFP protein. This result was even further confirmed in numerous melanoma cell lines expressing unique levels of MiTF. Cell lines with substantial ranges of MiTF accumulation survived better than cells with reduce or un detectable degree of MiTF.
This outcome is constant which has a latest locating that MiTF dose was correlated with cell survival right after broad band UV radiation, Like a tumor suppressor enjoying versatile roles in lots of aspects of cell cycle progression and DNA replication, p21WAF1 CIP1 is subjected to regulation of a number of tran scription factors such as p53, Rb, c Myc and MiTF, When it is actually effectively established selleckchem that p21WAF1 CIP1 inhibits CDK activities and therefore inhibits cell cycle progression, p21WAF1 CIP1 is also important for DNA replication initiation by binding to proliferating cell nuclear antigen, Consequently the exact position of p21WAF1 CIP1 in cell cycle progression is even more difficult and stays for being clarified. In A375 mela noma cell lines we observed a transient degradation of p21WAF1 CIP1 then a rapid recovery of this protein 12 hrs right after UVC. The early degradation occasion may well serve the objective of releasing PCNA from replication fork and consequently initiating a G1 arrest, as well as subsequent recovery could serve the purpose of inhibiting CKD activities for additional preserving the G1 arrest. CDK inhibitor p27Kip1 generally increases when cell cycle is arrested in G1 phase, yet in our experiment we observed that p27Kip1 degraded eight to twelve hours publish UVC radiation.
Intriguingly, though p21WAF1 CIP1 was degraded rapidly two to 4 hrs submit radiation, p27Kip1 maintained a comparatively unchanged level, when p27Kip1 was degraded 8 hrs publish radiation, p21WAF1 CIP1 levels started off to restore. It seems these two CDK inhibitors are orchestrated to be sure a G1 arrest in MiTF expressed A375 cells. Previously we showed that MiTF was temporarily degraded selelck kinase inhibitor immediately after elevation of cellular reactive oxygen species ranges, a practice that was also mediated by Erk1 two kinase. Contemplating that each UVC and ROS triggers very similar DNA damages and therefore could possibly utilize very similar repair pathways, the Erk1 two mediated phos phorylation and degradation of MiTF could possibly reflect a gen eral mechanism of MiTF mediated survival pathways and that is outlined in Fig seven. Upon UVR or ROS anxiety, MAP kinase is activated which leads to phosphorylation of MiTF on serine 73 and subsequent degradation of MiTF protein.
The temporary degradation was corre lated using a temporary G1 cell cycle arrest, correspond ing with p21WAF1 CIP1 degradation and re activation, which allows sufficient time for DNA injury repair and be sure of a far better cell survival, In response to UVB radiation, MiTF ranges were not transformed at the examined dose and time array, nor its phosphorylation standing, Nevertheless, MiTF was degraded without having clear band shifting following UVA treat ment, Pre remedy with U0126 also didn’t stop MiTF degradation right after UVA radiation, suggest ing that just after UVA MiTF was not phosphorylated by Erk1 2 kinase, nor was the degradation mediated by phosphorylation.

As illustrated in Fig. two, NF?B target genes are potently induced by PMA in each cell varieties. Remarkably, NF?B target genes are differentially expressed in K562 as when compared with K562 Adr cells. More notably, whereas IL6, IL8, MCP1 and A1 Bfl1 reveal stronger transcription in K562 cells, A20, cyclin D1, VEGF and P gp, are preferentially expressed in K562 Adr cells. Furthermore, repression of PMA inducible NF?B target genes can be observed in K562 and K562 Adr cells, irrespective of levels of Mdr1 P gp expression. Curiosity ingly, despite the fact that NF?B inhibitors can completely reverse the effect of PMA on P gp expression in K562 Adr cells, its basal transcription levels are not able to be additional reversed to your background P gp levels as observed in K562 cells. Eventually, efficacy of target gene repression would seem also to become compound and target gene distinct.
Altogether, these outcomes show differential inhibitory results of Sia mois polyphenols and withasteroids on target genes involved with irritation, metastasis, cell cycle, angio genesis, multidrug resistance, and anti apoptosis in doxo rubicin delicate or resistant K562 cells. Siamois polyphenols and withaferin A inhibit endogenous IL6 protein expression in K562 and selleck chemical K562 Adr cells, irrespective of doxorubicin sensitivity To assess no matter whether inhibition of endogenous NF?B tar get genes is additionally translated at the protein degree, we per formed IL6 ELISA of IL6 protein secreted in to the medium of K562 and K562 Adr cells, pretreated with dif ferent doses of quercetin or withaferin A for three h, both or not following 15 h treatment method of PMA, immediately after which medium was collected to determine IL6 protein amounts. As illustrated in Fig. 3, a comparable dose dependent decrease in IL6 protein amounts is often observed in both cell styles.
BMS-708163 In line with all the NF?B reporter gene results, inhibi tion of IL6 protein expression may be attained with decrease concentrations withaferin A than quercetin. Every one of the Siamois polyphenols and withaferin A avoid I?B degradation however the compounds selectively interfere with p38, ERK MAPK, MEK1 and Akt kinase activation As NF?B target gene expression encompasses several regulatory methods, together with I?B degradation, NF?B trans place, NF?B DNA binding and NF?B transactivation, we upcoming aimed to dissect which regulatory methods are impacted by Siamois polyphenols in K562 and K562 Adr cells. Since I?B degradation is needed for liberation and subsequent translocation of NF?B to your nucleus, we established Siamois polyphenol effects on PMA induced I?B protein degradation in K562 and K562 Adr cells. As maximal degradation of I?B is observed between 15 30 minutes just after PMA therapy, we up coming measured effects of Siamois polyphenols and withaferin A on I?B degradation following two h pretreatment and thirty minutes cotreatment with PMA.

The advent of molecular targeted therapies has spurred a search for pathological activation of receptors tyrosine kinase by way of many mechanisms in the number of malignancies such as OS. Among the RTKs KIT, Vascu lar endothelial development factor receptor 2, three and Platelet derived growth factor are identified for being concerned in OS progression and metastatiza tion, Two key pathways subsequently activated by RTKs will be the phosphatidylinositol 3 kinase AKT and also the mitogen activated protein kinases ERK 1 two.
Recent studies have demonstrated the cytoskeletal linker protein, ezrin, a member on the ezrin radixin moesin household of protein linkers among the actin cytoskeleton and plasma membrane, plays a significant function in the metastasis of OS kinase inhibitorNMS-873 and rhabdomyosarcoma, sug gesting that these metastasis linked molecules could possibly be prospective targets for treatment method, Matrix metallopro teinases play pivotal roles in tumour invasion by way of degradation of basement membranes and more cellular matrices, MMP 2 and 9 have already been observed to become involved in OS tumourigenesis and pulmonary metastasization, Sorafenib is surely an orally active biarylureic multi kinase inhibitor originally designed to block the ERK 1 two pathway by targeting Raf kinases, such as RAF one and B RAF, too as while in the presence of an V600E activat ing mutation. Off targets of this drug are other RTKs concerned in tumour progression and angiogenesis, Extra not long ago, it has been demonstrated that sorafenib induces apoptosis in human leukemia cells together with other human tumour cell lines through down regulation of the anti apoptotic protein myeloid cell leukemia one, a Bcl 2 loved ones member, Past its preclinical anti tumoural activity, sorafenib was established to be powerful in 3 different chemorefractory cancers. kidney, liver and thyroid carcinoma.
Sorafenib significantly prolongs progression absolutely free survival as well as overall survival of handled patients, Various molecular targets of sorafenib appear to be concerned during the pathogenesis or progression of OS. One pioneering perform demonstrated the amplification of Raf one in a single case of human OS, along with the expression of PDGF is associ ated with OS progression, Moreover, VEGF is in excess of expressed Ganetespib 888216-25-9 in 63% of untreated OS and it is predictive of pulmonary metastasis and bad prognosis, A broad immunohistochemical research on pediatric sound tumours, amongst them 18 cases of OS, demonstrated that KIT is expressed from the total case series, Inhibition with the ERK1 two pathway, mediated by statin treatment, induced apoptosis in OS cell lines, MCL 1 is expressed within a wide range of different human sarcoma cell lines, and MCL 1 antisense oligonucleotides combined with low dose cyclophosphamide gives a synergistic anti tumour result, and might qualify being a promising method to above come chemoresistance in human sarcoma, These studies propose that Sorafenib may very well be lively in OS.

reported that sufferers diagnosed with secondary GBM survived drastically longer than people with major GBM. Nonetheless, they correlated this obtaining to the younger age of situations with secondary GBMs than as being a reflection of various biologic habits, Quite a few research have emphasized the importance of age like a fac tor influencing survival in patients with glioblastoma, but a one of a kind getting in our examine was that for this popu lation analysis there was a survival benefit for younger age by ten 12 months increments and also when examining age being a constant variable. Race like a aspect in affecting the survival of sufferers with GBM has been studied prior to now. By far the most striking locate ing in our examine was that the Asian Pacific Islanders had a substantially superior survival when compared to your White major racial big difference in survival was seen during the Asian population when compared to white, black together with other populations.
Robertson et al. studied the inci dence of glioblastoma, astrocytoma and oligodendroglioma within the white and black population inside the Memphis Statis tical selelck kinase inhibitor Metropolitan region throughout the 1984 to 1994 time period. This review did not consist of the Asian race, but confirmed that there have been no major variations in survival be tween the white and black populations despite disparity inside the incidence costs. Our outcomes verify that racial differ ences in survival exist in patients diagnosed with glioblast oma, together with the Asian race having increased survival when compared to other races. The reasons for this are usually not plainly defined. Compact research have advised that genetic and molecular variations may perhaps play a role. There may very well be a greater incidence of principal glioblastomas overexpressing p 53 and PDGFR alpha much like sec ondary glioblastomas in Asians, Nevertheless, other un regarded molecular and biologic Thiazovivin things could perform a purpose and this needs to be more investigated.
The management of glioblastoma has progressively transformed and evolved over the program on the last two dec ades with new developments in engineering to assist with diagnosis, novel radiation techniques and advances in sur population. These information correlate with the review finished by Barnholtz Sloan et al, Among 1991and 1999, they studied one,530 patients during the SEER database diagnosed with glioblastoma aged greater than 65 years of age with the intention to analyze racial distinctions in survival. A gical procedures, Not surprisingly, many research have shown an improvement in outcomes above the final decade, An interesting obtaining in our research was the patients who have been diagnosed with glioblastoma throughout the many years 2000 to 2008 had a significantly enhanced survival when in contrast to other groups. The 2nd group by using a hazard ratio of 0. 93 and also the third group using a haz ard ratio of 0.

Significance was set at a P value of 0. 05. All statistical analyses had been performed with the software StatView v5. 1 from SAS Institute Inc, Cary, NC. Myeloproliferative neoplasms BCR ABL adverse are clonal, stem cell illnesses. Whilst JAK2 kinase will be the most regular mutation it is actually not the pri mary molecular event within this group of illnesses and quite a few other mutations are described, Usually these mu tations develop a rise in signaling pathways down stream of JAK2. Such as, STAT3 five is actually a central occasion within the pathogenesis of polycythemia vera, Recent treatment options only handle the signs of your dis ease and don’t provide the likelihood of a clinical molecular remission or cure, JAK2 inhibitors are emerging as promising new solutions on this disease.
Having said that, they don’t seem to attain finish molecular selleck or clinical remission, Proteomic screening solutions to seek out new physiopatho genic candidate proteins have not been broadly employed in cancer, even though a substantial amount of molecular genetic tests are carried out with variable benefits. One this kind of proteomic process is two dimensional big difference gel elec trophoresis, which assesses the protein profile in an accessible, economical, and higher resolution method. Nonetheless, numerous research present the resolution electrical power of 2D DIGE decreases when the cellular sort or even the volume and excellent from the protein samples will not be picked correctly, Molecular chaperones are necessary for stabilizing the fragile structures of a lot of receptors, protein kinases, and transcription aspects that take part in the pathways of normal cellular growth.
Heat shock proteins are re quired to maintain signaling proteins in an active con formation which can be swiftly triggered by development signals. As a result, HSP can be viewed as facilitators of authentic time responses to extracellular signals, particularly in develop ment and cell renewal. Lately, the selleckchem ABT-737 chaperone HSP90 has been implicated in safety of JAK2 from degradation from the MPN. As a result, the HSP90 inhibitor, PU H71, continues to be proposed as an alternate treatment to JAK2 inhibitors, Heat shock protein 70 is connected to HSP90 and blocks the apoptotic pathway at distinctive levels, HSP70 minimizes caspase activation and suppresses mitochondrial harm and nuclear fragmenta tion.
One particular from the ultimate targets of caspase three will be the transcrip tion issue GATA one, Overexpression of HSP70 can provide a selective survival benefit to tumor cells in part as a consequence of its means to inhibit several pathways of cell death, together with both intrinsic and extrinsic apoptosis. With regard to the intrinsic apop tosis pathway, HSP70 can bind straight towards the professional apoptotic BCL2 family members member BAX and protect against it from translocating to mitochondria, in which the latter disrupts mitochondrial membranes following an apoptotic stimulus.

Ionizing radiation upregulates mTOR in pancreatic cancer cells at the two transcriptional and protein amounts To identify no matter whether ionizing radiation modulates the ex pression and action of mTOR in human pancreatic can cer, PANC 1 cells have been cultured in normal ailment and taken care of with escalating doses of radiation for one h. As shown in Figure 2A, radiation induced a dose dependent increase of each mTOR and p mTOR at doses from 0 Gy to 10 Gy. To verify this, mTOR levels had been also examined in other two pancreatic cell lines, Capan two and BxPC 3, with radiation remedy at five Gy and also the related final results had been obtained, Furthermore, the mRNA degree of mTOR was detected and benefits showed that mTOR transcript was up regulated by radi ation in PANC one cells and also the peak worth appeared at five Gy by four.
36 fold, related data had been ob tained in BxPC 3 and Capan 2 cells, Meanwhile, Bcl two, Bcl XL and Mcl selleck chemicals CX-4945 one as principal mem bers of apoptosis family showed no large variation before and after radiation therapy, Collectively, ionizing radiation substantially selleck inhibitor induces mTOR expres sion and activation at mRNA too as protein levels, which potentially contribute to radioresistance in pancre atic cancer. mTOR is a significant issue in pancreatic cancer radioresistance To additional verify no matter if mTOR is really a direct aspect that is certainly associated with radioresistance of pancreatic cancer, PANC 1 irradiation resistant cell line was produced and colony formation assay was made use of to verify the radioresistance potential of PANC one RR, Intri guingly, increased ranges of mTOR and p mTOR have been ob served in PANC 1 RR cells as in contrast with PANC 1 P cells, To even more test that mTOR is indispens in a position inside the radioresistance,mTOR precise shRNA was transfected into PANC 1 cells.
Immediately after transfection, cells had been taken care of with radiation for 48 h, effects uncovered that endogenous mTOR in PANC one cells was remarkably downregulated and PANC 1 cells had been additional delicate to radiation in mTOR shRNA transfection group as in contrast using the manage shRNA group, Every one of these information collectively show that radiation in duced mTOR expression and activation contributes to radioresistance and knockdown of endogenous mTOR ef fectively overcomes the radioresistance of pancreatic can cer cells. Downregulation of miR 99b, a essential mediator of mTOR kinase, contributes to radiation induced mTOR upregulation It is well-known that miRNAs broadly take part in gene expression regulation and play vital roles in different phys iological and pathological processes.

In contrast, PEA3 depletion diminished the invasive cap abilities of OE33 by nearly 60%, indicating that PEA3 is significant for invasion by OE33 cells. To further extend our website link concerning PEA3, MMP one and invasion, we asked regardless of whether MMP one depletion in OE33 cells would also result in a lower in invasion. This was without a doubt the situation, albeit to a lesser extent, suggesting that PEA3 possible drives invasion by way of many targets additionally to MMP 1. Investigate on PEA3 has largely focused on its ability to manage MMPs and cell invasion. A former research in breast and ovarian cancer cells demonstrated that PEA3 controls the expression of cell cycle regulators this kind of as Cyclin D3 and p21 respectively, and therefore sug gested that it could possibly be concerned in controlling prolifera tion. We therefore investigated if PEA3 was crucial for oesophageal cancer cell proliferation. 1st we depleted PEA3 in Het1A cells.
Above a 96 hour time period, the proliferation of Het1A cells was just like cells trea ted with manage duplexes, In contrast, OE33 cells handled with either SMARTpool siRNA against PEA3 or even the deconvoluted Tosedostat structure siRNA constructs A and B, exhibited a sustained a development arrest, In summary, PEA3 is required for the proliferation and enhanced invasive properties of OE33 adenocarci noma cells. ERK MAP kinase signalling is essential for OE33 cell proliferation and invasion Prior studies have demonstrated that PEA3 activity is potentiated by ERK MAP kinase pathway signalling and that this signalling pathway plays an important purpose in cancer cell properties, including invasion and prolif eration, We for that reason investigated the activation standing of this pathway in oesophageal derived cell lines by western examination using an anti phospho ERK anti body.
Amongst the four lines studied, phospho ERK levels had been highest in OE33 cells, indicating the ERK pathway is lively in these cells, OE33 cells also contained higher levels of MMP 1 and MMP seven protein, which is consistent with their relative mRNA expression ranges, Even so, there appears to become additional submit selleck transcriptional events acting on MMP one as OE21 present much more MMP one protein than OE33 cells however consist of less MMP one mRNA, In contrast, Flo1 cells contained little MMP 1 mRNA or protein and very minimal ranges of phospho ERK, So the lack of ERK signaling in these cells most likely explains why MMPs are certainly not hugely expressed regardless of the presence of PEA3 household members. To test this hypothesis, we handled Flo1 cells with PMA to activate ERK pathway signalling. A considerable enhance in MMP one expression was observed, in maintaining with the notion that ERK pathway signalling is needed for MMP 1 induction also to PEA3 overexpression.

This was even more confirmed by MiTF S73A mutation which was not degraded after UVC. The degradation was inhibited by proteasome inhibitor MG132, suggesting that the sig naling pathways through Erk1 two activation soon after UVC and right after c Kit stimulation have been distinct from every other. We observed that re expression of MiTF WT within the A375 melanoma cell line restored a short-term G1 arrest soon after UVC, though handle cells expressing GFP or MiTF S73A cells didn’t, suggesting that degradation of MiTF after UVC may possibly be certain a right G1 cell cycle arrest and therefore allow DNA restore and enrich cell survival. In reality we observed that cells expressing MiTF WT showed much better overall survival right after UVC. While MiTF S73A mutant was existing continually after UVC, it was not able to set off the G1 arrest. As our data exhibits, a part of the reason could be the weak activation on p21WAF1 CIP1 professional moter by this mutant.
Nonetheless, it is actually also possible that there are other downstream genes differentially regu lated by MiTF WT and MiTF S73A, therefore affecting the cell cycle progression. The temporary G1 arrest mediated by MiTF WT appeared to boost cell survival just after UVC, as a replacement the cell death was decreased to about half of that in cells expressing MiTF S73A or handle GFP protein. This consequence was even more confirmed in different melanoma cell lines expressing different levels of MiTF. Cell lines with high ranges of MiTF accumulation survived superior than cells with reduced or un detectable degree of MiTF.
This result is consistent using a recent finding that MiTF dose was correlated with cell survival right after broad band UV radiation, As being a tumor suppressor enjoying versatile roles in many facets of cell cycle progression and DNA replication, p21WAF1 CIP1 is subjected to regulation of many tran scription elements which includes p53, Rb, c Myc and MiTF, Even though it’s effectively established that Odanacatib p21WAF1 CIP1 inhibits CDK activities and for that reason inhibits cell cycle progression, p21WAF1 CIP1 can be crucial for DNA replication initiation by binding to proliferating cell nuclear antigen, Hence the precise role of p21WAF1 CIP1 in cell cycle progression is a lot more complicated and stays to be clarified. In A375 mela noma cell lines we observed a transient degradation of p21WAF1 CIP1 and after that a speedy recovery of this protein 12 hrs after UVC. The early degradation event could serve the objective of releasing PCNA from replication fork and for that reason initiating a G1 arrest, and the subsequent recovery may serve the goal of inhibiting CKD routines for additional sustaining the G1 arrest. CDK inhibitor p27Kip1 commonly increases when cell cycle is arrested in G1 phase, nonetheless in our experiment we observed that p27Kip1 degraded eight to 12 hours post UVC radiation.

It particularly blocks FAK phosphorylation and thus targets FAK catalytic exercise. PF 228 is a extra specific system to lower FAK phosphorylation in contrast with FRNK overexpression. As a result, in our review PF 228 was even more utilized to verify the role of FAK phosphoryla tion during the chemoresistance of pancreatic cancer cells. We employed PF 228 to downregulate constitutive FAK phos phorylation in Panc one cells and LN induced FAK phos phorylation in Aspc one cells respectively. PF 228 could inhibit both constitutive and LN induced FAK phosphor ylation in a dose dependent method, 1M PF 228 was adequate to efficiently block the two constitutive FAK phosphorylation in Panc 1 cells and LN induced FAK phosphorylation in Aspc 1 cells. Consistent with the effects of FAK phosphorylation inhibition by FAK RNAi and purchase PTC124 FRNK overexpression, distinct inhibition of FAK phosphorylation by PF 228 led to the corresponding inhi bition of AKT but not ERK phosphorylation in Panc one cells and Aspc one cells.
The levels of complete FAK, Akt and ERK protein were not drastically impacted. We additional determined the effects of PF 228 on Gem induced apoptosis in pancreatic cancer cells. Cell apopto sis was established by techniques as described above. Con sistent with all the results of FAK Serdemetan structure RNAi and FRNK overexpression, PF 228 rendered Panc one cells far more sensi tive to Gem induced apoptosis, even though in AsPC one cells PF 228 treatment method antagonized LN mediated Gem chemoresistance, which was demon strated by an increased proportion of condensed nuclei, substantially larger of Annexin V positivity and even more cleaved caspase three protein expression. Having said that, PF 228 therapy alone didn’t significantly have an effect on the apop tosis of Panc one cells on plastic or Aspc 1cells on LN. Steady with all the results of FAK RNAi and FRNK more than expression, PF 228 decreased survivin expression and Poor phosphorylation at Ser136 in Panc 1 cells and antago nized the effects of LN on survivin expression and Bad phosphorylation at Ser136 in AsPC one cells, These results even more confirmed that, constitutive and LN induced FAK phosphorylation was not less than partially liable for the intrinsic chemoresistance to Gem in pancreatic cancer cells.

As a result, we may be capable to successfully use preclinical information to find clini cally pertinent biomarkers. Our system described over of combining preclinical information obtained in cell culture exper iments as very well using established xenograft versions may well create a robust gene expression signature that may be handy for both in vitro and in vivo research. We also utilized GSEA and determined the result of therapy and time in vivo. Compared to one day treatment method, 22 day remedy improved the expression of gene sets concerned in response to hypoxia and cancer. These obtain ings additional help importance of mTOR as being a central con troller integrating signals coming from separate pathways. Other researchers have also investigated the effect of treat ment with rapamycin and its analogues on gene expres sion. Gera et al. studied Akt activation and mTOR inhibition by rapamycin in prostate cancer and glioblast oma cell lines in vitro.
They recognized 62 regulated genes and expression of 29 them have been upregulated, nevertheless, none of these genes had been on our RMI listing. Majumder et al. utilised a transgene to produce activated Akt1 in lumi nal epithelial cells inside the selleck chemical ventral murine prostate. A pros tatic intraepithelial neoplasia phenotype designed in the transgenic mice, which was fully reversed by mTOR inhibition from the rapamycin analogue everolimus, by inducing apoptosis.They identi fied 571 genes or ESTs whose expression was altered by Akt expression and mTOR inhibition. Additional examination by using gene set enrichment analysis unveiled inac tivation of hypoxia inducible factor one and its target genes, like genes coding enzymes concerned in glycolysis pathway, which are all regulated by mTOR. We utilised our rapamycin responsive gene set to probe the gene set utilized in that review and recognized only endothelin 1 gene popular in each sets.
Interestingly, in our examine endothelin one gene expression was downregulated whereas in Majumder et al. study upregulated. Moreover, rapamycin treatment method doesn’t induce apoptosis in breast cancer cell lines, so the downstream results of rapamycin in these two models PD0332991 could be unique. Absence of concord ance may not be surprising thinking of this can be a compari son of gene expression in a breast cancer cell line with that of the model of Akt activated mouse PIN. As stated by Majumder et al, cell lines and xenografts display a more complicated genetic background than an Akt activation model as survival and adaptive events have previously taken area.