ATM s1981 in ICF cells was not connected with corresponding ranges of double strand breaks and didn’t result in phosphorylation of checkpoint and DNA repair proteins, together with p53, that are downstream targets on the ATM kinase. On top of that, we verify that ICF cells have intact cell cycle checkpoints; on the other hand, in contrast to a latest report , we provide you with proof that ICF cells react generally to ionizing radiation. Our findings suggest that despite the fact that ATM phosphorylation at serine 1981 plays an essential function while in the activation from the kinase, occasion along with this phosphorylation are necessary to render p53 and various downstream targets as phosphorylation substrates. Epstein Barr virus immortalized lymphoblastoid cell lines had been bought from Coriell Cell Repository : ICF patient GM08714A , her mother GM08728 and father GM08729A . Unrelated ICF male LCLs, ICF two and ICF three, were previously described as P5 and P1a , respectively. LCLs from ATM? ? cells previously described as AT203LA , though unrelated normal control LCL is line Nat9 was from the collection of Richard A. Gatti. Other LCLs bought from your Coriell Cell Repository were GM03317 and GM13164 .
RSTS LCLs RT 2644 and RT 1271 were previously described . The FHSD 1 and FHSD two LCLs had been a generous donation by Dr. Sara T. Winokur . In some experiments, ICF three was not employed as a consequence of the difficulty of developing this cell line. N 3 and a T cells have been put to use as favourable and detrimental controls, respectively, for every experiment. two.two. Western blot and antibodies Nuclear MLN9708 protein lysates from every single cell line were subjected to sodium dodecyl sulfate polyacrylimide gel electrophoresis , transferred to a charged PDVF membrane and immunoblotted with all the proper antibody recognizing both native ATM , ATM s1981 , native SMC1 , SMC1 s966 , NBS1 s343 , Rad 50 , p53 s15 or tubulin . Quantification of Western blotswas executed on with Typhoon 9410 by using ImageQuant five.2 application. Quantification of phosphorylated protein signals was determined by measuring the sum of pixels generated by every single band and normalizing every single band to the usual non irradiated sample. For this reason, each graph depicts the fold phosphorylation signal more than one.
0, the worth provided for the ordinary non irradiated sample. Error bars represent the normal error of 3 independent repetitions of every experiment. two.three. Chemical compounds utilized Wortmannin was resuspended in DMSO within a 10mM stock concentration. The ideal concentration was extra to the cell culture for 1h prior to irradiation. Chloroquine remedy was carried out for 6h as MG-132 133407-82-6 selleckchem described . 2.4. Immunofluorescence microscopy LCLs were harvested, washed when in PBS and dropped onto poly d lysine coated coverslips, and fixed with four paraformaldehyde, permeabilized using 0.5 Triton X 100 in PBS, then blocked with 10 fetal bovine serum in PBS.
The measurement of DNA strand breaks by FADU is determined by the partial denaturation of double stranded DNA beneath controlled alkaline and temperature circumstances. DNA strand breaks are internet sites wherever the unwinding of DNA can begin. Briefly, following infliction of DNA harm, cell lysis was carried out. Unwinding was terminated by including a neutralizing resolution. To quantify the quantity of DNA remaining double stranded, a commercially accessible fluorescence dye was used being a marker for double stranded DNA. 2.9. Statistical evaluation Information had been evaluated working with the Mann Whitney check. three. Final results three.1. Etoposide induces apoptosis of resting and proliferating T cells Previously it was proven by Tanaka et al. that human lymphoblastoid cells, which are in the G1 phase with the cell cycle, preferentially underwent apoptosis following therapy with etoposide . We had been interested regardless of whether cells which stay from the cell cycle can also be delicate to ETO remedy. To this finish, we carried out experiments on human T cells, which are resting cells and, for comparison, we made use of proliferating lymphoblastoid leukemic Jurkat cells.
We decided to complete our scientific studies utilizing an isolated pure population of T cells, as an alternative to peripheral blood lymphocytes usually utilized for dosimetry, that are the mixture of cells of different functions, lifespan and propensity to undergo apoptosis in vivo and beneath culture problem. Moreover, T cells derived from healthy folks are truly within the Rucaparib G0 phase. To display this we performed following analyses. To start with we checked DNA written content in resting T cells and Jurkat cells by movement cytometry. The outcomes presented in Fig. 1A demonstrate that the huge bulk of resting T cells were in G0 G1 phase , while inside the population of Jurkat cells only about half of them have been during the G0 G1 phase. Previously, we showed that PHA stimulation induced proliferation of resting T cells . Then again, DNA information measurement doesn’t discriminate amongst cells inside the G0 and G1 phase.
Thus, we carried out more analysis, namely the Ki67 expression was measured by immunocytochemistry in resting and PHA stimulated T cells. Ki67 is known as a standard marker of proliferating cells. As can be seen in Fig. 1B, ahead of stimulation Cinacalcet all cells have been Ki67 damaging, whilst just after PHA stimulation some cells were Ki67 optimistic. We measured the apoptotic index of ordinary T cells and Jurkat cells treated for 24 h with etoposide at several concentrations ranging from 1 to twenty M. Apoptosis was detected by flow cytometry applying the Annexin V seven AAD assay. The apoptotic index was defined since the sum from the percentage of cells which have been Annexin V good and seven AAD negative and these which were Annexin V and 7 AAD beneficial . Fig. 2A shows cumulative values from the apoptotic index for resting T cells.
This examine identifiedmore than proteins in these cell lines, as well as membrane and membrane associated proteins. The remaining proteins have been derived from organelles in the cell and various soluble proteins. A single strategy to overcome the issue of non precise protein contamination is careful choice of the biotinylating reagent. Sulfo NHS SS biotin which is made up of a cleavable di sulfide bridge has become reported to be even more cell surface exact . Cell surface membrane proteins in amurine T cell hydridoma cell and murine unfractionated splenocytes have been labelled in this method and remedy IEF and D SDS Page employed to more purify the biotinylated labelled proteins. Mass spectrometry identified proteins, of which had been plasma membrane proteins, and activation within the splenocytes with phorbol ester and ionomycin generated modifications in expression ranges of CD, MHC II molecules and glucocorticoid independent TNFR connected gene product . As a result, biotinylation of cell surface membrane proteins may be applied to detect plasma membrane proteome changes. Nonetheless, this examine also identifiedmany other proteins, which have been obviously not plasma membrane proteins.
The reasons for this are probably due to contamination from permeabilized cells and also non particular capturing of endogenous biotin containing proteins. An alternative supply of contamination is non specified binding for the beads themselves, being a latest study has highlighted many matrix support beads put to use for affinity purification can bind non particularly a variety of abundantly expressed proteins . Commonly, cell surface proteins happen to be biotin labelled with lipid pan JAK inhibitor selleck insoluble maleimide based thio reactive reagents or as a result of N linked carbohydrates employing hydrazide based reagents . These techniques yield labelled cysteine containing peptides or N linked glycosylated peptides, which may be predicted employing in silico methods and consequently could very well be made use of to determine whether a protein is possible to be surface labelled. Applying this in silico technique to the CD protein relatives, and together with the proviso that a minimum of two peptides have to be detected for high confidence dependable identification, CD proteins containing cysteine peptides and N linked glycosylated peptides were predicted for being identifiable by mass spectrometry .
Nevertheless, this examine also uncovered that CD proteins wouldn’t be recognized, and an illustration of such a protein is CD a popular B cell protein, which isn’t going to have N linked glycosylation online websites and in theory would only make a single cysteine containing peptide. In line with this particular, CD has not been detected by biotin labelling in any on the thus far published proteomics scientific studies on B or lymphoid Kinase Inhibitor Libraries selleckchem cells . Database in silico research happen to be utilised to estimate the amount of possible transmembrane proteins inside the human genome, and out of ?, transmembrane membrane proteins, are potentially glycoproteins .
Proteomic andmetabolomics data proof amodulation within the carbohydrate metabolism in absence of ATM activity, in particular a diverse glycolysis fee. Our findings are related using the emerging function of ATM as central regulator of cellular carbohydrate metabolism in response to oxidative worry. Lymphoma covers a broad number of heterogeneous cell varieties and diseases we refer to research with other leukemias lymphomas which have identified proteins which may perhaps also be related to B cell malignancies or demonstrate an experimental approach . For the purposes of this assessment, we briefly analysis the growth cycle of the B cell because the diverse B lymphoid malignancies can to some extent be classified in accordance to the corresponding regular B cell improvement stage. Consequently, most common lymphomas present cell surface and cellular markers indicating the stage during the advancement cycle from which they may be derived . B cell differentiation begins in the bone marrow with progression on the progenitor cell B cell, through the pre B cell stage to the immature B cell, which may possibly either be deleted by apoptosis or develop into a na?ve Bcell population that are regularly CD cells.
These tiny resting lymphocytes circulate in peripheral blood and are also resident in principal lymphoid follicles and follicle mantle zones. Mantle cell lymphoma , for instance is believed to derive from these CD na?ve B cells. Antigen stimulation of na?ve B cells results in proliferation and ultimately maturation into quick lived plasma cells that provide the early IgM antibody response. Antigen exposed Roscovitine cells migrate in to the primary follicle and fill the follicular dendritic cell network to form a germinal centre. Germinal centroblasts expressing low ranges of surface immunoglobulin down regulate BCL, rendering them susceptible to apoptotic cell death. CD and BCL are expressed in centroblasts but not in memory B cells and plasma cells. While in the germinal centre, somatic hypermutation within the variable chains on the hefty and light immunoglobulin gene loci provides rise to increased affinity antibodies.
BCL also undergoes somatic mutation, albeit at a lower frequency than the IGH locus. IGV area and BCL gene mutation are markers of B cells which have gone by the germinal centre. The majority of diffuse BMS-754807 large B cell neoplasms havemutated IGV and individuals with Burkitt lymphoma cells are BCL with mutated IGH genes, corresponding to a germinal centroblast. Consequently, DLBCL and Burkitt lymphomas are very quickly proliferating neoplasms and therefore are clinically aggressive tumours. Centroblasts mature to centrocytes predominantly during the light zone of your germinal centre, and these with somatic mutations and heavy chain class switching reexpress BCL and are rescued from apoptosis.
The concept of autophagy mediated cell death was, then again, supported in the s by experiments on neuronal death inside the target deprived isthmo optic nucleus in chick embryos. This neuronal death was characterized by abundant autolysosomes that in the end filled many of the cytoplasm, as well as from the loss of DNA through the nucleus to neighboring lysosomes. The fact that a cell?s own DNA was being degraded by autophagy went towards the view the autophagy was a survival marketing response to cellular strain. Prevention of Autophagic Cell Death by Pharmacological Inhibitors of Autophagy However, a death marketing position for autophagy gained only restricted acceptance until eventually it could be proved that inhibiting it prevented cell death. Preliminary proof for this was provided during the s by the death stopping results of methyladenine , an inhibitor with the formation of autophagic vacuoles which has been described as ?distinct? but only during the constrained sense that it does not alter the general level of protein synthesis.
Sandvig and van Beurs initial showed, in , that cell death, in this case toxin induced, may very well be prevented by mM MA. Subsequently, related doses of MAwere shown to prevent or delay cell death with autophagic traits in lots of scenarios including sympathetic neurons deprived of nerve growth component, telencephalic supplier Taxol neurons exposed to chloroquine, and cerebellar granule neurons deprived of serum and potassium. In all cases, the dying cells had been proven to include countless autophagic vacuoles, and their rescue by MAwas accompanied by a reduction inside their written content of autophagic vacuoles. The suppression by MA of autophagy is quite possibly attributable to its inhibition of class III phosphatidylinositol kinase , nevertheless it was uncertain no matter if this is certainly also the basis of its protection towards autophagic cell death, due to the fact its pharmacological profile is poorly characterized and it quite possibly has an effect on other enzymes. It had been for this reason crucial to check no matter whether better characterized inhibitors of PI K could have similar protective results.
In many situations, these inhibitors are proapoptotic, given that they inhibit the powerfully protective class I PI K pathway, so a protective effect as a result of inhibition of class III PI K can without difficulty be masked; but in serum deprived Computer cells, LY, wortmannin, and MA have all been Tivantinib kinase inhibitor proven to become protective, apparently as a result of the blockade of autophagy. Prevention of Autophagic Cell Death by Interference with Autophagy Genes Even so, even the improved characterized PI K inhibitors have an impact on other cellular processes too as autophagy, and definitive proof to the death mediating position of autophagy was provided only a short while ago, by scientific studies involving RNA interference of exact autophagy genes.
MDL72222 blocked emesis induced by ipecac in the dosedependent manner and presented partial protection towards cisplatin induced vomiting with the dose tested. Ondansetron and tropisetron completely protected only a few pigeons against mCPBG and emetine induced vomiting. Even so, the antiemetic possible of the two ondansetron and tropisetron might are constrained through the action of the two of these compounds to induce emesis within the pigeon. Portion of your obvious lack of effectiveness of the 5 HT, antagonists may be attributable to the all or none criteria employed because the dependent variable in parts within the existing research. This demanding criteria would not reveal any partial antiemetic effects, such as an improved latency to vomiting or perhaps a decrease in emetic episodes, which are commonly reported with 5 HT, receptor antagonists and were observed when MDL72222 was utilised to block cisplatininduced emesis inside the current research. So, use of these allor none criteria might possibly have induced the effectiveness of these compounds to become underestimated.
Species distinctions inside the emetic response could also account for your lowered efficacy within the 5 HT, receptor antagonists while in the present study and while in the review by Preziosi et al The vomiting reflex during the pigeon is initiated with obvious ease and, in addition to ridding Maraviroc UK-427857 selleck chemicals the body of feasible toxins, can be employed to feed the younger. Inside the existing examine, LY228729 was much more productive than the five HT, receptor antagonists to block emesis induced by uli, propose that 5 HT receptor agonists may have a broad cisplatin, ipecac, emetine, mCPBG, or ondansetron. These spectrum of antiemetic action that is not shared by five HT, information, as well as earlier information exhibiting the five HT receptor antagonists. This conclusion is further supported by agonists I OH DPAT and LY228729, but not the 5 HT, an the fact that five HT receptor agonists block a broad choice of tagonist tropisetron, prevented vomiting induced by ditolygu emetogenic stimuli in the amount of other species also anidine too as vomiting conditioned to environmental stim . Guinea pigs obtained from Marsh Farms, Gilroy, CA had been utilized.
The animals had been fasted overnight and sacrificed by a blow inside the neck; the abdomen was opened plus the ileum was isolated. Strips of ileum about two three cm extended were utilised as the intact intestine, or the longitudinal muscle myenteric plexus was ready as described by Paton and Zar . The tissues have been suspended in a twelve ml organ bath maintained at 37 C. The tissue was Capecitabine stored on a Krebs Ringer choice with the following composition : NaC1 118, KC1 four.75, CaC12 2.54, K2HPO4 1.19, MgSO4 one.20, NaHCO3 25.0, glucose 11, choline chloride 0.002, diphenydramine 0.001, gassed with 95 oxygen five carbon dioxide. Isometric muscular contractions were registered by way of a Grass force displacement transducer coupled to a Grass polygraph.
Elevated tail flick latencies had been evident in all remedy circumstances following defeat even though, yet again, the degree of inhibition of ache was reduced by remedy with medicines. In between groups comparisons indicated that whereas the groups were equivalent on pre defeat tail flick latencies, all doses of MDL 73 147EF appreciably attenuated defeat induced elevation in tail flick latencies several . A more study showed that smaller doses of MDL 73147EF were with no impact on defeat analgesia . MDL 72699 Statistical examination revealed a substantial foremost result for defeat I,86 109.17, P 0.011 in addition to a significant interaction in between drug and defeat . As proven in Fig. five , pretreatment with WAY 100135 prevented the lessen in extracellular 5 HT produced by imipramine . Pretreatment with an inhibitor of NA synthesis, aMPT was applied in an try to examine the influence of NA around the adjust in five HT generated by imipramine. As proven in Fig. 5 , there was no important distinction inside the impact of imipramine when administered 2 hr after olMPT. Extracellular 5 HT was decreased to about 70 of baseline irrespective of NA depletion. Whilst tissue levels of NA were not measured inside the current examine, ctMPT inside the reversible Proteasome inhibitor dose array used has become proven to deplete NA to about 50 of regular CNS ranges in two 4 hr right after injection , and in addition to lessen by 80 hippocampal dialysate DOPAC that is in all probability derived to a fantastic extent from NA neurons in this area ahab deh and Fillenz, 1992 . An alternative nonselective inhibitor of five HT and NA uptake, amitriptyline, was also tested. During neighborhood infusion of citalopram into the ventral hippocampus, systemic amitriptyline even at higher doses resulted in no sizeable alter in added llular 5 HT . In comparison to saline taken care of handle animals, at a dose of 10 mg kg, there was an obvious slight increase in extracellular five HT, while the difference was not considerable. The selective inhibitor of NA uptake, maprotiline, even at large doses had no significant result on extracellular five HT in comparison to saline manage levels . The decrease dose also tended to provide a rise in dialysate .five HT in comparison to saline Taxol control levels, but the big difference .was not sizeable. DISCUSSION Extracellular five HT from the ventral hippocampus of anesthetized rats was monitored by in vivo microdialysis. Various selective and nonselective monoamine uptake blockers were tested for his or her effects on 5 HT release. The results indicate the really selective five HT uptake blockers, citalopram, paroxetine and sertraline developed the largest inhibition of 5 HT release. Clomipramine blocks 5 HT uptake which has a potency only about lo fold higher than NA . Systemic administration of clomipramine developed a moderate inhibition of five HT release.
The animals had been then observed to the occurrence of clonic and or tonic flexion and tonic extension seizures through the subsequent 30 min. two.2.six. Neurological deficit check in mice 15 min following dosing, the mice have been individually placed on the 65 cm long 16 gauge copper wire suspended 40 cm above a table. The number of mice capable of remaining about the wire for at the very least ten s and also the complete time the mice remained to the wire, as much as a highest of 60 s were noted . two.three. Experimental layout Drug and vehicle treatments had been ready as coded administrations and their effects assessed within a ‘blind’ method. Mice have been made use of as soon as only in remedy groups of 5. Final results had been analysed using single factor analysis of variance and, wherever appropriate, followed by Dunnett’s procedure for comparing all treatment options with handle. Acute administrations had been provided i.p. or p.o. 40 min before testing and continual remedies had been provided twice everyday at 08:00 and 17:00 h, except for your pentylenetetrazole seizure check or even the neurological deficit test.
While in the latter cases, mice were dosed with RS 42358 197 or diazepam and left for 15 min before obtaining pentylenetetrazole or positioned about the suspended wire. Rats have been applied the moment only in therapy groups of ten or as 6 pairs and obtained car, diazepam or RS 42358 197 as acute therapies and had been examined forty min immediately after treatment method. Information obtained were analysed making use of single issue examination of variance followed by Dunnett’s t check. The protocols for Purmorphamine cost selleckchem the use of medication of abuse inside the rat have been the exact same as applied from the mouse. 93 A total of ten marmosets were made use of at four day intervals through the entire examine and had been subject to a random crossover of acute treatment options and assessed forty min just after therapy. Data obtained was analysed applying a paired t check. In any respect occasions, the behaviour of mice, rats and marmosets was routinely assessed for that presence of behaviours that will non specifically interfere with the expression of aversive behaviour and its inhibition, e.g. stereotyped movements, gross excitation, seizures or sedation.
two.4. Medicines Consuming water containing alcohol was freshly prepared on a daily basis. Nicotine hydrogen tartrate , cocaine hydrochloride , RS 42358 197 N two,four,five,6 tetrahydro 1 H benzo isoquinolin Sodium Danshensu one one hydrochloride and N 2,four,5,six tetrahydro l H benzo isoquinolin l 1 hydrochloride, synthesized while in the Institute of Natural Chemistry, Syntex Investigation, Palo Alto, USA, had been dissolved in typical saline. Pentylenetetrazole was dissolved in distilled water. Diazepam was dissolved from the minimal amount of polyethyleneglycol and ready to volume with distilled water. Doses of drugs are expressed as the base and were administered inside a volume of one ml kg or one ml 100 g by the i.p s.c. or p.o. route.
The decrease in dorsal raphe S HT neuronal firing produced by the microiontophoretic application of DO1 was related to the ejection current with complete inhibition of firing seen at 90 nA. iscussion Systemic administration of DOI produced a marked dose related decrease in dorsal raphe 5 HT neuronal firing. DOI also decreased the firing rate of 5 HT neurones in the dorsal raphe . Furthermore, the results compare with those obtained with the 5 HT agonist 8 OH DPAT which following either systemic or intra raphe administration inhibits both 5 HT neuronal firing and cortical 5 HT release . The decrease in the firing rate on i.v. administration of DOM has been suggested to be related to large increases in blood pressure rather than as a direct action of DOM on the raphe neurones . In this study blood pressure was not monitored. However, the systemic administration of DO1 produced an extremely rapid decrease in the firing rate of 5 HT neurones in the dorsal raphe and m.c re surprisingly the return to the basal rate occurred quickly after the period of cessation.
These findings may suggest that the effect of DO1 on the firing rate of the dorsal raphe neurones is mediated indirectly by change in another system and this might account for the on oft effects observed. One possible explanation is that DO1 has local anaesthetic properties; however this would not adequately explain the present results as DO1 only inhibited the firing of identified 5 HT neurones and not other ceils. Local administration of DOI into the raphe also produced a Pazopanib price decrease in 54HT neuronal firing ir?dicating that the DOI mediated inhibition was due to a direct effect in the dorsal raphe nucleus. The administration of DOI directly into the frontal cortex did not decrease frontal cortical concentration of extracellular 5 HT unlike systemic administration, suggesting that the effect of DOI on frontal cortex 5 I R release is not due to an action within the frontal cortex itself.
However, when DO1 was administered locally into the dorsal raphe the concentration of extracellular 5 HT in the frontal cortex Entinostat decreased. This result suggests that the decrease in 5 HT release in the frontal cortex on systemic administration of DO1 is due to an action of DO1 within the dorsal raphe and may possibly result from the decrease in the firing rate of 5 HT neurones. The decrease in extracellular concen tration of 5 H F in the frontal cortex, however, occurred considerably after the decrease in the firing rate of the 5 H I? neurones in the dorsal raphe and persisted after the firing rate had returned to pre drug value. The percentage decrease in extracellular .5 HT in the frontal cortex was also smaller than that of the firing rate of the 5 HT neurones in the dorsal raphe.
Phosphorylation regulation of some focal adhesion proteins such as FAK and paxillin are identified to become of utmost significance in the handle of focal adhesion construction turnover and anoikis . Regularly, FAK is stimulated by transmembrane integrin proteins that bind to fibronectin extracellularly and to actin filaments intracellularly. FAK, when activated by integrins, can suppress anoikis . Countless oncogenic development components, kinases, and prosurvival transcription factors, for instance, NF?B, inhibit anoikis by activation of FAK, independently of integrins . Since the actin filaments terminate at focal adhesion, plus the integrity of focal adhesion complicated requires right organization of ECM, integrins, actin, and a series of cytoskeletal proteins, anoikis is readily impacted through the reorganization of actin that processes RhoA modulation. Nonetheless, to date, the mechanism that RhoA regulates anoikis has not drawn ample review, though other two big members with the Rho loved ones, i.e Rac1 and Cdc42, are supposed to inhibit anoikis . Just lately, it has been reported that RhoA is activated within the ethanol induced anoikis in astrocytes .
Primarily based on our findings that RhoA is upregulated during the gastric cancer cells, and that overactivation of RhoA makes the gastric cancer cells resistant to anoikis, we hypothesize that, in these cells, RhoA confers anoikis resistance Ponatinib clinical trial kinase inhibitor via, at the very least in element, marketing F actin assembly and focal adhesion formation. It truly is regarded that active RhoA can initiate the assembly of a new actin filament from actin monomers , and vinculin is known as a essential protein in focal adhesion linking actin filament to integrin . We found in cells wherever endogenous RhoA activation is suppressed from the introduction of dominant damaging mutant that actin fails to organize on the fibers and that vinculin couldn’t localize to focal adhesion web sites. Additionally, these cells are sensitive to the autonomous and ATO induced apoptosis, at the same time as anoikis, compared to their parental cells. In contrast, in cells bearing the constitutively activated RhoA, focal adhesion is strengthened and cells are better spreading in culture, and moreover, cells are remarkably resistant to apoptosis and anoikis.
Consequently, our examine has verified for your very first time that RhoA activation is important Raltegravir for your upkeep of anoikis resistance phenotype in cancer cells in vitro, simultaneously suggesting that RhoA may very well be a handy therapeutic target for gastric cancer. Despite that RhoA activation may perhaps result in anoikis resistance parallel that of the non cytoskeletal pathway, as we also have noticed that NF?B activation is involved , the striking morphologic distinction presented from the two types of RhoA mutant transfected cells is proof of the predominant contribution from the cytoskeletal pathway.