When the balance of pro- and anti-apoptotic proteins adjustments to favor apoptosis, because it regularly does following efficient targeted therapy, two terminal members in the Bcl-2 household, Bak and Bax, homoand/ or hetero-dimerize to type porous channels in the mitochondria, committing the cell to apoptosis . Current information has demonstrated that the pro-apoptotic Bcl-2 loved ones member BIM can be a particularly important mediator of targeted therapy-induced apoptosis in the two blood and reliable tumor cancers . This BH3 only Bcl-2 relatives member directly binds on the antagonistic pro-apoptotic Bcl-2 members, this kind of as Mcl-1 and Bcl-2, by binding into their hydrophobic clefts, countering their pro-survival results . BIM also interacts straight with Bax, activating it to advertise cell death . In cancers addicted to receptor tyrosine kinases, the regulation of critical intracellular signaling pathways is underneath rigid control of your corresponding RTK. Pharmacological inhibition in the RTK with targeted therapies prospects to suppression of these signaling pathways and frequently effects in apoptosis .
In EGFR mutant NSCLC cells, we and many others have demonstrated that apoptosis is triggered by tipping the scale of pro- and anti-apoptotic Bcl-2 family members in favor of pro-apoptotic signaling . BIM amounts expand following MEK-ERK pathway suppression, and Mcl-1 selleck chemicals he has a good point levels reduce following PI3K-mTORC pathway inhibition downstream of EGFR . In most cases, the MEKERK pathway suppresses BIM expression by direct phosphorylation of BIM, leading to proteosomal degradation . The upregulation of BIM thanks to suppression of MEKERK signaling is important for the induction of apoptosis, but in EGFR mutant cancers, it isn’t adequate. Cutting down ranges of cellular BIM with siRNA and shRNA blocks TKI-induced apoptosis .
Other powerful targeted treatment paradigms have also demonstrated an integral position for BIM in advertising apoptosis in response to targeted therapies, together with BRAF mutant colorectal cancers , BRAF mutant melanoma cancers , Bcr-Abl translocated chronic myeloid leukemia cancers , and EML4-ALK translocated lung supplier PCI-24781 cancers . Importantly, proof from in vivo studies suggests, but does not show, that the apoptotic response may well be an crucial contributor to marked tumor regressions in vivo upon remedy with targeted therapies . So, we hypothesize that patients with oncogene-addicted cancers that undergo one of the most dramatic apoptotic responses to kinase inhibitors could get pleasure from the best advantage from kinase inhibitors. Even so, there continues to be no biomarker identified to date that accurately predicts which EGFR and HER2 addicted cancers are probably to undergo robust apoptosis in response to to TKIs.
Right here, we describe the identification of pre-treatment BIM amounts like a functional biomarker that predicts the induction of apoptosis in numerous oncogene-addiction paradigms.

To assess the likelihood that pharmacological inhibition of mTOR kinase inhibitor might be implemented to sensitize GBMs to cisplatin, and possibly other DNA-damaging chemotherapies, we tested the effect on the mTOR kinase inhibitor, PP242 on mediating cellular response to CDDP, along with other DNA damaging agents . PP242 significantly enhanced CDDP-mediated cell death of U87-EGFRvIII-expressing GBM cells , as did the IKK inhibitor BMS-345541 . PP242 also improved PARP cleavage of EGFRvIII-expressing GBM cells handled with temozolomide or etoposide , suggesting a potentially broader function for mTOR kinase inhibitors in sensitizing GBMs to DNA damaging chemotherapies through IKK/NF-|êB signaling. mTORC2 inhibition reverses cisplatin resistance in xenograft tumors To find out regardless of whether mTORC2 inhibition sensitizes EGFRvIII-expressing GBM cells to cisplatin in vivo, we created stable cell lines with shRNA-mediated knockdown of Rictor.
We employed this genetic technique, purchase INK1197 as opposed to pharmacological inhibition within the mTOR kinase, to unambiguously identify the significance of mTORC2 signaling on chemotherapy resistance in vivo, without having any direct suppression of mTORC1 signaling. We confirmed stable knockdown of Rictor and suppression of mTORC2 and NF-|êB signaling in U87 and U87/EGFRvIII cells, which also resulted in decreased cell proliferation . Rictor knockdown remarkably inhibited mTORC2 and NF-|êB signaling in xenograft tumors and decreased tumor size by about 50% , devoid of important induction of apoptosis. Importantly, Rictor knockdown reversed CDDP resistance, leading to about 80% tumor shrinkage .
In immunohistochemical analysis, Rictor knockdown led to lessen in p-p65 -positive tumor cells in addition to a 5-fold grow in apoptotic cells in the treatment of cisplatin. For that reason, mTORC2 inhibition can reverse chemotherapy resistance in vivo and acts synergistically with cisplatin to induce tumor cell death. To find out if the mTORC2-NF-|êB Rutoside pathway defined above is active in human GBM, we examined surrogate biomarkers of mTORC2 and NF-|êB in tumor tissue samples and adjacent normal brain from 140 sufferers arrayed on two tissue microarrays . Elevated phosphorylation of EGFR and Akt have been detected in 44% and 77% of GBMs respectively, as previously reported . These numbers are constant with all the independent findings of EGFR mutation and/or amplification in 45% and PI3K pathway activating mutations in 87% of GBMs, reported in the Cancer Genome Atlas scientific studies .
Importantly, elevated amounts of Rictor and phosphorylated NDRG1 , and p65 had been regularly detected in tumor samples relative to standard brain tissue . The detection of Rictor, phospho-Akt, phospho-NDRG1 and phospho-EGFR had been all drastically correlated with phospho-p65 .

4 groups of mice have been handled with either CIIDCAdTRAIL+DOX or, for management groups, with CIIDCAdTRAIL , CIIDCAdGFP+ DOX, or DCAdTRAIL+DOX , beginning two weeks soon after in vivo priming with CII in CFA twice per week for two weeks. Induction of TRAIL on these DCs was accomplished in three of these groups from the addition of DOX or 0.3% eyhanol like a handle for the consuming water with 4% sucrose for 10 weeks beginning on the time of administration of DCAdTRAIL treatment. At the very least ten mice have been integrated per group. Evaluation of advancement of arthritis and joint harm. A caliper was put to use to determine the diameter of each paw of each mouse everyday. Paw swelling was determined since the maximize in diameter compared with all the diameter with the initiation with the experiment. The severity of arthritis was graded based on the following scale: 0, typical without any swelling and erythema and no expand in joint diameter; 1, slight swelling and erythema with 0.
1 to 0.3 mm grow in joint diameter; 2, swelling and erythema with 0.three Telatinib to 0.6mm boost in joint diameter; three, considerable swelling and erythema with 0.six to 0.9mm raise in joint diameter; 4, pronounced swelling and erythema with 0.9 to one.2mm maximize in joint thickness or obvious joint destruction connected to visible joint deformity or ankylosis. Each limb was graded, resulting in a optimum clinical score of sixteen per animal and expressed since the mean score on a given day. Immediately after sacrifice, the joints had been harvested, fixed in 10% formaldehyde/PBS for not less than 24 hours, decalcified by using EDTA for 4 weeks, sectioned at 5?m thickness, deparaffinized, and stained with H&E . Immunohistochemical staining of T cell infiltration. The infiltration of T cells in the joint was established by staining tissue sections with an antiCD3 Ab.
Quenching of endogenous peroxidase was performed great post to read by incubating tissue sections with 3% H2O2 at RT for 15 minutes inside a humidified chamber. Just after washing with PBS, tissue sections have been incubated with 0.25% pepsin at 37C for 30 minutes to reveal fixed Ag epitopes. Tissue sections have been treated with blocking solution at RT for 30 minutes, followed by incubation with an HRPconjugated antiCD3 at RT for one hour. Slides have been incubated with a DAB staining kit for color visualization. Slides had been counterstained by incubation with methyl green at 65C for three minutes. Five fields had been randomly selected for every joint, and the average number of infiltrating T cells was established by adding the total number of T cells, then dividing by five to obtain the number of infiltrating T cells per field of each joint.
All four joints have been evaluated, and the average number of infiltrating T cells per field per joint for just about every mouse was established by adding the total number of infiltrating T cells in all four joints, then dividing by four.

Suzuki et al reported that a blend of nanobubbles and ultrasound could permeabilize cancer cells and potentiate the cytotoxicity of cisplatin and 5fluorouracil12 in 293T human kidney, MCF7 human breast adenocarcinoma, EMT6 murine mammary carcinoma, and colon 26 murine rectum carcinoma cell lines. Below optimum ailments, nanobubbles combined with ultrasound made major cytotoxicity that was not noticed with both ultrasound or the medication put to use alone. Cytotoxicity could possibly be further enhanced by growing the duty cycle ratio up to 80%. Fast collapse of the nanobubbles when mixed with ultrasound, led the authors to hypothesize that subnanobubbles, ie, cavitation bubbles, were made by collapse with the nanobubbles as well as the shock waves created by bubble cavitation, leading to transient membrane permeability, followed by entry of plasmid DNA and/or medication.
Influencing aspects and challenges Ultrasound wave and drug delivery The qualities from the ultrasound wave, ie, number of cycles per ultrasound pulse, peak adverse stress, and frequency, IPI145 play an essential position in ultrasonic drug delivery. In many reviews of URDDS, the frequency of ultrasound waves is set at 1 MHz simply because this frequency enables microbubbles within the 1¨C3 |ìm size range to respond to ultrasound. An ultrasound contrast agent exhibits inertial cavitation at larger acoustic pressure ,81 that may outcome in formation of shockwaves and microjets,82 generate a temporary pore in the cell membrane, and facilitate drug delivery on the cytoplasm from the cell.83,84 If reduced acoustic pressures are made use of , the bubbles oscillate linearly as well as the reflected frequency will be the identical as that in the frequency transmitted . If bubbles are located near the cell membrane, gentle oscillation may perhaps trigger the cell membrane to grow to be unstable, thereby enhancing endocytosis.
81,85 Improved acoustic strain Prasugrel will induce nonlinear growth and compression within the microbubbles, leading to the bubbles to end up a lot more resistant to compression than to growth. This really is referred to as noninertial cavitation , which results in emission of nonlinear harmonic signals.86 Harmonic imaging with microbubbles can be utilized to enhance the bubbletotissue backscatter signal ratio.two Larger acoustic strain will induce growth, compression, and disruption of the microbubbles . This inertial cavitation brought on microstreams or microjets to type close to bubbles implemented as flashreplenishment in a diagnostic reperfusion research .87 These microstreams can reach a peak velocity of 700 m/sec, triggering formation of transient pores which boost the permeability with the cell membrane.
88 A further important parameter will be the number of acoustic cycles . Bubbles will oscillate at reduced pressures when one hundred cycles are applied. On the other hand, when extra cycles are used in combination with greater pressures, the bubbles are destroyed at once.

Persistent Cardiotoxicity of Anthracyclines The morphologic alterations in chronic anthracycline cardiotoxicity are cardiac dilatation and, much less frequently, mural thrombosis; degeneration and atrophy of cardiac muscle cells; and interstitial edema and fibrosis. The very first two of those alterations are similar to these viewed in individuals with congestive cardiomyopathy. The degeneration of cardiac muscle cells can assume two kinds: the initial is characterized by loss of myofibrils, so that by light microscopy the affected cells appear palestaining but nonvacuolated, and also the 2nd is manifested by marked cytoplasmic vacuolization, often linked with myofibrillar reduction. These benefits are primarily similar in humans and in rat , mouse and rabbit models of DXR cardiotoxicity. These changes are associated with complete cumulative dose and in addition towards the time scheduling of the individual doses.
Such changes begin inside 24 hr after the administration of the big, single dose of DXR but get quite a few weeks or months to create when smaller, repeated doses are provided. No substantial variations are already found in the severity of morphologic alterations demonstrated by several individuals or experimental animals in response to a provided total TW-37 cumulative dose level. In histologic preparations the cytoplasmic vacuolization is detected far more effortlessly than could be the myofibrillar loss. In the light microscopic degree, each of those changes are noticed most obviously by examining tissues fixed with glutaraldehyde, embedded in plastic resins, sectioned at a thickness of 0.51.0 ,mm and stained with alkaline toluidine blue .
Electron microscopic research have proven that degeneration of cardiac muscle cells in chronic anthracycline toxicity is a complicated phenomenon that consists of the myofibrils, CGK 733 905973-89-9 the nuclei, the T tubules, the sarcoplasmic reticulum, the intercellular junctions, and also the mitochondria. The myofibrils display lysis of your myofilaments, improvements which account, no less than in component, for the cellular atrophy. The vacuolization from the cytoplasm is mainly as a result of pronounced swelling of the sarcoplasmic reticulum ; accumulation of lipid and dilatation from the transverse tubular program also contribute on the vacuolated appearance. The intercellular junctions undergo dissociation, with formation of hemidesmosomes, intracytoplasmic junctions and spherical microparticles. These three varieties of alter are also seen in myocardial degeneration of other triggers .
The mitochondria show pleomorphism, lower in dimension, alterations inside the density of the matrix, and concentric lamellae composed of electrondense materials. Calciumcontaining intramitochondrial inclusions have not been demonstrated unequivocally in DXR or DNR toxicity.

The McMaster University cdc series can also be presented. 2. Techniques 2.one Systematic Evaluation A systematic literature review was performed to evaluate management alternatives for cdc. Databases searched included Ovid medline, the Cochrane Library, embase, along with the Thomson Reuters Conference Proceedings Citation Index. Search outcomes have been filtered by 3 researchers. The literature searches had been performed on August one, 2012, and research with English abstracts from 1980 to August 2012 were integrated. Table ii summarizes the search approaches. Included studies reported on a minimum of ten topics which has a histopathologic diagnosis of cdc obtaining just one intervention. Series during which an evaluation of therapeutic effectiveness was not conceivable were excluded . Subgroups inside a larger research that fulfilled the inclusion and exclusion criteria were also integrated.
The primary final result was response charge . Secondary outcomes incorporated progressionfree selleck chemical DZNeP survival, total survival, and toxicity. Also recorded have been the study style, quantity of sufferers, spot of patients, patient and tumour demographics, criteria for diagnosis, and criteria for assessing response. Research quality was evaluated by assigning every single review a degree of proof based on the levels of evidence criteria set out by Oxford University?ˉs Centre for EvidenceBased Medicine10. Search terms, inclusion and exclusion criteria, and outcomes had been defined a priori. Kinase one presents an overview with the systematic critique searches. two.2 Case Series In the course of 2000¨C2012, six individuals treated at a single institution obtained a diagnosis of cdc.
Patient facts was retrospectively extracted in the workplace charts for all those patients and from regionallylinked electronic health care data. Extracted data incorporated PS-341 presenting historical past, staging, remedy, pathology, posttreatment program, and survival. Cytoreductive nephrectomy was defined as nephrectomy carried out soon after a diagnosis of metastatic ailment. 3. Results three.1 Systematic Assessment We recognized three research that met our specified inclusion and exclusion criteria. Excluded studies had been both isolated situation reviews, modest surgical series, or articles or blog posts not appropriate towards the search. three.1.1 Gemcitabine and Cisplatin Oudard et al. published a prospective multicentre phase ii research evaluating the impact of gemcitabine and both cisplatin or carboplatin on cdc11 .
This study, conducted at 6 French institutions, reported on individuals with metastatic cdc, confirmed by centralized histopathology overview, who had acquired no systemic therapy or radiotherapy while in the 4 weeks just before inclusion. Median patient age was 65 years , and 87% had previously undergone nephrectomy.

This stabilised complex poisons the cell by n with the intrinsic human topoisomerase II gene unexpectedly decreased . Examination from the time program of these events revealed the dexamethasoneinduced expression within the Dtopo II gene appeared as early as 6 h following dexamethasone addition and persisted for at the least 48 h . The related reduce in endogenous Htopo II expression essential somewhat longer to detect but in addition persisted for 48 h. HBT20parent and HBT20MAM control cells showed no significant modify in Htopo II gene expression with/without dexamethasone remedy for as much as 48 h . Effect of Dtopo II expression on amounts of Dtopo II and Htopo II protein The results of immunoblotting examination making use of antibodies for the Dtopo II and Htopo II have been steady together with the RNA final results.
In these cells that contained the Dtopo II gene and were exposed to dexamethasone for 24 h, Dtopo II protein was detected PF-562271 ic50 and also the quantity of immunoreactive Htopo II was decreased . DNA protein complex formation and DNA singlestrand breaks generated by etoposide in HBT20dTOP2 cells Indirect methods had been utilized to assess regardless if Dtopo II was growing the action of topoisomerase II most closely related to etoposide cytotoxicity, that’s production of topoisomerase IIDNA complexes following publicity of cells to etoposide. Little increases in etoposideinduced DNA protein crosslink formation or etoposideinduced DNA cleavage have been observed. Even so, these were not major.
Impact of Drosophila topoisomerase II gene expression around the sensitivity of HBT20 cells to etoposide along with other cancer chemotherapeutic agents In spite of the comparatively minor effect of your expression of exogenous topoisomerase II within the biochemical correlates of topoisomerase II drug i was reading this sensitivity, the HBT20dTOP2 cells have been significantly a lot more delicate towards the cytotoxic actions of 10 gM etoposide following gene induction with Dex . The lack of difference amongst the survival of transfected and nontransfected cells in the higher dose of etoposide might basically outcome through the loss of assay sensitivity at substantial drug concentrations. Dex treatment method didn’t alter the sensitivity of HBT20parent or HBT20MAM cells . Furthermore, the transfection procedure did not by itself alter etoposide sensitivity seeing that the clonogenic survival of HBT20parent, HBT20MAM, and HTB20dTOP2 not having Dex treatment was not statistically various .
Careful examination of this effect at distinct instances following gene induction revealed that the 24 h time level was the 1 at which highest sensitisation occurred .

While we now have not confirmed that there is a direct romance between improved IP-10 expression and leukocytic infiltration, it is noteworthy that peak IP-10 mRNA coincides with maximal leukocyte ranges inside the interstitium of ADR-treated rats. Also, we have now observed a parallel lessen in proteinuria, IP-10 mRNA expression, and interstitial cell infiltrate in ADR-nephrotic rats fed a very low protein weight loss plan, once again suggesting a shut romance amongst these occasions. A substantial protein diet didn’t appreciably modify interstitial irritation or IP-10 mRNA expression. As diet program has become previously shown to impact the immune system,47 we propose the advantageous effect of dietary intervention on proteinuria and glomerular injury may be related to diminished release of inflammatory mediators by renal cells. This is often not the 1st time the pattern of interstitial mononuclear cell infiltration is related to events while in the glomerulus.
4448 Cytokines as well as other chemoattractant Vismodegib molecules made by stimulated resident glomerular cells may diffuse down the mesangial stalk on the hilar place and attract cells from close by capillaries and venules.44’48 Then again, in vivo, IP-10 protein was uncovered not only in the glomeruli of nephrotic rats but additionally in tubular cells. Cytokines from the glomerulus may possibly also attain downstream tubular cells by way of glomerular ultrafiltrate or capillary blood. This kind of cytokines might then stimulate the expression of adhesion molecules and/or the release of chemokines by tubular epithelium.9,44,49 In this sense, it truly is noteworthy that, apart from glomerular cells, each cultured tubular epithelial cells and interstitial fibroblasts express IP-10 mRNA upon stimulation with cytokines and, for fibroblasts, with ADR.
TNF, which has become shown right here to be transcribed in glomeruli of ADR-treated rats and to be excreted from the urine of animals and people with nephrotic syndrome, 3435 increases P-10 mRNA in rat tubular epithelial cells. In ADR-induced Resveratrol nephrosis together with other glomerular illnesses, epithelial cell damage and/or the elevated permeability from the glomerular filtration barrier could notably encourage entry of cytokines into the urinary area. However, greater glomerular permeability also leads to increased exposure of cortical tubules to albumin and to lipids bound for the albumin. In turn, the tubular epithelium may perhaps generate chemotactic lipids.46 These lipids could act alone but may also synergize with other locally made chemokines. Conceivably, the chemotactic lipids could themselves elicit IP-10 expression.

Key outcomes showed that the maximal tolerated dose of Dox by athymic mice for a six week period was 6 mg/kg/week. Dox was ready in 0.9% sodium chloride and ip injections given twice weekly. The experimental procedure consisted of the pretreatment on the mice for 15 days with sodium chloride like a handle or six mg/kg/week Dox. MDA-MB- 435 cells have been then injected subcutaneously into their dorsal midline. Tumor development was determined 25 days soon after cell injection and sizes monitored by measuring two diameters with a dialcaliper. Tumor volume was calculated as Tv = length ?á 2 ?á |D/6. On the finish of your experiments, the mice had been sacrificed and the percentage of endothelial cells expressing P-gp within the liver, kidneys, heart, and tumor measured by flow cytometry. Tissues have been lower into about 1?á1-mm2 squares and rinsed in physiologic serum.
The pieces were incubated with two mg/ml collagenase at 37C for twenty minutes with regular agitation. The selleck chemical Seliciclib cell suspension obtained following extensive trituration by using a five ml pipette was filtered on the 70 |ìm nylon cell strainer followed by a second forty |ìm filtration. The second filtrates were centrifuged at 1200 rpm for five minutes as well as the pellets washed twice in one ml PBS containing 0.5% BSA. Endothelial cells had been isolated by immunoabsorption on magnetic beads coated with anti-mouse CD31 and CD105 IgG based on the recommended protocol . The isolated cells had been characterized by movement cytometry applying anti-mouse vWF IgG or C219 antibody. Labeling was unveiled by 2nd incubation with fluorescein-conjugated goat anti-mouse IgG. Immunohistochemical staining Immunohistochemical research have been carried out on 5 |ìm paraffin sections before and after therapy.
Major antibody selleck chemicals ACY-1215 towards P-gp C219 antibody was employed at 1:50 dilution. All of the immunostainings have been carried out in an automated immunostainer . The intensity and percentage from the cytoplasmic staining on tumor sections were mentioned. Statistical analyses Data were analyzed employing one-way ANOVA and Mann¨CWhitney U tests as ideal. The data of qPCR, invasion assay, and in vivo data are presented as mean ?à SEM. The rest of the information is presented as suggest ?à SD. A probability worth of ?ü 0.05 was regarded as statistically major. Benefits Multidrug resistance of endothelial cells Our experiments showed that HMEC-1 cells are at first sensitive to Dox treatment. In our try to research the induction of drug resistance in endothelial cells, we extra progressively rising doses of Dox into the culture media within the HMEC-1 cells while in a period of approximately twelve weeks.

Zebrafish Abcb1a/Abcb5 protein also associates with Abcb5 orthologs from other organisms inside a phylogenetic tree . In this tree, proteins from distinctive fish species designated as Abcb4±, Abcb1±, Mdr1± or P-glycoprotein± group to a cluster of fish P-glycoproteins that show near romantic relationship with tetrapod ABCB1 and ABCB4 . This suggests that tetrapod and teleost ABCB1/Abcb1 and ABCB4/Abcb4 possess a normal ancestor. Mammalian ABCB1 and ABCB4 sequences are tremendously comparable however the functions with the proteins largely vary. Whereas substrate recognition of ABCB1 is rather unspecific, enabling it to act as a versatile efflux pump of toxicants, ABCB4 may be a hugely specialized transporter that trans-locates selected phospholipids and does not efflux toxicants from cells . Variations in perform in spite of the large sequence similarity of ABCB1 and ABCB4 raise the question if functional properties in the closely connected fish P-glycoproteins are ABCB1- or ABCB4-like and if they are toxicant transporters.
Further, the capability of human ABCB5 to confer multidrug resistance of cancer cells could possibly suggest that the zebrafish Abcb5 ortholog could act as being a cellular efflux pump of toxicants. The absence of an abcb1 ortholog contradicted detection of an ABCB1-like efflux action and multixenobiotic resistance of zebrafish embryos . We, hence, investigated if instead selleck original site another P-glycoproteins, Abcb4 and Abcb5, act as toxicant transporters. Abcb4 and abcb5 transcripts are constitutively expressed throughout the very first 48 hours of zebrafish embryo advancement We examined constitutive expression levels of abcb4 and abcb5 transcripts in zebrafish embryos from 1 to 48 hpf for figuring out if these genes are transcribed and consequently are candidates to be associated with cellular toxicant defense in these stages.
Sodium Danshensu Transcripts of the two transporters had been existing in any way sampled time points of development . Abcb4 and abcb5 transcript ranges varied one to two orders of magnitude among developmental phases, but expression was usually minimal in comparison to, for instance, |-actin with transcript ranges that had been three to four orders of magnitude increased. Through the distinctive embryonic phases deemed transporter transcript ranges of the two transporters have been highest at one hpf; abcb4 was lowest at 12 hpf, growing from there on; abcb5 amounts varied from stage to stage . Transcripts in one and 6 hpf embryos are maternally transferred mRNA as de novo transcription is initiated concerning mid-blastula and gastrula phases .