Erlotinib and gefitinib happen to be the two most effectively developed and extensively used targeted EGFR focusing on medicines. Both gefitinib and erlotinib are actually implemented from the treatment of cancers that harbour an EGFR mutation, particularly non modest cell lung cancer. HER2 good breast cancer, by which the HER2 re ceptor is both overexpressed and or amplified, ac count for around twenty 30% of human breast cancers and therefore are associated with poorer prognosis. Non targeted breast cancer therapy possibilities may well include a single or much more of chemotherapy, radiation, and surgical procedure, whilst HER2 overexpressing breast cancers will normally involve trastuzumab based mostly treatment with newer agents this kind of as lapatinib, supplying a second line for treatment. Lapatinib was among the initial HER2 targetting tyrosine kinase inhibitors to get made use of during the clinic.
This dual kinase inhibitor which also targets EGFR was devel oped by GlaxoSmithKline and it is currently FDA accredited for the treatment inhibitor WP1130 of refractory breast cancer in combination with capecitabine. Identification of ro bust, reproducible predictive biomarkers is vital to the appropriate application of this kind of therapies. Several recent publications have found a correlation among pTEN AKT PI3K pathway activation and also the response the patient to either traztuzumab or lapatinib. The consensus of these reviews is that patients demonstrating very low pTEN expression are more likely to exhibit resistance to traztuzumab but sensitivity to lapatinib. A function for receptor autophos phorylation and down stream regulators of apoptosis has also been proven to get essential. These scientific studies have provided a important insight into intrinsic resistance during the HER2 target designs but have limited application as much more broadly clinically useful predictive biomarkers of response to therapy.
A lot more not long ago the little molecule TKI therapeutic arsenal has witnessed the addition of newer agents this kind of as, afatinib and neratinib. Afatinib is surely an irreversible inhibitor PD184352 EGFR HER2 inhibitor formulated by Boehringer Ingelheim currently becoming clinically evaluated in NSCLC. The aniline quinazoline framework of your inhibitor has the po tential to irreversibly bind to your EGFR and HER2 recep tors, which in turn prevents activation of the kinase domain. Much like afatinib, neratinib can be an irreversible in hibitor from the EGFR and HER2 receptors. Designed by Wyeth, this small molecule also inhibits the HER4 receptor. Neratinib interferes with phosphorylation by binding to your cytoplasmic domain with the receptors resulting in the inhibition of downstream phosphoryl ation of substrates. This inhibition in turn has an impact about the cells potential to proliferate and may ensure that the cell arrests in the accurate cell cycle transition to ensure cell death occurs.

Other datasets have been analyzed using a Mann Whit ney test for unpaired samples. In silico promoter evaluation within the Col3a1, Col5a1 and Col5a3 genes was carried out using the TFSearch and ALIBABA on the web software program, based mostly on the TRANSFAC algorithm. Stringent criteria have been utilized so that only the responsive components with a large homol ogy on the consensus sequence matched our search. Moreover, TCF LEF responsive factors, speci fic transcription components related with WNT signaling, were investigated employing the different consensus sequences as previously recognized. Result Major evaluation with the microarrays We had been able to dissect the subchondral bone and articu lar cartilage in a single piece. The heatmap from the RMA expression values through the microarray examination showed clustering on the transcriptomes into groups formed through the 3 wild variety and two from three Frzb mice, respectively.
The third presumed Frzb mouse clustered with all the wild forms and was sub sequently identified by re genotyping as a heterozygous animal. This sample was not utilized in the analysis. A total of 697 probe sets from 30,590 that had a current detection contact had been appreciably up regulated from the Frzb samples and one,524 had been appreciably down regu lated as in contrast towards the wild form mice. Cartilage particular and selleckchem NSC 74859 bone certain genes were located while in the highest percentiles of expressed genes from the microarray analysis, whereas genes specifi cally associated to T cells, B cells and platelets had been observed in reduced percentiles. possibly from RNA originating in the subchondral bone marrow. Applying the PANTHER resource, 493 mapped genes were identified as up regulated and 905 mapped genes had been identified as down regulated in Frzb mice. The 25 genes together with the greatest fold variation concerning Frzb and wild type mice are presented in Table one.
A com plete listing of all regulated genes and fold distinctions could be observed while in the supplemental products. Pathway Ginkgolide B analysis Distinctive bioinformatics tools had been utilised for analysis of your sizeable dataset with emphasis around the identification of pathways differentially regulated involving the Frzb and wild style mice. The PANTHER pathway analysis is shown in Table two. Among the up regulated pathways the ECM linked integrin pathway, the cadherin pathway, too as WNT signaling, were most striking from a biological viewpoint. Down regulated pathways pointed in the direction of irritation and immune cascades, the cell cycle, p53 activation and once again integrins. Associations within the differentially regulated gene set working with databases defining biological processes as ana lysed by PANTHER are proven within the more materi als. We also utilized the DAVID bioinformatics equipment spe cifically interrogating gene representation in KEGG and Biocarta databases.

Then, we carried out an unbiased, genome broad Cox regression survival analysis, evaluating the prognosis difference between those 3 groups. By doing this, poor prognosis asso ciated genes should display a poor prognosis during the higher expression group and also a superior end result from the low expression group. Inside the 2nd stage, we more assessed the bad prognosis correlation from the identified genes working with gene expression like a continuous variable and sought to correlate copy amount aberrations with gene expression by measuring if amplification was corre lated with substantial degree expression and deletion was asso ciated with lower level expression. Commencing with all the severe, we defined the lowest 10% of expression values throughout the entire 4,010 samples as reduced degree expression and the highest 10% of expression values as high level expression.
Employing death from breast cancer because the incident event, we carried out a genome broad Cox regression survival analysis and identified 152 genes whose large level expression was substantially asso ciated with increased danger of death from breast cancer. Furthermore, we assigned every single with the 4,010 samples into initially quartile, second quartile and third quartile subgroups according on the expression amounts of the selleck 152 recognized genes, and com pared prognosis distinctions amid these subgroups. On top of that, we applied expression signal being a continu ous variable to measure the distribution in the recognized genes. A total of 47 of your 152 genes showed linear cor relation among greater expression and bad prog nosis. The highest chance of death from breast cancer was observed in sufferers with both top 10% or 25% greater degree gene expression.
Considering the fact that amplifications or deletions are more likely to control the expression of genes inside the corresponding region, as well as correlation concerning copy amount and expres sion is recently suggested as an approach to pre dict the genuine molecular drivers in carcinogenesis, we then extended this evaluation of gene expression to assess the correlation amongst somatic copy selelck kinase inhibitor amount alterations and gene expression working with 481 invasive breast cancer samples obtained from TCGA. We identified that 26 of 47 poor prognosis connected genes showed a signifi cant correlation concerning copy quantity aberrations and mRNA expression. To assistance this modeling, we analyzed the expression of HER2, a renowned oncogene connected with bad prognosis primarily based on greater copy quantity and high gene expression. As expected, large level expression of HER2 was driven by coding area amplification and was drastically connected with bad prognosis. Importantly, we noticed each cytoplasmic HSP90 iso kinds, HSP90AA1 and HSP90AB1, had been amongst just about the most significant elements that led to increased danger of death from breast cancer, indicating that HSP90 plays a significant function in modulating bad prognosis pheno styles in breast cancer.

However, the perform for p21 downstream of TGFb has not been described in breast cancer. Within this examine, we located that large p21 expression corre lates with poor survival in breast cancer patients. The expression of p21 is required to promote tumor cell migration and invasion in vitro and community invasion in vivo. Furthermore, p21 expression is tightly regulated by TGFb Smad3 signaling in the panel of human basal like tri ple unfavorable breast cancer cell lines. We observed p21 to physically interact with Smad3 and also the histone acetyl transferase p CAF in response to TGFb and identified p21 and p CAF as vital regulators of TGFb mediated breast cancer cell migration and invasion. We also showed that p21 and p CAF regulate TGFb transcrip tional exercise on many tumor advertising target genes by controlling Smad3 acetylation and Smad3 occupancy on its DNA binding elements.
Immunohistochemical analysis of tissue arrays from breast cancer patients exposed a significant correlation concerning active TGFb Smad3 signaling and large expression levels of the two p21 and p CAF in lymph node positive invasive ductal carci nomas. Together, our findings identified p21 and p CAF as critical regulators of cell migration and invasion down stream selleck chemicals of TGFb Smad3 pathway in superior breast cancer. Techniques Cell culture and transfection Human breast carcinoma MDA MB231, SCP2 and SCP25 cells and HEK293 cells have been grown in DMEM supplemented with 10% fetal bovine serum and 2 mM L glutamine at 37 C in 5% CO2. SUM149PT, SUM159PT and SUM229PE had been grown in F 12 HAMS nutrient mixture supplemented with 5% FBS, five ?g ml insulin, 1 ?g ml hydrocortisone at 37 C in 5% CO2. SUM1315MO2 were grown in F 12 HAMS nutrient mixture supplemented with 5% FBS, five ?g ml insulin, 10 ng ml epidermal growth factor at 37 C in 5% CO2.
Cells have been transfected with distinct p21, p CAF, Smad2 and Smad3 siRNAs, six? myc Smad2, myc Smad3, p CAF and Flag tagged human p21 cDNAs implementing Lipofectamine 2000 reagent, according to your producers protocol. MDA chloroxine and SCPs cells have been serum starved for 24 hrs and stimu lated or not with 5 ng ml TGFb1 in DMEM supplemented with two mM L gluta mine. For stable cell line generation, SCP2 cells were transfected with p21 shRNA and pools of stable cells had been chosen with ten ng ml puromycin. SUM159PT cells were serum starved for 24 hrs from the absence of insulin and hydrocortisone in advance of TGFb1 stimulation. Western blot examination and immunoprecipitation Cells were lysed in cold extraction buffer containing protease inhi bitors. The lysates were then centrifuged at 14,000 rpm for 15 minutes at 4 C.

The outcomes showed that MEK inhibitor U0126 and PLCc1 inhibitor U73122 partially blocked EGF EGFR induced migration exercise of AGS cells, indicating that each signal transduction pathways participat ed from the regulating practice. To elucidate the potential inhibitory action of PKG II on these signal transduction pathways, we to begin with explored the inhibitory impact of PKG II on EGF initiated PLCc1 mediated signal transduction pathway. The outcomes showed that PKG II prevented each of the key occasions in this signal transduction pathway, which includes the Tyr 992 phosphorylation activation of EGFR, the phosphorylation activation of PLCc1, the formation of second messenger DAG, the release of calcium into cytoplasma, PKG II on EGF EGFR induced MAPK ERK mediated signal transduction pathway, our previous function have shown that PKG II inhibits the activation of all critical elements from the pathway induced by EGF in gastric cancer cell line BGC 823.
Within this paper, we investigated the inhibitory effect of PKG II on EGF EGFR induced activation of crucial components on this pathway. The outcomes confirmed that PKG II inhibited EGF induced activation of Ras protein and MAPK ERK in AGS cells, suggesting that PKG II also inhibited EGF EGFR induced signal transduction of MAPK ERK mediated inhibitor Dovitinib pathway within this cancer cell line. These success systematically uncovered that PKG II inhibited EGF induced migration of gastric cancer cells by way of blocking EGF EGFR initiated signal transduction of PLCc1 and MAP ERK mediated pathways. The signal transduction of the two PLCc1 and MAP ERK mediated pathways can activate compact G protein Rac1, which can be a vital component in regulating cell migration. To confirm selleck inhibitor the inhibition of PKG II on this vital occasion in EGF induced migration of Gas cells, we utilized pull down method to check out the activity of Rac1 in differently taken care of AGS cells.
The results showed that through EGF induced migration, Rac1 was activated and activation was associated with the two PLCc1 and MAP ERK mediated signaling. In addition, PKG II inhibited the EGF induced activation of Rac 1. This even more confirmed the inhibitory result of PKG II on EGF EGFR initiated cell migration. EGFR is closely associated with tumorigenensis. In excess of expres sion and mutation of EGFR frequently take place in many cancers. Investigate information showed that over 50% 70% of lung cancer, colon cancer and breast cancer have large expression of EGFR. In addition, cancer patients with over expression of EGFR ordinarily have bad prognosis. For instance, EGFR more than expression was detected in 60% of non little cell lung cancer sufferers plus the prognosis of your sufferers have been poor, by using a survival of four 5 months only. In vitro experiments confirmed that above expression of EGFR triggered transformation of NIH 3T3, Rat one and NRK cells and blocking EGFR activation inhibited proliferation of some tumor cells.

05 for your determination as to no matter if a big difference is sizeable or not. This p value would be the probability of gaining a false constructive consequence, so on typical we would count on to acquire a false optimistic consequence about the moment every twenty occasions the check was applied. As a result on this study the level of statistical significance was defined as alpha,0. 01 i,e. there was a 1% optimum possibility of incorrectly rejecting the null hypothesis that there is no association in between vitamin D supplementation and genetic expression. The FDR,0. one, was utilized to a listing of genes, not any distinct gene. We carried out suitable correction for multiple testing that incorporated using a one. five fold modify of gene expression mixed with false discovery fee, 0. one in our principal examination. Using a 1. five fold transform of gene expression combined with ANOVA in our subgroup evaluation. Using P,0. 01 for reducing false positive and strategy validation together with the actual replicate time PCR.
Major enrichment of GO biological approach classes had been tested for working with EASE program with P,0. 05. Results Demographic Mocetinostat molecular weight and various baseline characteristics Eight subjects who met the inclusion exclusion criteria have been enrolled. No recruited topics refused to present consent. Sixteen microarrays from eight subjects passed the excellent manage filters and normalized with the RMA method. Suggest of age, BMI and serum 25 D amounts were 26. 564 years, 2765. 9 kg m2 and 21. 868. 6 ng ml respectively and all of them have been white. 3 participants acquired 400 IUs of vitamin D3 each day and 5 participants obtained 2000 IUs of vitamin D3 each day. Immediately after eight weeks of vitamin D3 supplementation serum 25 D levels while in the group that obtained day by day 2000 IUs had a two fold maximize in contrast to subjects who obtained and had an increase of 5. 664. 9 ng ml.
Affect of vitamin D3 supplementation on expression of genes in human white blood cells To check out gene expression relationships concerning and inside of the 400 IU and 2000 IU groups principal element examination was performed. Complete variability of personal chips right after normalization is illustrated in Figure 2. There GSK2126458 wasnt a significant difference as a way to take a look at gene expression relationships among and inside of the 400 IU and 2000 IU groups. Relating to all participants, with false discovery charge,0. one, and also a one. five fold transform, 291 genes have been identified to have a statistically substantial distinction in expression from baseline to adhere to up right after vitamin D3 supplementation. The record of those 291 genes is shown in Table S4. There was a minimum of a 1. five fold inhibition of 82 genes whose expression was substantially reduced and at the very least a 1. 5 fold induction of 209 genes whose expression was substantially enhanced right after supplementation with either 400 or 2000 IU of vitamin D3 for two months. For verification candidate of gene expression improvements serious time PCR was performed for 4 genes which include CD83, TNFAIP3, KLF10 and SBDS.

These data show that whilst Myt3 expression is minimal from the producing pancreas it is relatively abundant in mature islets. Provided the substantial degree of similarity among the MYT loved ones members, and their possible functional redundancy, we needed to find out which family members member was most abundant in producing pancreas tissues and in adult islets. Applying our SAGE data we established that whereas Myt1 is extra extremely expressed in Ngn3 expressing endocrine precursor cells, Myt3 is even more abundant in mature islets. Myt1l could not be assessed as it doesn’t create any SAGE tags that uniquely map to it. In agreement, qPCR analysis of Myt1, Myt1l and Myt3 in building pancreas tissues and grownup islets showed that Myt1 was clearly much more abundant than Myt1l or Myt3 within the building pancreas, notably at E15. five and E18. 5.
Nonetheless, all three family members showed maximal expression in grownup islets, probable due selleck chemical for the higher proportion of cells expressing these things in islets as in contrast to inside of the whole establishing pancreas. To clarify which within the MYT family members is dominant in adult islet cells, we established the complete copy number of Myt1, Myt1l, and Myt3 transcripts in islets, likewise as in MIN6 and aTC1 cells using absolute quantification qPCR. Myt3 was expressed at a 15 fold increased level in islets, a four fold greater level in MIN6 cells, and a two. 5 fold higher degree in aTC1 cells than Myt1, along with a 23 fold greater level in islets, a 3 fold increased degree in MIN6 cells, along with a 2. five fold greater degree in aTC1 cells than Myt1l. Last, we sought to find out the expression level of Myt3 in human islets relative to mouse islets, and identified that although Myt3 is expressed in human islets, Myt3 expression is 4 fold increased in mouse islets.
Together, these data show that Myt3 is a lot more abundantly expressed in mature pancreas endocrine cell sorts than both Myt1 or Myt1l, and verify its expression in the and b cells. Myt3 is Expressed in Maturing and Adult selleckchem Endocrine Cells The above information indicate that Myt3 expression happens predominantly in grownup islet cell kinds. To find out regardless of whether Myt3 protein amounts match this pattern, and to determine the cell forms that Myt3 is expressed in, we formulated an antibody towards it. Making use of this antibody we identified no evidence of Myt3 protein within the creating pancreas at either E14. five or E16. 5. At E18. five nevertheless, Myt3 protein was noticed in both insulin and glucagon expressing cells. Similarly, Myt3 staining was evident throughout the islet in adult pancreas sections, whilst no Myt3 staining was evident while in the surrounding exocrine tissue. Similar to what we observed in our in situ experiments with full islets, co staining of grownup sections with endocrine cell markers showed that Myt3 co localizes in cells expressing insulin, glucagon, somatostatin and pancreatic polypeptide.

Benefits demonstrated that CX3CL1 gene expres sion is induced in normal breast epithelium of ER2 tumors compared to usual breast epithelium of ER tumors, suggesting that CX3CL1 expression could possibly be an early marker of ER2 tumor formation. On top of that, these information are supported by proof from previously published breast cancer gene expression information sets through which enhanced CX3CL1 gene expression was linked to ER tumors. Conversely, current research demonstrat ed that although CX3CL1 expression didn’t significantly correlate with ER status, substantial levels of CX3CL1 was linked with far better patient end result. Even further research are obviously necessary to absolutely have an understanding of whether or not CX3CL1 expression could possibly help define patient danger and aid in distinguishing among susceptibility to molecular tumor subtypes through early cancer advancement before histolog ical abnormalities are detected.
selective Aurora Kinase inhibitors Additionally to studying CX3CL1, past research have examined expression of CX3CR1 on breast cancer cells and the autocrine effects within the CX3CL1 CX3CR1 axis on regulating breast cancer cell migration. For instance, it was proven that exposure to proinflammatory cytokines greater the expression of CX3CR1 on human breast cancer cells therefore improving migration of those cells towards CX3CL1. Especially, TNFa triggered a substantial improve in mRNA transcript amounts of CX3CR1 in each MCF7 and MDA MB 231 cells. Also, treatment method of MCF7 cells, that are minimally invasive and also have reduced metastatic probable, with interleukin 1 and TNFa increased cell surface expression of CX3CR1. It has also been proven that CX3CR1 is involved in homing of breast cancer metastases to the brain. So, expression of CX3CR1 in tumor cells may serve as being a predictor for that occurrence of brain metastases.
In spite of precisely what is identified with regards to the autocrine effects of CX3CL1 CX3CR1, minimum investigate is executed to examine the position of CX3CL1 secretion by tumor cells plus the paracrine mechanisms by which tumor cell secreted CX3CL1 interacts with CX3CR1 expressing cells from the surrounding tumor microenvironment while in early mammary tumor formation. On this paper we have now shown for the 1st time that iFGFR1 Cyclopamine induced CX3CL1 regulates the migration of macrophages through the preliminary phases tumor formation. Considering that tumor connected macrophage infiltration has previously been proven to correlate with poor patient prognosis in quite a few tumor varieties, which include breast tumors, its crucial that you identify the macrophage population that is certainly existing in the principal tumor web-site and what regulates the recruitment of this population. By blocking CX3CR1 in vivo we had been able to reduce macrophage infiltration towards the mammary epithelium of MMTV iFGFR1 mice.

Information from each species indicated unique expression of NKX3 one in prostate cells and its absence within the hematopoietic compartment. For examination of NKX3 1 expression in T ALL we screened 24 human T ALL cell lines by RQ PCR. Seven T ALL cell lines demonstrated detectable NKX3 1 expression with distinct in tensities. Western blot examination showed sizeable NKX3 1 protein levels in JURKAT, PER 117 and RPMI 8402 as in comparison to the prostate cell line LNCAP. Of note, the t beneficial cell line KARPAS 45 expressing MLL AFX fusion protein showed no NKX3 1 expression, discount ing direct activation by MLL fusion proteins. Thereafter, NKX3 1 transcript amounts of JURKAT and PER 117 were in comparison with that with the prostate cell line LNCAP, indicating about 9 fold greater expression in prostate cells than in T ALL cells. Interestingly, heart cells expressed about eight fold increased amounts of homeobox gene NKX2 five than t favourable T ALL cell lines CCRF CEM and PEER.
These information show aberrant and ectopic expression of the two NKL homeobox genes in T ALL cells at comparable amounts when in comparison with their physiological tissue controls. Absence of Chromosomal Aberrations at NKX3 one in T ALL To investigate if the ectopic expression of NKX3 one in T ALL cells was chromosomal in origin, we carried out FISH analyses on metaphase selleckchem Bicalutamide chromosomes of all seven NKX3 one optimistic T ALL cell lines using flanking and straddling probes. However, no chromosomal rearrangements have been detected, in dicating a wild kind configuration throughout. We then analyzed copy variety variations in JURKAT and PER 117 by genomic profiling. Again in both cell lines no improvements in genomic copy variety on the NKX3 1 locus at 8p21 have been detected. We concluded that ectopic NKX3 1 expression in T ALL cells will not be chromosomally mediated contrasting with NKX2 five and also other leukemic NKL homeobox genes in T ALL.
For this reason, we postulated that deregulated expression of NKX3 one in T ALL could be because of aberrant pursuits of signalling pathways and or TFs. Analyses of TFs and Signalling Pathways of Prostate and T ALL NKX3 one is physiologically expressed and regulated in prostate cells. To examine probable aberrant activities of prostate specific activatory TFs, we analyzed special info the roles of FOXA1, ETS1 and SOX4 in T ALL cell lines. Array information indicated vital expression ranges of ETS1 and SOX4 in T ALL cell lines even though that of FOXA1 appeared inconspicuous. Nonetheless, the NKX3 one beneficial cell line RPMI 8402 expressed the highest levels of FOXA1 as analyzed by RQ PCR.

We next examined the distribution of your PCFT as well as the RFC of rat pancreas plasma membrane on Optiprep density gradient. We have now validated this strategy earlier by measuring the particular activity of alkaline phosphatase in the many fractions collected from gradient utilizing colon apical membranes. The pattern of specific exercise of alkaline phosphatase in all these fractions exposed a gradient with considerable activity from the prime floating fractions, indicating that these fractions contained lipid rafts. So the fractions isolated from the gradient employing the pancreatic plasma membrane have been subjected to western blotting for that PCFT along with the RFC. We noticed the presence with the PCFT and RFC protein from the leading 5 floating fractions with negligible or no expression thereafter of Optiprep density gradient. Together, these information offer strong proof that the vast majority with the PCFT along with the RFC pool from the pancreas plasma membrane are linked with the DI lipid raft microdomains.
In addition, there was much less expression of the two the PCFT as well as RFC during the lipid rafts in persistent alcoholism. The extent of lower was 25 to 45% for PCFT and 15 to supplier Deforolimus 46% for RFC respectively, which is in accordance using the decreased amounts of those transporters while in the PPMV. Localization of PCFT and RFC in pancreas Studying the localization of these transporters in pancreas by immunohistochemistry exposed the localization from the PCFT and RFC in the basolateral side of pancreatic plasma membrane. In ethanol fed rats there was a marked reduction in the intensity of RFC and PCFT favourable cells in pancreas. Serum, RBCs, and tissue folate levels As this examine handled hindered folate absorption while in alcoholism, the determination of folate levels was of prime significance.
The results showed that a significant lower in serum, RBCs and pancreatic tissue folate amounts in continual alcoholism, confirming the association of alcoholism with folate deficiency. Because of the limitations from the technique employed U0126 to measure launched folate from the tissue, the tissue folate levels presented here certainly are a comparison of relative folate extracted from the manage and ethanol fed rats and these levels might not always reflect the real pancreatic folate. Methylation of your PCFT and also the RFC gene It had been of interest to evaluate that how alcohol associated lowered folate levels within the pancreas would impact the promoter area methylation on the folate transporters. Consequently we deter mined the CpG island methylation of PCFT and RFC gene. As shown in Fig eight there was a substantial lower in methylation of promoter area of RFC gene in ethanol fed rats as compared to regulate, having said that no major adjust in methylation of PCFT promoter region was observed.