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The epidermal growth factor receptor (EGFR) is considered an important therapeutic target in pancreatic cancer, but it is currently impossible to identify those patients who are most likely to benefit from EGFR-directed therapy. We examined the biological effects of the EGFR tyrosine kinase inhibitor gefitinib (ZD1839, Iressa) in a panel of nine human pancreatic cancer cell lines. The drug strongly inhibited DNA synthesis and induced low levels of apoptosis at clinically relevant concentrations in a subset of three of the lines (L3.6pl, BxPC3, and Cfpac1). Sensitivity to gefitinib correlated directly with ligand [transforming growth factor-α (TGF-α)] expression (r2 = 0.71, P = 0.004) but not with surface EGFR expression. The gefitinib-sensitive cells displayed constitutive baseline EGFR phosphorylation, whereas the gefitinib-resistant cells did not. Exposure to gefitinib or a small interfering RNA construct specific for TGF-α reversed the constitutive EGFR phosphorylation and downstream target [extracellular signal-regulated kinases (ERK), AKT] phosphorylation in the gefitinib-sensitive cells but had no effects on ERK or AKT phosphorylation in gefitinib-resistant cells. Baseline EGFR phosphorylation was lower in a subclone of L3.6pl selected for low TGF-α expression, and these cells were also resistant to gefitinib-mediated growth inhibition. Gefitinib blocked the growth of tumor xenografts derived from L3.6pl cells but had no effect on the growth of tumors derived from EGFR-independent MiaPaCa-2 cells. Together, our data show that TGF-α expression identifies a subset of human pancreatic cancer cells that is dependent on EGFR signaling in vitro and in vivo. Quantification of TGF-α expression may therefore represent an effective means of identifying EGFR-responsive primary tumors. (Cancer Res 2006; 66(7): 3802–12)
The epidermal growth factor (EGF) receptor (EGFR) is a transmembrane glycoprotein with an extracellular ligand-binding domain, a hydrophobic membrane-spanning domain, and a cytoplasmic domain that harbors tyrosine kinase activity. Binding of specific ligands, such as EGF, transforming growth factor-α (TGF-α), or amphiregulin, to the extracellular domain results in EGFR dimerization, autophosphorylation of the tyrosine kinase domain, and internalization, resulting in activation of downstream signaling pathways that are involved in cell proliferation and survival (1–4). The idea that the EGFR pathway plays a central role in solid tumor progression is suggested by the observation that many human epithelial cancers, including breast, head and neck, non–small cell lung cancer, renal, ovarian, colon and gastric cancer, and gliomas, exhibit amplification/overexpression of the EGFR and increased expression of receptor ligands (2, 5–9). EGFR overexpression occurs in 30% to 50% of human pancreatic adenocarcinomas, correlating with rapidly progressive disease, resistance to chemotherapy, and poor prognosis (10–14).
Gefitinib (also known as ZD1839 or Iressa, AstraZeneca, Macclesfield, United Kingdom) is an orally active, reversible, competitive inhibitor of the EGFR tyrosine kinase ATP-binding site that was developed for cancer therapy (15). Unfortunately, recent clinical trials showed that therapy with gefitinib did not improve patient outcomes when the drug was combined with cytotoxic agents in first-line therapy and did not prolong survival compared with placebo in pretreated patients with advanced non–small cell lung cancer (16–18). These observations and others have undermined enthusiasm for the drug and other EGFR inhibitors in solid tumor therapy. However, other recent work showed that a subset of non–small cell lung cancers, colorectal cancers, and glioblastomas are highly sensitive to single-agent therapy with gefitinib and that many of these tumors contain somatic activating mutations in exons 18 to 21 within EGFR ATP binding domain (19–22), raising the possibility that subsets of solid tumors with the appropriate biological characteristics might be exquisitely sensitive to the drug. Importantly, disease stability and symptomatic improvement can be achieved in non–small cell lung cancer patients whose tumors do not contain EGFR mutations, indicating that mechanisms aside from mutation of the EGFR tyrosine kinase domain must also dictate drug sensitivity.
Here, we examined the effects of gefitinib on cell growth in a panel of nine human pancreatic cancer cell lines. We then examined the biological properties associated with gefitinib sensitivity in an attempt to identify biomarkers associated with sensitivity to the EGFR antagonist. Our data indicate that a subset of cell lines is dependent on TGF-α-mediated activation of the EGFR for cell proliferation and strongly suggest that pancreatic tumors expressing high levels of TGF-α and phosphorylated (activated) EGFR are EGFR-dependent in vitro and in vivo.
Materials and Methods
Reagents. Gefitinib was purchased from The University of Texas M.D. Anderson pharmacy. Stock solutions were prepared in DMSO and stored at −20°C. Recombinant human EGF and TGF-α ELISA kits were purchased from EMB Biosciences (San Diego, CA). The antibody used for immunohistochemistry and Western blot analyses were from the following sources: mouse anti–proliferating cell nuclear antigen (PCNA) clone PC10 was from DAKO (Copenhagen, Denmark); rabbit anti-total EGFR, rabbit anti-pEGFR (Tyr1068), rabbit anti-total AKT, rabbit anti-pAKT (Ser473), rabbit anti-total mitogen-activated protein kinase (MAPK), and mouse anti-pMAPK (Thr202-Tyr204) were from Cell Signaling Technology (Beverly, MA); mouse anti-TGF-α was purchased from EMB Biosciences; and lamin A/C was from Santa Cruz Biotechnology (Santa Cruz, CA).
Cell lines and culture conditions. The human pancreatic cancer cell lines Aspc1, BxPC3, Capan2, Cfpac1, Hpaf2, HS766T, MiaPaCa-2, and Panc-1 were obtained from the American Type Culture Collection (Manassas, VA). The L3.6pl human pancreatic cancer cell line was established from COLO-357 cells as previously described (23). L3.6pl clones expressing low levels of TGF-α as measured by Northern blot and ELISA analyses were generated by one of us (C.H. Baker) by limiting dilution. The BxPC3 cell line was maintained in RPMI 1640 (Life Technologies, Inc., Gaithersburg, MD). All of the other cell lines were maintained in MEM. The media were supplemented with 10% fetal bovine serum (FBS; Life Technologies), sodium pyruvate (Bio Whittaker, Rockland, ME), L-glutamine (Bio Whittaker), nonessential amino acids (Life Technologies), vitamins (Life Technologies), and antibiotics (penicillin/streptomycin; Bio Whittaker). Adherent monolayer cultures were incubated at 37°C in a mixture of 5% CO2 and 95% air.
Analysis of EGFR mutations. PCR was used to amplify exons 18, 19, 20, and 21 from the human EGFR gene using genomic DNA isolated from each of the cell lines. PCR amplicons were purified using QIAquick PCR purification kit (Qiagen, Valencia, CA). PCR products were sequenced, and the results were blasted and compared with the wild-type EGFR sequence from Genbank.
Detection of EGFR cell surface expression by flow cytometry. Cells were harvested, incubated for 1 hour at 4°C with the anti-EGFR monoclonal antibody (mAb) C225 (ImClone Systems Incorporated, Somerville, NJ), and washed twice with ice-cold PBS containing 0.5% bovine serum albumin (BSA). After 1-hour incubation at 4°C in the dark with AlexaFluor-488-conjugated goat anti-human IgG antibody (Invitrogen Corp., Carlsbad, CA), cells were washed, resuspended in ice-cold PBS/BSA, and analyzed by flow cytometry (Beckman-Coulter, Miami, FL). The Coulter software was used to determine mean fluorescence intensities.
[3H]thymidine proliferation assay. Cells were plated in 96-well plates at a density of 1 × 104 per well. Cells were exposed 24 hours later to various concentrations of gefitinib (0.1-10 μmol/L) in serum-free MEM. After 24 hours, the medium was removed and replaced with fresh MEM containing 10% FBS and 10 μCi/mL [3H]thymidine (Amersham Biosciences, Piscataway, NJ). The cells were pulsed with [3H]thymidine for 1 hour, lysed by the addition of 0.1 N KOH, and harvested onto fiberglass filters. The incorporated tritium was quantified in a scintillation counter.
Cell cycle analysis. Cells were growth in six-well plates in the presence of 10% MEM. After reaching 70% confluence, the cells were exposed to various concentrations of gefitinib for 24 hours. Cells were harvested by trypsinization and pelletted by centrifugation. The pellets were then resuspended in PBS containing 50 μg/mL propidium iodide, 0.1% Triton X-100, and 0.1% sodium citrate. Samples were stored at 4°C for 1 hour before analysis. Propidium iodide fluorescence was measured by fluorescence-activated cell sorting (FACS) analysis (FACScan FL-3 channel; Becton Dickinson, Mountain View, CA) as described previously (24). Cells displaying a hypodiploid content of DNA indicative of DNA fragmentation were scored as apoptotic.
Small interfering RNA–mediated knockdown of TGF-α. L3.6pl cells were grown in six-well plates until they reached 40% confluence and then transfected with small interfering RNA (siRNA) targeting endogenous TGF-α and unrelated targets using OligofectAMINE (Invitrogen). All the siRNA constructs were obtained from Dharmacon Research, Inc. (Lafayette, CO). The TGF-α siGENOME SMARTpool reagent was used to knockdown TGF-α expression. Functional nontargeting siRNAs (siCONTROL Non-Targeting Pool) was used to show that transfection did not induce nonspecific effects on gene expression. We used siRNA directed against lamin A/C as an off-target control.
Immunoblot analyses. Cells were scraped from tissue culture plates with a rubber policeman into Triton lysis buffer [1% Triton X-100, 150 mmol/L NaCl, 25 mmol/L Tris (pH 7.5), 1 mmol/L glycerol phosphate, 1 mmol/L sodium orthovanadate, 1 mmol/L sodium fluoride, and one Complete Mini Protease Inhibitor Cocktail tablet (Roche, Indianapolis, IN)] and were incubated for 1 hour on ice. The lysates were then clarified by centrifugation before resolution by 10% SDS-PAGE. Polypeptides were transferred to nitrocellulose membranes. Membranes were blocked in 5% nonfat milk in a TBS containing 0.1% Tween 20 (TBS-T) for 2 hours at 4°C, incubated overnight with relevant antibodies, washed, and probed with species-specific secondary antibodies coupled to horseradish peroxidase (Amersham Biosciences), and detected by enhanced chemiluminescence (Renaissance, New England Nuclear, Boston, MA).
Immune complex cdk-2 kinase assay. Cells were cultured in 10 cm dishes and exposed to 1 μmol/L gefitinib for various periods of time. Cells were then harvested with trypsin and lysed for 1 hour at 4°C in the Triton X-100 lysis buffer described above. After centrifugation, the clarified supernatants were incubated with an anti cdk-2 antibody for 2 hours followed by overnight incubation at 4°C with protein G-Sepharose beads (Santa Cruz Biotechnology). The immune complexes were washed twice with lysis buffer and once with kinase buffer [25 mmol/L Tris (pH 7.2) and 10 mmol/L MgCl2]. Immunoprecipitates were incubated with 1 μg histone H1, 150 μmol/L ATP, and 10 μCi [γ-32P]ATP in 50 μL kinase buffer for 15 minutes at room temperature. SDS sample buffer was used to terminate the reactions, samples were boiled for 5 minutes, and the mixtures were loaded onto 12% SDS-PAGE gels. The gels were stained with Coomassie blue, destained, and dried, and radiolabeled histone H1 was detected by autoradiography.
Effects of gefitinib on tumor growth. Six-week-old male athymic nude mice (NCr-nu) were purchased from the Animal Production Area of the National Cancer Institute-Frederick Cancer Research and Development Center (Frederick, MD). L3.6pl cells and MiaPaCa-2 (1 × 106/100 μL HBSS) were injected s.c. into the dorsal flank areas of the mice. When the tumors reached 0.3 cm3, mice were randomized into two treatment groups of 10 mice each: The first control group received vehicle solution, whereas the second group received 100 mg/kg gefitinib, both for 5 d/wk by i.p. injection. Tumor volumes were determined weekly using a caliper and the following formula: V = π (smaller diameter)2 (larger diameter)/6. Mice were euthanized and primary tumors were excised and weighed. For immunohistochemistry, one part of the tumor tissue was formalin-fixed and paraffin-embedded, another part was embedded in optimum cutting temperature compound (Miles, Inc., Elkhart, IN), frozen rapidly in liquid nitrogen, and stored at −80°C.
Immunohistochemical analysis. Paraffin-embedded tissues were used for identification of PCNA, total and phosphorylated EGFR, TGF-α, and total and phosphorylated Akt. Sections (4-6 μm thick) were mounted on positively charged Superfrost slides (Fisher Scientific, Houston, TX) and dried overnight. Sections were deparaffinized in xylene, treated with a graded series of alcohol [100%, 95% and 80% ethanol/double-distilled H2O (v/v)], and rehydrated in PBS (pH 7.5). Sections analyzed for PCNA were microwaved for 5 minutes for antigen retrieval (25). All samples were incubated with 3% hydrogen peroxide in methanol (v/v) for 12 minutes to block endogenous peroxidase, washed with PBS (pH 7.5), and incubated in protein blocking solution (5% normal human serum, 1% normal goat serum in PBS, v/v) for 20 minutes. The sections were incubated with the appropriate primary antibody in a humidified chamber overnight at 4°C, rinsed with PBS, and incubated in protein blocking solution for 10 minutes. Sections were then incubated for 60 minutes with the corresponding peroxidase-conjugated secondary antibody at room temperature. Positive reactions were visualized by incubating the slides with stable 3,3′ -diaminobenzidine (Research Genetics, Huntsville, AL) for 7 to 10 minutes. The reactions were terminated by rinsing the slides with distilled water. The slides were counterstained with Gill's hematoxylin (Sigma Chemical) for 10 seconds, mounted with Universal Mount (Research Genetics), and dried on a 56°C hot plate. Sections stained for TGF-α were preincubated with goat anti-mouse IgG F(ab)2 fragment (1:10 dilution in PBS) overnight at 4°C before the incubation with the primary antibody. Frozen tissues used for identification of total and phosphorylated MAPK were sectioned (8-10 μm), mounted on positively charged Plus slides (Fisher Scientific), and air-dried for 30 minutes. Frozen sections were fixed in cold acetone (5 min), acetone/chloroform (v/v; 5 min), and acetone (5 min), and washed with PBS. Slides were blocked with PBS supplemented with 5% normal human serum and 1% normal goat serum for 20 minutes and incubated with the primary antibody in a humidified chamber overnight at 4°C. The slides were then rinsed with PBS and incubated with goat anti-rabbit Alexa 488 for 1 hour at room temperature. After PBS washes, Hoechst nuclear counterstain was applied. To quantify PCNA expression, the number of positive cells was counted in 10 random 0.159 mm2 fields at ×100 magnification. To quantify the intensity of immunohistochemical reactions, the absorbances of six positive cells per field in 10 random 0.159 mm2 fields at ×100 magnification of different treated tumor tissues were measured. Microscopy was done using a Nikon Microphot-FXA microscope (Nikon, Inc., Garden City, NY), and images were captured using a cooled charge-coupled device Hamamatsu 5810 camera (Hamamatsu Corp., Bridgewater, NJ) and Optimas Image Analysis software (Media Cybernetics, Silver Spring, MD). Immunofluorescence microscopy was done using a ×20 objective on an epifluorescence microscope equipped with a narrow band-pass excitation filter mounted in a filter wheel (Ludl Electronic Products, Hawthorne, NY). Photomontages were prepared using Adobe Photoshop software (Adobe Systems, Inc., San Jose, CA).
Statistical analysis. All assays done in this study were conducted at least thrice. Numerical data are presented as mean ± SE. Comparisons between test and control groups were evaluated using Student's t test. Statistical significance was set at P < 0.05.
Effects of gefitinib on the growth of pancreatic cancer cell lines in vitro. Previous studies showed that EGFR inhibitors arrest responsive cell lines within the G1 phase of the cell cycle in vitro (26, 27). We therefore used [3H]thymidine incorporation assays to measure rates of DNA synthesis in cells exposed to increasing concentrations of gefitinib (0.1-10 μmol/L) as a means of identifying the responsive cell lines within our panel. We observed significant heterogeneity in drug responsiveness, with only three of nine cell lines (L3.6pl, BxPC3, and Cfpac1) displaying at least a 50% inhibition of DNA synthesis (IC50) at concentrations of ZD1839 ≤1 μmol/L (Table 1). We obtained independent confirmation of these results by measuring the accumulation of cells in G1 by propidium iodide staining and FACS analysis. As shown in Table 2, gefitinib induced increases in G1 with corresponding decreases in S phase in the three cell lines that displayed significant decreases in DNA synthesis in the [3H]thymidine incorporation assays (L3.6pl, BxPC3, and Cfpac1), whereas it had no effect on cell cycle progression in the MiaPaca-2 or Panc-1 cells. Other work has shown that EGFR inhibitors can induce apoptosis in drug-sensitive tumor cells (28–30). However, the percentage of cells in the sub-G0-G1 population was relatively modest, suggesting that gefitinib exerts largely cytostatic effects on these cells (Fig. 1A).
In an attempt to identify the molecular events responsible for the G1 block observed in the gefitinib-sensitive cells, we measured the effects of the drug on expression of the cyclin-dependent kinase inhibitor p27Kip1 and on cdk-2 kinase activity. Gefitinib induced a time-dependent accumulation of p27Kip1 in the drug-sensitive cell lines L3.6pl, BxPC3, and CfPac1, but not in the drug-resistant MiaPaCa-2 or Panc-1 cells (Fig. 1B). Accumulation of p27Kip1 was associated with an inhibition of cdk-2 kinase activity (Fig. 1B).
Expression of EGFR and TGF-α. We used the antihuman EGFR antibody C225 and flow cytometry to determine the levels of surface EGFR expressed by our cells. EGFR expression was highly variable, with Aspc1 and Panc-1 displaying the highest levels (mean fluorescence intensities of 100 and 59.5, respectively) and HS766T and MiaPaca-2 the lowest (mean fluorescence intensities of 11.7 and 4.3, respectively; Fig. 2A). Thus, there was no relationship between surface EGFR expression and gefitinib sensitivity, consistent with previous conclusions (31–34). To determine whether L3.6pl, BxPC3, or Cfpac1 harbored activating EGFR mutations that might render them more sensitive to gefitinib, we isolated genomic DNA and sequenced exons 18 to 21 of the EGFR expressed by these cells and all of the other lines in the panel. These exons encode the tyrosine kinase domain and contain all of the activating mutations identified in the previous studies (19–21). However, we found no mutations in any of the cell lines (data not shown).
Because we did all of our [3H]thymidine assays with cells cultured in serum-free medium (i.e., no exogenous ligand), the simplest explanation for our results was that the three gefitinib-sensitive cell lines were dependent on autocrine ligand–EGFR interactions for cell cycle progression and the other six were not. The major autocrine ligand for the EGFR that is expressed by epithelial tumors is TGF-α (8, 9). Therefore, to test our hypothesis, we measured autocrine TGF-α, EGF, and HB-EGF production in our panel of cell lines by ELISA (Fig. 2B). Consistent with the [3H]thymidine and cell cycle analyses, L3.6pl and BxPC3 produced the highest constitutive levels of TGF-α (mean = 1,034.21 ± 200 and 561 ± 82 pg/μg protein, respectively). Cfpac-1 produced intermediate levels of TGF-α that were similar to those observed in four of the gefitinib-resistant cells, and the remaining gefitinib-resistant lines (MiaPaCa-2 and Panc-1) produced very little to no detectable TGF-α (Fig. 2B). Overall, we observed a direct linear correlation between gefitinib sensitivity and TGF-α production (r2 = 0.71, P = 0.004). In contrast, we observed no correlation between cellular EGF or HB-EGF production and gefitinib sensitivity in the panel (data not shown).
Effects of gefitinib on EGFR-related signaling pathways. The observation that constitutive TGF-α expression correlated with gefitinib sensitivity strongly suggested that the EGFR signal transduction pathway was constitutively "on" in the gefitinib-sensitive subset of pancreatic cancer cell lines and not in the others. To directly address this possibility, we compared the levels of baseline EGFR phosphorylation in the cells (Fig. 3A). We also stimulated the cells with exogenous EGF and measured the concentration-dependent effects of gefitinib on EGFR phosphorylation to determine whether differences in gefitinib responsiveness could be shown (Fig. 3B). Consistent with our hypothesis, the gefitinib-sensitive L3.6pl, BxPC3, and Cfpac1 cells all displayed significant baseline EGFR phosphorylation when they were grown in serum-free medium, whereas the gefitinib-resistant MiaPaCa-2 and Panc-1 cells did not (Fig. 3A). Gefitinib reversed the basal EGFR phosphorylation observed in all three drug-sensitive cell lines (Fig. 3A). The effects of gefitinib on EGF-mediated EGFR phosphorylation were very similar across the panel (Fig. 3B), indicating that major differences in the receptor responsiveness to gefitinib-mediated inhibition did not underlie the differential drug sensitivity observed.
EGF-induced progression through the G1-S cell cycle checkpoint is mediated by activation of the extracellular signal-regulated kinase (ERK) and AKT signal transduction pathways (35–37). Therefore, we compared the concentration-dependent effects of gefitinib on ERK and AKT phosphorylation in the gefitinib-sensitive and gefitinib-resistant cell lines. Significant constitutive ERK and AKT phosphorylation were observed in all of the cell lines, consistent with the general importance of these pathways in driving tumor cell proliferation (Fig. 3A). Interestingly, exogenous EGF failed to increase phosphorylated ERK or phosphorylated AKT levels in the L3.6pl and BxPC3 cells (Fig. 3C), strongly suggesting that the EGF-EGFR pathway was already optimally activated in these cell lines. However, exogenous EGF triggered further increases in ERK and AKT phosphorylation in the Cfpac1, MiaPaCa-2, and Panc-1 cells that were reversed by gefitinib in a concentration-dependent fashion (Fig. 3C). Conversely, ZD1839 strongly inhibited constitutive ERK and AKT phosphorylation in L3.6pl, BxPC3, and Cfpac1 cells grown in serum-free medium, whereas it had no effect on baseline ERK and AKT phosphorylation in the MiaPaCa-2 and Panc-1 cells (Fig. 3A). Together, these data strongly suggest that constitutive EGFR activation drives ERK and AKT phosphorylation in the gefitinib-sensitive pancreatic cancer lines. Other mechanisms must account for the constitutive ERK and AKT phosphorylation observed in the gefitinib-resistant cells.
Effects of TGF-α modulation on EGFR pathway activity and sensitivity to gefitinib. To more directly determine the relationship between TGF-α expression and sensitivity to gefitinib, we knocked down TGF-α expression in L3.6pl cells via RNA interference and compared cellular responsiveness to gefitinib in silenced cells and controls. Quantification of cellular TGF-α levels by ELISA confirmed that silencing was very efficient (80% inhibition; Fig. 4A). Constitutive EGFR, ERK, and AKT phosphorylation were strongly reduced in L3.6pl cells transfected with the TGF-α-specific siRNA construct relative to the levels observed in the untreated cells, cells transfected with a nontargeting control, or cells transfected with an off-target control (lamin A/C), confirming that TGF-α was directly responsible for receptor and downstream pathway activation (Fig. 4A). Furthermore, baseline [3H]thymidine uptake was lower in the TGF-α-silenced cells versus control siRNA-treated cells (2,612 ± 322 cpm/105 cells versus 3,509 ± 669, P < 0.05), and gefitinib had no further effect on [3H]thymidine incorporation in the TGF-α-silenced cells (Fig. 4B).
In a second series of experiments, we compared the gefitinib responsiveness of the parental L3.6pl cells to a subclone of L3.6pl that spontaneously expresses reduced levels of TGF-α (130.75 ± 32 pg/μg protein in the subclone versus 639.95 ± 6.6 pg/μg protein in the parental L3.6pl cells, or an 80% reduction). The subclone expressed lower levels of phosphorylated EGFR, ERK, and AKT at baseline than the parental cells (data not shown) and was also significantly less sensitive to gefitinib-mediated growth inhibition (Fig. 4C).
Effects of gefitinib on the growth of L3.6pl and MiaPaCa-2 xenografts. Previous studies have shown that EGFR antagonists inhibit angiogenesis in preclinical models of human pancreatic cancer (38, 39). Thus, it is conceivable that EGFR-directed therapy can be effective whether the tumor cells themselves are dependent on EGFR signaling for proliferation. To address this possibility, we compared the effects of gefitinib therapy on the growth of drug-sensitive L3.6pl and drug-resistant MiaPaca-2 tumors growing in vivo. We initiated therapy when the tumors had reached 0.3 cm3 and treated the mice daily via i.p. injection with a fairly high dose of the drug (100 mg/kg). As shown in Table 3 and Fig. 5A, gefitinib did not produce cumulative toxicity or changes in body weight at this dose but did significantly inhibit the growth of L3.6pl tumors compared with those isolated from mice treated with vehicle alone (P < 0.05). All mice in the control group showed progressive disease and were sacrificed early because of tumor burden. Gefitinib seemed to exert largely cytostatic effects on the L3.6pl tumors, as has been reported previously (26, 27). The mean number of PCNA-positive cells per high power field in the L3.6pl tumors was reduced from 190.8 ± 35 in the control group to 57.1 ± 13 in tumors exposed to 100 mg/kg gefitinib (P < 0.001). On the other hand, gefitinib had no effect on the growth of the MiaPaCa-2 tumors, and all mice were sacrificed after 2 weeks because of tumor burden. Gefitinib also had no detectable effect on proliferation in the MiaPaCa-2 tumors, as the mean number of PCNA-positive cells per high power field was 85.4 ± 12 and 84.2 ± 9 in tumors harvested from control and gefitinib-treated tumors, respectively.
Finally, we assessed the effects of gefitinib therapy on EGFR, ERK, and AKT phosphorylation in L3.6pl and MiaPaCa-2 tumors by immunohistochemistry. Initial studies confirmed that TGF-α levels were high in the L3.6pl tumors but were nearly undetectable in the MiaPaCa-2 tumors (Fig. 5B), analogous to the patterns of TGF-α expression observed in the two cell lines in vitro (Fig. 2B). Furthermore, the effects of gefitinib on protein kinase activation in vivo were nearly indistinguishable from the effects observed in the two cell lines in vitro. Specifically, in L3.6pl tumors, we observed marked reductions in EGFR, ERK, and AKT phosphorylation in response to gefitinib therapy, whereas the drug had no significant effects on any of the markers in the MiaPaCa-2 tumors. Quantitative analyses of immunohistochemical staining intensities are summarized in Table 4.
Cancer of the exocrine pancreas is the fourth leading cause of cancer-related death in the United States (40). Our inability to detect pancreatic cancer at an early stage, the inherent aggressiveness of the disease, and the lack of effective systemic therapies are responsible for the nearly identical incidence and mortality rates observed. Adjuvant therapy has little effect on mortality, and the current frontline agent (the deoxycytidine analogue gemcitabine) provides symptomatic improvement but does not extend median survival beyond 6 months (41). Thus, the development of more effective, biology-based therapies is desperately needed.
The EGFR is overexpressed by a large percentage of primary pancreatic tumors in a manner that correlates with disease progression and resistance to conventional therapy (10–14). Furthermore, previous studies with EGFR antagonists in orthotopic L3.6pl xenografts showed that they strongly inhibit tumor growth via mechanisms that involve direct effects on tumor cells and indirect effects exerted through inhibition of angiogenesis (38, 39). Unfortunately, recent clinical experience with gefitinib and other EGFR antagonists in pancreatic cancer patients has been largely disappointing (42). Therefore, we wondered whether tumor heterogeneity might limit the number of primary tumors that could potentially respond to EGFR-directed therapy. Identification of the factor(s) that engender EGFR dependency could enable us to prospectively identify those tumors that are most likely to respond to gefitinib and other EGFR antagonists.
In an effort to address this hypothesis, we investigated the growth inhibitory effects of gefitinib in a panel of nine human pancreatic cancer cell lines. Our results confirmed that a minority (three of nine) of the cell lines displays significant sensitivity to pharmacologically and clinically relevant concentrations of gefitinib (≤1 μmol/L) as measured by two independent assays for G1-S cell cycle arrest. Importantly, we observed a linear correlation between gefitinib sensitivity and TGF-α expression in our cell lines, strongly suggesting that autocrine TGF-α production drove EGFR pathway dependency. We obtained additional evidence for the existence of this relationship by comparing the gefitinib sensitivities of parental L3.6pl cells and a subclone that constitutively produced lower levels of TGF-α, in that the former was much more sensitive to gefitinib-mediated growth arrest than the latter. Finally, using a siRNA construct directed against TGF-α, we confirmed that a causal relationship exists between cellular TGF-α production and EGFR pathway activation and proliferation in the L3.6pl cells. Thus, our results show that TGF-α expression identifies those human pancreatic cancer cell lines that exhibit constitutive EGFR pathway activation and are sensitive to EGFR antagonists in vitro. Our data would predict that TGF-α expression would also directly correlate with constitutive EGFR, ERK, and AKT phosphorylation and sensitivity to gefitinib-mediated growth arrest in primary patient tumors, and we plan to test this hypothesis in future studies.
In agreement with previous reports, our data show that the level of total EGFR expression does not correlate with gefitinib sensitivity. For example, we found that the Panc-1 and Aspc1 express relatively high surface EGFR levels, but both displayed EGFR resistance (gefitinib IC50 >10 μmol/L) in all of our functional assays (31–34). The lack of a relationship between surface EGFR levels and sensitivity to EGFR inhibition stands in contrast to our findings in human bladder cancer cell lines, where we found that gefitinib-sensitive cells expressed significantly higher levels of surface EGFR than did gefitinib-resistant cells.4 The situation is also different from that observed with another erbB family member in human breast cancer, where expression of erbB2/HER-2 correlates with responsiveness to the anti-HER2/neu receptor mAb trastuzumab (43, 44). Therefore, it seems that some human pancreatic cancer cell lines express high EGFR levels but the receptor is not activated at baseline. Whether ligand provided by the in vivo microenvironment would render them EGFR-dependent and gefitinib-sensitive is an issue that requires additional investigation.
Although gefitinib did induce increases in apoptosis in the three cell lines that were most strongly growth arrested by the drug, the levels of cell death were much lower than the degree of growth arrest observed. Furthermore, gefitinib produced largely cytostatic effects on the growth of L3.6pl tumors in vivo, a finding that is consistent with the conclusions of others (26, 27). Therefore, the most obvious effects of gefitinib seem to involve cell cycle arrest in G1 or at the G1-S checkpoint associated with accumulation of p27 and inhibition of cdk-2 activity. This cell cycle arrest could have important implications for the outcome of single-agent therapy in that stable disease rather than disease regression might be the more typical response to successful intervention. Furthermore, it is possible that the cell cycle arrest produced by EGFR-directed therapy could affect the outcome of combination therapy with conventional cytotoxic agents (gemcitabine, taxanes, etc.) that tend to act on cells at later stages in the cell cycle (45–47). Importantly, we have obtained evidence that G1-arrested cells may be more sensitive to apoptosis induced by tumor necrosis factor–related apoptosis-inducing ligand (TRAIL; ref. 48), a cytotoxic cytokine that is currently being evaluated in phase I clinical trials. Indeed, we have confirmed that gefitinib induces synergistic cell killing when it is combined with TRAIL in EGFR-dependent cell lines in vitro (49).4
Perhaps not surprisingly, we observed significant levels of ERK and AKT phosphorylation in our cell lines regardless of whether or not they were gefitinib sensitive. However, in the sensitive cells, ERK and AKT phosphorylation were not further increased by exogenous EGF but were exquisitely sensitive to gefitinib, whereas EGF produced strong increases in ERK and AKT phosphorylation in the resistant cells, yet baseline phosphorylation of the kinases was completely insensitive to gefitinib. These observations are perfectly consistent with our hypothesis that EGFR-mediated signaling networks are "on" in the gefitinib-sensitive cells and "off" in the resistant ones, driven by availability of autocrine ligand production. Interestingly, ERK and AKT phosphorylation do not seem to correlate with the presence of activating K-ras mutations, in that BxPC3 is the only cell line in our panel that retains wildtype K-ras. We would expect that direct targeting of the ERKs and/or AKT would produce more global effects on tumor growth than are observed by targeting EGFR alone, but these strategies also seem to be associated with more systemic toxicity (49, 50). It is tempting to speculate that other autocrine growth factor–growth factor receptor interactions are required for cell cycle progression in some or most of the gefitinib-insensitive cell lines within our panel. Identifying them could yield additional therapeutic targets.
In conclusion, we have shown that the antiproliferative activity of gefitinib in human pancreatic cancer cell lines is linked to TGF-α production and inhibition of EGFR-mediated signaling but does not correlate with the degree of EGFR expression. Prospectively identifying potentially responsive tumors by their expression of TGF-α and exploiting the cytostatic effects of this class of drugs by combining them with TRAIL and other agents that preferentially kill G1-αrrested tumor cells may represent promising new approach to the treatment of pancreatic cancer.
Grant Support: Regina Elena Cancer Institute and the M.D. Anderson Specialized Programs of Research Excellence (SPORE) in Pancreatic Cancer.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Isaiah J. Fidler for critical review of the manuscript; Menashe Bar-Eli for providing the specific primers for exons 18 to 21 used in this study; Yasemin D. Yazici for her helpful advice; and Carol Oborn, Donna Reynolds, and Joseph Douglas for their assistance with histology and immunohistochemistry used in this study.
↵4 M. Shrader, in preparation.
- Received October 17, 2005.
- Revision received January 20, 2006.
- Accepted January 30, 2006.
- ©2006 American Association for Cancer Research.


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So this morning on the very day the Mydays promised I was going to start, I roll out of bed, headed to the bathroom...and NOTHING. So what do I do? I do what any infertile woman who is officially one day LATE does, I squeal and reach over to a drawer that contains no less that 5 pregnancy tests. On this particular morning though I didn't have any cheapies, no Dollar Tree tests, not even a regular two pink line test. All I had were digitals, expensive $15 a pop digitals. I weighed my options of waiting until tomorrow to test so I could pick up a few of the handy dandy $1 tests from the Dollar Tree or I could suck it up and waste the digital on a more than likely BFN.
The Decision!
(Did you really think I could wait?)
I wasted $15 on NOT PREGNANT!! Thanks AF, that was a great way to add insult to injury! I got PUNKED! I guess I will start my period at some point. At least I can have that "AF is here" celebratory glass of wine tonight without a silly pregnancy getting in the way. **SIGH**
*AF- Aunt Flo aka: My Period

subset: commoncrawl


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An Introduction to GAO's Key Issues
Each year, we issue hundreds of reports on topics spanning the full breadth and scope of the federal government's responsibilities. In fiscal year 2013, we issued 709 reports, made 1,430 new recommendations, and testified at 114 congressional hearings. We developed … Continue reading
Image February 21, 2014 Tagged audits, BRAC, cybersecurity, financial reform, financial stability, food safety, government accountability office, issues, Key Issues, managing for results in government, medical products, military bases, multimedia, oversight, reports, SI, subject, summar, topics, usgao, website 1 Comment


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Please note: This course's title has changed from 'Dynamics 365 Unified Operations Core' to 'Dynamics 365: Core Finance and Operations'.
Get the skills you need to become Microsoft Dynamics 365: Core Finance and Operations certified on this 3-day accelerated, all-inclusive course. Gain knowledge on how to configure and implement applications to meet your business requirements.
Learn how to analyse business standards and requirements in order to design appropriate business solutions as well as implement best practices for your industry. You'll immerse yourself in a distraction-free learning environment, using concepts from finance, operations, customer service, supply chain management and more over your 3-day course.
Through Firebrand's unique Lecture | Lab | Review methodology, you'll absorb the official curriculum fast as you're taught by Microsoft Certified Trainers (MCTs). With Firebrand, you'll certify return to the office certified 40% faster than traditional training.
You'll learn how to:
You'll be prepared for and sit exam MB-300 at the Firebrand Training Centre during the course - this is covered by your Certification Guarantee. Plus, when you pass MB-300, you'll only need to pass one additional exam to achieve any of the following certifications:
Course MB-300T00-A: Microsoft Dynamics 365: Core Finance and Operations
As part of your accelerated course, you'll sit the following exam at the Firebrand Training Centre, covered by your Certification Guarantee:
On this course, you'll get access to the following Microsoft Official Curriculum (MOC):
Microsoft Official Exams:
Exam MB-300: Microsoft Dynamics 365 Core Finance and Operations
Your accelerated course includes:
Before attending this accelerated course, you should have:
There are no official prerequisites but Microsoft strongly recommend you hold the following certification:
Unsure whether you meet the prerequisites? Don't worry. Your training consultant will discuss your background with you to understand if this course is right for you.
Here's the Firebrand Training review section. Since 2001 we've trained exactly 76572 students and asked them all to review our Accelerated Learning. Currently, 96.73% have said Firebrand exceeded their expectations.
Read reviews from recent accelerated courses below or visit Firebrand Stories for written and video interviews from our alumni.
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Ontario's Waterloo Public Health reported 10 new positive tests for the coronavirus on Friday, bringing the total number of cases in the region to 1,066.
There were no new deaths reported Friday leaving the total number of COVID-19-related deaths in the area at 112.
441 new coronavirus cases, 28 deaths in Ontario as total deaths surpass 2,000
Another 10 people have recovered from the virus pushing the total number of resolved cases to 697 or 65 per cent. Provincially, 76 per cent of all cases have been cleared.
Thirty-two people remain in hospital as a result of the virus, a number which had risen as high as 48 on May 13.
New cases have been reported at several of the major hot spots in the area over the past couple of days.
There have now been 91 positive tests for the coronavirus among employees of Conestoga Meats in Breslau, Ont.
There have also been more positive tests among residents and staff of Forest Heights Long Term Care, peoplecare AR Goudie and Trinity Village in Kitchener which have all seen multiple residents suffer COVID-19-related deaths.
Archived: Live updates on coronavirus in Canada
Ontario reported 441 new cases of the novel coronavirus on Friday, the highest case number reported in the last two weeks, bringing the provincial total to 24,628 cases.
The death toll has risen to 2,021 as 28 more deaths were reported.
© 2020 Global News, a division of Corus Entertainment Inc.


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The return was brought about by touching base with a friend from years ago. It turns out that my friend had a familiarity with YouTube videos. We decided that we would pool our resources and try to create two channels. I would reboot my channel, and help my friend with a Make Up channel for her 15 year old.
My friend has a creative eye, and can create thumbnails, Flash Intro clips, and funky visuals for the channels. While thinking about restarting my channel, I had focused on Drone Tours of US Cities. But I saw a clip of a roller coaster and it has a bunch of views. I was in Los Angeles when I saw the clip, and remembered reading that Disneyland had a new Star Wars ride called Rise of the Resistance.
I extended my business trip in LA and dropped by Universal Studios Hollywood, Disney California Adventure and Disneyland. I filmed with my cell phone camera. When I got home, I went on Amazon and picked up a GoPro Hero 7 Black and Kamre Video Glasses. Two weeks later, I went to film at Knott's Berry Farm, and a month after than I filmed Magic Mountain.
Now, I am ready to add Amazon Affiliate links for the goodies I purchased.
This channel earns a commission for any purchases made thru the Affiliate Link. Thank you for your support.
GoPro Hero 7 Black Edition with Two Extra GoPro USA Batteries + Sandisk Extreme 64GB MicroSD + Free Polaroid 16GB MicroSD (80GB Total) - https://amzn.to/3fEYIGH
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DJI Phantom 4 PRO Professional Drone, Hobby RC Quadcopter & Multirotor, White, CP.PT.000488 - https://amzn.to/2SWzqu2


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New Year, New You?
Erminia Errante, Staff Writer
January 31, 2013
People all around the world make New Year resolutions every single year. Some people make resolutions to lose weight, quit smoking or become healthier; most of these resolutions in Mercy College are very hard to commit to. The Lessing's food company in the Mercy College Cafeteria makes it very difficult...
10 Mistakes You're Making At The Gym
Kristin McGovern, News Editor
April 5, 2012
Most people have a love/hate relationship with the gym. I can't tell you the amounts of people I see come into the gym after I do, and leave before I do. I mean, sometimes I can see why people do a quick visit at the gym. It's too crowded most of the time. I hate going to a packed gym, so I always...


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There's a very famous quote which says, "We are what we repeatedly do. Excellence, therefore, is not an act, but a habit." Aristotle said that way back when (like before Christ) and it rings just as true today as it did in ancient Greece. The truth is that we are not in control of most of our actions; we are simply slaves to our habits - and our habits will either give us the results we want, or hold us back from the life we could be living.
Normally, I'd take this topic on habits into a weight loss setting. Creating habits around the foods you eat, your exercise, and your sleep is a great way to ensure that you see physical improvement. But what if our mental health is holding us back from even beginning to create those habits?
I know I have set too big of a goal before. It's inspiring in the moment to say something completely outside of your reach - you get a dopamine rush just from setting a goal. It is a taste of the success you want, but when it is too far outside of your reach, you simply never begin.
Rather than trying to set boring goals, because no one gets excited about boring goals, just break your goals down into very simple steps. For example, instead of saying, "I am going to lose thirty pounds by June," and ending your process with the goal, break down the goal into easily accomplished steps. These steps could look like:
When you've graduated from that list and are craving more to get to your goal, you can make a new list:
And so on, and so on. You get the picture. Here's the thing, however: if you never show up to step one on that list, you're never going to make it to step three, four, five, or your new list.
Take off the responsibility hat for a second. You don't have to fix your nutrition completely in one night. You don't have to go to the gym seven days a week, four weeks a month, twelve months a year. You don't have to suddenly start sleeping eight hours per night and develop a meditation practice.
All you need to do is show up in the right amount of consistency for your goals. For each and every person, there is a different definition of consistency. Your coach should work with you to define the correct program which will help you to be consistent. Consistent in your workouts; consistent in your nutrition; consistent in your self-care and mental health.
If going to the gym five days per week gives you the feeling that you can eat anything you want, congratulations, you don't need to be at the gym five days per week. Regardless of who you are, eating whatever you want will not end well. You will negatively impact your health. Similarly, if eating clean at every meal throughout the week makes you feel deprived and leads to binge eating everything in sight on Saturday, eating clean throughout the week is the wrong plan because it has made your consistency fall apart. If you hate meditation and doing it every day for 20 minutes makes you resent healthy living, 20 minutes is too long. Start with 1 minute and build from there. Do what you can do consistently, and work on leveling up over time.
The name of the game is showing up. Just show up consistently, and your results happen naturally.
Get Results


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These gorgeous Ruby Kiss Freeze Dried Rose Petals add just the right touch to any occasion.
These fabulous rose petals are one of our favourites! The petals are a deep, rich, dark red, all over colour and are a perfect shape. A very classic rose petal colour and absolutely stunning!
Our 5 Litre Bag of Ruby Kiss Freeze Dried Rose Petals is a simple plastic bag, tied with Truly Madly Deeply ribbon.
Our 5 Litre Bag contains enough petals for about 38 handfuls or cones, or enough to decorate 8 to 10 tables seating 8 guests.
Our lovely petals are freeze dried from the highest quality roses, they are soft, natural, biodegradable and keep all their curves and colour.
What they don't do is wilt, become soggy or go brown!
The Freeze Dried Rose Petals are also stain resistant and therefore cause no problems if they get wet…essential for dresses, table linen and wooden floors.
Perfect for using at any venue where only biodegradable petals are permitted.


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|View all
MayView clinics to merge with Ravenswood, ending 47 years of operation
Nurse pratitioner Jessie Peng examines Maria Gonzalez at the MayView Community Health Center in Mountain View on Nov. 25. A merger between MayView and Ravenswood Family Health Center was announced ON Dec. 6. Photo by Sammy Dallal.
© 2020 Almanac Online.
All rights reserved.


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Wedding Planner
A Guide To Planning Your Wedding
Image Credit: Terri & Lori Fine Art Photography & Film Studio Let's cut straight to the chase, as romantic and fun as wedding planning sounds, the harsh reality is it can be stressful and overwhelming at times. You have been riding high in engagement heaven, to then come back down to reality with an almighty…Read More
Inviting Children To Your Wedding
Image Credit: Terri & Lori Fine Art Photography & Film Studio As a wedding planner, it's vital that I establish a couples intentions regarding inviting children to their nuptials in the early stages of planning their wedding. This will not only allow me to recommend appropriate wedding venues, as it is not unusual…Read More
Do I Need A Wedding Planner
Image Credit: Terri & Lori Fine Art Photography & Film It's inevitable that couples will experience a whole host of emotions throughout their wedding planning journey, ranging from pure excitement through to utter overwhelm. For some, a brand new venture that they can project manage is beyond exciting. Gathering inspiration, creating spreadsheets, reaching out to…Read More
The Planning Platform
I am very excited to share with you today a brand new wedding planning package that I am now offering to couples planning their wedding day. The Planning Package has been created to provide assistance to couples who are planning their own wedding, but require guidance with a particular element of planning their big day.…Read More
A Bride's Speech
Image Credit: Terri & Lorri Fine Art Photography & Film With many couples now choosing to incorporate a variety of non traditional elements into their wedding day. The bridal speech is an increasingly popular and much welcomed addition to the modern wedding. Gone are the days that you can expect to hear only from male…Read More
Choosing Your Wedding Day Suppliers
Photo Credit: Terri & Lori Fine Art Photography & Film For anyone new to the world of wedding planning it can be a daunting place. With so many wonderful wedding suppliers available, couples are often left overwhelmed and at a loss at where to begin. To help you along the way I have put together…Read More
Wedding Drink Trends for 2019
When Heather from The White Mule Company asked if she could share her wedding drink trends for 2019 on my blog, I of course jumped at the chance. Having run this beautifully converted horse trailer since 2017, I could think of nobody better to put us in the know about all things cocktails, bars and well…Read More
Starting Your Wedding Planning Journey
I'm hoping that you are now obsessed with wedding blogs and are enjoying your daily fix of wedding prettiness. I am also hoping that you have confirmed your budget. Which means one thing, your wedding planning can commence….. Venue A large portion of your budget will have been allocated to your venue, so securing this…Read More
So You're Engaged
Photo Credit: Terri & Lori Fine Art Photography & Film If you are reading this blog post, then I'm guessing you are newly engaged. So, many congratulations to you both, and welcome to the wonderful world of wedding planning. As you have probably gathered, I am an Essex based wedding planner who is completely obsessed…Read More
Hiring A Wedding Planner
The number of couples choosing to reach out to a wedding planner has increased considerably over recent years. In fact, an article recently published on Hitched suggests that 26% of couples planning their wedding are now choosing to work alongside a planner. Considering that hiring a wedding planner was once thought of as a luxury…Read More


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ProShow Gold Free Download
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OS: Win98,WinME,WinXP,Windows2000
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Poké bowls are so versatile – you can switch out the protein and all the veggies for whatever you like best. Serves 2.
- 2 salmon fillets, or 2 chicken thighs, or 2 small steaks – cut into bite-sized chunks
- 1 tbsp soy sauce
- 1 tsp sugar
- 1 tsp sesame oil
- 150 grams sushi rice
- 210 ml water
- 1/2 tsp salt
- 3 tbsp rice vinegar
- Choose any 3 or 4 of the following:
- 1 small mango
- 1 avocado
- 10 radishes
- 100 grams edamame beans
- 12 cherry tomatoes
- half a cucumber
- 1 red pepper
- 2 large tablespoons kimchi
- To garnish (optional): coriander leaves, red chilli, black sesame seeds
- Mix together the soy sauce, sugar and sesame oil in the bowl to create a marinade. Coat your salmon, chicken or steak in the marinade and set aside while you do the rest.
- Put the sushi rice, water and salt into a saucepan and bring to the boil. Cover with a lid and simmer for 10 mins. Then take off the heat and let sit for 15 mins – do not remove the lid.
- After the 15 mins is up, fold the vinegar gently into the rice and set aside.
- While the rice is cooking, prepare the rest of the ingredients: chop the vegetables/fruit into bite-sized pieces (obviously you don't need to chop up the edamame beans!).
- Heat a frying pan to a high temperature, and quick-cook your salmon, steak or chicken. The salmon and steak will cook extremely quickly as you want them still pink in the middle. The chicken may take a little longer to cook through. Throw in the marinade towards the end to make sure your meat or fish is nicely coated in the sweet, sticky glaze.
- To assemble: put the rice in the bottom of two bowls, and divide up the meat or fish and veggies/mango between the two bowls (see picture).
- Garnish with a few coriander leaves, sliced red chilli and black sesame seeds if using.


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New Delhi: A comment on an Instagram post Smriti Irani shared last night reads, "Sometimes I doubt you are a minister, you are so normal like a common man." Whether or not the actress-turned-politician qualifies as a 'common man,' she did share this one common experience with the rest of us - at least, most of the rest of us. Smriti Irani, minister of Information and Broadcasting, sat patiently through the end credits of Avengers: Infinity War rolled, waiting for the post-credits scene. "You know you are an Avengers fan when the credits roll and you still wait for the post-credits scene," Ms Irani revealed on Instagram. Ms Irani, who has a teenaged son and daughter, didn't say who she was watching the film with.
- "You are the coolest minister ever," read one comment
- She didn't reveal who she was watching the film with
- The post-credits scene is key to Avengers 4
Her followers were instantly floored. "You are the coolest minister ever," read one comment - and the sentiment was repeated in several others. One sad comment from a resident of Jhansi complained about a cinema switching off as soon as the credits began rolling. "Please take action against this serious issue," the disappointed Avengers fan begged.
See Smriti Irani's post:
It's a good thing Smriti Irani stayed till the end because that post-credits scene is key to Avengers 4 (currently destroying the box office globally) - SPOILER (if you haven't watched the film yet, don't read any further). Before the credits roll, Thanos the Mad Titan halves the population of the universe, disintegrating several superheroes along with regular folks. After the credits roll, S.H.I.E.L.D agents Nick Fury and Maria Hill are also disintegrated on the streets of New York - but, and it's a big but, not before Director Fury manages to send out a red alert on an intergalactic beeper.
But who was the alert meant for? Who could be powerful enough to swoop in and rescue the decimated Avengers when some of the strongest heroes - Black Panther, Star-Lord and Spider-Man among them - fell to Thanos? The thrilling answer is Captain Marvel, a female air force pilot stuck in space with alien DNA melded with hers. Actress Brie Larson is onboard to play Captain Marvel who, at some point in the comic books, absorbs the powers of a binary star.
If that's not badass and not worth waiting through a few minutes of credit roll for, we don't know what is.


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We rely on the generosity of our community to meet the needs of our youth. If you are interested in donating new clothing or other items, we ask that you follow the guidelines before making a donation. Due to limited storage space, we are unable to accept donations without an appointment. We are no longer accepting used clothing with the exception of GENTLY used business attire.
Amazon Wish List
Donate from anywhere by ordering from our Amazon Wish List! Items will be delivered directly to our shelters. When placing an order on Amazon, please email Patricia Wojtowicz (firstname.lastname@example.com) with your name, address and contact information. We would love to thank you for your gift!
In-Kind Donation Drop-offs
Covenant House Michigan, Detroit campus:
- Due to the COVID-19 crisis and limited storage space, we only accept donations by appointment until further notice.
- In order to drop off a donation, please schedule an appointment with Patricia Wojtowicz, who can be reached at email@example.com or 313.463.2014.
The Detroit campus is currently accepting new items such as jeans, shorts, athletic/jogger pants, hoodies, t-shirts, bras, underwear, shoes, bathrobes, belts (smaller sizes needed) and college-sized backpacks. We will also accept gently used jeans and shoes.
If you decide to ship directly from a vendor, please make sure that you label it with Patricia Wojtowicz's name so we know it is a donation.
Covenant House Michigan, Grand Rapids campus:
- Due to the COVID-19 crisis and limited storage space, we only accept donations by appointment until further notice.
- In order to drop off a donation, please schedule an appointment with Kirsten Emerson at email@example.com or 616.608.1388 X2.
The Grand Rapids campus is currently accepting only new items including men and women's sweat pants and hoodies, solid color tee shirts, belts, boots, gloves, coats, ladies hygiene products, pillows, thermal lunch bags, twin sheets, comforters and blankets, and backpacks.


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Statement Regarding Portland Events
Deviation has been made aware that someone claiming to be a Deviation employee is alleged…Continue Reading
What are you looking for?
Deviation has been made aware that someone claiming to be a Deviation employee is alleged…Continue Reading
Behind the scenes with athlete and LP designer Lupe Hagearty: Lupe joined our team of…Continue Reading
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There are still a few more stops on our demo tour! The tour schedule is…Continue Reading
With Mt. Hood finally getting some coverage and officially open, we're proud to introduce you…Continue Reading
Do you have the Ski Fever? If you're getting stoked for the upcoming season but…Continue Reading
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While most people are spending the summer hiking, biking, swimming or hiding from the heat…Continue Reading
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What if, together, we could imagine hundreds of civic innovations to improve our communities?
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The plot thickens in the case of NXIVM and that of Seagram heiress, Clare Bronfman's trial, for human trafficking and child sex trafficking charges, after the sex cult leader collapsed in court during a hearing when it was revealed that she had secretly hired Michael Avenatti as her attorney, although Avenatti had never submitted any paperwork to the court, declaring that he was involved in her legal proceedings.
Clare Bronfman of DOS and NXIVM, appeared in court for an emergency hearing, alongside her other attorney, Mark Gregaros, who is the co-conspirator in Michael Avenatti's Nike $25million extortion case, which saw Avenatti arrested only some days ago.
Michael Avenatti is currently facing a lengthy jail sentence on extortion charges in relation to attempting to blackmailing Nike, amidst an alleged College basketball scandal, along with Gregaros, and now that Avenatti faces fresh charges on his own, on Wednesday, Judge Nicholas Garaufis wasted no time in asking Clare Bronfman, if she was also being represented by Michael Avenatti.
Clare Bronfmann didn't answer the judges question, prompting Garaufis to demand a simple "yes or no" answer, which was when Clare stumbled back to her table in the courtroom and collapsed. Paramedics and EMTs were on hand to examine Bronfman, as she remained conscious, before being led out of the court room on a stretcher, despite never answering the judges question.
However, Clare Bronfman will be back in the courtroom on Thursday, to continue proceedings and answer more questions, about failing to declare to the court that Michael Avenatti is one of her attorney's in the case, and also that there will be no delays in her case, should Michael Avenatti go to jail.
So, it's interesting now that we begin to explore the connection, once again of Michael Avenatti and his former client, Stormy Daniels, who was once a cult member of NXIVM herself, to that of Clare Bronfmann, one of the leaders of the sex cult, along with Keith Raniere, aka Vanguard, who has been exposed for having sex with minors, and which was facilitated by and through members of NXIVM.
Stormy Daniels has now distanced herself from Michael Avenatti, but in that he has been secretly representing Clare Bronfman, shows the extent of the connection of NXIVM to Democrat politicians and pawns, and to that of certain elite figures.
Yes, NXIVM has been cited numerous times for its ties to the Clinton Foundation, and at least ten years ago, Anthony Weiner had also been spotted at a NXIVM cocktail mixer alongside Stormy Daniels, by a witness in circa 2007, named Ben Szemkus. All of these people are related, do you see?
Yes, hoards of extra incriminating evidence was found on Anthony Weiner's laptop, linking the Clinton Foundation to the child trafficking dealings of NXIVM.
It's also worth noting, the trips that NXIVM members took and held at Sir Richard Branson's Necker Island, a place where Barack Obama also spent time in, with the Virgin business mogul.
NXIVM have also been cited as having involvement in that private Barack Obama White House party, where $65,000 worth of Hot Dogs, were flown in from Chicago for a special event.
Furthermore, NXIVM have also been mentioned in the SNC-Lavalin case in Canada, where Justin Trudeau's former cabinet member resigned, after it was revealed that she was asked not to prosecute the engineering firm, for acquiring prostitutes for Muammar Gaddafi's son, Al-Saadi Gaddafi.
And, on Al-Saadi Gaddafi's extended visit to Canada, he procured services totalling $30k Canadian dollars worth of prostitutes and unknown services, linked back to NXIVM, as part of a payoff from SNC-Lavalin in acquiring lucrative contracts in the re-building of Libya.
Who is a key top aide to Justin Trudeau? Stephen Bronfman, of course, who is family of Clare Bronfman, which is why Justin Trudeau asked SNC-Lavalin not to be prosecuted, when the money spent on prostitutes was discovered by Jody Wilson Raybould, which all links back to sex cult, NXIVM.
Do you see how all of these people are connected, back to Clare Bronfman and Keith Raniere of NXIVM? Are these the sorts of people you want in political office, or in political circles, with ties to a child sex trafficking cult?
Now we have the revelation of Michael Avenatti being hired as one of Clare Bronfman's lawyers, and it's undeniable that there's so much more to the NXIVM case than is being revealed to the public, with the Clinton Foundation's actions in Haiti, which should also very much up for public scrutiny, and yet, the mainstream media try keep the Clare Bronfman, Keith Raniere and NIXVM members' full charges, as secret as possible from public knowledge.
Last week, NXIVM member Nancy Salzman, pleaded guilty to one count of racketeering, stating that "I am pleading guilty because I am in fact guilty," whilst it remains unclear of just who else will be seeking a plea deal in the NXIVM case, and who else will plead guilty. Allison Mack has reportedly been trying to get her own court case in the NXIVM trial, in order to separate herself from the rest of the NXIVM group, away from Keith Raniere and Clare Bronfman, in seeking leniency.
Nancy Salzman is a member of the Clinton Global Initiative and she will serve time for her crimes in NXIVM, and she's currently facing twenty years in prison for one charge of racketeering. Which really makes you wonder as to the justifiable amount of years will be given for the charges of human trafficking of children, doesn't it?
Recently, it was also revealed that Clare Bronfman and Keith Raniere had an intimate relationship, and that one of Nancy Salzman's former employees, who was 15-years old, regularly had sex with Raniere, and she later became his first sex slave in DOS, a master to slave subsidiary of the NXIVM sex cult.
All four other NXIVM members, apart from Nancy Salzman, whom are on trial, are facing human trafficking charges, but what's not being mentioned is the child sex trafficking part, or the sexual abuse of children and the procurement of children, and this also links back to the Clinton Foundation, also to top level Democrats and to that of some of NXIVM's celebrity clients.
NXIVM catered to the sickening demands of their client book, with the information recovered in several raids, and via Anthony Weiner's laptop. Numerous big names were found inside NXIVM's client book. One of which, was the name of Anthony Bourdain, who's rumoured preference was for little girls, between the ages of 6 to 9 years old, which may explain why he hung himself?
Do you now see the breadth of the NXIVM case, and just why these people should be prosecuted to the full extent of the law, for their crimes against children, and that absolutely nobody should be immune from prosecution, or free from judicial judgement for such child crimes, after being involved in, and or participating in crimes against children, including those that trafficked, and ordered children, in order to sexually abuse them, or even worse.
Under the Obama administration, organisations like NXIVM could flourish, however, under the Trump administration, it saw the NXIVM organisation run south of the border and continue their operations from Mexico, but Raniere was later arrested there by Federal Police, and brought in by the authorities, over one year ago.
This sparked the investigations into NXIVM, with charges now on the table for Clare Bronfman, Keith Raniere, Allison Mack and Kathy Russell.
So, do you think it's merely a coincidence that Michael Avenatti and Stormy Daniels, with their now exposed ties to NXIVM, were apart of a major failed Democrat coup, against the American President? Why do you think that is? Perhaps to cover up for the crimes of NXIVM, and its links to the Clinton Foundation and the Clinton Global Initiative in human trafficking?
Story by The Narrator
Featured Photo Credit: AP


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Spectators will be discouraged, but not strictly prohibited, when the city's recreational facilities reopen this fall.
The City of Fort St. John's department of recreation gave a presentation to city council during the meeting on Monday, August 10, regarding rec facilities reopening and the guidelines and protocols that will be followed. The issue of whether or not spectators would or wouldn't be permitted at events was one of the main takeaways from the presentation.
Ultimately, the city will not be prohibiting spectators, but is strongly discouraging them from attending events, and is advising user groups to do the same. However, so long as the number of people at an event doesn't exceed 50, spectators may be able to attend certain events, and it will be up to each group to manage that.
For the city's ice rinks at the Pomeroy Sport Centre and North Peace Arena, only 20 people will be allowed on the ice at one time, but up to 50 people can be present at once, which includes city staff, people in the stands, and coaches on the bench.
The speed skating oval will be limited to 50 people at one time, between the actual ice service and spectator area, while the walking track will be limited to 30 people at a time. Up to 40 people are allowed across both pools at the North Peace Leisure Pool at a time.
The 50 person limit is per event, not per building. This means that each ice surface in the Pomeroy Sport Centre could have 50 people around it at a time, while another 50 people are upstairs using the oval, for example.
The Pomeroy Sport Centre and Kid's Fieldhouse Arena will open on September 8, and the North Peace Leisure Pool on September 14. The North Peace Arena will open in October, though no official date has been announced.
The use of face-masks will not be mandated or enforced, though facility staff may have to wear them depending on the event taking place.
Drop-in times will still exist , but they will now need to be booked in advance, to ensure the 50-person limit isn't exceeded and people aren't waiting outside the buildings to get in for prolonged periods of time during the winter months.
The city plans to release detailed guidelines for the use of each building, as well as an informative video series, ahead of the reopening of each building.
Email sports reporter Dillon Giancola at firstname.lastname@example.com.


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Our Keepers program is designed to minister to all of those who want to STOP committing crimes! If you have a desire to stop what you are doing we can help! If you are on probation and/or parole and you desire to change your life, this membership is for you! There is no cost to be a member, lifetime membership, free gifts, spiritual guidance, job training, job leads, housing referrals, and more. Sign up today!!!
Prodigal Families
(For those who have family in & out of jail). Prodigal Families is a new program for our families who have loved ones in prison and/or loved ones who struggle living a life of committing crimes. We will provide supports for our Prodigal families. God wants to restore your relationship and we are here to help! Weekly support group meetings are coming soon! We know that you have been praying for your love one to be delivered from a life of crime. Our support group meetings are designed to help you forgive and forget the past and help you move forward, using the Word of God! Sign up now!
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(For Teens & Juveniles)
Our teen ministry is committed to ministering to every area that impacts our teens! Today, teenagers are dealing with a number of issues from sexual sins, peer pressure to commit crimes, pressure to use and sell drugs, skip school, and live only for today. Our program uses the Word of God to deliver Hope and Deliverance to prevent teens from living a life of self destruction!
We are doing our best to prepare the content of this site. However, Blessed Behind Bars Ministry cannot warranty the expressions and suggestions of the contents, as well as its accuracy. In addition, to the extent permitted by the law, for Blessed Behind Bars Ministry shall not be responsible for any losses and/or damages due to the usage of the information on our website. The links contained on our website may lead to external sites, which are provided for convenience only. Any information or statements that appeared in these sites are not sponsored, endorsed, or otherwise approved by for Blessed Behind Bars Ministry. For these external sites, for Blessed Behind Bars Ministry cannot be held liable for the availability of, or the content located on or through it. Plus, any losses or damages occurred from using these contents or the internet generally.
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EZ Groom Puppermint shampoo is designed to deeply cleanse your pet's coat without stripping its natural moisture. This fresh, mint-scented shampoo is formulated to eliminate nasty pet odors, leaving your dog or cat smelling fresh and clean.
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- Also safe for cats
- Made in the USA
CAUTION: Be extra careful to keep this shampoo out of pet's eyes.


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Não Vai Sair
One Pager counting down to the 2014 FIFA World Cup to be held in Brazil… except the catch is the microsite points out government promises vs reality. Each time you refresh/visit the site a random promise loads against a relevant background image. By looking at the social shares, they are not the only ones disappointed.
Countdown to the World Cup that will took place in Brazil. Many promises were made by the politicians, but the reality is a little bit different. In this site you can see what was promised and what is the reality.
Categories Informational Sport
- Need Hosting? I'd highly recommend Bluehost
- Broken link? Please Email or Tweet me
- Credits: Fernando Leite
- Reviewed: 2 Jun 2014 by Rob Hope


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Goals of the Human Genome Project determine the entire sequence of human DNA identify all the genes in human DNA store this information in databases improve tools for data analysis transfer related technologies to the private sector address the ethical, legal, and social issues (ELSI) that may arise from the project.
Obtain Genomic DNA Sample Sequence genomic DNA Assemble sequences in order Annotate sequence Sequencing a genome
Chemical reaction that includes: DNA polymerase DNA primer Nucleotide bases (A, T, G, C) Nucleotide bases that are 'labeled' Addition of labeled bases stops reaction. Repeated many times. Sanger Sequencing
DNA separated by size using a gel and an electric current _+_+ DNA moves towards positive charge Short DNA moves faster Sequenced sample put in well
How do we sequence a genome? For the HGP, two approaches were used: 1. Hierarchical sequencing 2. Shotgun sequencing
CCCATTAGATGCGATGGGTTAAAA GGTTAAAAATCGATCCCATTTTACG How do we put the sequences together in the right order? Genome assembly - based on finding regions of overlap between individual sequencing fragments Very, very difficult problem for complex genomes!!
Genome Annotation Annotation – identifying what part of DNA corresponds to genes, etc. Compare to known genes: Gene already described and sequenced Expressed Sequence Tags (EST), essentially randomly sequenced mRNA Predict genes: Computer predictions
Genome made of two types of DNA Euchromatic –Comprises 93% of your DNA –Contains most of the genes in your genome –99% has been sequenced Heterochromatic DNA –Comprises ~7% of your DNA –Highly repetitive –Some parts are structural: contains centromeres, telomeres –Gene sparse –Very difficult to sequence, largely unexplored.
Euchromatic DNA 2.8 Billion base pairs ~30,000 genes –Many fewer than expected, initial guesses were ~100,000 genes –50% have unknown function –Less than 2% of the total genome 98% "junk" DNA –Does not code for genes –Function is unknown - but potentially very important!!! –Many (~50%) repeated sequences (e.g. AGAGAGAGAGAG) and transposable elements
What does the draft human genome sequence tell us? How the genome is arranged Genes occur in gene-dense "jungles" and gene poor "deserts". Genes appear to be concentrated in random areas along the genome, with vast expanses of noncoding DNA between. Chromosome 1 has the most genes (2968), and the Y chromosome has the fewest (~231).
HapMap An NIH program to map genetic variation within the human genome Begun in 2002 Construct a map of the patterns of variation that occur across human populations. Facilitate the discovery of genes involved in complex human traits and diseases.
Evolutionary Genomics - comparing genomes of different species to learn about genome evolution and function OrganismGenome Size (Bases) Estimated Genes Human (Homo sapiens)3 billion30,000 Laboratory mouse (M. musculus)2.6 billion30,000 Mustard weed (A. thaliana)100 million25,000 Roundworm (C. elegans)97 million19,000 Fruit fly (D. melanogaster)137 million13,000 Yeast (S. cerevisiae)12.1 million6,000 Bacterium (E. coli) 4.6 million3,200 Human immunodeficiency virus (HIV) 97009 Gene number does not directly scale with complexity of organism!
What do evolutionary comparisons tell us? How the Human Compares with Other Organisms? Humans have 3X as many kinds of proteins as the fly or worm mRNA transcript "alternative splicing" and chemical modifications to the proteins. This process can yield different protein products from the same gene. Large portions of non-genic DNA highly conserved, suggesting the serve some function.
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