To study the hypothesis that gemcitabine treatment augments the chemoresistance to gemcitabine by clusterin (sCLU) upregulation. Clusterin inhibition could augment the chemosensitivity of human pancreatic cancer cells by inhibition of clusterin-dependent pERK1/2 activation.
Clusterin was silenced by serial concentration of OGX-011 transfection in pancreatic cancer MIAPaCa-2 and BxPC-3 cell lines, then treated with serial concentration of gemcitabine. After the cells were treated with OGX-011 for 8 h, the cells were then treated with 5 μM ERK inhibitor PD98059 for 18 h or transfected with a wt-pERK-expressing plasmid into these cells for 24 h, after which the cells were treated with 1.0 uM gemcitabine for 24–72 h. Cell proliferation was determined by MTT. Apoptosis was quantified by flow cytometry,.sCLU and pERK1/2 production was analyzed by western blot, and sCLU mRNA was analyzed by RT-PCR. Xenograft of established tumors was used to evaluate primary tumor growth and apoptosis after treatment with gemcitabine alone or in combination with OGX-011. Phosphorylated ERK1/2 and sCLU levels in tumor tissues were measured by TUNEL analysis.
As detected by MTT and FACS assay, a combination of gemcitabine + OGX-011 reflected the chemotherapeutic sensitivity and increased the gemcitabine -induced apoptosis in MIAPaCa-2 and BxPC-3 cells. Western blotting and RT-PCR analysis revealed that the expression of clusterin was higher in gemcitabine -resistant MIAPaCa-2 cells, however, decreased significantly after pretreatment with OGX-011. Furthermore, the OGX-011 or combination of gemcitabine + OGX-011 decreased the gemcitabine -induced activation of pERK1/2. wt-pERK-re-expression decreased OGX-011+ gemcitabine -induced apoptosis. Finally, OGX-011 in combination with gemcitabine substantially decreased the in vivo tumor growth and promoted apoptosis. Taken together, clusterin confers gmcitabine resistance in pancreatic cancer cells.
Knockdown of clusterin by OGX-011 transfection sensitizes pancreatic cancer cells to gemcitabine by inhibition of gemcitabine -induced clusterin-pERK1/2 activation.
Pancreatic cancer has the worst prognosis of all major cancers, with an overall 5-year survival rate of around 5%. The current clinical standard of care for advanced pancreatic cancer is gemcitabine, a cytotoxic nucleoside analogue. Gemcitabine results in a tumor response rate of 12% and offers a median survival time of 5 months. Unfortunately, this means that the best current treatment offers very modest benefits. Recent studies have indicated that targeted therapies in combination with gemcitabine can have statistically significant benefits. However, the results to date remain meager, and new approaches to improving the effectiveness of gemcitabine are needed. One of the targets considered for combination therapy that has generated wide attention is clusterin.
Clusterin, also known as testosterone-repressed prostate message-2 (TRPM-2), sulfated glycoprotein-2 (SGP-2), apolipoprotein J (Apo J) or SP40, is a ubiquitous heterodimeric-secreted glycoprotein of 75–80 kDa. A single-copy gene in humans of nine exons, spanning over 16 kb and located on chromosome 8p21-p12, encodes an mRNA of approximately 2 kb, which directs the synthesis of a 449-amino acid primary polypeptides chain. Recent focus has turned to clusterin as a key contributor to chemoresistance to anticancer agents. Its role has been documented in prostate cancer for paclitaxel/docetaxel resistance as well as in renal, breast, and lung tumor cells. Moreover, it is abnormally upregulated in numerous advanced stage and metastatic cancers spanning gastric cancer, bladder, cervical, breast,ovarian, hepatocellular, colorectal, renal, prostate, head and neck, lung carcinomas, melanomaand lymphoma.It is noteworthy that only the cytoplasmic/secretory clusterin form (sCLU), and not the nuclear form, is expressed in aggressive late stage tumors, which is in line with its antiapoptotic function.
Many reports also document that sCLUc inhibits mitochondrial apoptosis. For example, sCLUc suppresses p53-activating stress signals and stabilizes cytosolic Ku70-Bax protein complex to inhibit Bax activation. sCLUc specifically interacts with conformationally altered Bax to inhibit apoptosis in response to chemotherapeutic drugs. sCLU sliencing alters the ratio of anti-apoptotic Bcl-2 family members, disrupting Ku70/Bax complexes and Bax activation[24, 25]. In addition, sCLU increases Akt phosphorylation levels and cell survival rates. sCLU induces epithelial-mesenchymal transformation by increasing Smad2/3 stability and enhancing TGF-β-mediated Smad transcriptional activity. sCLU also promotes prostate cancer cell survival by increasing NF-κB nuclear transactivation, acting as a ubiquitin-binding protein that enhances COMMD1 and I-kB proteasomal degradation via interaction with E3 ligase family members. sCLU sliencing stabilized COMMD1 and I-κB, suppressing NF-κB translocation to the nucleus, and suppressing NF-κB-regulated gene signatures. Thus, sCLU has a key role in preventing apoptosis induced by cytotoxic agents and has the potential to be targeted for cancer therapy.
It has recently reported sCLU was overexpressed in pancreatic cancer tissues and sCLU overexpression confered gmcitabine resistance in pancreatic cancer cells,. Furthermore,sCLU silencing sensitized pancreatic cancer cells to gemcitabine chemotherapy, however the mechanism is still unclear.
ERK1/2 is an important subfamily of mitogen-activated protein kinases that control a broad range of cellular activities and physiological processes. ERK1/2 can be activated transiently or persistently by MEK1/2 and upstream MAP3Ks in conjunction with regulation and involvement of scaffolding proteins and phosphatases. There is abundant evidence that survival factors can use the ERK1/2 pathway to increase the expression of several pro-survival BCL-2 proteins, notably BCL-2, BCL-xL and MCL-1, by promoting de novo gene expression in a variety of cell types. Clearly the ERK1/2 pathway can regulate several members of the BCL-2 protein family to achieve cell survival. ERK1/2 signalling can provide protection against chemotherapeutic cytotoxic drugs.
It has shown previously sCLU plays an important role in astrogliosis by stimulating the proliferation of astrocytes through activation of the extracellular signal-regulated kinase 1/2 signaling pathway. Shim and Chou et al. also found significant relation between sCLU and ERK1/2 expression[33, 34]. We therefore suggested that sCLU silencing sensitized pancreatic cancer cells to gemcitabine chemotherapy may via ERK1/2 signaling pathway.
sCLU is not a traditional druggable target and can only be targeted at mRNA levels. An antisense inhibitor targeting the translation initiation site of human exon II CLU (OGX-011) was developed at the University of British Columbia and out-licensed to OncoGeneX Pharmaceuticals Inc. OGX-011, or custirsen, is a second-generation antisense oligonucleotide with a long tissue half-life of ~ 7 days, which potently suppresses sCLU levels in vitro and in vivo. OGX-011 improved the efficacy of chemotherapy, radiation, and hormone withdrawal by inhibiting expression of sCLU and enhancing apoptotic rates in preclinical xenograft models of prostate, lung, renal cell, breast, and other cancers[35–39].
In this study, we study the effect of sCLU silencing by OGX-011 on sensitizion of pancreatic cancer cells to gemcitabine chemotherapy, and eluated the mechanisms.
The human pancreatic cancer MIAPaCa-2 cells resistant to gemcitabine and BxPC-3 cells sensitive to gemcitabine were purchased from American Type Culture Collection. They were routinely cultured in DMEM supplemented with 10% fetal bovine serum in a 37°C incubator in a humidified atmosphere of 5% CO2.
OGX-011 was purchased from OncoGenex Technologies. The antisense oligonucleotides were second-generation 21-mer antisense oligonucleotides with a 2′-O-(2-methoxy)ethyl modification. The antisense oligonucleotide clusterin sequence corresponding to the human clusterin initiation site was 5′-CAGCAGCAGAGTCTTCATCAT-3′ and designated OGX-011 (OncoGenex Technologies). The MEK inhibitor PD98059 was products of Calbiochem (San Diego, CA, USA), Antibodies for sCLU, and phospho-specific or the total form of antibodies against ERK1/2,GAPDH were purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Total RNA was extracted from PANC-1 cells using TRIzol reagent (Invitrogen, CA, United States), according to the manufacturer's protocol. The cDNAs were synthesized using the TaKaRa RNA polymerase chain reaction (PCR) Kit (TaKaRa, Japan). A full-length cDNA encoding human wt-pERK was cloned by PCR using 500 ng cDNA as a template and primers containing HindIII and BamHI restriction enzyme sites. The PCR products were ligated into pcDNA3.1 (Invitrogen, CA, United States) to create the plasmid pcDNA3.1- wt-pERK. MIA PaCa-2 and BxPC-3 cells were transfected with the pcDNA3.1 vector or pcDNA3.1- wt-pERK using FuGENE (Roche Diagnostic GmbH, Mannheim, Germany), according to the manufacturer's protocol.
MIA PaCa-2 and BxPC-3 cells were treated with OGX-011(400,800,1000,1200 nM) for 24 h, then the cells were cultured overnight in 6-well plates and transfected with pcDNA3.1- wt-pERK using Lipofectamine Plus (Invitrogen) in 1 ml serum-free medium according to the manufacturer's instructions. Four hours post-transfection, each well was supplemented with 1 ml of medium containing 20% FBS. Twenty-four hours post-transfection, media were removed and the cells were harvested or treated with gemcitabine for a further 24 hours.
About 25 μg protein was extracted, separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene fluoride membranes, and then reacted with primary rabbit antibodies against sCLU(1:100), pERK1/2(1:100) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)(1:200). After being extensively washed with PBS containing 0.1% Triton X-100, the membranes were incubated with alkaline phosphatase-conjugated goat anti-rabbit antibody for 30 minutes at room temperature. The bands were visualized using 1-step™ NBT/BCIP reagents (Thermo Fisher Scientific, Rockford, IL, USA) and detected by the Alpha Imager (Alpha Innotech, San Leandro, CA, USA).
The mRNA extraction and RT reaction for synthesizing the first-strand cDNA was carried out according to the manufacturer's instructions. Primer sequences were below: 5′-CCAACAGAATTCATACGAGAAGG-3′ and 5′-CGTTGTATTTCCTGGTCAACCTC-3′ for sCLU;5′-TGATGGGTGTGAACCACGAG-3′, 3′-TTGAAGTCGCAGGAGACAACC-5′for GAPDH. The PCR conditions consisted of an initial denaturation at 95°C for 3 min, followed by 28 cycles of amplification (95°C for 15 s, 58°C for 15 s, and 72°C for 20 s) and a final extension step of 5 min at 72°C. PCR products were analyzed on a 1.2% agarose gel. The significance of differences was evaluated with Student's t-test. The mean ± SD are shown in the figures. P < 0.05 was considered to be statistically significant.
To identify the induction of apoptosis, cells underwent propidium iodide (PI) staining and fluorescence-activated cell sorting (FACS) as to the manufacture's instruction. In brief, cells were plated at a density of 1 × 105 cells/ml. After allowing 24 hours for cell adherence, cells were transfected and/or treated. Cells were collected by gentle trypsinization, washed in phosphate-buffered saline (PBS), pelleted by centrifugation and fixed in 70% ethanol. Immediately prior to staining, cells were washed twice in PBS and resuspended in PBS containing RNAse A (20 μg/ml). Cells were stained with propidium iodide (final concentration 10 μg/ml) for 10 min at room temperature. Samples were analyzed by FACS (FL-3 channel) using a Beckman Coulter Counter Epics XL flow cytometer (Beckman Coulter, Miami, FL, USA). For each sample, 50,000 events were collected and stored for subsequent analysis using EXPO software (version 2.0; Applied Cytometry Systems, Sheffield, UK). The percentage of cells in the sub-G0 phase was quantitated as an estimate of cells undergoing apoptosis.
Cells were plated at 2 × 103 cells per well in 96-well plates for six days. Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT, Trevigen, Inc., Gaithersburg, MD) in accordance with the manufacturer's instructions. Plates were read using a Vmax microplate spectrophotometer (Molecular Devices, Sunnyvale, CA) at a wavelength of 570 nm corrected to 650 nm and normalized to controls. Each independent experiment was done thrice, with 10 determinations for each condition tested. At identical time points,cells were trypsinized to form a single cell suspension. Intact cells, determined by trypan blue exclusion, were counted using a Neubauer hemocytometer (Hausser Scientific, Horsham, PA). Cell counts were used to confirm MTT results.
MIAPaCa-2 or BxPC-3 cells (107) were injected into the pancreas of SCID mice. Four weeks after tumor implantation, the mice were assigned to one of the following four treatment groups (n = 10 each): (a) vehicle control; (b) gemcitabine, biweekly treatment 80 mg/kg/injection; (c) OGX-011, biweekly treatment 0.35mg/kg/injection; (d) gemcitabine plus OGX-011, with gemcitabine on Monday and Thursday and OGX-011 on Wednesday and Saturday. All groups received treatment via i.p. injection. Mice in all groups were killed after 5 weeks of treatment. Orthotopic tumors were harvested and weighed.
Five serial sections (5 um thick) were obtained for each frozen tumor, mounted on glass slides, and then fixed in 4% paraformaldehyde. The first section was processed for H&E staining. Apoptosis was evaluated by terminal transferase dUTP nick end labeling [TUNEL] staining using the Apoptag Peroxidase In Situ Detection Kit S7100 [Chemicon] according to the manufacturer's instructions.
All statistical analyses were performed using the SPSS13.0 software. The results were presented as means ± SD of two-three replicate assays. Differences between different groups were assessed using X2 or t-test. A P value of <0.05 was considered to indicate statistical significance.
To investigate whether upregulation of sCLU expression is a cause or a result of gemcitabine -induced resistance, both MIAPaCa-2(resistant to gemcitabine) and BxPC-3 (sensitive to gemcitabine) cells cells were treated with gemcitabine at 0.5uM for 2–24 h (Figure1A) or at concentrations 0.1-1.0 uM for 12 h (Figure1B). Sensitive BxPC-3 cells rapidly responded (sCLU up-regulation peaked by 12 h and began decreasing by 16 h by increasing sCLU expression level under 1.0 uM doses of gemcitabine. MIAPaCa-2 cells already expressing higher sCLU levels, did not further express sCLU following gemcitabine treatment. Considering that changes in sCLU expression seem to be independent of sCLU mRNA, which did not change significantly as indicated by real-time PCR (data not shown). These results suggested that post-translational modification of sCLU may be altered in response to gemcitabine treatment.
Induction of sCLU in a time and dose dependent fashion by gemcitabine treatment. A. Western analysis showing sCLU expression after 2–24 hours treatment with 0.5 nM gemcitabine. Induction of sCLU is evident in chemo-sensitive BxPC-3 cells when treated with high doses of gemcitabine but not in MIAPaCa-2, in which the high levels of sCLU remained unchanged. B. Western analysis showing sCLU expression in cell extracts after 12 hours treatment with 0.1-1.0 nM gemcitabine. sCLU increased in gemcitabine -sensitive BxPC-3 cells at different doses. At difference, expression of sCLU was unchanged in the MIAPaCa-2-resistant cells. The data shown are representative of three independent experiments.
Resistance to anticancer agents is one of the primary impediments to effective cancer therapy. Both intrinsic and acquired mechanisms have been implicated in drug resistance but it remains controversial which mechanisms are responsible that lead to failure of therapy in cancer patients.
In the present study, MIAPaCa-2 and BxPC-3 cell lines were treated with 1.0 uM of gemcitabine for 24 hours, significant apoptosis (21%) was shown in BxPC-3 cell lines,compared with control(P < 0.05). However, in MIAPaCa-2 cells, 1. 0uM of gemcitabine treatment did not induce significant apoptosis (P > 0.05). It has shown above only low levels of apoptosis were detected in pancreatic cancer cells following 1.0 uM of gemcitabine treatment. This might be due to the intrinsic and simultaneous induction of clusterin by gemcitabine. Indeed, knockdown of sCLU by 1200 nM OGX-011(maximally reduced sCLU expression) led to a significant increase in gemcitabine-induced apoptosis in both MIAPaCa-2 cells and BxPC-3 cells by FACS analysis (Figure2A,*P < 0.05). However, knockdown of sCLU itself did not affact apoptosis of MIAPaCa-2 cells and BxPC-3 cells (Figure2A).
Targeting sCLU by OGX-011 sensitizes pancreatic cancer cells to gemcitabine treatment. A, BxPC-3 and MIAPaCa-2 cells were transfected either with OGX-011 (1200nM) and then challenged with gemcitabine dose of 1.0 uM at 24 h. FACS analysis demonstrating that OGX-011 enhanced gemcitabine toxicity in both of the cells. B, Comparative viability of MIAPaCa-2 cells and BxPC-3 cells before and after sCLU sliencing. Cells were cultured in 96-well plates, then transfected either with OGX-011. Twenty-four hours after last transfection, cells were treated with gemcitabine. Seventy-two hours after drug addition ,cell viability was estimated. The data shown are representative of three independent experiments (*P < 0.05).
On the other hand, cellular viability was studied under experimental conditions similar to this described above. Figure2B shows significantly less viability of MIAPaCa-2 cells and BxPC-3 cells pre-treated with 1200nM OGX-011(*P < 0.05). Together, the aforementioned data indicate that silencing sCLU by OGX-011 enhanced gemcitabine toxicity in the pancreatic cancer cells. Control oligodeoxynucleotide did not have obvious effect on apoptosis or growth in both cells (data not shown).
Studies were then performed to assess the effects of gemcitabine on ERK1/2 activation in BxPC-3 and MIAPaCa-2 cells. Exposure to 0.5-1.0 μM gemcitabine (18 hr) induced ERK1/2 activation in BxPC-3 cells (Figure3A).In MIAPaCa-2 cells, 0.5-1.0 μM gemcitabine treatment did not affact ERK1/2 activation (Figure3A). However, co-administration of the 5 μM ERK inhibitor PD98059 essentially abrogated expression of pERK1/2 in both untreated and gemcitabine -treated BxPC-3(Figure3B) and MIAPaCa-2 cells (Figure3B). These findings indicate that in breast cancer cells, 5 μM ERK inhibitor PD98059 essentially abrogate basal ERK1/2 activation as well as gemcitabine -mediated ERK1/2 activation.
ERK inhibitor PD98059 inactivate ERK1/2 in untreated and gemcitabine-treated breast cancer cells. A, BxPC-3 and MIAPaCa-2 cells were exposed to the indicated concentrations of gemcitabine for 18 hr. The cells were then lysed and subjected to WB analysis to monitor pERK1/2 (Thr42/Tyr44) expression as described in Materials and Methods. B, BxPC-3 and MIAPaCa-2 cells were exposed (18 hours) to either 5 μM PD98059, 0.5-1.0 μM of gemcitabine, or the combination, after which proteins were prepared and subjected to WB as described above to monitor pERK1/2 expression. For (A) and (B), lanes were loaded with 25 μg of protein; blots were then stripped and re-probed with GAPDH to ensure equivalent loading and transfer. Representative results are shown; two additional studies yielded equivalent results.
To determine whether ERK1/2 protects pancreatic cancer cells from gemcitabine -induced cell death or not, 5 μM PD98059 was used to inhibit pERK1/2. BxPC-3 and MIAPaCa-2 cells was treated with 1.0 μM of gemcitabine. The results shown both BxPC-3 and MIAPaCa-2 cells were significantly more sensitive to gemcitabine -mediated apoptosis compared to cells exposed to gemcitabine in the absence of PD98059 (P < 0.05; Figure4). It also shows significantly less viability of MIAPaCa-2 cells and BxPC-3 cells pre-treated with 5 μM PD98059 ,then treated with 1.0 nM gemcitabine(data not shown). These findings argue that ERK1/2 inactivation plays a significant functional role in the potentiation of gemcitabine lethality.
Inhibition of ERK1/2 sensitizes BxPC-3 and MIAPaCa-2 cells to gemcitabine -induced apoptosis. BxPC-3 and MIAPaCa-2 cells were treated with 5 μM PD98059 for 18 hours ,then the cells were exposed to 1.0 μM gemcitabine for 24 hours. Gemcitabine -induced cell death was determined by FACS. All values represent the means ± SD for duplicate determinations performed on three separate occasions. * Significantly greater than values obtained for cells cultured in the absence of PD98059; P <0.05).
We first evaluated the effect of sCLU silencing on the pERK1/2 activation in MIAPaCa-2 cells. MIAPaCa-2 cells were treated with 1200 nM OGX-011 for 24 hours. Figure5A shows significant decrease in pERK1/2 activation in the two cells. BxPC-3 has no basic pERK1/2 expression, so it only used for pERK re-expression. It has shown sCLU silencing itself did not affact apoptosis and growth of MIAPaCa-2 cells and BxPC-3 cells. However, sCLU silencing combined with 1200 nM OGX-011 treatment led to a significant increase in gemcitabine-induced apoptosis in both MIAPaCa-2 cells and BxPC-3 cells by FACS analysi (Figure2A).We next explored whether pERK re-expression could eliminate the effects of sCLU silencing on gemcitabine-induced apoptosis. BxPC-3 and MIAPaCa-2 cells were treated with 1200 nM OGX-011 for 8 hours, then a wt-pERK-expressing plasmid was transfected into these cells, after transfection for 24 hours ,the cells were treated with 1.0 uM gemcitabine for another 24 hours. While vector transfection did not decrease gemcitabine-induced apoptosis in both MIAPaCa-2 and BxPC-3 cells (data not shown). However wt-pERK-re-expressing in BxPC-3 and MIAPaCa-2 cells significantly decrease in gemcitabine-induced apoptosis (Figure5B). These data demonstrated knockdown of clusterin sensitizes pancreatic cancer cells to gemcitabine via pERK1/2 dependent pathway.
Knockdown of clusterin sensitizes pancreatic cancer cells to gemcitabine via pERK1/2 inactivation. A, MIAPaCa-2 cells were treated with 1200 nM OGX-011 for 24 hours, after which proteins were prepared and subjected to Western blot as described above to monitor pERK1/2 expression. B, BxPC-3 and MIAPaCa-2 cells were treated with 1200 nM OGX-011 for 8 hours, then a wt-pERK-expressing plasmid was transfected into these cells for 24 hours, after which the cells were treated with 1.0 uM gemcitabine for 24 hours. Gemcitabine -induced cell death was determined by FACS. Representative results are shown; two additional studies yielded equivalent results (*P < 0.05).
Four, two, and three deaths were noted in the vehicle control, gemcitabine-, and OGX-011-treated groups, respectively, before the end of the 5-week treatment period because of large tumors. Conversely, all mice receiving gemcitabine and OGX-011 in combination were alive and exhibited a healthier appearance. Orthotopic tumors were dissected free of surrounding normal tissues and weighed. As shown in Figure6A, gemcitabine alone did not significantly reduced tumor weights in BxPC-3 and MIAPaCa-2 cells compared to the controls,however, gemcitabine in combination with OGX-011 significantly reduced tumor weights by 5-fold (P < 0.001) in MIAPaCa-2 cell relative to the vehicle control, and 3-fold (P < 0.001) in BxPC-3 cell relative to the vehicle control. The further decrease in tumor weights observed in the combination treatment group was significantly different from the gemcitabine monotherapy group (P < 0.001). OGX-011 alone failed to inhibit tumor growth.
To investigate if the mechanisms involved in the induction of apoptosis in targeted lesions of tumor xenografts represented a phenotypic response of BxPC-3 and MIAPaCa-2 tumors, the TUNEL assay was performed. Representative results are shown in Figure6B. In the combination treatment groups of BxPC-3 and MIAPaCa-2 tumors, TUNEL-positive cells in tumor sections presented with fragmented nuclei. As shown in Figure6B, gemcitabine (80 mg/kg) or OGX-011 alone did not produce significant increases in apoptosis compared with the vehicle control. However, the extent of apoptosis was significantly increased by 5-fold (P < 0.002) in MIAPaCa-2 tumors ,and 3-fold (P < 0.001) in BxPC-3 tumors, treated with gemcitabine and OGX-011 in combination.
To determine whether inhibition of Clusterin by OGX-011 enhances sensitivity to gemcitabine via pERK1/2 inactivation, we detected the pERK1/2 expression by western-blotting assay. As shown in Figure6C, gemcitabine treatment did not activate pERK1/2 in the MIAPaCa-2 tumors, and gemcitabine treatment signicantly activated pERK1/2 in the BxPC-3 tumors. However, gemcitabine in combination with OGX-011 significantly inhibited pERK1/2 activation.We therefore think that sCLU sliencing sensitizes pancreatic cancer cells to gemcitabine chemotherapy by inhibiton of ERK1/2 activation.
Pancreatic cancer is one of the most difficult human cancers to treat due to the inability to detect disease at an early stage and the lack of effective therapies. Although there has been some progress in the use of improved diagnostic methods and development of novel targeted therapies, the overall survival rate has not improved over the last decade. The most commonly used chemotherapy for pancreatic cancer, gemcitabine, has modest clinical benefit and may not improve overall survival to a clinically meaningful degree[40, 41]. The lack of significant clinical response of pancreatic cancer patients to chemotherapy is likely due to the inherent chemoresistance of pancreatic cancer cells as well as impaired drug delivery pathways. Understanding the underlying mechanisms of drug resistance in pancreatic cancer is critical to develop new effective treatments for this deadly disease.
sCLU expression has been implicated in chemoresistance in several other cancer types[43–45], including pancreatic cancer. Because the resistance of tumor cells to various available chemotherapeutic agents has been one of the major factors leading to poor survival in pancreatic cancer patients, we therefore hypothesized that sCLU confers chemoresistance to pancreatic cancer cells.
In this study, we demonstrated that sCLU was correlated with inherent resistance both in vitro and in vivo. We found that high levels of sCLU in pancreatic cancer MIAPaCa-2 cell line was correlated with gemcitabine resistance, low levels of sCLU in BxPC-3 cells was sensitive to gemcitabine .To demonstrate the role of sCLU in gemcitabine resistance, we manipulated the endogenous level of sCLU in a gemcitabine -sensitive BxPC-3 cell line and a gemcitabine -resistant MIAPaCa-2 cell line. We found that gemcitabine -sensitive BxPC-3 cells became more resistant to gemcitabine when endogenous sCLU expression was up-regulated. Conversely, gemcitabine -resistant MIAPaCa-2 cells became more sensitive to gemcitabine and more apoptotic in vitro and in vivo when endogenous sCLU expression was down-regulated by GOX-011 treatment. These results indicated that high levels of endogenous sCLU were involved in the gemcitabine resistance of ovarian cancer cells.
Acquired drug resistance is also thought to be a reason for the limited benefit of most pancreatic cancer therapies.In the present study, we found treatment by gemcitabine increased sCLU expression in BxPC-3 cells, suggesting that sCLU upregulation is likely to be an adaptative response that mediates chemoresistance.We also investigated whether anticlusterin treatment sensitized BxPC-3 cells to gemcitabine.
GOX-011 efficiently inhibited sCLU expression in BxPC-3 cell lines, and this activity was associated with a increase in cell apoptosis in gemcitabine-treated BxPC-3 cells in vivo and vitro. This was indicated that increased sCLU, expression was correlates with gemcitabine resistance in pancreatic adenocarcinoma cells. These results provide preclinical proof of principle for the use of OGX-011 as a novel therapeutic strategy for gemcitabine resistance in the treatment of pancreatic cancer.
Though sCLU confers gmcitabine resistance in pancreatic cancer cells, however, the signaling pathway was unclear. ERK activation has been identified as a potential survival pathway in several tumor types, and recent studies show that ERKs may also be activated in response to chemotherapeutic drugs[47–50], and pERK1/2 played critical roles in drug resistance. Our in vitro and in vivo studies here indicated that pERK1/2 play significant roles in gemcitabine resistance to pancreatic cancer cells. Most importantly, we demonstrated that blocking pERK1/2 enhanced the chemotherapeutic potential of gemcitabine in pancreatic cancer cells in vitro. ERK1/2 inhibitors in combination with chemotherapeutic drugs might be a better option to treat patients with pancreatic cancer than drugs alone.
It has shown previously sCLU plays an important role in regulating ERK1/2 signal[32–34].We next study whether sCLU silencing sensitized pancreatic cancer cells to gemcitabine chemotherapy may via ERK1/2 signal. Our results shown sCLU sliencing by OGX-011 sensitizes pancreatic cancer cells to gemcitabine treatment,followed by inhibition of pERK1/2 activation. Conversely, transfection with a constitutively active wt-pERK1/2 construct promotes gemcitabine resistance. These data demonstrated sCLU sliencing sensitizes pancreatic cancer cells to gemcitabine via pERK1/2 dependent signaling pathway.
In conclusion, gemcitabine may influence pancreatic cancer behavior via the upregulation of sCLU, which might play a major role in the effects of gemcitabine, protecting pancreatic cancer cells from the effects of gemcitabine. Inherent chemoresistance of pancreatic cancer cells to gemcitabine may be correlated to sCLU. Blocking sCLU, on the other hand, reverses the drug's unwanted effects on cancer cell apoptosis and survival. In addition, our studies have firmly established a role for sCLU as a cell survival gene that is increased after gemcitabine chemotherapy to inhibit tumor cell death. The inhibition of sCLU, using OGX-011, enhances the cytotoxic effects of chemotherapy agents via pERK1/2 dependent signaling pathway.
Yong Tang, Fenghua Liu contributed equally to this work.
An erratum to this article is available at http://dx.doi.org/10.1186/1756-9966-32-42.
TY, LFH, ZCN and JYS performed the majority of experiments; SSC and TY designed the study and wrote the manuscript; TY and JYS edited the manuscript. All authors read and approved the final manuscript.


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AIS researchers use a wide variety of innovative research techniques to address these questions. These include design science approaches to construct new artifacts, case studies, field studies, surveys, experiments, and archival research as well as fundamental theoretical analysis.
The AIS discipline also plays a significant role in educating future accountants and auditors about the implications of information technology for the practice of accounting and auditing.
Joining the AIS section allows accounting and auditing faculty and practitioners to be exposed to leading-edge research and teaching practice
Greetings AIS Members,
Thank you for entrusting me with the role of President over the last year. I am so excited for the future of our Section. From the 2020
Annual Meeting, to the Midyear, to the AIS Bootcamp, I had the pleasure of Zoom meeting with many energetic, engaged, collaborative, and collegial members. It has been an inspiring year!
The slides provide a brief Business Report.
We will have a full update of Section Business during the Midyear Meeting in January 2022. I look forward to seeing you all in person ...
Journal of Information Systems
The Journal of Information Systems (JIS) is the academic journal of the Accounting Information Systems (AIS) Section of the American Accounting Association. Its goal is to support, promote, and advance Accounting Information Systems knowledge. The primary criterion for publication in JIS is contribution to the accounting information systems (AIS), accounting and auditing domains by the application or understanding of information technology theory and practice.
Calls for Nominations
Notable Contribution to the Accounting Information Systems Literature Award
The AIS Section Outstanding Dissertation Award
Journal of Information Systems Best Paper Award
AISwebmaster@aaahq.org · Webinars Page


Please join us on Monday, November 26 to welcome our guest speaker James Boutilier. He will address our Society on "Whither International Support for China".
We seem to be entering a new era in the Chinese saga with the recent push back against Chinese activities, whether it be Trumpian tariffs, espionage trials in the United States that target Chinese Americans, anti-United Front legislation in Australia, Malaysia's recalibration of its relationship with China, warnings from a senior Singaporean commentator about Chinese debt traps, new skepticism about President Xi's Belt and Road Initiative, Canada's unwillingness to permit a Chinese takeover of Aecon or Canada's imposition of tariffs on Chinese steel.
Prior to his appointment at MARPAC, Dr. Boutilier spent twenty-four years on staff at the Royal Roads Military College in Victoria as Head of the History Department and then as Dean of Arts. During his time at RRMC, he was instrumental in establishing the military and strategic studies degree program at the college and taught courses on naval history, contemporary Asia, the history of the Pacific, and strategic issues. He is also an adjunct professor of Pacific and Asian Studies at the University of Victoria and the President of the Maritime Awards Society of Canada. Dr. Boutilier, his charming wife, Ping, and brilliant daughter, Janou, live in Victoria, British Columbia.
Location: Royal Vancouver Yacht Club, 3811 Point Grey Road, Vancouver, B.C.


A personal coach is a trusted partner who can help you identify and uncover what keeps you from reaching your fullest potential. It is a one-on-one relationship that provides a supportive and non-judgmental space to help you create what you want for yourself. The Core Energy Coaching process helps you tap into your inner purpose and passion and connect that with outer goals and tasks to bring about extraordinary and sustainable results.
What types of people have personal coaches?
People from all walks of life work with professional coaches to help attain their goals. Personal coaches work with people in all areas of their life including relationships, career, finance, health and more. Working with a personal coach is not limited to any type of person, but is best for those who want to take initiative in their own life. People who hire a personal coach are committed to a more fulfilled life and are willing to do the work needed to get there.
How will personal coaching help?
The results will vary depending on what brings you to a personal coach. If you hire a coach to help you out professionally, personal coaching can help you make your current job more gratifying, gain better focus on managing your work, and market yourself more effectively. If your reasons are personal, you can find a coach to help you improve your relationships, resolve ongoing negative behaviors, improve your overall health, and grow spiritually. With personal coaching you can live a more balanced and enhanced life, and make your dreams come true!
What happens in a personal coaching session?
Each session is tailored to your needs. In the beginning, we will discuss what you are committed to accomplishing through our work together and we will begin to create a plan to achieve your goals. Periodically, I will give you assignments to help direct your progress. We will work together to acknowledge obstacles, explore alternate perspectives and celebrate successes. With the conclusion of each session, we will revisit your goals and evaluate the next steps that are needed to achieve these results.
Coaching can take you above and beyond your own personal limitations. More often than not, people try to achieve their greatest potential, but don't even realize what stands in their way. A personal coach can offer an unbiased and objective perspective, and can help you realize your patterns and habits. Most people experience that they are more efficient when working with a professional coach and that this provides the opportunity for them to better realize their potential. Personal coaching can make your dreams attainable and will give you support and encouragement along the way.
While the personal coaching I provide can be therapeutic, it is not therapy. Coaching is distinct from traditional therapy because it is not designed to address psychological issues. While emotional issues may arise, the primary focus of coaching is to help you identify your goals, achieve them, and develop constructive habits and skills. There is little emphasis placed on the past with coaching, but instead the focus is on creating what you want now and in the future.
How long do people generally work with a personal coach?
To experience the effectiveness of a personal coaching program, I suggest you make an initial three-month commitment. At the end of the three months, we will evaluate progress and discuss options for continued coaching.
What we discuss will be held in confidence and not shared with others. However, our discussions are not privileged for legal purposes. The privilege of confidentiality that exists for the clergy, therapists and attorneys is not granted to coaching professionals. That said, personal coaching is a confidential process, and your personal information will always be protected.


I have loaded a session archive (.saz file), but it's not displayed anywhere (file name).
Add right click option to decode base 64 on any filed, such like request field, or Jason response field, it might be also xml field or even entire response. right click and base64 decode.
It's difficult to compare request information by not being able to tearoff multiple request details windows. Currently, the tearoff only pops out the request details window, but doesn't allow for multiple per request.
Could you please clarify if fiddler can be used in Windows terminal server without any license cost?


text:
<?php

namespace {
    interface Twig_Extension {}
}

namespace Foo {
    class Bar implements \Twig_Extension {}
}

redpajama_set_name: RedPajamaGithub


text:
'use strict';

var mongoose    = require('mongoose'),
    async       = require('async'),
    utils       = require('../utils/utils'),
    flowUtils   = require('../utils/flowUtils'),
    paths       = require('../models/paths'),
    templates   = require('../models/templates'),
    constants   = require('../models/constants'),
    db          = require('../app').db.models;

function GET_entry(req, res) {
    var model = {};
    flowUtils.ensureEntryIdParam(req, 'issue');
    var ownerQuery = flowUtils.createOwnerQueryFromQuery(req);
    flowUtils.setEntryModels(ownerQuery, req, model, function (err) {
        ownerQuery['screening.status'] = constants.SCREENING_STATUS.status1.code;
        ownerQuery.parentId = null;
        async.parallel({
            opinions: function (callback) {
                flowUtils.getTopOpinions(ownerQuery, model, callback);
            }
        }, function (err, results) {
            flowUtils.setModelOwnerEntry(req, model);
            res.render(templates.wiki.issues.entry, model);
        });
    });
}

function GET_index(req, res) {
    var model = {};
    var query = flowUtils.createOwnerQueryFromQuery(req);
    if(query.ownerId) {
        flowUtils.setScreeningModel(req, model);
        flowUtils.setEntryModels(query, req, model, function (err) {
            //query = flowUtils.createOwnerQueryFromModel(model);
            query['screening.status'] = model.screening.status;
            db.Issue
                .find(query)
                .sort({title: 1})
                .lean()
                .exec(function (err, results) {
                flowUtils.setEditorsUsername(results, function() {
                    results.forEach(function (result) {
                        result.issueType = constants.ISSUE_TYPES['type' + result.issueType];
                        flowUtils.appendEntryExtras(result);
                    });
                    model.issues = results;
                    flowUtils.setModelOwnerEntry(req, model);

                    // screening and children count
                    flowUtils.setScreeningModelCount(model, model.entry.childrenCount.issues);
                    res.render(templates.wiki.issues.index, model);
                });
            });
        });
    } else {
        // Top Issues
        db.Issue
            .find({ ownerType: constants.OBJECT_TYPES.topic, 'screening.status': constants.SCREENING_STATUS.status1.code })
            .limit(100)
            .lean()
            .exec(function(err, results) {
                flowUtils.setEditorsUsername(results, function() {
                    results.forEach(function (result) {
                        result.issueType = constants.ISSUE_TYPES['type' + result.issueType];
                        result.topic = {
                            _id: result.ownerId
                        };
                        flowUtils.appendEntryExtras(result);
                    });
                    model.issues = results;
                    flowUtils.setModelContext(req, model);
                    res.render(templates.wiki.issues.index, model);
                });
            });
    }
}

function GET_create(req, res) {
    var model = {};
    flowUtils.setEntryModels(flowUtils.createOwnerQueryFromQuery(req), req, model, function (err) {
        model.ISSUE_TYPES = constants.ISSUE_TYPES;
        flowUtils.setModelOwnerEntry(req, model);
        res.render(templates.wiki.issues.create, model);
    });
}

function POST_create(req, res) {
    var query = { _id: req.query.issue || new mongoose.Types.ObjectId() };
    db.Issue.findOne(query, function(err, result) {
        var dateNow = Date.now();
        var entity = result ? result : {};
        entity.title = req.body.title;
        entity.content = req.body.content;
        entity.contentPreview = flowUtils.createContentPreview(req.body.content);
        entity.friendlyUrl = utils.urlify(req.body.title);
        entity.issueType = req.body.issueType;
        entity.editUserId = req.user.id;
        entity.editDate = dateNow;
        if(!result) {
            entity.createUserId = req.user.id;
            entity.createDate = dateNow;
            flowUtils.initScreeningStatus(req, entity);
        }
        entity.private = req.params.username ? true : false;
        if(!entity.ownerId) {
            //delete req.query.opinion;
            delete req.query.issue;
            var q = flowUtils.createOwnerQueryFromQuery(req);
            entity.ownerId = q.ownerId;
            entity.ownerType = q.ownerType;
        }
        flowUtils.syncCategoryId(entity, { entryType: constants.OBJECT_TYPES.issue }, function () {
            db.Issue.findOneAndUpdate(query, entity, {
                upsert: true,
                new: true,
                setDefaultsOnInsert: true
            }, function (err, updatedEntity) {
                var updateRedirect = function () {
                    var model = {};
                    flowUtils.setModelContext(req, model);
                    var url = model.wikiBaseUrl + paths.wiki.issues.entry + '/' + updatedEntity.friendlyUrl + '/' + updatedEntity._id;
                    res.redirect(url);
                };
                if (!result) { // if new entry, update parent children count
                    flowUtils.updateChildrenCount(entity.ownerId, entity.ownerType, constants.OBJECT_TYPES.issue, function () {
                        updateRedirect();
                    });
                } else {
                    updateRedirect();
                }
            });
        });
    });
}

module.exports = function (router) {

    /* Issues */

    router.get('/', function (req, res) {
        GET_index(req, res);
    });

    router.get('/entry(/:friendlyUrl)?(/:friendlyUrl/:id)?', function (req, res) {
        GET_entry(req, res);
    });

    router.get('/create', function (req, res) {
        GET_create(req, res);
    });

    router.post('/create', function (req, res) {
        POST_create(req, res);
    });
};

module.exports.GET_entry = GET_entry;
module.exports.GET_index = GET_index;
module.exports.GET_create = GET_create;
module.exports.POST_create = POST_create;


This thesis introduces the physical simulation of surgical thread for usage in a microsurgical training simulator for the education of medical students. To allow interactive simulation the thread must be real time capable. Importantly, in the simulation, the thread must behave in a way that it looks like a real thread to the user. The user can then "dive into" the simulation, because for the user, the simulation of the thread appears real. We refer to this "diving into" the simulation as "immersion". The physical model of the thread is a mass-spring model based on the Kirchhoff theory for elastic rods. One challenge is the stiffness constraint of the thread. A real world thread does not change it's length signiffcantly even under high stress. In a mass-spring model this property can be obtained by using high spring constants. But if an explicit integration method is applied the so called "overshooting" effect presents a problem. It causes the system to diverge when the spring constants are too high. In this thesis the problem is addressed by applying an implicit integration method. A key property of implicit integration methods is that it is unconditionally stable and thereby allows a large time step in the numerical integration. But it also requires that a linear system of size equal to the number of degrees of freedom in the system is solved. If the number of degrees of freedom is high this conflicts with the real-time requirement of the simulation. In this work it is shown that for the case of the thread the matrix in the linear system is banded and can therefore be solved in linear time. Another advantage of the implicit integration is that forces are propagated along the complete thread within one time step. For the simulation of microsurgical sutures knots have to be modeled. A knot causes numerous contacts of the thread with itself. The contact forces are modeled and calculated using a physical model. Because all forces propagate along the whole thread within one time step all contacts interact with each other. A force applied at one contact affects all other contacts. Because of this all contact forces have to be solved for simultaneously. The interaction of the contacts due to the implicit integration are calculated resulting in a linear system which describes the relation between the contact forces and the relative movement of the thread at the contacts. Physically correct contact forces have to be found with this linear system. Similar to the simulation of rigid bodies, a linear complementary problem or a nonlinear complementary problem results depending on the model that is used for the contact forces. In case of rigid body simulation the "projected Gauss-Seidel" is a proven method for solving the problem. In this thesis the nonlinear conjugate gradient (NNCG) method from Silcowitz-Hansen et al. is applied. This method was originally developed for rigid body simulations. The thread has been integrated into the microsurgical training simulator "MicroSim". Which is to say, interactions between the thread and tissue and forceps have been modeled and incorporated into "MicroSim". These interactions have to be compatible with the implicit integration of the thread. In a joint work with Sismanidis and Schuppe a training module for MicroSim has been developed. This training module allows for training of a microsurgical anastomosis of blood vessels.


Throughout my time in America, I have gained a growing awareness of sweeping perceptions that have presented themselves to me in contemporary media, current events, and day-to-day social interactions.
White People is a sardonic take on the existence of racism in the present-day. By digitally whitewashing any perceivable signs of Caucasian skin, I hope to communicate the caustic effects of racial profiling through an exaggerated lampoon. The choice to alter only Caucasian skin stems from my minority status as a "person of color", and is based on my personal experiences with the "white majority" and white culture. The pictures attempt to reconcile the immediacy of a passing glance with the pervasiveness of subconscious perceptions brought on by race and skin color.
As it stands, the existence of an "us and them" mentality in today's society is a problem that must be tackled eventually. These images reflect a genuine effort to understand and stem our instantaneous and sometimes unintentional process of "color-coding" the world around us, and attempt to prompt a discussion on racism through a distorted and sarcastic view of white people in America.


text:
Daniel Maldini (* 11. října 2001 Milán, Itálie) je italský fotbalový útočník hrající od začátku své kariéry za AC Milán. Od sezony 2022/23 je hráčem Spezie, kde je na hostování. Jeho dědečkem byl fotbalista a trenér Cesare Maldini a jeho otcem je obránce Paolo Maldini.

Statistiky

Úspěchy

Klubové
 1× vítěz italské ligy (2021/22)

Odkazy

Poznámky

Reference

Externí odkazy
 Profil hráče na Transfermarkt.it

Italští fotbalisté
Fotbalisté AC Milán
Narození v roce 2001
Narození 11. října
Narození v Miláně
Žijící lidé
Muži

redpajama_set_name: RedPajamaWikipedia


Hold down + + W+S then hit or or A or D. The car will start to rotate; to get gold, look for the burn marks on the tarmac and use it to align your outer tire.
Essentially this is just a handbrake turn, accelerate into the half-circle of cones and press the handbrake button while still holding down W. Turn the car left or right and it will start to spin. When the nose of the car is pointing around the cone (so you won't hit it) release the handbrake and accelerate back to the starting point.
Due to the time limit you might need to use the handbrake again to come to stop within the cone box.
For this test you have to do a 90° handbrake turn, then come to a stop. The two issues are the turn and the parking. To maximize turn, try to be a close to edge of cones so that you can get a better time. After you get the turn down (you can try as many times as you want until you get it), you will have to be good at parking.
Speed up then slow down just before the tires pop, then speed up and park the car in the cones without touching them.
This one is tough but straight forward. The trick is to keep your laps as tight as possible to the inside cones using the handbrake to spin the car 180°. First-person view makes this much easier. Get back on the gas as quickly as possible, as you cannot afford to let the car lose too much speed if you want to get all five laps in under 36 seconds, which is required for gold.
Again, first person view is useful to get the timing right. Stay towards the inside of the track. Start your turns when the right (or left if you're going counterclockwise) edge of the screen is about 3/4 of the way between the penultimate and last cones. Picture your right (left) wheel just skimming around the edge of that last cone. If you knock it over, that doesn't count as damage.
This one is also pretty tough until you learn how to do it right. All you need is to accelerate past the first two cones, then let go of the gas. And then once you pass the third cone, hit the gas and make the 180° turn. Then again, just like before, hit the gas through the first two, then just release it for the third. If you do this right, you should have about four or five seconds to get back to where you started. Going really fast then stomping on the brakes doesn't usually work here. You can go a little slow because you have some time if you did the first part correctly.
This one as well can be timed well in first-person mode. Reverse your turn between the penultimate and last cones. No need to brake; just let up on the gas.
Slow down at about two or three seconds, by letting go of W then use reverse to make the 90° turn.
This is only tough because you have to understand one thing. If you are on two wheels, you cannot turn in the direction you are slanted in. If you do so, you will go down. It will take a couple tries, but it's not really that hard.
In this test you have to back up, reverse direction, and park in the cone box.
Obviously, first-person mode is not going to help here. Instead, use third-person view and use Q and E to swivel the point of view around so you are looking at the front of the car, and the course behind it. Now all you have to remember is that in reverse, you turn the wheel the opposite way you want the rear wheels to go.
Hit the police car on the back tire and it will spin. Land up next to it without damaging yourself to achieve gold.
Speed up and only let the left side wheels hit the jump, as soon as the car is near the end of the jump and half way in the air, release W and try to turn right. The car will then do a barrel roll and land on its wheels, then try to park the car straight.
To complete driving school you have to drive across the city in under two minutes without damaging the car too badly and without stopping.
It terms of what you have to do, this is a relatively easy task but there are two things to keep in mind: firstly never stop. If you skid out or hit something keep the accelerator down and keep driving you can spin out and still complete the course. As for damage, grazing another vehicle or lamppost is not all that important if it saves you a couple of seconds. For gold you'll need no damage, but to just complete the School mission you can afford a few knocks here and there.
This is tricky due to the requirement not to stop, but otherwise you are probably pretty familiar with driving through the city at high speed. First-person view is a big help on this one to help avoid clipping turning or stationary cars. Take care, because the Super GT has a tendency to oversteer. Try not to accelerate into a turn, instead out of a turn.
When the test starts, make a quick left and right to get onto the street going past your garage. You can cut the first corner; there are no curbs to worry about. Drive as fast as you can along the street until you get to the rise in front of the cathedral (the trolley track is usually clear, except in the unlucky case you hit a trolley head-on). Turn right and then turn left at the next junction. Note that the "racing line" through this point is into the oncoming traffic so you'll have to see whether the line is clear before you decide to take it.
Now continue along the street until you get to the steep, twisting decline. This is the trickiest part of the course because it is easy to either hit something or lock your wheels and spin to a stop. If in doubt: slow down, because you are going downhill you are not going to lose momentum.
At the bottom of the hill turn right through the checkpoint and the drive as fast as you can down the Esplanade to Easter Basin Docks, turn right up the hill and stop outside the driving school.
Note: this course can also be completed in reverse to the description above, and claim the gold award. If the downhill near the checkpoint is a problem, take the course in reverse.
Another option is to get onto the rail track nearby, and head towards the tunnel. As you come out of the tunnel, turn left onto the grass, onto the road and follow to the checkpoint. This seems to allow for faster times, at least on the PC.
Two other things to note are that on the PC version you need to be wary of the game's habit of spawning vehicles directly in front of you that are too close to avoid, and there are several places were the sidewalks are more than wide enough to fit the GT. Since the game registers this as a "car" mission, fewer pedestrians spawn, so if traffic is too heavy take the sidewalk.
This page was last edited on 21 April 2015, at 05:04.
This page has been accessed 61,566 times.


Amazon India Announces The New Fire TV Stick 4K & The All New Alexa Voice Remote Supporting TV controls.
Yesterday Amazon India announced its new Fire TV Stick 4K in India as a successor to the 2nd Generation Fire TV Stick. This new Fire TV Stick 4K comes with an all-new Alexa Voice Remote with dedicated voice controls buttons which were found to be missing on the earlier generation remote. The entire bundle is available for the price of Rs.5,999. However, if you wish to buy the new Alexa Remote, it's available for Rs.1,999 as it is backwards compatible with the 2nd Gen Fire TV Stick.
The new Fire TV Stick 4K is the first streaming stick from Amazon to support Dolby Vision and HDR10+ for achieving the optimum viewing experience.
The new Alexa Voice Remote now houses a dedicated Volume, Power and Mute buttons with which you can now easily control your TV.
Fire Stick 4K comes with a quad-core processor which is clocked at 1.7GHz which in turn delivers faster loading times and provides a good streaming experience. There are also a vast number of 4K titles including 4K Ultra HD, Dolby Vision & HDR+ supported content. Just say, "Alexa show me 4K TV shows".
The users can also experience 3D surround sound with the compatible home theatre equipment systems.
Mr Marc Whitten, VP - Amazon Fire TV said, "We've listened to our customers and pushed to deliver a higher performance Fire TV. The new quad-core processor delivers a fast and fluid experience, and support for Dolby Vision and HDR10+ provides an amazing picture quality," He then added, "It is a complete 4K solution our customers are looking for, all within a compact stick form factor. The team invented an entirely new antenna technology & combined that with a powerful 802.11ac Wi-Fi to optimize for the best possible 4K UHD streaming experience, even in congested network environments. Plus, the all-new Alexa Voice Remote lets you control your complete entertainment experience & use your voice to find the content you want quickly. Just say, 'Alexa, find comedies', or 'Alexa, play The Grand Tour'.
Not to forget about the thousands of apps and Alexa Skills catered for the Fire Stick and Alexa Remote. Same goes for the vast amount of content which includes new and upcoming movies and TV episodes present on Prime Video, Netflix, Hotstar and others. Users can even get the glimpse of their social media channels with the supported FireFox or the preloaded Silk Browser. Not to mention the swift workaround to access the YouTube content. The users can also enjoy listening to their favourite music via several apps such as Amazon Prime Music, Saavn and TuneIn.
Finding anything on the Fire TV Stick 4K now becomes much easier with the help of the new Alexa Remote. Users can press and hold the microphone button to search Movies, TV shows, Apps, check sports scores and even order food, book a cab, view live camera feeds, control your smart home - all thanks to the Alexa integration in the Fire TV Stick. Users also have the novelty to enable the Far Field Voice control on their Fire TV Stick 4K with the help of their existing Echo devices. Now imagine integrating the recently announced Echo Plus and Echo Subdevices, that'll be like bringing a party to your home. Integration of Echo devices also translates to the fact that the user can now watch and enjoy movies & songs without even touching the Remote. Just say, "Alexa, play ******* on my Fire TV."
The new Fire Stick 4K is available for pre-order on Amazon India at a price of Rs.5,999 including the new Alexa Remote. While the standalone Alexa Remote costs about Rs.1,999 only. Both of these devices will start shipping from starting November 14.
All the existing Fire TV devices in India will soon receive a software update starting from 14th November which will enable Alexa support on Fire TV which includes the Far Field Control support as well.


Ohhoo, someone is mid-rolling out there!
Congrats mate! Awesome win indeed, even without the 5 scatters which I'm pretty sure is only a matter of time before you nail during free spins feature!
Yep...with a healthy wallet, I'm back to playing with slightly bigger bets...oooh okaaay...more like Mid-rolling, hehehe. Like back to the good old days of some years ago.
Jammin Jars - dead in the water!
Napoleon - dead in the water!
Don't worry mate...one day, someone else will be mopping up for you...it's a vicious circle!
Some days you're the windshield, some days you're the bug.. some day's you're the unicorn flying overhead and laughing. thank you drugs..
Congrats Guru-san...excellent win there...but take it from me...forget about Jammin Jars, Napoleon and whatever new...just stick with your favourite paying oldies and you'd be better off!
And that's exactly what I'm planning to do, you bet mate!
Wish I could confirm, but the reality is quite different unfortunately... The beginning of the year wasn't exactly what I was expecting and definitely had nothing to do with with the word "luck". Most of the big wins were either helping me get even or in a small profit but nowhere near to what one used to expect when on a winning streak.
Anyway, sincerely hope we will all encounter many fantastic eye-to-eye meetings with our beloved Lady Luck in 2019!
Well my phone has been going crazy with notifications so I came to see what the fuss is all about and now I see!! Congrats Guru!
Well, I don't know a real man /including myself/ who would refuse such eye-to-eye meeting with a gorgeous girl like you anyway.
It's just your explosive, stormy and totally unpredictable typical Macedonia temper which bothers me, that's why preferring meetings with Lady Luck...For the time being.
Ha! Now that's quite an interesting strategy!
Playing with only 1 line? Oh geees...and what a damn good win that was...imagine what the win would have been if played with the full 10 lines...but...congrats anyway mate!
Okaaaay...Guru had his excellent win...My P had his unbelievable 1-line win...so Afi has his nice 15-lines win too...hehehe.
In fact, it's a win that I have been waiting for so long to happen from this favourite game of mine...until tonight!
You probably already know that games like Gemix, Runecraft, Moon Princess, etc, are not my kind of games, but once in a while, I would give them a shot anyway. On my lucky day, the Gargantuan gave me a nice surprise...hehehe.
Bonanza: 800x base game hit (probably the same as v's) with a board of diamonds.
Congrats on the nice wins Mr P...so, were you in the kitchen, or in the toilet, or in the garden whilst the reels were spinning mate?
Finally back to this thread! Unfortunately this time the winnings were Lost, but still, nevertheless a good Play time.
Immortal Romance at Slotjoint casino - it's been a long time since I had a win like this on IR.
Extra Chilli at Casino Heroes- I have a love/hate relationship with it. It gets addicting to buy the features and for the most part it gives low payouts, however the winning potential is definitely there.
Happy to see you posting here once again and hope this was just the beginning of a loooong, amazing winner streak!
Gave that Multi fruit 81 a decent stab and ended up 250 up on that - nothing amazing but gave a decent amount of wins.


text:
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Outsourcing and artificial intelligence: Re-engineering outsourcing agreements
Global | Publication | January 2020
The AI Revolution
In the 1990s and 2000s, as everything from banking to buying books moved online, "brick and mortar" businesses were suddenly required to have an online presence and to design new business processes to accommodate the new online world. Through a practice called business process engineering (BPE), banks had to design online services and appropriate authentication procedures, retailers designed online payment processes, and businesses in many sectors implemented processes for back office activities (such as invoicing and accounts payable) and customer-facing activities (such as responding to queries and complaints).
The generation of new online businesses that flourished during this period enjoyed a natural advantage in this environment. Their business processes had been engineered from the start for the online world, whereas the established players often ran their old pre-internet processes and the new online processes in parallel. Over time, through business process reengineering (BPR), these parallel processes were redesigned so that the brick and mortar businesses could compete in the new environment.
The revolution in AI poses a similar challenge today to that posed by the internet revolution. AI will be able to drive efficiencies through further labour arbitrage and by generating and allowing customers to react to richer insights into their business processes. A new wave of BPE will create processes centred around AI, and no doubt in time, old processes will need to redesigned through BPR.
In almost every outsourcing that we have seen over the past two or three years, the transformation plan includes the deployment of automation and AI to drive efficiency. Rules-based automation has been around for some time (it is not the focus of this article), but the autonomous nature of AI and the specific requirements for training an AI solution present a new set of challenges for the outsourcing practitioner.
This article asks a simple question: Is the traditional outsourcing agreement, "engineered" for the pre-AI world, suitable in a world in which AI displaces both people and rules-based methods of automation?
The answer to that apparently simple question is somewhat complex. This article addresses the question by: (1) looking at the ways in which outsourcing agreements may need to be redesigned for the deployment of AI, whether one is looking to amend an existing outsourcing agreement through change control or contracting for an entirely new outsourcing; and (2) focusing on the impact of AI on a few key provisions in outsourcing agreements:
IP and customer data
Liability, relief and remediation
The exit schedule
Maintenance arrangements
The charging schedule
AI will necessarily affect other provisions in the agreement (for example, the data protection provisions), with consequences in some sectors more so than in others (for example, regulated outsourcings in the financial services sector). More information on these topics can be found on our Inside Tech Law blog. This article will then consider the downstream impact of AI within an outsourcing context on the customer's other third-party vendors, and will conclude with some observations on contracting for disruptive technologies (such as AI) more generally.
The full article was published by educational charity the Society for Computers and Law (SCL).
Zayed Al Jamil
zayed.aljamil@nortonrosefulbright.com
Sourcing and technology
Trends and opportunities for digital infrastructure operators and investors in the APAC region
The dramatic and unpredictable events of 2020 have fuelled the growth of digital infrastructure development and investment in the Asia-Pacific region and we expect that opportunities will continue to abound for operators and investors alike.
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Beyond COVID-19: Crisis response or road to recovery?
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The EU adopts its first global human rights sanctions regime
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At O'Donnell Metal Deck, we are committed to providing the fastest and easiest service for metal decking in Elizabeth and the surrounding New Jersey area. O'Donnell Metal Deck is conveniently located a few hours down 95, just past Baltimore. We offer fast, weekly delivery of our roof deck products to the Elizabeth area.
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Over 50 firefighters hospitalised after extinguishing a blaze
Update: November, 30/2015 - 18:54
More than 100 firefighters took part in extinguishing a fire in Hai Phong City' Nam Hai Port on Friday. — Photo dantri.com.vn
HAI PHONG (VNS) — More than 50 firefighters were hospitalised after an operation to extinguish a blaze at Nam Hai Port, an official of the Fire Fighting and Prevention Department of Hai Phong said.
Initial results showed that soldiers had inhaled the toxic phosphorus fumes, and were suffering manifestations of respiratory failure, Brigadier Le Quoc Tran, director of the department said yesterday.
He said a crane dropped a container onto Contship ACE ship which was carrying about 20 containers of phosphorus docked at Nam Hai Port of this northern port city to complete a goods delivery on Friday afternoon.
The collision was so strong that it cracked a phosphorus container on board which caught fire.
Immediately after the fire occurred, the unit sent more than 100 soldiers to extinguish the blaze. As the fire spread to a large area, many of the soldiers did not have the respiratory mask, but still took part in the operations.
After the fire was put out, dozens of firefighters reported symptoms such as headaches, nausea and breathlessness. Of these, two soldiers had severe respiratory problems and were taken to emergency department at Viet Tiep Hospital, while 50 other policemen were transferred to the Hai Phong Medical University Hospital for treatment. — VNS


text:
Mike McCracken
Mike McCracken began his career at Far West Recycling as a part-time warehouseman in the summer of 1989 to earn money for college. He was hired full-time in June of 1994 as a Broker Assistant. In 1997, he was promoted to Plant Manager at our Northeast plant. In 2005, Mike was promoted to CFO/Vice President. In 2021 Mike became the President and CEO.
Mike graduated from Sunset High School in 1987. He received his BS in General Science at the University of Oregon in 1993. He then went to night school for two years while working at Far West Recycling to obtain his BS in Information Technology from the University of Phoenix in 2002. Shortly thereafter, he went back to school to the University of Portland to obtain his MBA in 2004.
As President, Mike provides leadership for the company by working with the board and other executives to establish short and long-term goals, plans and strategies. Mike presides over the entire workforce, manages budgets and makes sure resources are allocated properly. Mike ensures that departments meet their individual goals and are responsible for overall accountability to shareholders and the general public.


Whether you live north of the Lehigh River in the Historic Downtown area or near Lafayette College in College Hill, all Easton residents need a reliable home cooling system. When you choose Jack Lehr Heating, Cooling & Electric, that's exactly what you'll get! With Jack Lehr, you can rest easy knowing all air conditioner services, including installation, repair, maintenance, and replacement services, will be completed by skillfully trained technicians.
Contact us at 888-454-7365 today for a free estimate on your new central air conditioner or other cooling system in the Easton area!
Shopping for a new central air conditioner for your home? From Prospect Park to Palmer Heights, our certified technicians can help you determine the best size and system for your home's unique cooling needs! If you're looking to upgrade your aging system with a newer, more efficient model, it will not only improve your home's comfort no matter how hot it gets outside, but it could also save you money on your energy bills! Plus, if you're interested in installing a dehumidifier, WiFi-enabled programmable thermostat, or other HVAC part or accessory, we can help with that too.
Give us a call at 888-454-7365 for all of your central air conditioner needs in the Easton area! We provide superior air conditioning repair services for all types of systems.
Do you want to cool only certain areas of your home? Consider installing a ductless system! At Jack Lehr, we're proud to be a Mitsubishi Diamond Dealer and sell and install Mitsubishi ductless mini-split systems throughout Northwood Heights and Lincoln Terrace, among other districts in the Easton area. These systems offer easy installation, as well as superb energy efficiency and savings.
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Don't let the name fool you—heat pumps work all year long to heat and cool your home. So whether you're rushing home from the Easton Farmer's Market in the summer or bundling up for a long walk through the downtown area in the winter, switching to an electric heat pump is a great way to realize long-term energy savings and increased comfort.
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For residential air conditioner services in Easton and the Greater Lehigh Valley area, call the experts at Jack Lehr to schedule a service or request a free system replacement estimate. We look forward to serving you!


Jackie! Here's to you, girl!
Never saying, "No, no. No!
forty before she said the word.
Jacqueline Henry is a New York-based writer and editor. Her work has appeared in or is forthcoming from The New York Times, TSR: The Southampton Review, Clarion, The Cape Rock, The North Atlantic Review, Round, and Writer's Digest magazine. She won first place in the 2009 Writer's Digest Poetry Contest for "The Undertaker's Wife." She holds an MFA in Creative Writing and Literature from Stony Brook University where she has taught creative writing. In 2014, Jacqueline walked six hundred miles across Spain on the Camino de Santiago.


81 Community score What's this?
Earn 63 seeds What's this?
Zesty with magnolia, grapes and grapefruit; very refreshing bitter-sweetness, definitely on the lighter end and a good tea for the summer.
Sunflowers, white grapes, and hay.
There's definitely the earthy note. I also get the magnolia in the aftertaste, and the sweetness and the olive oil throat sensation.
Very zesty, it was hard not to be focused on that. Maybe aging it would be great but on the other hand, it would be a pleasing and refreshing tea to drink in summer!
Sugar sweetness and creaminess as the base notes; mint, grapes skin and citrus sweetness as the high notes; all of that transferring into a pleasant and refreshing bitterness.
Silver artichoke green furry buds with crocodile green and dark cherry leaves.
Milk chocolate and salted caramel with dried cranberries.
Car showroom and cherry drops. Magnolia cream.
Bright grapefruit yellow and orange.
Moves from palm sugar and cream through magnolia sweetness to pink grapefruit zest and juniper berries.
Mineral dry and quenching with an olive oil catch in the throat leading to a botanical bitter-sweetness.
Dried lime and artichoke green buds and young leaves.
Chatty and energising like a great aperitif.
A really small batch tea sourced from the ancient forest conservation base of the Gushu Institute in Yunnan. I recommend grabbing this little fella while it is available as we have less than 200 cakes of this Yiwu available.
Yiwu is a revered area of Yunnan and specialises in producing top shelf, traditionally handmade raw PuErh. Anyone with the slightest interest in PuErh should know about this area as it carries with it a very high reputation (it is known as the Queen of PuErh with Lao Banzhang being called the King).
Yiwu sits on the Eastern most part of the southern tip of Yunnan very close to the Laos border. It comprises 6 large mountainous areas all with their own villages. The most easterly of these areas is called Yiwu (yes there is a 'Yiwu' in Yiwu) which is the origin of the 'Tipple Twister'.
The characteristics of Yiwu PuErh are teas with very low bitterness which are soft, thick and easy drinking and have a predominance of high, fruity aromas. Yiwu's are often described as prune tasting, especially when aged properly. But, because the area of Yiwu is very large and even tea from outside Yiwu is often faked to increase the price tag, finding an authentic ancient tea Yiwu can be challenging. We have tasted many Yiwu's over the years but for one reason or another we have never purchased any - the taste did not match the price or we could not verify the origin.
The 'Tipple Twister' is a tea that we bought the moment we tasted it because it was so unique - the botanicals and zest dances over the warmer, creamier notes and the aftertaste is sweet and citrusy. It will be really fascinating to find out how this tea changes over the years and we are definitely going to hoard a few for ageing.
NO TASTERS AVAILABLE: Due to the amount of waste when breaking into these 100g cakes (we do not give Beengholes as tasters) and the limited quantity available, we are not offering tasters of this tea.
Potent Gushu raw PuErh from Lao Man E village in Bulang. Exclusive batch, house roasted by Mei Leaf in London. Quince, blow torched rice and pears. Sweet Limoncello bitters.
Raw old tree PuErh from the He Kai mountain region with signature creamy notes of brown bread ice cream, sultana and lemon curd fruitiness and a hot laundry aroma.


Carrier Corp, a division of United Technologies Corp, announced that the European Commission (EC) in Brussels, Belgium, has approved the company's intended acquisition of Linde Refrigeration, a division of Linde AG.
Under the agreement, Carrier will acquire Linde for about $390 million (or approximately 325 million euros), including estimated debt upon closing. The transaction is expected to close withing three months.
"Refrigeration is a core business at Carrier and this acquisition will be a synergistic fit to our existing operation. The Carrier/Linde combination will allow us to provide a more comprehensive range of products and services," said Geraud Darnis, president of Carrier.
Linde employs 6,300 people and has annual sales of approximately $1 billion (866 million euros, including the German dry shelving business with sales of 28 million euros, which is not part of this transaction). Based in Cologne, Germany, it has manufacturing facilities in Europe, Asia, and South America. Products include industrial and commercial refrigeration systems, and refrigerated display cases and subsystems.


Living World® Critter Playtime is a portable playpen that can be set up indoors or outdoors to create a spacious play area for non-jumping small pets.
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The amount of exhibition space already booked for imm cologne 2018 has exceeded all of the fair organisers' expectations. The international interiors show will return to Cologne for 7 days from January 15 to 21, 2018.
This positive development is driven by a double-digit percentage increase in registrations from abroad. "Not only does northern Europe represent an attractive market environment for furnishings companies from all over the world – imm cologne is also a business platform where new interior design trends and products must be showcased if these businesses are to remain competitive", notes Arne Petersen, giving his assessment.
In addition to the large number of returning exhibitors from previous years, a marked increase in interest from exhibitors from eastern and south-eastern Europe has been observed, especially from Poland, Greece and Turkey. Furniture companies from southern Europe also value the market in Germany – a double-digit increase in exhibitors from Italy, Spain and Portugal has likewise been reported. And more and more exhibitors from the United States are happy to make the long journey to the Rhine. The number of registrations from within Germany is stable, according to Petersen.
In total, the Cologne-based organisers expect over 1,200 exhibitors, over 70% of whom will be travelling from every corner of the globe. Exhibitors at imm cologne can hope to attract new markets, because the audience is also increasingly international. In Cologne, buyers from Germany, Europe and overseas are presented with an unparalleled variety of international companies from all interiors product categories and price brackets.
For imm cologne's trade visitors, this development means an even greater choice and variety of products and suppliers from the international interiors sector. In Koelnmesse's 11 halls, they can not only find out about the latest interior design trends and global product innovations, but also effect purchases easily. There is no other trade fair platform in the world where business can be conducted more effectively or efficiently in the interior design sector.


text:
Prague 1/2 predictions? Rupp ftw? - Official 2017 Prague Half Live Discussion Thread
Pro News/Info/Results
Last post 8 minutes ago
1 2 3 4 5 6 ... 13 14
scorpion_runner
4 years ago 03/30/2017 7:43pm CDT
re: douglas burke
douglas burke wrote:
Bhabbi wrote:
Man, I don't get all the Rupp hate. I don't like how NOP does things either, but Rupp is by far the GOAT American runner EVER at 5,000 and up.
But sub-59, um, no.
Why Not? 26:44 on the Track is better than most who have gone sub 59, and Salazar's runners are often best at half marathon, examples Goucher and Hasay best event has been the half marathon (so far for Hasay) and Ritz's best race was the 12:56 for 5000 after that his 60:00 and 60:12 Half marathons are his best performances at any distance (except for the mentioned 12:56), and while Rupp has not ran a fast marathon yet, he got 3rd in the Olympics, I have a feeling Rupp will excel at Half Marathon.
You're forgetting Dathan's 2:07 marathon!...
Rupp got third in the olympics, but he ran 2:10...and faded badly in the end..Rupp is a solid runner, but he is highly overrated.
However, he might have that special Alberto juice behind him, so he just might put up something impressive...
use your brain
re: scorpion_runner
scorpion_runner wrote:
How fast did Dathan and Ryan run in the Olympics and what place did they finish? Moron
re: ThatAverageRunner
ThatAverageRunner wrote:
No he isn't. He is not better than lagat at 5'000 meters who holds the american outdoor record.
And he is not better than Ryan Hall at the marathon, because ryan holds the american record, and ryan holds the american half marathon record as well. Rupp will never in his life run 2:04!
I even put Dathan ahead of rupp in the marathon, because Dathan ran 2:07. Rupp has never broken 2:10. Also, Dathan has the second fastest american half marathon time
So how is rupp the greatest american runner at 5,000 meters and up??..
And I haven't even talked about Meb and Khalid for the marathon level.
Hall doesn't have the marathon record.
It's a pretty safe bet to say OVERALL that Rupp is the greatest american distance runner. Lagat has beaten him in quite a few 5ks, but not in many other distances. Rupp's time is only a little behind Lagat's, and Lagat spent half his career as a Kenyan during the EPO era. Not to knock Lagat specifically at all, but he doesn't beat out Rupp for overall best american distance runner. And Rupp has some time to get some good results at the marathon distance. He already has a global medal at it. I'm not sure I follow your argument. Who's a better overall american distance runner?
Ryan hall does have the american marathon record, which is what I stated....go re-read what I wrote.
Secondly, then you need to say that rupp is the greatest all around american runner. By saying he is the greatest AT 5,000 and up is basically saying that he is better than other american runners in those individual events, which is not the case.....What you should have said is that he has achieved on so many different levels of running, making him the best all around american runner.
re: use your brain
use your brain wrote:
Your post makes no sense. You said nathan's best run is his half marathon run...and I was clearly letting you know that you forgot his 2;07 marathon run, which is the third fastest american marathon time if I am not mistaken.
So I don't understand your response.
My statement on Rupp's olympic time has nothing to do with Nathan, and vice versa. I was just stating the fact the Rupp faded badly, and was hurting towards the end of that race, which wasn't a face course.
Nice to know that you're great at name calling, though
correction: Dathan
cwarcarblue11
Agreed he is the greatest all around American runner. He's definitely the best American 10k runner ever, and is already super close to being greatest American marathoner ever thanks to his bronze medal. The one thing he needs is a fast time, and I think he will definitely run 2:04-2:05 sometime in his career.
Sad to see how many people hear hate on the only American-born runner who has proven he can hang with the east Africans from 10k-marathon.
ThatAverageRunner
Ryan Hall's marathon record doesn't count you fool. Everyone knows that.
And saying he's the greatest at 5,000 and up basically implies that he's the greatest american distance runner. No one person wins every single race they ever run in every event. 5,000 and up was used because anything lower is considered middle distance to most. And even then, Rupp has some solid mile / 3k times. I think most people will agree when it's said that Rupp is the greatest american distance runner, like him or not.
re: cwarcarblue11
cwarcarblue11 wrote:
Please explain this to me: and is already super close to being greatest American marathoner ever thanks to his bronze medal.
Frank Shorter ran 2:10 in 1972! That's Rupp's personal best right now! FRank won gold in the olympics, and was ranked number 1 in the world for three straight years. And was ranked number 1 in america for 5 straight years!
And this is why Rupp is highly, highly overrated!
and when you say greatest american marathon runner, are you talking american born I guess? Because rupp is not seeing Khalid ever in his life at marathon running.
" Ryan Hall's marathon record doesn't count you fool."
What? How in the hell does it not count, and it is in the record books, as well as his half marathon.
Jeffery Mutai said that he broke the marathon record because ryan hall set the pace!
so now it does not count because you say so?...lol
Agree with some of this but no, he is not the only American runner with that capability. Ritz got bronze in the half marathon world champs, so he can indeed hang with the top guys.
Queens Queens
Rupp only ran two marathons, both in championship races under not very stellar weather conditions. No time trial yet.
Not dismissing Shorter's career, but the field was definitely much weaker than today.
Are you serious? You're proving that you have no idea what you're talking about. Times don't count at Boston. He also had an insane tailwind that day. His record is not ratified. I like Ryan Hall. He's a great runner and seems like a great guy. He does have the half marathon record and it's a decent record, but he does not have the marathon AR. Khalid Khannouchi has the american record at 2:05:38.
Slow Bro
Bruh, get outta here with that argument. You either know better but are pretending you don't, or you're not the sharpest tool in the shed.
In case you need it spelled out - Galen has only raced two tactical marathons in hot conditions. He only did what he needed to do in LA to win on a brutally hot day, and then in the Olympics he was obviously racing for place, not time. And that was a hot day as well - remember the ice hats?
Put him on a course like Chicago or Berlin, in a race with rabbits, and I have no doubt he'll run in the 2:05 range. He surely has 2:07 in him at the very least, so it's laughable to put Ritz above him as an OVERALL runner when Dathan only has the advantage in an event Rupp has only raced twice, and never time-trialed.
You don't say...I'm saying he RAN 2:04, and set a serious fast pace, and yes it is an asterisk....but he ran 2:04 and that is a record fast time for an american ..period.
a time Rupp will never see in his existence. period.
re: Slow Bro
Slow Bro wrote:
WHICH IS EXACTLY MY POINT ...so how could he be on his way to being the greatest marathon runner of all time?
Thank you for showcasing his inexperience.
Secondly, we shall see what he is made of very soon . I have seen so many posts on this board regarding rupp will break the american half record for an upcoming half marathon race, only to see that not happen.
So here we are again at another half marathon race.....we shall see...we shall see
Whats the day and time and how do we watch?
4 years ago 03/31/2017 1:52am CDT
Khalid Khannouchi has the american record at 2:05:38.
Hell yeah...real american!
Gotta bee
Using Africans? P.S Galen has two Olympic medals other guys 0
Speaking for 1
Will you think differently about Rupp if his coach is given a 4 year ban?
Be honest Speaking for 1
re: Speaking for 1
Speaking for 1 wrote:
Will you think differently if his coach is never banned?
Showing 61 to 80 of 273 posts


right for her to take medical marijuana at school.
and the Americans with Disabilities Act (ADA).
disorders and epilepsy, according to the suit.
to receive medical marijuana to treat her epilepsy, the suit stated.
prohibitions in state law, according to the suit.
from using medical marijuana at home.
allowing pot on school grounds.
to school, her attorney, Steve Glink, said.
of marijuana that can make users high.
tongue or wrists to regulate her epilepsy, the suit stated.
teacher's aide, according to the suit.
prosecution for possession or distribution of the drug.
A hearing has been set for the matter Friday in federal court.
let schools allow the drug for medical use, but no schools did so initially.
parents or caregivers to give qualified students medical marijuana.
cerebral palsy who died that year, after the law was passed.
to conflict with your right to an education," Springer said.
their kids high. It's to keep them alive and healthy."