Recombinant epithelial cell adhesion molecule extracellular domain (EpEX) has a high potential as a candidate for passive and active immunotherapy as well as cancer vaccination. In the present study, ...EpEX was expressed as a thioredoxin fusion protein in Escherichia coli (E. coli). The effect of different hosts and expression conditions on the expression level of the fusion protein was also evaluated. Moreover, the effect of temperature and isopropyl-ß-d-thiogalactopyranoside (IPTG) concentration on protein solubility was assessed. The codon optimized-synthetic gene was cloned into pET32a (+) expression vector and transformed into E. coli BL21 (DE3), RosettaTM (DE3), and OrigamiTM (DE3). The protein expression was confirmed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Lowering the expression temperature to 16 °C and IPTG concentration to 0.5 mM also dramatically increased the volumetric productivity of the fusion protein. In optimum culture condition, high-level expression of the target fusion protein was detected in RosettaTM (DE3) and OrigamiTM (DE3) (207 and 334 μg/mL, respectively), though they were expressed as inclusion bodies. No improvement was observed in the solubility of the fusion protein by reducing the temperature or IPTG concentration even when expressed in a TrxB/gor mutant strain. Results showed that Trx tag combined with other strategies utilized here could be effective to achieve high level of protein production but not effective in solubility improvement. However, new approaches might be necessary to enhance the solubility of EpEX in the E. coli system.
One month-ahead electricity prices open up a great potential for manufacturing companies that aim at reducing energy costs. On the machine level, an energy cost-oriented scheduling can be realized ...through determining the least electricity cost-intensive launch times for job processing during a day. To obtain reliable price information at production planning stage in advance, well-performing forecast models are required. In this paper, we apply a Weighted Nearest Neighbor (WNN) approach, a TBATS approach, and a Deep Feedforward Neural Network (DFNN) approach to forecast EPEX Spot day-ahead electricity prices of the European Energy Exchange in Germany/Austria, while accounting for a forecast horizon of up to one month. Model performances are compared by means of established error measures as well as by adjusted error measures to better interpret results from a production planner's point of view. For evaluation purposes, 1357 different forecasts were computed for each of the methods, allowing valid conclusions as to their performance. The results show that all approaches lead to acceptable accuracies. However, the DFNN outperforms WNN and TBATS, providing well-fitting forecasts even 29 days ahead.
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•Artificial Neural Networks outperform TBATS and Weighted Nearest Neighbor.•Forecast results are highly applicable for mid-term production planning purposes.•Neural Network forecasts for longer terms are almost as precise as for shorter ones.•On average, only 5% of the adjusted forecasts exceed the limit of a 10.00 EUR error.
•The anti-EGFR antibody Cetuximab has resistance issues in head and neck cancers.•High expression of EpCAM is a poor prognostic factor in head and neck cancers.•Soluble EpEX of EpCAM binds with EGFR ...to activate the ERK signaling pathway.•Soluble EpEX enhances cetuximab resistance in head and neck cancers.•EpCAM is a potential biomarker for clinical efficacy and resistance to cetuximab.
Cetuximab (Cmab) is a molecularly targeted monoclonal antibody drug for head and neck squamous cell carcinoma (HNSC), although cetuximab resistance is a serious challenge. Epithelial cell adhesion molecule (EpCAM) is an established marker for many epithelial tumors, while the soluble EpCAM extracellular domain (EpEX) functions as a ligand for epidermal growth factor receptor (EGFR). We investigated the expression of EpCAM in HNSC, its involvement in Cmab action, and the mechanism by which soluble EpEX activated EGFR and played key roles in Cmab resistance.
We first examined EPCAM expression in HNSCs and its clinical significance by searching gene expression array databases. We then examined the effects of soluble EpEX and Cmab on intracellular signaling and Cmab efficacy in HNSC cell lines (HSC-3 and SAS).
EPCAM expression was found to be enhanced in HNSC tumor tissues compared to normal tissues, and the enhancement was correlated with stage progression and prognosis. Soluble EpEX activated the EGFR-ERK signaling pathway and nuclear translocation of EpCAM intracellular domains (EpICDs) in HNSC cells. EpEX resisted the antitumor effect of Cmab in an EGFR expression-dependent manner.
Soluble EpEX activates EGFR to increase Cmab resistance in HNSC cells. The EpEX-activated Cmab resistance in HNSC is potentially mediated by the EGFR-ERK signaling pathway and the EpCAM cleavage-induced nuclear translocation of EpICD. High expression and cleavage of EpCAM are potential biomarkers for predicting the clinical efficacy and resistance to Cmab.
Background and purpose: The epithelial cell adhesion molecule (EpCAM), is one of the first cancer- associated markers discovered. Its overexpression in cancer stem cells, epithelial tumors, and ...circulating tumor cells makes this molecule interesting for targeted cancer therapy. So, in recent years scFv fragments have been developed for EpCAM targeting.
Experimental approach: In this study, an scFv against EpCAM extracellular domain (EpEX) derived from 4D5MOC-B humanized mAb was expressed in Escherichia coli k12 strain, and in order to obtain the optimum culture conditions in chemically defined minimal medium, response surface methodology (RSM) was employed. According to the RSM-CCD method, a total of 30 experiments were designed to investigate the effects of various parameters including isopropyl-b-D-thiogalactopyranoside (IPTG) concentration, cell density before induction, post-induction time, and post-induction temperature on anti EpEX-scFv expression level.
Findings/Results: At the optimum conditions (induction at cell density 0.8 with 0.8 mM IPTG for 24 h at 37 °C), the recombinant anti EpEX-scFv was produced at a titer of 197.33 μg/mL that was significantly consistent with the prediction of the model.
Conclusion and implication: The optimized-culture conditions obtained here for efficient production of anti EpEX-scFv in shake flask cultivation on a chemically defined minimal medium could be applied to large- scale fermentation for the anti EpEX-scFv production.
Epithelial cell adhesion molecule (EpCAM) is known to highly expression and promotes cancer progression in many cancer types, including colorectal cancer. While metastasis is one of the main causes ...of cancer treatment failure, the involvement of EpCAM signaling in metastatic processes is unclear. We propose the potential crosstalk of EpCAM signaling with the HGFR signaling in order to govern metastatic activity in colorectal cancer.
Immunoprecipitation (IP), enzyme-linked immunosorbent assay (ELISA), and fluorescence resonance energy transfer (FRET) was conducted to explore the extracellular domain of EpCAM (EpEX) and HGFR interaction. Western blotting was taken to determine the expression of proteins in colorectal cancer (CRC) cell lines. The functions of EpEX in CRC were investigated by proliferation, migration, and invasion analysis. The combined therapy was validated via a tail vein injection method for the metastasis and orthotopic colon cancer models.
This study demonstrates that the EpEX binds to HGFR and induces downstream signaling in colon cancer cells. Moreover, EpEX and HGF cooperatively mediate HGFR signaling. Furthermore, EpEX enhances the epithelial-to-mesenchymal transition and metastatic potential of colon cancer cells by activating ERK and FAK-AKT signaling pathways, and it further stabilizes active β-catenin and Snail proteins by decreasing GSK3β activity. Finally, we show that the combined treatment of an anti-EpCAM neutralizing antibody (EpAb2-6) and an HGFR inhibitor (crizotinib) significantly inhibits tumor progression and prolongs survival in metastatic and orthotopic animal models of colon cancer.
Our findings illuminate the molecular mechanisms underlying EpCAM signaling promotion of colon cancer metastasis, further suggesting that the combination of EpAb2-6 and crizotinib may be an effective strategy for treating cancer patients with high EpCAM expression.
Epithelial Cell Adhesion Molecule (EpCAM) has been discovered as one of the first tumor-specific antigens overexpressed in epithelial cancer. The present review focuses on the role of EpCAM in ...physiology and homeostasis of epithelia. Recent research pointed to a close interaction of EpCAM with other cell-cell contact molecules like E-cadherin and claudins and an intimate crosstalk with Wnt and TGF-beta signaling in the regulation of cell growth. Moreover, EpCAM has been shown to modulate trans-epithelial migration processes of white blood cells. Mutations of the EpCAM gene lead to disturbances of epithelial homeostasis and cellular differentiation from the stem cell compartment. In the intestinal tract EpCAM mutations contribute to congenital tufting enteropathy. Regarding tumorigenesis EpCAM can act as an oncogene still depending on additional driver mutations and epithelial phenotype of tumor cells. Tumor cells display increased EpCAM expression that often correlates with the loss of strict basolateral localization. Many tumors show enhanced regulated intramembrane proteolysis (RIP) of EpCAM and loose EpCAM expression under conditions of epithelial to mesenchymal transition. The resulting extracellular EpEX and intracellular EpICD fragments mediate proliferative signals to the cell. Resulting fragments can be validated either by sensitive enzyme-linked immune-sandwich assays (EpEX) or by immunohistochemistry (EpICD). The present review gives an overview on the detection of EpCAM fragments as predictive markers for disease progression and survival of cancer patients.
We analyze liquidity costs on continuous and auction-based intraday power markets using a cost-of-round-trip measure that works for both market designs. We use data from the Italian auction-based ...intraday market and the German continuous market and present descriptive statistics as well as multivariate regression models to analyze determinants of liquidity costs in both markets. To test for differences in liquidity due to market design, we employ a double machine learning technique controlling for several confounding variables. We show that weekly patterns, yearly seasonality, electricity demand, as well as the influence of temperatures significantly affect liquidity costs. Comparing liquidity costs in both market, we find that, overall, liquidity costs are lower on the Italian market. However, Italian costs increase towards later auctions, while the costs on the German continuous intraday market decrease and reach their low close to physical delivery, where costs are lower than on the last Italian market trading the corresponding products.
•We compare liquidity cost in auction and continuous intraday electricity markets.•We use volume-based cost of roundtrip measure to quantify liquidity cost.•We use Italian and German markets as model for continuous and auction markets.•Costs are on average smaller for the auction market than the continuous market.•Continuous trading has smaller costs close to delivery and for large volumes.
The burden of excess energy from the high renewable energy sources (RES) share creates a significant reduction of residual load for the future, resulting in reduced market prices. The higher the ...share of stochastic RES, the more often the price will be 0 €/MWh. The power market needs new methods to solve these problems. The development of virtual power plants (VPPs) is aimed at solving techno-economic problems with an increasing share of RES in the power market. This study analyses a possible implementation of stochastic and deterministic RES in a VPP to generate secured power, which can be implemented in the European Power Exchange (EPEX)/European Energy Exchange (EEX) power market using existing market products. In this study, the optimal economic VPP configuration for an RES-based power plant is investigated and implemented into standard power market products. The results show that the optimal economic VPP configuration for different market products varies, depending on the energy availability and the marginal costs of the VPP components. The size of the VPP components is positively correlated to the components’ share of the energy generated. It was also found that projecting or implementing VPPs in Germany at current market prices (EPEX/EEX prices) is not yet economically feasible for a small share of market products. However, the secured power can be marketed on the SPOT and in the futures market with higher and more stable prices compared with the status quo.
The dominating view in the literature is that renewable electricity production increases the price variance on spot markets for electricity. In this paper, we critically review this hypothesis. Using ...a static market model, we identify the variance of the infeed from intermittent electricity sources (IES) and the shape of the industry supply curve as two pivotal factors influencing the electricity price variance. The model predicts that the overall effect of IES infeed depends on the produced amount: while small to moderate quantities of IES tend to decrease the price variance, large quantities have the opposite effect. In the second part of the paper, we test these predictions using data from Germany, where investments in IES have been massive in the recent years. The results of this econometric analysis largely conform to the predictions from the theoretical model. Our findings suggest that subsidy schemes for IES capacities should be complemented by policy measures supporting variance absorbing technologies such as smart-grids, energy storage, or grid interconnections to ensure the build-up of sufficient capacities in time.
Aim: This study was conducted to evaluate the effect of preflaring and file size on the accuracy of the Root ZX and E-PEX Pro electronic apex locators (EALs).
Materials and Methods: The actual ...working length was set 1 mm short of the apical foramen in the forty extracted mandibular premolars. The teeth were embedded in an alginate mold, and two examiners performed the electronic measurements using #10, #15, and #20 K-files. The files were inserted into the root canals until the "0.0" or "APEX" signals were observed on the light-emitting diode or display screens for the E-PEX Pro and Root ZX, respectively, retracting to the 1.0 mark. The measurements were repeated after the preflaring using the S1 and SX Pro-Taper instruments. The mean differences between the actual length and electronic length values were analyzed by the Wilcoxon signed-ranked test with a significance level of P < 0.05. The factors evaluated were "Accuracy of EALs," "size of file," and "presence of preflaring.
Results: No significant differences were observed in the accuracy of both EALs when compared with the actual lengths. On the size of the file, significant difference noticed with #20 K-file. The preflaring procedure significantly (P < 0.05) increased the accuracy of the measurements for the Root ZX and Epex Pro.
Conclusions: The tested EALs showed acceptable accuracy, whereas the preflaring procedure revealed a more significant effect than the used file size.
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