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The theranostic sodium iodide symporter (NIS) gene allows detailed molecular imaging of transgene expression and application of therapeutic radionuclides. As a crucial step towards clinical application, we investigated tumor specificity and transfection efficiency of epidermal growth factor receptor (EGFR)-targeted polyplexes as systemic NIS gene delivery vehicles in an advanced genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDAC) that closely reflects human disease. PDAC was induced in mice by pancreas-specific activation of constitutively active KrasG12D and deletion of Trp53. We used tumor-targeted polyplexes (LPEI-PEG-GE11/NIS) based on linear polyethylenimine, shielded by polyethylene glycol and coupled with the EGFR-specific peptide ligand GE11, to target a NIS-expressing plasmid to high EGFR-expressing PDAC. In vitro iodide uptake studies in cell explants from murine EGFR-positive and EGFR-ablated PDAC lesions demonstrated high transfection efficiency and EGFR-specificity of LPEI-PEG-GE11/NIS. In vivo 123I gamma camera imaging and three-dimensional high-resolution 124I PET showed significant tumor-specific accumulation of radioiodide after systemic LPEI-PEG-GE11/NIS injection. Administration of 131I in LPEI-PEG-GE11/NIS-treated mice resulted in significantly reduced tumor growth compared to controls as determined by magnetic resonance imaging, though survival was not significantly prolonged. This study opens the exciting prospect of NIS-mediated radionuclide imaging and therapy of PDAC after systemic non-viral NIS gene delivery.
Pancreatic ductal adenocarcinoma (PDAC) is currently the fourth leading cause of cancer-related mortality in developed countries despite its comparably low incidence of less than 3 %, clearly demonstrating the lack of effective therapeutic strategies. The five-year survival rate is around 7 % for all stages of the disease and drops to below 2 % and a median survival of less than a year for patients with metastatic disease, mostly due to late diagnosis at the stage of inoperability and the unusually high resistance of PDAC to conventional radiation and chemotherapy [1, 2]. Despite intensive scientific and industrial efforts, so far no significant extension of survival could be achieved by any of the numerous therapy approaches tested .
The genetic and morphological changes in the carcinogenesis of PDAC are well known and include the initiation and progression of premalignant lesions to invasive and metastatic pancreatic cancer [3–6]. The genetic hallmark of PDAC development is an activating mutation in the KRAS oncogene, followed by other genetic changes, commonly including inactivation of the tumor suppressors TP53, CDKN2A (P16INK4A) and SMAD4, and activation of several growth factor receptors, such as the epidermal growth factor receptor (EGFR) [3, 7].
Against this background, several complex genetically modified mouse models of PDAC that mirror the typical changes found in human patients, have been generated in recent years [3, 4, 8–10]. One such model is the Ptf1a+/Cre;Kras+/LSL-G12D;Trp53loxP/loxP (Kras;p53) mouse. Here, PDAC is induced by pancreas-specific activation of constitutively active KrasG12D in combination with conditional deletion of Trp53 . To restrict these genetic modifications to the pancreas, mice with the mutated alleles are interbred with animals that express the Cre recombinase driven by the pancreas-specific promoter for Ptf1a, a subunit of pancreas transcription factor 1 (Ptf1) that is required to commit cells to a pancreatic fate during embryonic development [4, 11]. Thus, the activation of the oncogenic KrasG12D via excision of a transcriptionally inhibitory LSL (loxP-STOP-loxP) construct and deletion of the floxed tumor suppressor Trp53 occur in the pancreas only, leading to ductal lesions with complete penetrance [4, 12]. The development of endogenous mouse models away from the usual transplant models represents a significant step in the evolution of preclinical models . The morphological and molecular composition of endogenous tumors far better reflects human disease, making them highly suitable to predict the clinical effectiveness of a specific treatment strategy.
The sodium iodide symporter (NIS; SLC5A5) mediates the uptake of iodide into thyroid follicular cells allowing both diagnostic and therapeutic application of radioiodide in thyroid cancer patients [14, 15]. In our previous work, we have extensively investigated the dual reporter/therapy capacity of NIS in various non-thyroidal tumors and have proven the feasibility of extrathyroidal radioiodide therapy after tumor-selective NIS gene transfer [16–26]. Transfection of cancer cells with the NIS gene allows non-invasive monitoring of functional NIS expression and in vivo biodistribution before the application of a therapeutic dose of radioiodide. One of the major hurdles of efficient and safe application of the NIS gene therapy concept in the clinical setting is optimal tumor-specific targeting in the presence of low toxicity and high transfection efficiency of gene delivery vectors, with the ultimate goal of systemic vector application.
In a previous study, we used synthetic polyplexes based on pseudodendritic oligoamines with high intrinsic tumor affinity for NIS gene therapy in a syngeneic neuroblastoma mouse model as well as a subcutaneous human hepatocellular carcinoma mouse model [16, 18]. After systemic NIS gene transfer, the tumor-selective accumulation of radioiodide was sufficient for a significant therapeutic effect. In addition to an intrinsic tumor affinity due to the so-called enhanced permeability and retention (EPR) effect based on "leaky" tumor vasculature, the tumor targeting of polyplexes can be further optimized by the attachment of tumor-specific ligands. To this end, in a subsequent study, we used LPEI-PEG-GE11 polymers composed of linear polyethylenimine (LPEI), shielded by polyethylene glycol (PEG) and coupled to the synthetic peptide GE11 as an EGFR-specific ligand for NIS gene delivery . After systemic application of these polymers condensed with NIS DNA, tumor-specific radioiodide accumulation demonstrated effective and EGFR-specific tumor targeting in a high EGFR-expressing xenograft mouse model of hepatocellular carcinoma. After the injection of a therapeutic dose of 131I, tumoral iodide uptake was sufficiently high for a significant delay of tumor growth and prolongation of animal survival .
Based on our previous work and the well-known characteristic upregulation of EGFR in PDAC, we investigated the potential of EGFR-targeted polyplexes for systemic NIS gene therapy in an advanced endogenous mouse model of PDAC as a next step towards clinical application.
In order to optimize transfection conditions for LPEI-PEG-GE11 polymers condensed with the NIS plasmid (LPEI-PEG-GE11/NIS) in high EGFR-expressing PDAC cell explants derived from Kras;p53 mice (Figure 1A), radioiodide uptake activity was evaluated 24 h after polyplex application (data not shown). A conjugate to plasmid (c/p) ratio of 0.8 resulted in highest transfection efficiency at lowest cytotoxicity. Therefore, this c/p ratio was used in all subsequent experiments. Twenty-four hours after transfection with LPEI-PEG-GE11/NIS, cell explants from three different mice showed a 22-26-fold increase in 125I accumulation as compared to cells incubated with the empty control vector LPEI-PEG-GE11 (Figure 1B). Transfection with untargeted LPEI-PEG-Cys/NIS (targeting ligand GE11 replaced by a cysteine residue) resulted in significantly lower iodide uptake activity compared to EGFR-targeted LPEI-PEG-GE11/NIS (Figure 1B). In both cases, iodide uptake was blocked upon additional treatment with the NIS-specific inhibitor perchlorate.
Figure 1: Iodide uptake in PDAC cell explants in vitro. Kras;p53 mice develop PDAC that occupies a large portion of the abdominal cavity below the stomach (A). (B) PDAC cell explants from three separate mice (three technical replicates per mouse) transfected in vitro with LPEI-PEG-GE11/NIS showed a significant increase in perchlorate- (ClO4--) sensitive 125I accumulation compared to transfection with LPEI-PEG-Cys/NIS (mean ± S.E.M.; *p<0.05; **p<0.01; ***p<0.001). No iodide uptake above background levels was observed in cells transfected with LPEI-PEG-GE11 alone. (C) Transfection of EGFR-ablated PDAC cell explants from two mice (three technical replicates per mouse) with LPEI-PEG-GE11/NIS and LPEI-PEG-Cys/NIS showed no significant differences between transfection with targeted or untargeted polyplexes, demonstrating EGFR-specificity of the targeting ligand GE11 (mean ± S.E.M.).
To further verify EGFR-specificity of the targeting ligand GE11, we performed additional iodide uptake studies in EGFR-ablated PDAC cell explants from Ptf1a+/Cre;Kras+/LSL-G12D;Trp53loxP/loxP;Egfrfl/fl (Kras;p53;Egfr) mice. No significant difference between transfection with targeted LPEI-PEG-GE11/NIS or untargeted LPEI-PEG-Cys/NIS polyplexes was observed (Figure 1C). Polyplex-mediated NIS gene transfer did not affect cell viability for any of the treatment conditions compared to untreated cells as measured by MTS assay (data not shown).
Functional NIS expression in mice with high EGFR-expressing PDAC was imaged by whole body 123I gamma camera and 124I PET imaging. Polyplexes were administered intravenously (i.v.) either 24 or 48 h before injection of the respective radionuclide for imaging.
In vivo 123I gamma camera imaging revealed high levels of NIS-mediated radionuclide accumulation in pancreatic tumors both at 24 and 48 h after systemic injection of EGFR-targeted LPEI-PEG-GE11/NIS (Figures 2A, 2B). Tumors accumulated 10.8 ± 0.7 % of the injected dose per gram (ID/g) with an average biological half-life of 4 h at 24 h and 14.2 ± 1.4 % ID/g with an average biological half-life of 4.5 h at 48 h (Figure 2E). For 131I, a tumor-absorbed dose of 74.7 mGy/MBq/g tumor with an effective half-life of 3.2 h (24 h after polyplex administration) and 96.5 mGy/MBq/g tumor, effective half-life 4.5 h (48 h after polyplex administration), was calculated. In contrast, injection of non-coding control polyplexes LPEI-PEG-GE11/antisenseNIS (NIS sequence back to front) resulted in no significant tumoral radioiodide accumulation (Figure 2C). In addition to 123I uptake in the tumor, radioiodide accumulation was also observed in the stomach, the thyroid and the salivary glands, as they physiologically express NIS, as well as in the urinary bladder due to renal radionuclide elimination (Figures 2A-2C). To further confirm that tumoral iodide uptake was indeed NIS-mediated, LPEI-PEG-GE11/NIS-injected mice were additionally treated with the competitive NIS-inhibitor perchlorate 30 min before 123I administration, which completely blocked polyplex-mediated tumoral iodide accumulation in addition to physiological uptake in the stomach, the thyroid gland and the salivary glands (Figure 2D).
Figure 2: In vivo imaging of NIS-mediated iodide uptake. 123I scintigraphy revealed pancreatic tumoral radioiodide uptake 24 h (A) and 48 h (B) after injection of mice with LPEI-PEG-GE11/NIS that was not seen after injection with non-coding LPEI-PEG-GE11/antisenseNIS (C) Iodide uptake was perchlorate-sensitive (D) and therefore indeed NIS-mediated. (E) Radionuclide retention time in tumors was determined by serial scanning over 10 h (mean ± S.E.M.; 24 h: n=9, 48 h: n=7). 124I PET-imaging confirmed findings of scintigraphy and allowed better differentiation between tumoral and stomach radioiodide uptake (F, H) After injection of the control vector LPEI-PEG-GE11/antisenseNIS (G, I), no pancreatic iodide uptake activity above background levels could be detected. Significantly higher radioiodide accumulation 48 h after gene transfer as compared to 24 h was confirmed by PET (mean ± S.E.M.; n=5 each; *p<0.05) (J) SG: salivary glands.
To better distinguish tumoral uptake from iodide accumulation in the stomach, we additionally employed three-dimensional high-resolution 124I PET to image radioiodide biodistribution. Again, systemic injection of LPEI-PEG-GE11/NIS resulted in strong transfection of tumor tissue at both time points (Figures 2F, 2H), an effect that was not seen in mice treated with LPEI-PEG-GE11/antisenseNIS (Figures 2G, 2I). Quantification of tumoral 124I uptake again revealed significantly higher radioiodide accumulation 48 h after i.v. injection of LPEI-PEG-GE11/NIS as compared to 24 h after NIS gene transfer (Figure 2J).
48 h after polyplex administration, mice were sacrificed and dissected. Tumors and non-target organs (liver, lung) were analyzed for NIS mRNA expression by quantitative real-time PCR (qPCR). A 20-fold increase in NIS mRNA expression was detected in PDAC lesions from mice injected with LPEI-PEG-GE11/NIS as compared to untreated tumors (Figure 3A). In contrast, no significant NIS mRNA expression above background levels was observed in non-target organs and tumors of mice treated with the control vector LPEI-PEG-GE11/antisenseNIS (Figure 3A). In tumors from LPEI-PEG-GE11/NIS-treated mice, areas of NIS-specific immunoreactivity were observed surrounding ductal lesions by immunohistochemical and immunofluorescence staining using a human NIS-specific antibody (Figure 3B). NIS staining was found to be both cell membrane-associated and cytoplasmic. In contrast, tumors from mice treated with the control vector LPEI-PEG-GE11/antisenseNIS showed no NIS-specific immunoreactivity (Figure 3B).
Figure 3: Analysis of NIS mRNA and protein distribution ex vivo. NIS-specific qPCR analysis revealed a 20-fold increase of NIS mRNA expression in pancreatic tumors of mice injected with LPEI-PEG-GE11/NIS as compared to tumors of untreated mice. In contrast, NIS mRNA was not increased in non-target organs and in tumors of mice injected with the control vector LPEI-PEG-GE11/antisenseNIS (mean-fold change ± S.E.M.; ***p<0.001) (A) Both immunohistochemical (B, upper three panels; magnification: 10×, 20× and 40×) and immunofluorescence staining (B, bottom panel; magnification: 200×) of sections of pancreatic tumors revealed areas of NIS-specific immunoreactivity after systemic application of LPEI-PEG-GE11/NIS. In contrast, tumors treated with the control vector LPEI-PEG-GE11/antisenseNIS showed no NIS-specific immunoreactivity.
PDAC-bearing mice were treated with three cycles of LPEI-PEG-GE11/NIS followed by 131I 48 h later – the optimal time point for radionuclide injection based on the imaging studies. Controls were injected with non-coding LPEI-PEG-GE11/antisenseNIS and 131I or saline only. Tumor progression was monitored by magnetic resonance imaging (MRI). Mice in the therapy group showed a significant stabilization of tumor growth and, in two cases, even a reduction in tumor volume (Figures 4A, 4D), while aggressive tumor growth was observed in both control groups (Figures 4B-4D). This led to an enhanced survival in the therapy group that lived up to 28 days post therapy start with a median survival of 25 days, as compared to the antisenseNIS group that survived up to 13 days with a median survival of 11 days and saline controls that lived up to 21 days, median survival 21 days (Figure 4E). The effect on mouse survival was, however, not significant.
Figure 4: Therapeutic application of 131I after NIS gene transfer in vivo. Kras;p53 mice were treated with three cycles of i.v. injection of polyplexes on days 0/4/7 followed by i.p. injection of 55.5 MBq 131I 48 h later, on days 2/6/9. Tumor sizes were monitored weekly by MRI. Exemplary MRI images of endpoint tumor sizes from an LPEI-PEG-GE11/NIS + 131I- (A), an LPEI-PEG-GE11/ antisenseNIS + 131I- (B) and a NaCl + NaCl-treated Kras;p53 mouse are shown (C). Tumors are highlighted by red dotted lines. (D) Mice treated with LPEI-PEG-GE11/NIS + 131I (n=6) showed a stabilization in tumor volume compared to control groups LPEI-PEG-GE11/antisenseNIS + 131I (n=3; mean ± S.E.M.; *p<0.05) and NaCl + NaCl (n=4; **p<0.01). Mean tumor volumes (solid lines) and volumes for individual mice (dotted lines) are shown. (E) Injection of LPEI-PEG-GE11/NIS + 131I led to an increased overall and median survival in the therapy group (n=6) compared to control groups injected with LPEI-PEG-GE11/antisenseNIS + 131I (n=3; n/s) or NaCl + NaCl (n=4; n/s).
While the incidence of PDAC is gradually increasing, the prognosis of patients with pancreatic cancer has not significantly changed over the last 20 years – despite numerous advances in diagnostic imaging, surgical techniques and chemotherapeutic strategies [3, 27]. Intensified chemotherapy protocols in patients with advanced pancreatic cancer show a significant, yet still unsatisfactory survival benefit . So far, no targeted agent or approach has changed this fatal course of the disease, even though preclinical trials in in vitro cell culture systems and in vivo xenograft models had shown promising results [3, 28]. These set-backs can mainly be attributed to the complexity of the disease. The homogeneous molecular equipment, simple stromal architecture and immune deficiency of xenograft models limits their transferability to the clinical setting. Endogenously grown tumors, in contrast, are genetically and morphologically heterogeneous, less vascularized and harbor a far more complex microenvironment with high immunosuppression and extensive desmoplasia [29, 30]. Genetically engineered mouse models that closely reflect the key aspects of pancreatic carcinogenesis have been shown to correlate well with data from clinical trials and provide an exciting new platform to predict human tumor responses to treatment .
After the proof-of-principle of our polyplex-mediated NIS gene therapy concept in different subcutaneous xenograft tumor models [16–18], the genetically engineered mouse model of PDAC used in this study provides an important step towards further development towards clinical application. Based on the known activity of EGFR in PDAC and this model, we chose EGFR-targeted LPEI-PEG-GE11 polymers as delivery vehicles for the NIS gene [17, 31, 32].
Transfection of PDAC explant cell cultures with LPEI-PEG-GE11/NIS led to significant perchlorate-sensitive and therefore NIS-mediated radioiodide accumulation. The empty vector LPEI-PEG-GE11 did not result in iodide accumulation above background levels, further confirming NIS-dependency of radioiodide accumulation. Iodide uptake was significantly reduced after transfection with non-targeted LPEI-PEG-Cys/NIS, demonstrating improved transfection efficiency using the targeting ligand GE11. EGFR-specificity of targeting was further substantiated by the observation that in EGFR-negative cultures derived from Kras;p53;Egfr mice, no significant difference between transfection with EGFR-targeted or non-targeted vectors was observed. Translating these promising in vitro results to systemic vector application in vivo, intravenous administration of LPEI-PEG-GE11/NIS resulted in a significant perchlorate-sensitive tumor-specific iodide uptake in mice harboring endogenous PDAC tumors, as demonstrated by 123I gamma camera imaging. Three-dimensional 124I PET imaging with increased sensitivity and resolution was employed for more accurate quantification of tumoral radioiodide uptake, as radionuclide signals from pancreatic lesions partially overlap with stomach signals based on physiological gastric NIS expression. Results from PET imaging confirmed gamma camera imaging results with strong radioiodide signals in pancreatic tumors. Control experiments with LPEI-PEG-GE11/antisenseNIS showed no significant tumoral radioiodide accumulation above background levels, confirming NIS-specificity of tumoral tracer uptake. These molecular imaging data were further corroborated by NIS-specific immunohistochemistry and immunofluorescence as well as qPCR analysis.
Both the abundance and the permeability of the tumor's vasculature are crucial for sufficient transgene delivery into the tumor [33, 34]. One of the main factors thought to hamper efficient drug delivery to PDAC, is its highly desmoplastic stroma alongside its high interstitial pressure and poor vascularization . Thus, the enhanced permeability and retention effect that is caused by the irregular, "leaky" tumor vasculature and is usually exploited for passive targeting of therapeutic agents to tumor sites, is limited in PDAC [34, 36]. For this reason, an additional tumor-targeting strategy is particularly important. Our imaging data convincingly demonstrate that targeting our polyplexes to EGFR allows strong transfection of pancreatic tumors with NIS. In a previous study, using the same vector construct in a subcutaneous hepatocellular carcinoma xenograft model, a tumor-absorbed dose of 47 mGy/MBq/g was calculated for 131I 24 h after polyplex administration , while in the current study, a dose of 74.7 mGy/MBq/g tumor 24 h post polyplex injection was achieved. We mainly attribute this significantly enhanced tumoral radioiodide uptake to the very high EGFR expression in PDAC. NIS staining was restricted to areas of high EGFR expression surrounding ductal lesions . This focal pattern of transgene expression further underlines the advantage of NIS as therapy gene in this setting, as the high radionuclide bystander effect allows destruction of tumor cells beyond transfected cells.
Building on these promising results, the next logical step was to evaluate the therapeutic effectiveness of 131I in the PDAC mouse model after LPEI-PEG-GE11-mediated systemic NIS gene delivery. We were able to demonstrate stabilization, and, in two cases, even a pronounced reduction, of tumor growth after application of three cycles of LPEI-PEG-GE11/NIS followed by 131I. Mouse survival was prolonged in the therapy group, especially compared to the non-coding LPEI-PEG-GE11/antisenseNIS-treated control group, although without reaching statistical significance, despite the strong effects on tumor growth. Interestingly, while animals in the saline group had to be sacrificed due to compromised well-being owing to excessive tumor growth, the non-coding control group showed signs of ill health at much lower tumor volumes and had to be sacrificed. Similarly, effects on animal health were observed in the therapy group, though to a lower extent. We attribute this observation to toxicity of the LPEI-based conjugates, possibly combined with effects from 131I. Due to the stabilization of tumor growth in therapy animals, they fared better than the non-coding control group that was potentially affected by side effects from polyplex and radioiodide injection in addition to rapid tumor growth. To date, the use of LPEI-based polymers did not affect animal health in any of our previous studies, nor was viability of PDAC cell explants affected in the current study. LPEI has been shown to exhibit certain cytotoxic effects both in vitro and in vivo [37–41], though LPEI-based polyplexes have already been tested in a clinical trial for bladder cancer therapy and no adverse effects were reported . Similarly, we have so far only encountered side effects from 131I in one previous study with the objective to radioablate mouse thyroids under intentional stimulation of thyroidal radioiodide uptake . Symptoms developed with a delay of seven days after radioiodide application, while in the current study, animal health deteriorated from the beginning of treatment . However, our earlier work was done in subcutaneous xenograft models, where tumor growth per se has no impact on animal health. In contrast, Kras;p53 mice with their extremely aggressive pancreatic tumor growth and subsequent rapid health deterioration, seem to react more unfavorably to the polyplexes and/or radioiodide treatment. LPEI is seen as the "gold standard" for non-viral DNA delivery, as it shows such high transfection efficiency and flexibility at relatively low toxicity, compared to other viral and non-viral gene delivery approaches. To further refine our approach and solve the toxicity issue, we are currently developing sequence-defined polymers with higher biocompatibility for targeted NIS gene delivery .
In conclusion, our data clearly show the high potential of EGFR-targeted nanoparticle vectors to target the NIS gene to PDAC. After systemic application of LPEI-PEG-GE11/NIS, we were able to reach sufficient iodide concentrations at the tumor site to (1) produce a strong enough signal to image pancreatic tumors in situ and (2) provoke a therapeutic effect. Based on its role as potent and well characterized reporter gene, NIS allows non-invasive imaging and detailed characterization of in vivo biodistribution of functional NIS expression as an essential prerequisite for exact planning and monitoring of clinical gene therapy trials with the aim of individualization of the NIS gene therapy concept in the clinical setting.
Establishment of the Kras;p53 (Ptf1a+/Cre;Kras+/LSL-G12D;Trp53loxP/loxP) and Kras;p53;Egfr (Ptf1a+/Cre;Kras+/LSL-G12D;Trp53loxP/loxP;Egfrfl/fl) strains has been described previously [4, 11, 12, 44, 45]. Mouse strains were maintained on a mixed C57BL/6;129/Sv background. Animals were kept under specific pathogen-free conditions with access to mouse chow and water ad libitum. Both male and female mice at 5-7.5 weeks of age were used for experiments. The experimental protocol was approved by the regional governmental commission for animals (Regierung von Oberbayern, Munich, Germany).
Cell explants from primary PDAC of Kras;p53 and Kras;p53;Egfr mice were isolated as described previously  and cultured in DMEM high glucose medium (Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal bovine serum (v/v; PAA, Colbe, Germany), 100 U/mL penicillin/100 μg/mL streptomycin (Invitrogen) and 1% non-essential amino acids (v/v; Invitrogen). Cells were maintained at 37°C and 5% CO2 in an incubator with 95% humidity. Cell culture medium was replaced every second day and explants were passaged at 85% confluency.
The human NIS-encoding plasmid and LPEI-based conjugates were cloned and synthesized, respectively, as described previously . Plasmid DNA was condensed with polymers at indicated c/p ratios (w/w) in HEPES-buffered glucose (HBG: 20 mmol/L HEPES, 5% glucose (w/v), pH 7.4) as described previously  and incubated at room temperature for 20 min before use. Final DNA concentrations were 2 μg/mL for in vitro studies and 200 μg/mL for in vivo studies.
For in vitro transfection experiments, PDAC cell explants were grown to 60-80% confluency. Explants were incubated for 4 hours with polyplexes in the absence of serum and antibiotics followed by incubation with complete growth medium for 24 h. Either LPEI-PEG-GE11/NIS (EGFR-targeting of NIS due to the EGFR-specific ligand GE11), LPEI-PEG-Cys/NIS (no active targeting of NIS to EGFR, as the ligand GE11 is replaced by a cysteine residue), or LPEI-PEG-GE11 alone (polymer without NIS DNA) were added in c/p ratios as indicated. Transfection efficiency was evaluated by measurement of iodide uptake activity as described below. Transfections were done in triplicate for each separate explant.
Following transfections, iodide uptake of PDAC cell explants was determined at steady-state conditions as described previously [48, 49]. Results were normalized to cell viability that was measured using the commercially available MTS-assay (Promega, Mannheim, Germany) as described previously .
For the proof-of-principle of NIS-mediated tumor-specific radioiodide accumulation in vivo, polyplexes (LPEI-PEG-GE11/NIS, c/p 0.8) were applied via the tail vein at a DNA dose of 2.5 mg/kg (50 μg DNA in 250 μL HBG). Mice received 18.5 MBq 123I (GE Healthcare, Braunschweig, Germany) intraperitoneally (i.p.) 24 h (n=9) or 48 h (n=7) after polyplex injection and radioiodide distribution was monitored by serial imaging on a gamma camera (Forte, ADAC Laboratories, Milpitas, CA, USA) equipped with a VXHR (ultra-high resolution) collimator as described previously . Regions of interest were quantified and expressed as a fraction of the total amount of applied radionuclide per gram tumor tissue. The retention time within the tumor was determined by serial scanning after radioiodide injection. A subset of mice (n=2 for each time point) was pretreated i.p. with 2 mg of the competitive NIS inhibitor sodium perchlorate (NaClO4; Sigma-Aldrich, Taufkirchen, Germany) 30 min before 123I administration. Dosimetric calculations for 131I were made using the Medical Internal Radiation Dose (MIRD) technique and a RADAR dose factor (http://www.doseinfo-radar.com). In order to achieve better discrimination between uptake in the tumor and the adjacent stomach, 24 or 48 h after i.v. injection of polyplexes (LPEI-PEG-GE11/NIS, each time point n=5; LPEI-PEG-GE11/antisenseNIS, each time point n=1) mice received 10 MBq 124I (Perkin Elmer, Waltham, MA, USA) i.p. and radioiodide biodistribution was monitored by static acquisition 3 h post injection using a micro PET system (Inveon, Siemens Preclinical Solutions, Erlangen, Germany). Mean tumoral radioiodide uptake was calculated in MBq/mL by manually placing 3D regions of interest in the tumor.
Total RNA was isolated from PDAC or non-target tissues (liver, lung) using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's recommendations. Single-stranded oligo (dT)-primer cDNA was generated using Super Script III Reverse Transcriptase (Invitrogen). qPCR was performed with the cDNA from 1 μg RNA using SYBR Green PCR master mix (Qiagen) in a Rotor Gene 6000 (Corbett Research, Morthlake, New South Wales, Australia). The following primers were used: NIS, forward 5′-ACACCTTCTGGACCTTCGTG-3′, reverse 5′-GTCGCAGTCGGTGTAGAACA-3′ and GAPDH, forward 5′-GAGAAGGCTGGGGCTCATTT-3′, reverse 5′-CAGTGGGGACACGGAAGG-3′. Relative expression levels were calculated using the comparative ΔΔCt method and internal GAPDH for normalization.
Immunohistochemical and immunofluorescence staining of NIS was performed using a mouse monoclonal antibody directed against human NIS (kindly provided by John C Morris, Mayo Clinic, Rochester, MN, USA) as described previously [20, 51].
Starting when mice were around 30 d of age, tumor sizes were assessed weekly by high resolution MRI on a 3T clinical scanner (Philips Ingenia 3.0T; Royal Philips Electronics, Eindhoven, The Netherlands). Once tumors reached the inclusion size of 200-450 mm3, therapy trials were started. To this end, 48 h after systemic administration of LPEI-PEG-GE11/NIS or, as control, LPEI-PEG-GE11/antisenseNIS, a therapeutic dose of 55.5 MBq 131I (GE Healthcare) was administered i.p. (LPEI-PEG-GE11/NIS + 131I n=6; LPEI-PEG-GE11/antisenseNIS + 131I n=3). A second control group received saline only (n=4). The cycle consisting of systemic NIS gene transfer followed by radioiodide was repeated for a total of three times on days 0/2, 4/6 and 7/9. Mice from all groups were sacrificed when at least one endpoint criterion was reached. Endpoint criteria included a tumor volume >1000 mm3, a body weight loss >15%, as well as a number of general physical, clinical and behavioral criteria. Body condition was monitored by independent animal care personnel blind to treatment and hypothesis.
Results are reported as mean ± S.E.M., mean-fold change ± S.E.M. or, for survival plots, percent. Statistical significance was generally tested by two-tailed Student's t-test except for the therapy study. For tumor volumes, one-way ANOVA was performed, followed by Tukey's Honestly Significant Difference test. Statistical significance of Kaplan-Meier plots was analyzed by log-rank test. p-values <0.05 were considered statistically significant (*p<0.05; **p<0.01; ***p<0.001; n/s not significant).
We are grateful to Sissy M Jhiang (Ohio State University, Columbus, OH, USA) for supplying the full-length human NIS complementary DNA and to John C Morris (Mayo Clinic, Rochester, MN, USA) for providing the NIS mouse monoclonal antibody. We also thank Nathalie Schwenk (LMU Munich, Munich, Germany) for her help with in vitro and ex vivo studies, Sybille Reder, Stefan Ambros, Klemens Scheidhauer and Jakob Allmann (Klinikum rechts der Isar der Technischen Universität München, Munich, Germany) for assistance with PET and MRI studies, Wolfgang Rödl (LMU Munich, Munich, Germany) for conjugate synthesis as well as Doris Mayr (LMU Munich, Munich, Germany) for preparation of paraffin-embedded slides.
This work was supported by grants from the Deutsche Forschungsgemeinschaft within the Collaborative Research Center SFB 824 to C.S. (project C8) and J.T.S. (project C4) and within the Priority Programme SPP1629 to C.S. and P.J.N. (SP 581/6-1, SP 581/6-2, NE 648/5-2), as well as a grant from the Wilhelm-Sander-Stiftung to C.S. (2014.129.1), a grant within the European Commission Seventh Framework Program (FP7/CAM-PaC under grant agreement n°602783) and a grant from the German Cancer Consortium (DKTK) to J.T.S., a grant from the excellence cluster Nanosystems Initiative Munich to E.W. as well as a grant from the Center for Nanoscience (CeNS) to M.O. The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
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22. Knoop K, Schwenk N, Schmohl K, Muller A, Zach C, Cyran C, Carlsen J, Boning G, Bartenstein P, Goke B, Wagner E, Nelson PJ, Spitzweg C. Mesenchymal stem cell-mediated, tumor stroma-targeted radioiodine therapy of metastatic colon cancer using the sodium iodide symporter as theranostic gene. J Nucl Med. 2015; 56:600-606. doi: 10.2967/jnumed.114.146662.
23. Grunwald GK, Klutz K, Willhauck MJ, Schwenk N, Senekowitsch-Schmidtke R, Schwaiger M, Zach C, Goke B, Holm PS, Spitzweg C. Sodium iodide symporter (NIS)-mediated radiovirotherapy of hepatocellular cancer using a conditionally replicating adenovirus. Gene Ther. 2013; 20:625-633. doi: 10.1038/gt.2012.79.
24. Grunwald GK, Vetter A, Klutz K, Willhauck MJ, Schwenk N, Senekowitsch-Schmidtke R, Schwaiger M, Zach C, Wagner E, Goke B, Holm PS, Ogris M, Spitzweg C. Systemic image-guided liver cancer radiovirotherapy using dendrimer-coated adenovirus encoding the sodium iodide symporter as theranostic gene. J Nucl Med. 2013; 54:1450-1457. doi: 10.2967/jnumed.112.115493.
25. Grunwald GK, Vetter A, Klutz K, Willhauck MJ, Schwenk N, Senekowitsch-Schmidtke R, Schwaiger M, Zach C, Wagner E, Goke B, Holm PS, Ogris M, Spitzweg C. EGFR-Targeted Adenovirus Dendrimer Coating for Improved Systemic Delivery of the Theranostic NIS Gene. Mol Ther Nucleic Acids. 2013; 2: e131. doi: 10.1038/mtna.2013.58.
26. Urnauer S, Morys S, Krhac Levacic A, Muller AM, Schug C, Schmohl KA, Schwenk N, Zach C, Carlsen J, Bartenstein P, Wagner E, Spitzweg C. Sequence-defined cMET/HGFR-targeted Polymers as Gene Delivery Vehicles for the Theranostic Sodium Iodide Symporter (NIS) Gene. Mol Ther. 2016; 24:1395-1404. doi: 10.1038/mt.2016.95.
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34. Cabral H, Matsumoto Y, Mizuno K, Chen Q, Murakami M, Kimura M, Terada Y, Kano MR, Miyazono K, Uesaka M, Nishiyama N, Kataoka K. Accumulation of sub-100 nm polymeric micelles in poorly permeable tumours depends on size. Nat Nanotechnol. 2011; 6:815-823. doi: 10.1038/nnano.2011.166.
35. Miller BW, Morton JP, Pinese M, Saturno G, Jamieson NB, McGhee E, Timpson P, Leach J, McGarry L, Shanks E, Bailey P, Chang D, Oien K, et al. Targeting the LOX/hypoxia axis reverses many of the features that make pancreatic cancer deadly: inhibition of LOX abrogates metastasis and enhances drug efficacy. EMBO Mol Med. 2015; 7:1063-1076. doi: 10.15252/emmm.201404827.
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text:
A Local Inspiration, the Chiefs Are Sending 18-Year-Old P.J. Allred to the Super Bowl
Allred hasn't let cerebral palsy hinder his love for sports and life
The Smith-Cotton Tigers trailed by 40 points on this September night in mid-Missouri, but the score couldn't have mattered less. The story here was about the young man coming in at quarterback – 18-year-old P.J. Allred – and the dream that he was about to make a reality.
Allred took the snap from under center and moved to his right, using every ounce of adrenalin to keep his legs moving downfield. Allred made a jab toward the sideline before cutting back inside, revealing 80 yards of grass ahead of him.
This was his chance to score.
A senior at Smith-Cotton, Allred has been involved in just about every sport throughout his life, from football, baseball and basketball to running track. Athletics are simply a part of who he is.
He also happens to have cerebral palsy - presenting Allred with a variety of physical challenges - but his condition has no effect on his spirit.
"He's never backed down from anything. The kid is fearless," said P.J.'s mother, Valerie Corpe. "He would be outside for hours and hours throwing the football around if he could."
After all, Allred is no stranger to beating the odds.
Born premature, Allred was initially given days to live before pulling through and making it home by Christmas. Still, doctors warned that he would remain entirely deaf, was unlikely to ever walk and had a life expectancy of just 18 years.
The prognosis would seem daunting to many, but Allred and his family welcomed the challenge.
"We started setting goals for him since the day he was diagnosed, and he's always surpassed everything," Corpe explained. "As a baby, he would go an hour when the other kids could only do 45 minutes of therapy. From the beginning, he was trying so hard. That's always been a part of his life."
That effort paid dividends, too. Allred was walking by his fourth birthday and was showing signs of functional hearing a year later. Now already beyond his initial life expectancy, doctors expect Allred to live just as long as anybody else.
"He eats miracles for breakfast," Corpe said.
And those miracles hadn't gone unnoticed. In fact, in partnership with The Dream Factory, the Kansas City Chiefs wanted to provide this special young man with an experience worthy of his unwavering spirit.
That meant a trip to Arrowhead Stadium on Sunday as the Chiefs took on the rival Oakland Raiders, and as the players warmed up, Allred was right there on the sideline to see it all in person.
It was simply an unforgettable afternoon, from Pro Football Hall of Famer Bobby Bell congratulating Allred on his football career at Smith-Cotton to Chiefs' quarterback Patrick Mahomes - who Allred was hoping to meet – running over to the sideline specifically to meet his biggest fan.
"He was just grinning from ear-to-ear taking it all in," Valerie said. "When I saw Mahomes hug him and give him a ball, his face was just unforgettable."
That moment alone was already one to last a lifetime, but Chiefs Chairman & CEO Clark Hunt had yet to reveal one more surprise as he presented a giant ticket bearing the numerals "LIV."
Allred was going to the Super Bowl.
"He felt so much love," Corpe said. "It was just amazing. He still hasn't come down."
Which brings us back to that fateful night in September at Smith-Cotton High School.
The crowd erupted as Allred lunged toward the end zone, completing his 80-yard trek down the field. He fell onto the turf, his mission complete, as players from both teams swarmed around.
Allred - who fought off death as an infant, shattered limitations as a child and inspired countless others as a young man - had scored a touchdown.
Word of the moment spread like wildfire, even landing on SportsCenter. A dream was now a reality, and in the coming months he'd meet the reigning NFL MVP and receive tickets to the greatest sporting event on the planet, but for those that know Allred, none of this is a surprise.
It's simply who he is, and he has plenty left to achieve.
"He dreams big," Valerie said. "He's a true reason to never give up."

redpajama_set_name: RedPajamaCommonCrawl


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After the 2008/2009 crisis, the automotive industry is reverting to growth. And the growth is likely to continue in the coming years. Up to 2015, global car sales will increase to even more than 90 million vehicles annually, an increase of 20 percent compared to today's figures. In particular, the BRIC countries (Brazil, Russia, India and China) and emerging markets such as Thailand, Malaysia, Indonesia and the Philippines will contribute to this growth. Despite these positive signs, the automotive industry is facing an interminable and far-reaching transformation process. In addition to the continuous improvement of the internal combustion engine and the integration of alternative fuels, in particular new mobility concepts (such as car-sharing) and the electrification of the powertrain (in the broadest sense: e-mobility) will fundamentally change the traditional automotive industry.
Although, during the transformation process of the automotive industry, the transition from conventional drive technologies to battery electric vehicles ("BEVs"), which appear to reduce CO2 emissions and noise in urban areas, will be highlighted, the market penetration of BEVs will take a long time coming. In particular, the infrastructure problems, especially in large cities, consumers' lack of detailed knowledge about BEVs and the fears of limited range and long charging times leading to a low willingness to pay and acceptance for BEVs. But the market diffusion of BEVs can be accelerated by linking BEVs with alternative mobility concepts. It is possible (I.) to offer BEVs without other mobility concepts (in the narrow sense: traditional purchase and ownership of the vehicle), (II.) to offer BEVs in conjunction with the use of other modes of transport such as train, bicycle and others ("intermodal" transport solutions), (III.) to combine owning BEVs with renting traditional internal combustion engine vehicles for occasional trips (more than 150 kilometres) or (IV.) to offer BEVs within a car-sharing concept (in the narrow sense: no ownership of the vehicle).
Against this background, we conducted an exploratory analysis of consumer acceptance and willingness to pay for BEVs with or without a link to the above mentioned alternative mobility concepts. Our results show that BEVs already find acceptance. More than half of respondents were positive about BEVs. The other half were either undecided or critical about BEVs. The willingness to pay for BEVs was 25,000 euros, and around 5,000 and up to 10,000 euros below current market prices. Only eight percent of respondents were sufficiently willing to pay current market prices. However, our results also show that acceptance and willingness to pay for BEVs can be stimulated by a link with alternative mobility concepts. A link between BEVs and the possibility of the use of other modes of transport not only increases acceptance, but also leads to a six percent higher willingness to pay for BEVs. Linking BEVs with the rental of traditional internal combustion engine vehicles for occasional trips (more than 150 kilometres) shows the highest acceptance and willingness to pay. Due to this link, respondents are willing to accept eight percent higher prices for BEVs. Abandoning BEV ownership by using a car-sharing solution shows the lowest acceptance and willingness to pay. Compared to the current prices of existing car-sharing concepts, respondents rated their willingness to pay around 15 percent lower. Our results show initial evidence that acceptance and willingness to pay for BEVs can be increased by linking BEVs intelligently with alternative mobility concepts.
The analysis and its results will be summarized in the conference paper. Following that, further investigation of the market penetration of BEVs and alternative mobility concepts as well as possible consumption patterns will be conducted.


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Levi & Korsinsky, LLP announces that a OLO class action lawsuit has been filed on behalf of investors who purchased Olo Inc. (OLO) Class A common stock between August 11, 2021 and August 11, 2022. For more on the OLO Lawsuit please contact us today.
According to the Olo Inc. lawsuit, throughout the Class Period defendants made false and/or misleading statements and/or failed to disclose that:
(1) Subway was ending its contract with Olo; (2) Olo's key business metric – active locations – could not continue to grow as defendants touted due to the loss of Subway's business; and (3) as a result of the above, defendants' statements about Olo's business, operations, and prospects were false and misleading and/or lacked a reasonable basis.
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At 4:05am local time today, an atypical plane landed on a tarmac in Abu Dhabi: Si2, a futuristic aircraft entirely powered by solar energy. It was imagined and built by the two Swiss explorers Bertrand Piccard and André Borschberg, who founded Solar Impulse to promote the use of clean energies. They set the goal of circumnavigating the world by air, powered by the sun, with no fuel or polluting emissions. Starting in 2004, it took the team more than a decade to design and proof test this unique aircraft. Si2 took off in March 2015 for a 17-leg journey, spanning over 26,000 miles and using 11,000 kWh worth of solar energy. After 510 flying hours, Si2 has set 19 world records, according to the Fédération Aéronautique Internationale (FAI), on this historic expedition.
Google helped build and host Solar Impulse's digital presence, and on the first day of their round-the-world journey, we jointly launched the #FutureIsClean initiative, a platform to encourage the world to support the adoption of necessary clean technologies.
We're deeply committed to powering the world with clean energy. Our goal is 100% renewable power, and so far we've committed to purchase nearly 2.5 gigawatts of renewable energy—equivalent to taking more than 1 million cars off the road—making us the largest non-utility purchaser of renewable energy in the world.
But commitment also comes through advocacy. That's why in 2013, Google became the internet and technology partner of Solar Impulse: to raise awareness for what's possible with clean technology and renewable energy. Everybody could use the plane's technologies on the ground to reduce our world's energy consumption, save natural resources and improve our quality of life.
A global community formed to join the #FutureIsClean movement, following the progression of the Si2 during its travel around the world on www.solarimpulse.com, and tuning in for the pilot's conversations with the Mission Control Center in Monaco (MCC). A virtual cockpit, built with the help of Google engineers and platforms, provided the telemetrics of Si2 (altitude, speed, battery level, equipment on board, etc.) and immersed children and supporters in the technical and human challenges that Solar Impulse embarked upon.
Today, we join the rest of the world in congratulating the Solar Impulse team for this outstanding accomplishment. Solar Impulse's pioneering spirit enabled them to push human boundaries and demonstrate that clean technologies can achieve goals we once thought were impossible.
From smartphone assistants to image recognition and translation, machine learning already helps us in our everyday lives. But it can also help us to tackle some of the world's most challenging physical problems -- such as energy consumption. Large-scale commercial and industrial systems like data centers consume a lot of energy, and while much has been done to stem the growth of energy use, there remains a lot more to do given the world's increasing need for computing power.
Reducing energy usage has been a major focus for us over the past 10 years: we have built our own super-efficient servers at Google, invented more efficient ways to cool our data centers and invested heavily in green energy sources, with the goal of being powered 100 percent by renewable energy. Compared to five years ago, we now get around 3.5 times the computing power out of the same amount of energy, and we continue to make many improvements each year.
Major breakthroughs, however, are few and far between -- which is why we are excited to share that by applying DeepMind's machine learning to our own Google data centers, we've managed to reduce the amount of energy we use for cooling by up to 40 percent. In any large scale energy-consuming environment, this would be a huge improvement. Given how sophisticated Google's data centers are already, it's a phenomenal step forward.
The implications are significant for Google's data centers, given its potential to greatly improve energy efficiency and reduce emissions overall. This will also help other companies who run on Google's cloud to improve their own energy efficiency. While Google is only one of many data center operators in the world, many are not powered by renewable energy as we are. Every improvement in data center efficiency reduces total emissions into our environment and with technology like DeepMind's, we can use machine learning to consume less energy and help address one of the biggest challenges of all -- climate change.
The equipment, how we operate that equipment, and the environment interact with each other in complex, nonlinear ways. Traditional formula-based engineering and human intuition often do not capture these interactions.
The system cannot adapt quickly to internal or external changes (like the weather). This is because we cannot come up with rules and heuristics for every operating scenario.
Each data center has a unique architecture and environment. A custom-tuned model for one system may not be applicable to another. Therefore, a general intelligence framework is needed to understand the data center's interactions.
To address this problem, we began applying machine learning two years ago to operate our data centers more efficiently. And over the past few months, DeepMind researchers began working with Google's data center team to significantly improve the system's utility. Using a system of neural networks trained on different operating scenarios and parameters within our data centers, we created a more efficient and adaptive framework to understand data center dynamics and optimize efficiency.
We accomplished this by taking the historical data that had already been collected by thousands of sensors within the data center -- data such as temperatures, power, pump speeds, setpoints, etc. -- and using it to train an ensemble of deep neural networks. Since our objective was to improve data center energy efficiency, we trained the neural networks on the average future PUE (Power Usage Effectiveness), which is defined as the ratio of the total building energy usage to the IT energy usage. We then trained two additional ensembles of deep neural networks to predict the future temperature and pressure of the data center over the next hour. The purpose of these predictions is to simulate the recommended actions from the PUE model, to ensure that we do not go beyond any operating constraints.
We tested our model by deploying on a live data center. The graph below shows a typical day of testing, including when we turned the machine learning recommendations on, and when we turned them off.
Our machine learning system was able to consistently achieve a 40 percent reduction in the amount of energy used for cooling, which equates to a 15 percent reduction in overall PUE overhead after accounting for electrical losses and other non-cooling inefficiencies. It also produced the lowest PUE the site had ever seen.
Because the algorithm is a general-purpose framework to understand complex dynamics, we plan to apply this to other challenges in the data center environment and beyond in the coming months. Possible applications of this technology include improving power plant conversion efficiency (getting more energy from the same unit of input), reducing semiconductor manufacturing energy and water usage, or helping manufacturing facilities increase throughput.
We are planning to roll out this system more broadly and will share how we did it in an upcoming publication, so that other data center and industrial system operators -- and ultimately the environment -- can benefit from this major step forward.


Alpine Pullover by Stüssy. Mesh lined wind and water resistant nylon shell. Featuring kangaroo pocket, drawstring waist and hood with contrasting trim. Patch and printed logos.
In the late '80s and early '90s, Stüssy was born from the Southern California surf scene and swept through the clothing landscape to redefine the look and ideology of casual-wear.


The multiplexer has never work for me. I tried the multiplexer and I think than I accidentally do a short circuit that has burn the chip and one of my Pi camera (the second was correct). It seem like normal than it's doesn't work, as we can see on their GitHib in the "issues" where only 4 of them has been resolved and a lot of peoples than have the same troubles. A person have also open a other GitHub to try to do reverse engineering to see why it's doesn't work.
Seriously, don't buy this and chose a other method to have multiple camera, like using a other type of board (like the "Raspberry Pi Compute Module I/O Board" of Adafruit) or use multiple USB/IP camera or any other type of camera. Personally, I choose to have one Pi Camera and multiple USB camera for my stereo system and it work very easily without any problem.


As an inConfidenceclient, you have access to our training and development services. With many highly relevant training topics to choose from, our training services help organizations to address the wide range of challenges faced by employees and management in today's work environment.
inConfidenceuses recognized best practices for designing and presenting learning programs for adults. Our sessions are highly interactive with exercises, discussions, activities and other proven training strategies. The inConfidencesession facilitators are skilled professionals with relevant subject matter experience, and are successful at bringing concepts to life and enhancing learning and retention. Backed by more than 20 years of training experience, our program provides clients with maximum value for its training investment.
We continually add new topics to our catalogue, revising and upgrading the content. When requesting training, please refer to this webpage to ensure you are accessing the most up to date version of our training catalogue.
Please allow a minimum of 4 weeks' notice for booking of all seminars. Seminars will be booked no further than 90 days in advance.
A completed Request Form can also be emailed to wellness@medavie.bluecross.ca.
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Remember: inConfidence is here for you 24 hours a day, seven days a week, with support for work, life, and everything in between. Call us, or log in on the website anytime!


The NZ Sock Company has been making great socks for generations. The commitment for the business has been passed on and allowed the company to grow into one of the world's foremost innovators of sock technology.
When NZ Sock reached out to Solutionists in 2017, the brief was to create an online solution that could cater for three specific target markets: The end consumer as an ecommerce platform, international and local businesses as a showroom and to retailers as an online ordering platform.
As part of the website project, the brand got an entirely new fresh look incorporating the needs of the B2C, B2B and wholesale market. The solution supports customer-specific logins for individual product pricing options, specials and promotions.
The website is fully integrated with the in-house business system Fabrix, which means sales transactions and customer updates are automated, streamlining business processes.
The B2C solution provides customers with automated freight calculations, access to promotional offers, recommended complimentary products and an inbuilt search engine to make browsing and more importantly shopping easy.
Registered wholesale customers can log in with their own login to view additional information and content unique to them including their very own product catalogues and collections. The B2B bulk order form allows for quick creation of sales orders.


There are a lot of people that have cars that are worth a lot of money, but they do not really take good care of it. For you to know what your car wants and is in need of and why it is acting up a certain way, you need to be aware of the different parts of the car and also about their proper functionality. This is really important as well. Even if you are about to buy your new car, there are a lot of things that need to be considered and kept in mind.
Following are some of the things that you need to know about a car.
If you are really rich, then saving up money is not much concern of yours. However, if you are someone who lives on a normal salary, then you might want to start saving money to get a car that you really like. It is the best option to start to save money and pay for the car in full instead of paying through an auto loan. Loans must be looked at as your last option only. That is why it is really important that you start saving up money from an early age and when you reach your twenties, you might be able to get a reasonable car.
If you do not have the amount to pay for the car in full, then you might need to consider to get an auto loan. There is no harm in taking out a loan, but the problem is that when you take out a car on loan, you will be paying extra money as an interest on the loan as well. So, isn't it better that you pay for the car only and not the loan with its interest.
If you are someone who has a lot of knowledge about cars, and you want to add things likes seats, mirrors, engine from different companies and you want to install that in your car, this is also possible. You just have to look at the different retailers and get the equipment or different parts of your car from these sites. You can look for the best tires of different companies at Tires in Abu Dhabhi and you will find the goodyear tyres. These tires are the best quality and there is such a wide range of tires that can be on their site. All you have to do is just look at what type of tire suits best for you. You can also get it shipped if you live in some other area.
This point is very necessary because you need to get your car insured in case something bad happens to it or it gets in an accident. This way, you can use the insurance to cover for any expenses.


If you ever have a problem, you can count on your friendly local Berserker to solve it as quickly and efficiently as possible. Provided, of course, that your problem lies somewhere in the realm of "How exactly am I going to splatter this evil wizard's innards all over the dungeon?" Hobbies include wearing bearskins, drinking mead and saying unkind things about magic users.
The Orc Berserker is the paragon of all things savage and furious. By combining the berserker's high situational attack bonuses with the boosted base attack of an orc, you can create a fearsome offensive powerhouse. Both the berserker and orc are well-suited for fighting enemies who are stronger than yourself. The Orc Berserker is very reliant on BLOODLUST, so avoiding becoming a L10 character is preferable. This can actually be difficult on some of the longer dungeons. The Humility can be helpful in this regard.
The Human Berserker is generally inferior to the Orc, but is situationally useful on dungeons where you cannot avoid becoming a 10th level character. With items to boost his base attack he can exceed the damage of the Orc berserker, but only at the 10th level. If you have not yet unlocked the Orc, the Human is a perfectly acceptable substitute.
Halfling Berserker can use their potions to tank damage from higher level enemies you are looking to fight anyway. Due to your high resists you get more out of your health and thanks to all the extra health potions from item conversion you will be able to use them more liberally while leveling up while still leaving resources for the boss. An additional benefit is that with some glyph use ( CYDSTEPP or GETINDARE) you're perfectly capable of fighting even physical damage enemies if necessary, and you're also highly resistant to poison. The Trisword is a superb item for the Halfling Berserker, giving a sizable situational boost to base attack that synergizes with the Berserker's attack bonus.
The Berserker one of the more specialized classes, with massive magic resistance, attack bonuses, and a significant penalty to spellcasting. Because of his MAGESLAY class feature, repeatable glyphs such as HALPMEH, PISORF, and BURNDAYRAZ have limited usefulness, but single-use glyphs such as APHEELSIK or GETINDARE glyphs are extremely helpful. The heightened mana cost is manageable if you're only using the glyph once. As a result, the Berserker tends to favor utility glyphs. Although the CYDSTEPP glyph works well for him, he needs to acquire two mana boosters before it's usable, which can be a bit of a problem.
The berserker works best when fighting enemies with magic damage, which he can carve apart with great ease. Bosses such as Aequitas the Warlock are easy prey to the Berserker. He has considerably more difficulty fighting physical-resist enemies, and will need to leverage his advantage against those with magic damage to prevail overall. By fighting powerful dragonspawn or warlocks, a Berserker can earn large amounts of bonus experience and level up quite quickly. This enables him to leave large numbers of weaker monsters on the board for later, which he can kill to level-up mid-battle against the boss.
Base attack is the most important attribute for a berserker, so equipping items to boost it is a very good idea. The Trisword is a highly recommended item in this regard, especially on a Halfling Berserker. Don't hesitate to power it with Mana Potions, either.
Glowing Guardian: One curious aspect about the Berserker is that he's often stronger as a L9 character than he is as a L10 character. This is because he gains a damage bonus against monsters that are higher-level than him, and once he reaches L10 he loses this bonus against the boss. In addition, his favorite spells are not affected by character level. As a result, Humility is very useful for the Berserker, and he also benefits nicely from the Absolution boon. Since the Berserker is not very effective with Fireballs, Cleansing is one of the best ways to deal with physically-resistant bosses.
Taurog: Taurog and Orc Berserker makes one obvious combo. The Berserker casts spells sparingly, and most glyphs can be converted at low levels to gain significant boost to anything. However, you should be cautious as Berserkers who use the APHEELSIK glyph can do poorly with Taurog and should delay worship or else worship a different deity entirely. Taurog's Helm will max out your Magic resist and you can effectively regen-fight 3-4 levels higher than yourself even without any glyph.
The Earthmother: High mana cost is not much a problem for IMAWAL, and Entanglement is a great boon for strikers. Early Vine Form spam greatly helps you. After you are done with the help of Slowed, you will eventually convert to better god, since Clearance is not very powerful for Berserkers outside of when you need tons of BURNDAYRAZ. Don't hesitate to wipe plants out with Clearance at mid levels. Desecrating her altar to please your new god also a nice move.
This page was last modified on 20 November 2017, at 15:51.


text:
Home / National / Over 100 Applicants for Café Job After Wage Hike
Over 100 Applicants for Café Job After Wage Hike
Posted on January 19, 2018 by Korea Bizwire in National, Social Affairs, Top News with 0 Comments
An individual referred to only as Hwang, who owns a café in Suwon, was surprised to learn that an online job ad he placed on recruiting website Albamon this month saw dozens of job seekers apply to work at his café. (Image: Kobiz Media)
SUWON, Jan. 19 (Korea Bizwire) – More than 100 people responded to a job advertisement for café staff the day the it was posted.
An individual referred to only as Hwang, who owns a café in Suwon, was surprised to learn that an online job ad he placed on recruiting website Albamon this month saw dozens of job seekers apply to work at his café.
"Only five or six months ago, it was difficult to find part-time workers, but so many people have applied all of a sudden this year," Hwang said.
"As the minimum wage has gone up to 7,530 won per hour, I think few coffee shops are looking for employees," the café owner added.
Contrary to last year, when an ad Hwang placed drew fewer than 10 applicants, the coffee shop owner had to shortlist 10 candidates and conduct thorough interviews before deciding to work with a man in his mid-20s this year.
Another coffee shop in Pyeongtaek, where finding workers is traditionally difficult, saw 15 people apply to fill a vacancy.
"Though my bet is that it's the result of the minimum wage hike, I'm not entirely sure what the cause is," the owner of the café in Pyeongtaek said.
The minimum hourly wage in South Korea went up 16.4 percent this year, and experts are divided over the impact of the increased wage on the job market.
According to data from Alba Heaven, job openings dropped nearly 11 percent after the wage hike came into force this year.
However, one company official says it's hard to say whether the decrease is a direct result of the wage hike or if other circumstances have also played a role.
"It is true to an extent that some employers are considering downsizing in the wake of the minimum wage hike. But to say that there are fewer jobs because of the wage hike is not fair, as many factors are at play here," the official noted.
Kim Yong-ki, a professor of business administration at Ajou University, said the decrease in job growth due to wage hikes can be solved via commercial rent prices and better relationships with franchise companies and affiliates.
"Many studies show that a wage hike doesn't directly affect the job market negatively," Kim said.
"As well-intended economic policy can have side effects in the beginning, issues like job losses stemming from a wage hike must be solved by ensuring affordable commercial rental prices and building better relationships between franchisors and shop owners," he added.
Hyunsu Yim (hyunsu@koreabizwire.com)
Café Job
Wage Hike
S. Korea to Boost Exports through New Manufacturing Boom
S. Korea to Hike Security Budget for Diplomatic Missions in 2016
Samsung to Sponsor Crufts 2015, Exhibiting Smart Doghouse
Parental Leave Still a Dream for Many Koreans


text:
Q: I cannot install Ruby's DevKit well on windows 8.1
I'm on windows 8.1 x64
I cannot install ruby DevKit well, what to do next?
ruby -v

1.9.2p290<2011-07-09[i386-mingw321]>

I want to configure octopress, seems stuck here now.
----"E:\Ruby192\lib\ruby\gems\1.9.1\gems\rdiscount-2.1.6\ext\mkmf.log"

have_func: checking for random()... -------------------- no

"gcc -o conftest -IE:/Ruby192/include/ruby-1.9.1/i386-mingw32 -IE:/Ruby192/include/ruby-1.9.1/ruby/backward -IE:/Ruby192/include/ruby-1.9.1 -I.     -O3 -g -Wextra -Wno-unused-parameter -Wno-parentheses -Wpointer-arith -Wwrite-strings -Wno-missing-field-initializers -Wno-long-long conftest.c  -L. -LE:/Ruby192/lib -L. -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\tk\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\tcl\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\libyaml\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\pdcurses\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\openssl\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\zlib\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\iconv\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\gdbm\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\libffi\\lib      -lmsvcrt-ruby191-static  -lshell32 -lws2_32  "
checked program was:
/* begin */
1: #include "ruby.h"
2:
3: #include <winsock2.h>
4: #include <windows.h>
5: int main() {return 0;}
/* end */

"gcc -o conftest -IE:/Ruby192/include/ruby-1.9.1/i386-mingw32 -IE:/Ruby192/include/ruby-1.9.1/ruby/backward -IE:/Ruby192/include/ruby-1.9.1 -I.     -O3 -g -Wextra -Wno-unused-parameter -Wno-parentheses -Wpointer-arith -Wwrite-strings -Wno-missing-field-initializers -Wno-long-long conftest.c  -L. -LE:/Ruby192/lib -L. -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\tk\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\tcl\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\libyaml\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\pdcurses\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\openssl\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\zlib\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\iconv\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\gdbm\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\libffi\\lib      -lmsvcrt-ruby191-static  -lshell32 -lws2_32  "
conftest.c: In function 't':
conftest.c:8:53: error: 'random' undeclared (first use in this function)
conftest.c:8:53: note: each undeclared identifier is reported only once for each function it appears in
checked program was:
/* begin */
1: #include "ruby.h"
2:
3: #include <winsock2.h>
4: #include <windows.h>
5:
6: /*top*/
7: int main() {return 0;}
8: int t() { void ((*volatile p)()); p = (void ((*)()))random; return 0; }
/* end */

"gcc -o conftest -IE:/Ruby192/include/ruby-1.9.1/i386-mingw32 -IE:/Ruby192/include/ruby-1.9.1/ruby/backward -IE:/Ruby192/include/ruby-1.9.1 -I.     -O3 -g -Wextra -Wno-unused-parameter -Wno-parentheses -Wpointer-arith -Wwrite-strings -Wno-missing-field-initializers -Wno-long-long conftest.c  -L. -LE:/Ruby192/lib -L. -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\tk\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\tcl\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\libyaml\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\pdcurses\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\openssl\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\zlib\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\iconv\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\gdbm\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\libffi\\lib      -lmsvcrt-ruby191-static  -lshell32 -lws2_32  "
E:\Users\HYPERG~1\AppData\Local\Temp\cce9IqGL.o: In function `t':
E:\Ruby192\lib\ruby\gems\1.9.1\gems\rdiscount-2.1.6\ext/conftest.c:8: undefined reference to `random'
collect2: ld returned 1 exit status
checked program was:
/* begin */
1: #include "ruby.h"
2:
3: #include <winsock2.h>
4: #include <windows.h>
5:
6: /*top*/
7: int main() {return 0;}
8: int t() { random(); return 0; }
/* end */

--------------------

have_func: checking for srandom()... -------------------- no

"gcc -o conftest -IE:/Ruby192/include/ruby-1.9.1/i386-mingw32 -IE:/Ruby192/include/ruby-1.9.1/ruby/backward -IE:/Ruby192/include/ruby-1.9.1 -I.     -O3 -g -Wextra -Wno-unused-parameter -Wno-parentheses -Wpointer-arith -Wwrite-strings -Wno-missing-field-initializers -Wno-long-long conftest.c  -L. -LE:/Ruby192/lib -L. -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\tk\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\tcl\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\libyaml\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\pdcurses\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\openssl\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\zlib\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\iconv\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\gdbm\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\libffi\\lib      -lmsvcrt-ruby191-static  -lshell32 -lws2_32  "
conftest.c: In function 't':
conftest.c:8:53: error: 'srandom' undeclared (first use in this function)
conftest.c:8:53: note: each undeclared identifier is reported only once for each function it appears in
checked program was:
/* begin */
1: #include "ruby.h"
2:
3: #include <winsock2.h>
4: #include <windows.h>
5:
6: /*top*/
7: int main() {return 0;}
8: int t() { void ((*volatile p)()); p = (void ((*)()))srandom; return 0; }
/* end */

"gcc -o conftest -IE:/Ruby192/include/ruby-1.9.1/i386-mingw32 -IE:/Ruby192/include/ruby-1.9.1/ruby/backward -IE:/Ruby192/include/ruby-1.9.1 -I.     -O3 -g -Wextra -Wno-unused-parameter -Wno-parentheses -Wpointer-arith -Wwrite-strings -Wno-missing-field-initializers -Wno-long-long conftest.c  -L. -LE:/Ruby192/lib -L. -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\tk\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\tcl\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\libyaml\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\pdcurses\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\openssl\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\zlib\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\iconv\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\gdbm\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\libffi\\lib      -lmsvcrt-ruby191-static  -lshell32 -lws2_32  "
E:\Users\HYPERG~1\AppData\Local\Temp\ccumIxqZ.o: In function `t':
E:\Ruby192\lib\ruby\gems\1.9.1\gems\rdiscount-2.1.6\ext/conftest.c:8: undefined reference to `srandom'
collect2: ld returned 1 exit status
checked program was:
/* begin */
1: #include "ruby.h"
2:
3: #include <winsock2.h>
4: #include <windows.h>
5:
6: /*top*/
7: int main() {return 0;}
8: int t() { srandom(); return 0; }
/* end */

--------------------

have_func: checking for rand()... -------------------- yes

"gcc -o conftest -IE:/Ruby192/include/ruby-1.9.1/i386-mingw32 -IE:/Ruby192/include/ruby-1.9.1/ruby/backward -IE:/Ruby192/include/ruby-1.9.1 -I.     -O3 -g -Wextra -Wno-unused-parameter -Wno-parentheses -Wpointer-arith -Wwrite-strings -Wno-missing-field-initializers -Wno-long-long conftest.c  -L. -LE:/Ruby192/lib -L. -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\tk\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\tcl\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\libyaml\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\pdcurses\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\openssl\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\zlib\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\iconv\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\gdbm\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\libffi\\lib      -lmsvcrt-ruby191-static  -lshell32 -lws2_32  "
checked program was:
/* begin */
1: #include "ruby.h"
2:
3: #include <winsock2.h>
4: #include <windows.h>
5:
6: /*top*/
7: int main() {return 0;}
8: int t() { void ((*volatile p)()); p = (void ((*)()))rand; return 0; }
/* end */

--------------------

have_func: checking for srand()... -------------------- yes

"gcc -o conftest -IE:/Ruby192/include/ruby-1.9.1/i386-mingw32 -IE:/Ruby192/include/ruby-1.9.1/ruby/backward -IE:/Ruby192/include/ruby-1.9.1 -I.     -O3 -g -Wextra -Wno-unused-parameter -Wno-parentheses -Wpointer-arith -Wwrite-strings -Wno-missing-field-initializers -Wno-long-long conftest.c  -L. -LE:/Ruby192/lib -L. -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\tk\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\tcl\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\libyaml\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\pdcurses\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\openssl\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\zlib\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\iconv\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\gdbm\\lib -LC:\\Users\\Luis\\Projects\\oss\\oneclick\\rubyinstaller\\sandbox\\libffi\\lib      -lmsvcrt-ruby191-static  -lshell32 -lws2_32  "
checked program was:
/* begin */
1: #include "ruby.h"
2:
3: #include <winsock2.h>
4: #include <windows.h>
5:
6: /*top*/
7: int main() {return 0;}
8: int t() { void ((*volatile p)()); p = (void ((*)()))srand; return 0; }
/* end */

--------------------

check_sizeof: checking size of unsigned long... -------------------- int

"gcc -IE:/Ruby192/include/ruby-1.9.1/i386-mingw32 -IE:/Ruby192/include/ruby-1.9.1/ruby/backward -IE:/Ruby192/include/ruby-1.9.1 -I.     -O3 -g -Wextra -Wno-unused-parameter -Wno-parentheses -Wpointer-arith -Wwrite-strings -Wno-missing-field-initializers -Wno-long-long   -c conftest.c"
checked program was:
/* begin */
1: #include "ruby.h"
2:
3: #include <winsock2.h>
4: #include <windows.h>
5: typedef unsigned long rbcv_typedef_;
6: static rbcv_typedef_ *rbcv_ptr_;
7:
8: /*top*/
9: int conftest_const[(sizeof((*rbcv_ptr_)) == sizeof(int)) ? 1 : -1];
/* end */

--------------------

check_sizeof: checking size of unsigned int... -------------------- int

"gcc -IE:/Ruby192/include/ruby-1.9.1/i386-mingw32 -IE:/Ruby192/include/ruby-1.9.1/ruby/backward -IE:/Ruby192/include/ruby-1.9.1 -I.     -O3 -g -Wextra -Wno-unused-parameter -Wno-parentheses -Wpointer-arith -Wwrite-strings -Wno-missing-field-initializers -Wno-long-long   -c conftest.c"
checked program was:
/* begin */
1: #include "ruby.h"
2:
3: #include <winsock2.h>
4: #include <windows.h>
5: typedef unsigned int rbcv_typedef_;
6: static rbcv_typedef_ *rbcv_ptr_;
7:
8: /*top*/
9: int conftest_const[(sizeof((*rbcv_ptr_)) == sizeof(int)) ? 1 : -1];
/* end */

--------------------

A: You can install Ruby 1.93, some tutorials are too old, and I've installed Ruby 1.93, then it's ok Now.

redpajama_set_name: RedPajamaStackExchange


This snippet could be used when a text input has a default value. Thus, when user clicks on the input to write, the default value disappears and allows the user to write, but if the user does not type anything, the default value will appears again. It also adds a class to the input when the user is typing on it and is removed when the input loses focus.
This snippet must be inside the ready event.
For the HTML markup, the only thing to take care is that you must use the same class in all the inputs that you want to give this functionality. In the example I use \"input-text-js\", but you can use whatever you want.


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