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Knockdown of SALL4 expression using RNA interference induces cell cycle arrest, enhances early apoptosis, inhibits invasion and increases chemosensitivity to temozolomide in U251 glioma cells
- Authors:
Affiliations: Department of Neurosurgery, Changzheng Hospital, Second Military Medical University, Shanghai 200003, P.R. China
- Published online on: August 4, 2017 https://doi.org/10.3892/ol.2017.6722
- Pages: 4263-4269
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Glioma is the most common subgroup type of brain tumor, with an incidence of 5–6/100,000 cases/year in the United States (1). Among all types of glioma, glioblastoma multiforme (GBM) accounts for ~50% of cases and has the most malignant phenotype (2). Despite the extensive developments that have been invested in surgical techniques and therapeutic agents, 88% of patients with GBM succumb to this disease within 3 years (3). GBM remains one of the most challenging malignancies worldwide.
Embryonic stem cells (ESCs) are known for their potent pluripotency and are able to differentiate into >220 cell types in the adult body. The human homologue of the drosophila spalt-like transcription factor 4 (SALL4) is a zinc-finger transcription factor, which is responsible for maintaining pluripotency and longevity of ESCs (4–6). Previously, cancer stem cells (CSCs) have been identified in various types of malignancies (7,8) and demonstrate high self-renewal capabilities able to sustain tumor growth (9). Thus far, ESCs and CSCs have been revealed to share numerous biological similarities with the SALL4 expression pattern being one of them. In various types of tumors, high SALL4 expression level has been associated with increased malignancy, including increased metastasis, enhanced proliferation (10–12) and poor differentiation (13,14).
In the authors' previous study, it was demonstrated that SALL4 was highly expressed in glioma and significantly associated with poor survival (15). The present study further investigated the biological role of SALL4 in the tumorigenesis of glioma and explored the underlying mechanism of action. It was revealed that SALL4 regulated the cell cycle, apoptosis, tumor cell invasion and temozolomide (TMZ) treatment response in the U251 GBM cell line. Furthermore, decreased SALL4 expression level was associated with a decreased expression level of core transcription factors, including POU class 5 homeobox 1 (OCT4), SRY-box 2 (SOX-2) and Nanog homeobox (NANOG). Additionally, knockdown of SALL4 inhibited O-6-methylguanine-DNA methyltransferase (MGMT) and adenosine triphosphate-binding cassette subfamily G member 2 (ABCG2) expression levels, which may serve essential roles in GBM chemoresistance. These results suggest that SALL4 serves an important role in tumorigenesis and may be a useful therapeutic target for GBM.
Materials and methods
Cell culture, construction and transfection
The human malignant glioma cell line U251 was purchased from the Type Culture Collection of the Chinese Academy of Science (Shanghai, China). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 8% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and antibiotics (100 U/ml penicillin G and 100 µg/ml streptomycin). A total of four different short interfering RNA (siRNA) sequences against SALL4 (GeneBank accession no. NM-020436) and one scrambled siRNA were purchased from Shanghai GenePharma Co. Ltd. (Shanghai, China). The siRNA sequences were as follows: siRNA-1, 5′-GCTAGACACATCCAAGAAAGG-3′; siRNA-2, 5′-GCCGAAAGCATCAAGTCAAAG-3′; siRNA-3, 5′-GCCGACCTATGTCAAGGTTGA-3′; siRNA-4, 5′-GGAAGTTGGCCATCGAGAACA-3′; and scrambled siRNA, 5′-TTCTCCGAACGTGTCACGT-3′. Cells were transfected with siRNAs using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Following knockdown efficacy evaluation, the 3rd and 4th siRNA variants at 72 h were selected for subsequent experiments. The groups were named as blank control (BC; untransfected), negative control (NC; scrambled siRNA), SALL4/siRNA-1 and SALL4/siRNA-2. Details of cell culture, construction and transfection siRNA, and evaluation of the siRNAs intervention efficacy were performed as previously described (15).
Protein extraction and western blotting
U251 cells were washed with 1X PBS and pelleted at 12,000 × g for 10 min at 4°C. Total protein concentration was measured using a Bicinchoninic Assay Protein Assay kit (Beyotime Institute of Biotechnology, Haimen, China). Proteins extracted from cells (40 µg per lane) were separated using 10% SDS-PAGE gel (Beyotime Institute of Biotechnology) and transferred to a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA) at 100 V for 1 h. Following blocking with 5% non-fat milk in PBS with 0.1% Tween (PBST) for 1 h at room temperature, the membranes were incubated at 4°C overnight with primary antibodies and washed three times with 1X TBST. Next, cells were incubated at room temperature for 2 h with the secondary antibody. The primary antibodies used were as follows: Mouse polyclonal anti-SALL4 (Abgent, Inc., San Diego, CA, USA; cat no. AP1488b, dilution, 1:500); mouse polyclonal anti-OCT4 (cat no. 11263-1-AP), mouse polyclonal anti-NANOG (cat no. 14295-1-AP), mouse polyclonal anti-SOX2 (cat no. 20118-1-AP), mouse polyclonal anti-ABCG2 (cat no. 10051-1-AP) and mouse polyclonal anti-MGMT (cat no. 17195-1-AP) (all from ProteinTech Group, Inc., Chicago, IL, USA; dilution, 1:500); and polyclonal rabbit anti-β-actin (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat no. sc-130656, 1:1,000) used as a gel loading control. The secondary antibody used was horseradish peroxidise-conjugated Goat Anti-Rabbit IgG (H&L) AP (BioVision, Inc., San Francisco, CA, USA; cat no. 93-6923-100, dilution, 1:2,000). Immunoblotting bands were visualized visualized using ECL Substrates (Tanon Science and Technology Co., Ltd, Shanghai, China) and quantified using ImageJ software version 1.48 (National Institutes of Health, Bethesda, MA, USA).
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis
Total RNA was extracted from U251 glioma cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Reverse transcription was performed using the Thermoscript RT-PCR system (Invitrogen; Thermo Fisher Scientific, Inc.) with random hexamer primers and the Superscript II Reverse Transcriptase kit (Invitrogen; Thermo Fisher Scientific, Inc.), for 30 min at 25°C, 30 min at 42°C and 10 min at 85°C. qPCR was performed using the quantitative Real Time PCR 7500 sequence detection system (Applied Biosystems, Foster city, CA, USA). RT-qPCR was performed as follows: 40 cycles of denaturation at 95°C (12 sec) and annealing/extension at 60°C (40 sec). Primers were designed using Primer 5.0 software (Applied Biosystems). The primer sequences were as follows: SALL4 forward (F), 5′-ATAGTCAAGCCGAAAGCATCAAGTC-3′ and reverse (R), 5′-CTCCGACCTTCCATCTCAGTGC-3′; OCT4 F, 5′-ACCTATTCAGCCAAACGACCAT-3′ and R, 5′-CTGCTTCCTCCACCCACTTCT-3′; SOX2 F, 5′-GCTCGCAGACCTACATGAAC-3′ and R, 5′-GGGAGGAAGAGGTAACCACA-3′; NAN OGF, 5′-ATACCTCAGCCTCCAGCAGATG-3′ and R, 5′-TTCTGCCACCTCTTAGATTTCATTC-3′; MGM T F, 5′-TCTTCACCATCCCGTTTTCCAG-3′ and R, 5′-CTTCTCCGAATTTCACAACCTTCAG-3′; ABCG2 F, 5′-GAAACCTGGTCTCAACGCCATC-3′ and R, 5′-ACTTGGATCTTTCCTTGCAGCTAAG-3′; and GAP DH F, 5′-CATGAGAAGTATGACAACAGCCT-3′ and R, 5′-AGTCCTTCCACGATACCAAAGT-3′. SALL4 mRNA expression was normalized to GAPDH and analyzed using the 2−ΔΔCq method (16).
Cell cycle analysis
Cell cycle analysis was performed by flow cytometry using transfected and control U251 cells in log-phase growth. Cells were washed with PBS, fixed with 90% ethanol overnight at 4°C and incubated with RNase at 37°C for 30 min. Cell nuclei were stained with propidium iodide (PI) at 4°C for an additional 30 min. The stained nuclei were then analyzed using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The populations of cells distributed in the G0/G1, S and G2/M cell cycle phases were evaluated using WinMDI version 2.9 software (The Scripps Research Institute, San Diego, CA, USA).
Apoptosis assay
Apoptosis was evaluated using an annexin V fluorescein isothiocyanate (FITC)/PI apoptosis detection kit (Thermo Fisher Scientific, Inc.), according to the manufacture's protocol. Briefly, following incubation (16 h) at 37°C in a 5% CO2 humidified atmosphere, U251 cells cultured in 6-cm dishes were digested with trypsin without EDTA, washed twice with PBS and suspended in 100 µl binding buffer, followed by staining with 5 µl Annexin V-FITC and 5 µl PI for 15 min in the dark at room temperature and then analyzed by flow cytometry as aforementioned.
Invasion assay
Equal numbers (1×105) of transfected and control U251 cells were seeded in separate 24-well cell culture inserts coated with Matrigel with 8 µm pores. 500 µl DMEM supplemented with 10% FBS was added into the lower chamber as a chemoattractant. Following a 24-h incubation at 37°C with 5% CO2, cells that were adhered to the upper surface of the filter were removed using a cotton applicator. The cells on the lower surface of the membrane (the migrated cells) were fixed with 3.7% formaldehyde for 10 min at −20°C, stained with 0.1% crystal violet (Beyotime Institute of Biotechnology) for 30 min at 37°C. The cell number was determined in at least five randomly selected fields under a light microscope (magnification, ×200; SZ61; Olympus, Tokyo, Japan). The invasion rate was determined for three independent experiments as follows: No. of migrated cells/total no. of cells ×100.
Cytotoxicity assay
The cytotoxicity of TMZ (Merck & Co., Inc., Whitehouse Station, NJ, USA) on glioma cells was detected using a Total Superoxide Dismutase Assay kit and WST-8 assay according to manufacturers' protocol. Briefly, U251 cells were plated at a density of 5×103 cells/well in 96-well plates and allowed to attach overnight at 37°C. Various concentrations of TMZ (2, 4, 8, 16, 32, 64, 128 and 256 µg/ml) were subsequently added, and the cells were cultured for 72 h at 37°C. A total of 4-h prior to harvest, 10 µl/well of the Cell Counting kit-8 reagent (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added and the cells were incubated for 2 h at 37°C. The optical density (OD) at 450 nm was recorded using a microplate reader (Bio-Rad). The cell survival rate was determined by comparing the OD values of the treated samples with those of the untreated controls within each group. The concentration of TMZ required to inhibit cell growth by 50% (the toxic concentration, TC50) was evaluated using survival curves.
Statistical analysis
Data are expressed as the mean ± standard deviation of three independent experiments. Statistical analysis was performed using the Student's t-test between two groups, whereas the comparison between ≥ three groups was performed using one-way analysis of variance. Post hoc tests were used for comparisons between groups. Student-Neuman-Keuls method was used when equal variances assumed. If equal variances not assumed, Dunnett's T3 method was used. All analyses were performed using SPSS version 13.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.
SALL4 knockdown induces cell cycle arrest, enhances apoptosis and invasion inhibition
The cell cycle distribution in various transfection groups of U251 glioma cells were analyzed by flow cytometry 72 h after transfection. No significant differences in the fraction of G1 phase cells were identified between the NC group (52.11±1.92%) and BC group (42.30±0.42%) (P=0.14; Fig. 1A). However, following treatment with siRNAs, the G1 phase cell fraction in the SALL4/siRNA-1 (77.17±1.77%) and SALL4/siRNA-2 (76.67±1.12%) groups were significantly higher compared with that of the BC (both P<0.001) and NC groups (siRNA-1, P=0.002; siRNA-2, P=0.005) (Fig. 1A). Correspondingly, the fraction of cells in the S phase were 36.75±0.95, 31.03±3.88, 10.54±1.08 and 9.76±1.16% in the BC, NC, SALL4/siRNA-1, and SALL4/siRNA-2 groups, respectively. Additionally, the reduction in the fraction of cells in the G2-M phase were 18.41±2.10, 17.17±2.01, 12.28±2.84 and 13.58±1.10% in the BC, NC, SALL4/siRNA-1, and SALL4/siRNA-2 groups, respectively. SALL4/siRNA-1 and SALL4/siRNA-2 demonstrated significantly lower S phase cell proportions compared with those in the BC (both P<0.001) and NC groups (siRNA-1, P=0.047; siRNA-2, P=0.041). However, no significant differences in the G2-M cell cycle phase were identified in the SALL4/siRNA-1 and SALL4/siRNA-2 groups, when compared with the BC (siRNA-1, P=0.24; siRNA-2, P=0.21), and NC (siRNA-1, P=0.39; siRNA-2, P=0.36) groups.
Figure 1.
SALL4 knockdown induced cell cycle arrest, enhanced early apoptosis and inhibited invasion. (A) Inhibition effects of SALL4 on cell cycle distribution. The cells were analyzed by flow cytometry at 72 h after transfection. Histograms reveal G0/G1 cell cycle phase arrest and a decline in S phase in siRNA groups. (B) Knockdown of SALL4 increased the early apoptosis rate in U251 cells. Apoptosis rate was evaluated in fluorescein isothiocyanate-annexin V and propidium-stained cells by flow cytometry. Apoptotic cell numbers were significantly higher in siRNA groups. (C) Knockdown of SALL4 expression level decreased cell invasive capacity of U251 cells. Matrigel assays demonstrate the effect on the invasive potential in each group. The histogram indicated the mean number of invaded cells at 72 h after top-side cell seeding, which revealed that the capacity of invasion was significantly suppressed in both siRNA groups. BC: untransfected; NC: scrambled siRNA. *P<0.05 siRNA-1 vs. BC, ▲P<0.05 siRNA-1 vs. NC, §P<0.05 siRNA-2 vs. BC, #P<0.05 siRNA-2 vs. NC. SALL4, spalt-like transcription factor 4; siRNA, short interfering RNA; BC, blank control; NC, negative control.
Subsequently, the present study compared the apoptosis rate prior to and following SALL4 knockdown (Fig. 1B). It was revealed that SALL4/siRNA-1 (85.88±0.54%) and SALL4/siRNA-2 (86.41±0.87%) induced significantly increased early apoptosis rates compared with that in the BC, and NC groups (all P<0.001).
The Transwell migration was performed to further examine the effect of SALL4 on glioma cell invasion (Fig. 1C). The mean invasion cell number was 94.33±3.51 and 91.33±1.53 in the BC, and NC groups, respectively, compared with 45.00±1.00 and 47.67±3.06 in the SALL4/siRNA-1, and SALL4/siRNA-2 groups, respectively. The siRNA treatment groups significantly reduced the migratory ability of cells compared with that in BC (siRNA-1, P=0.005; siRNA-2, P<0.001) and NC (siRNA-1, P=0.001; siRNA-2, P<0.001) groups.
The Janus kinase (Jak)-signal transducer and activator of transcription 3 (Stat3) signaling pathway is utilized by SALL4 to fulfill its function
In order to investigate the reduction of malignancy of glioma detected in the present study, the expression levels of OCT4, SOX2 and NANOG were analyzed, which are essential factors of the Jak-Stat3 signaling pathway. It was demonstrated that SALL4/siRNA-1 and SALL4/siRNA-2 suppressed the expression levels of OCT4, SOX2 and NANOG, which were all statistically lower compared with the BC and NC groups (Fig. 2A). Compared with the BC group, the inhibition rates of SALL4/siRNA-1 on OCT4, SOX2 and NANOG mRNA expression levels were 59% (P=0.004), 38% (P=0.001) and 39% (P=0.002), respectively, whereas the inhibition rates of SALL4/siRNA-2 were 65% (P<0.001), 26% (P=0.038) and 29% (P=0.036), respectively. Compared with the NC group, the inhibition rates of SALL4/siRNA-1 were 65.54% (P<0.001), 37.37% (P=0.038) and 40.20% (P=0.013), respectively, whereas the inhibition rates of SALL4/siRNA-2 were 70.59% (P<0.001), 25.25% (P=0.037) and 30.40% (P=0.032) (Table I; Fig. 2A). Western blot analysis revealed similar results (Fig. 2B).
Figure 2.
SALL4 knockdown altered the expression levels of OCT4, SOX2, NANOG, MGMT and ABCG2. (A) The mRNA expression levels of OCT4, SOX2, NANOG, MGMT and ABCG2 in siRNA groups was markedly lower compared with in BC and NC groups at 72 h post-transfection. (B) Western blotting revealed that changes in expression levels at the protein level were qualitatively similar to the changes in mRNA expression levels. BC group; NC group; SALL4/siRNA-1 group; SALL4/siRNA-2 group. *P<0.05 siRNA-1 vs. BC, ▲P<0.05 siRNA-1 vs. NC, §P<0.05 siRNA-2 vs. BC, #P<0.05 siRNA-2 vs. NC. SALL4, spalt-like transcription factor 4; OCT4, POU class 5 homeobox 1; SOX2, SRY-box 2; NANOG, Nanog homeobox; MGMT, O-6-methylguanine-DNA methyltransferase; ABCG2, adenosine triphosphate-binding cassette subfamily G member 2; siRNA, short interfering RNA; BC, blank control; NC, negative control.
SALL4 knockdown increases chemosensitivity to TMZ
Subsequently, the present study aimed to investigate the effect of SALL4 on chemotherapeutic TMZ during glioma treatment. It was revealed that the TC50 of TMZ in SALL4/siRNA-1 and SALL4/siRNA-2 groups were 68.34±3.52, and 67.44±4.71 µg/ml, respectively. Compared with that in the BC (113.66±23.07 µg/ml) and NC groups (114.93±20.91 µg/ml), the TC50 value of SALL4/siRNA-1 (BC, P=0.047; NC, P=0.030) and SALL4/siRNA-2 were significantly decreased (BC, P=0.043; NC, P=0.026) (data not shown).
In order to describe this phenomenon, the present study hypothesized that the knockdown of SALL4 suppressed glioma tumor resistance via MGMT and ABCG2 downregulation. The present study revealed that SALL4/siRNA-1 decreased MGMT and ABCG2 mRNA expression levels by 39, and 28%, respectively, which were significantly lower compared with that of the BC (MGMT, P=0.021; ABCG2, P=0.032) and NC (MGMT, P<0.001; ABCG2, P=0.024) groups (Table I; Fig. 2A). Similarly, when compared with the BC and NC groups, SALL4/siRNA-1 decreased MGMT protein expression level significantly (BC, P=0.009; NC, P=0.010), and SALL4/siRNA-2 significantly decreased expression levels of MGMT (BC, P=0.007; BC, P=0.009) and ABCG2 (BC, P=0.009; BC, P=0.006) (Fig. 2B).
Since SALL4 was first detected as an oncogene in leukemia (17), the function of SALL4 had been studied in various types of cancer. For example, in lung, breast, gastric and colorectal cancer cells (10–12), SALL4 has been demonstrated to regulate cell viability, apoptosis, and tumorigenicity (12). In the authors' previous study, it was reported that SALL4 served an essential role in glioma and its expression was negatively associated with prognosis (15); however, the detailed mechanisms remain unclear. The present study aimed to investigate the detailed mechanisms underlying SALL4 in glioma tumorigenesis. Following knockdown of SALL4, cell cycle arrest, significantly increased levels of early apoptosis and invasion inhibition were observed in GBM cells. Furthermore, decreased SALL4 expression level significantly decreased mRNA and protein expression levels of OCT4, SOX2, and NANOG. In addition, inhibition of SALL4 decreased the TC50 of chemotherapeutic agent TMZ. These results partially elucidated the specific mechanisms utilized by SALL4, indicating that SALL4 may serve an important role in GBM tumorigenesis.
The CSC theory hypothesizes that CSCs and ESCs share numerous malfunctioned proliferation signal pathways (18), and the Jak-Stat3 signaling pathway is considered as one of the most important (19). At the top hierarchy of the Jak-Stat3 signaling pathway, OCT4, SOX2 and NANOG, are considered to serve the most important function in maintaining ESC properties (20). Previously, increased glioma progression was identified to be associated with upregulated OCT4, SOX2 and NANOG (21,22). Additionally, combinatorial expression levels of OCT4, NANOG and SOX2 were positively associated with increasing glioma malignancy (22). The present study demonstrated that knockdown of SALL4 significantly downregulated the mRNA and protein expression levels of OCT4, SOX2, and NANOG, which supports the results regarding SALL4 involvement in cell cycle arrest, enhanced apoptosis and invasion inhibition. These data suggest that SALL4 inhibits glioma tumorigenesis by participating in the Jak-Stat3 signaling pathway. However, it remains unclear how and which factors SALL4 interacts with in the Jak-Stat3 signaling pathway; therefore, further research is required for elucidation.
Notably in the present study, the knockdown of SALL4 significantly reduced the TMZ TC50. As previously demonstrated, cancer utilizes various mechanisms enabling resistance to anticancer drugs, including DNA repairing and reducing toxin uptake via pumping-out chemotherapeutics (23). TMZ is a classical chemotherapy drug for glioma, which is normally used by patients with GBM followed by surgical resection and radiotherapy (24). However, the majority of patients with glioma develop resistance to TMZ during treatment (25). Thus, it was hypothesized that glioma may use or develop 'DNA repairing' and 'toxin pumping-out' strategies to remain resistant. MGMT is a DNA repair protein, which removes alkylating adducts from the O6 position of guanine and protects cells from cytotoxic, and mutagenic effects, resulting in a resistance of tumor cells to alkylating agent-based chemotherapy (26). Following the knockdown of SALL4, a significant decrease in the TC50 of TMZ with simultaneous inhibition of MGMT expression was observed. A previous study demonstrated that the expression level of SALL4 was positively associated with histone deacetylase activity in EpCAM-positive hepatocellular carcinomacell Hep3B and HuH7 cell lines (27). As the present study knocked down SALL4 in glioma cells, there would be less potent histone deacetylase activity, which may inhibit various protein expression levels. Although it remained unclear whether MGMT promoter would be deacetylated, the present study observed a lowered TC50 for TMZ as well as decreased MGMT protein expression levels simultaneously. Thus, it was reasonable to hypothesize that the knockdown of SALL4 decreased histone deacetylase activity, which inhibited MGMT expression and the subsequent TMZ TC50 in glioma cells. This hypothesis is supported by the results of a previous study that also used glioma cells (28) and the results of the present study. Additionally, pumping out chemotherapeutics is another popular mechanism used by various types of cancer. ABC drug transporters have been accepted to reduce the efficacy of chemotherapeutics by pumping out toxic drugs and thus contribute to aggressive tumor behaviors, and poor prognosis (29). As previously demonstrated to be enriched in GBM (30), ABCG2, which is a member of the ABC family, could also be involved in tumor resistance. The present study demonstrated that following knockdown of SALL4, ABCG2 exhibited significantly reduced expression levels, which may explain the reduction of TMZ TC50. Thus, the results of the present study suggest that MGMT and ABCG2 serve important roles in GBM resistance, and SALL4 is responsible for maintaining high expression and activity levels of MGMT and ABCG2. Further studies should focus on where and how SALL4 interacts with MGMT and ABCG2.
In conclusion, the present study revealed that the knockdown of SALL4 significantly decreased the malignancy and increased the sensitivity of TMZ to U251 cells. These results indicate that SALL4 may serve an essential role in the tumorigenic properties of glioma cells. Thus, SALL4 may be a potential therapeutic target for glioma, particularly for GBM.
The present study was supported by the National Natural Science Foundation of China (grant no. 30930094) and the Natural Science Foundation of Shanghai (grant no. 13ZR1414200).
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Omelette recipe from the Ministry of Food campaign. www.jamieoliver.com Jamie's Ministry of Food first aired on Channel4 in the UK in September 2008 Stay up to date with Jamie Oliver: Follow him on Twitter http://twitter.com/JAMIEOLIVER Facebook http://www.facebook.com/jamieoliver Website http://www.jamieoliver.com/
Main Dishes
You asked for it so we had to give it! Gennaro's bolognese, this is one hell of a recipe that is worth every minute of love, care, and attention. Give it a go, you won't be disappointed! Gennaro in Italy Over the coming months we will be posting a series of Gennaro's recipes from Italy,
A very special moment and tribute, to a very special man. x Gennaro in Italy Another video in a series of Gennaro's recipes from Italy, some of which you, the audience, have specifically asked for, and others are Gennaro doing what the Italians do best – sourcing good ingredients and making simple, bold dishes with
Another veg packed recipe from Me & Tesco for you guys! This is a paid ad by Tesco. (AVAILABLE IN UK ONLY) Thanks for subscribing! : https://www.youtube.com/user/jamieoliver?sub_confirmation=1 Links from the video: Traybake Smoky Beans | Jamie Oliver | UK | AD Cannellini Bean & Pasta Soup | Jamie Oliver | UK | AD For more
The most delicious and light fettuccine you can imagine. It is full of traditional garden veggies which give it an incredible freshness whilst the smoked trout gives it a wicked twist. Make sure you serve this STRAIGHT away with a generous serving of Parmesan for the ultimate dish and accompanied with an awesome dressed salad…
A lovely Tomato salad from Jamie Does which first aired in the UK on C4 in 2010. All the food made in this video was shared out and eaten among the very lucky crew. Nothing goes to waste. For more nutrition info, click here: http://jamieol.com/D3JimM Jamie Oliver's Food Tube | http://goo.gl/EdJ0vK Subscribe to Food Tube
Why pay out for shop bought pastry when it's so easy to make your own? Jamie has a recipe for sweet, crumbly shortcrust pastry that's so easy even Buddy can make it! It's perfect for making ahead of time and freezing until you're ready to fill it with something delicious. So, you've got your sweet
This simple and healthy recipe packs a punch full of flavour and is a great alternative to an indulgent curry for any night of the week. Succulent chicken thighs cooked in mustard seeds, curry leaves and turmeric with chunky aubergine, tomatoes, peppers and potatoes. It's on the front cover of this month's Jamie Magazine and
Trust us, you're going to love this one! It might sound unusual, but Jamie's method of cooking a whole chicken will blow you away. The milk creates lovely curds and a delicious sauce, all with wonderful flavour from garlic, sage, lemon and a hint of cinnamon. As Jamie says, it's not like anything you've had
Another cracking recipe from Jamie's new TV show Keep Cooking and Carry on. In these times when it's difficult to get some products never fear as Jamie has your back! His new Channel 4 show in the UK features many handy hints and tips and swaps so you can #KeepCookingandCarryOn Catch up on all of
The wonderful Akis Petretzikis has a fantastic moussaka recipe using baked vegetables instead of fried to make it that little bit lighter. Delicious layers of potato, aubergine, courgette and beef, all topped off with a creamy béchamel sauce and a handful of parmesan. The perfect tasty teatime treat. If you want more recipes from Akis,
Jamie introduces the wonderful Maunika Gowardhan to the Food Tube family with a bang, as she teaches him how to master the most delicious and tender Indian butter chicken recipe. Recreate the tandoor feel right in your own home. This'll certainly make you think twice before getting take out… It's not often you see Jamie
Let's get ready to Crumble! If you don't know what a crumble is, then today is your lucky day, as Jamie shows us three fantastic ones. They're a fruity dessert with crispy, crunchy, buttery topping, perfect to serve with cream, ice cream or delicious homemade custard! Jamie also shows us how to take these crumbles
If you're looking for an Italian meat-free recipe for your family that's tasty and quick to make, Gennaro has the answer with this delicious aubergine parmigiana. Homemade tomato sauce layered with slices of griddled aubergine, parmesan and finished with a crisp breadcrumb and mozzarella topping. Teatime will never be the same again. For this and
We're transporting you back in time now Food Tubers to when Jamie turned 30 and took a road trip through Italy cooking all the way. Most of it made it's way onto TV but this little gem has been tucked away ever since. Here he is in Sicily cooking pasta and learning from the masters.
Happy Independence Day to all our brothers and sisters across the pond. x Find the recipe here: http://www.jamieoliver.com/recipes/cheese-recipes/my-nyc-cheesecake All the food made in this video was shared out and eaten among the very lucky Food Tube crew. Nothing goes to waste. For more nutrition info, click here: http://jamieol.com/D3JimM Jamie Oliver's Food Tube | http://goo.gl/EdJ0vK Subscribe
This absolutely scrumptious dish is all about getting the good stuff in a classic comfort food dish. Cauliflower adds a healthy twist to this gorgeous, crispy family favourite. In these times when it's difficult to get some products never fear as Jamie has your back! His new Channel 4 show in the UK features many
King of Caribbean food Levi Roots kick starts summer with this awesome Jerk Chicken recipe. Pieces of chicken marinated in spicy pimento, chilli flakes and the famously hot Scotch Bonnet chilli – this dish is not for wimps! Get in the party mood and make a batch for your friends this weekend. Just have a
This is guilt-free comfort food at it's very best! Jamie's 'Happiness Pasta' is made with aubergine, tomatoes, chilli & garlic, all cooked down into a beautiful pasta sauce with ricotta. Serve it up with some wholewheat pasta and you're making a great, healthy meal for four people. Not only that, but it's only 470 calories
Chocolate! As a naughty little treat who doesn't love a beautiful, delicious chocolate brownie? Jamie has a perfect recipe for an indulgent one, made with top quality ingredients; no flavouring, no nuts, no fruit – just chocolate, chocolate and more chocolate! It's gooey in the middle and crisp on top, all served up with ice
Jamie spent months perfecting this recipe, and wants to share it with you! All the usual chilli, garlic and onion is combined with Jamie's secret addition of apple juice and cider vinegar to make this wonderful sauce. Make a big batch and use it on everything! Links from the video: Breakfast Tortilla | http://jamieol.com/BreakfastTortilla Hot
Get cosy for the new year with a warming slow-cooked lamb recipe from the father of flavour – Gennaro Contaldo. Filmed in beautiful Malta and made with love and passion, you'll see why it's his favourite homecoming meal. What's your favourite slow-cooked stew? Please share your great ideas in the comments box below – we'd
Jamie is back with another recipe from his new TV show Keep Cooking and Carry On. Recipes to do while you are stuck at home that will fill you with the good stuff and use up whatever you have to hand. Just be adaptable and don't be scared of making a swap out if you
Jamie shows you a brilliant way of cooking pork using balsamic vinegar. Try it and let us know what you think in the comments box. This pork goes really well with these beans. http://youtu.be/BbIwGEMVw40 All the food made in this video was shared out and eaten among the very lucky Food Tube crew. Nothing goes
Fresh pasta is the simplest thing in the world and all you need is strong flour (00) and water. In no time at all you can knock together a delicious meal like this Carbonara that uses minimal ingredients but has maximum flavour. #stayhome If in UK watch Jamie's new show tonight at 5.30pm on Channel
This super-speedy potato dauphinoise recipe is a great idea if you're short on time but still want great flavour. Creamy, cheesy and delicious. Jamie shows you his clever cheats and takes you through every step of the way. This easy recipe is from Jamie's 30 Minute Meals TV series first aired on Channel 4 in
We all know that bread is hard to find or you can't get to the store to get it but there is nothing that lifts the soul like the smell of fresh bread baking. If you can, grab some flour or hunt in the cupboards and have a go. Great to do with the kids
If you're a parent you'll love our new channel dedicated to help you feeding your family: http://jamieol.com/FamilyFoodTube. We've got great meals for all ages to save you time, money and get the good stuff into your kids. Things like this simple and versatile ragu recipe which is the base for Jamie's Family lasagne : https://youtu.be/SalyS66njMY
Burger fans get ready to feel those taste buds tingling! Jamie and DJ BBQ have a recipe for The Ultimate Beef Burger. Mustard brushed patties stacked high on toasted brioche buns with two pickles, a spicy mayo sauce, crisp lettuce, smoky bacon and slices of crumbly red Leicester. Woohoo! This one will definitely make you
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- Dairy Milk Oreo Mint 120g
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You have no obligation to purchase the product once you know the price. You can simply remove the item from your cart.
Dairy Milk Oreo Mint 120g
- Description
New! Try the yummy Cadbury Dairy Milk chocolate with a creamy mint filling and Oreo biscuit pieces.
- Contains
1 x Cadbury Dairy Milk with Oreo Mint Flavour 120g
- Allergy & Nutritional Information
Name: Cadbury Dairy Milk with Oreo Mint Chocolate Bar 120g
Allergen Information
Allergens can be found in the ingredients list
Contains Milk, Soya, Wheat. May Contain Nuts.
Typical values Per 100 g Per 3 Chunks (15 g) *Reference Intakes Energy 2325 kJ / 557 kcal 349 kJ / 84 kcal 8400 kJ / 2000 kcal Fat 35 g 5.2 g 70 g of which Saturates 20 g 2.9 g 20 g Carbohydrate 54 g 8.2 g 260 g of which Sugars 49 g 7.3 g 90 g Fibre 1.5 g 0.2 g - Protein 5.9 g 0.9 g 50 g Salt 0.42 g 0.06 g 6 g *% reference intake of an average adult (8400 kJ / 2000 kcal)
- Delivery
- Orders up to £10£3.95
- Orders over £10£5.50
- Next day DPD£6.50
- Weekend (mainland)£8.00
- European deliveryVarious
- Reviews
Feefo Reviews - Average 88% (33 reviews)
Date Score Customer Comment Our Response 06-Jun-2020 Product:
Exactly as ordered for a birthday gift, just tasty! 11-May-2020 Product:
Did not receive item...... 07-May-2020 Product:
Delicious, delicious, delicious! 29-Apr-2020 Product:
purchased as a gift but he loves chocolate so he was very happy 28-Apr-2020 Product:
Sent as gift, loved by recipients 28-Apr-2020 Product:
Great will definitely use again 23-Apr-2020 Product:
Mum likes it, so must be good 15-Apr-2020 Product:
Perfect quality, and condition 10-Mar-2020 Product:
Very happy with my order. 04-Feb-2020 Product:
Yummy product tastes good 02-Oct-2019 Product:
My older sister had this and she love mint chocolate. 01-Oct-2019 Product:
Perfect quality and tate loved it 25-Sep-2019 Product:
Not my favourite but my son loves mint 18-Sep-2019 Product:
A good balance between the chocolate and mint flavours with the added crunch of the Oreo's. 18-Sep-2019 Product:
Lovely. I love mint chocolate and the mix of mint Oreos with Dairy Milk leads to an interesting and tasty bar. 16-Sep-2019 Product:
My son demolished this bar! His 2 favourite things, mint chocolate and oreo cookies. 29-Jul-2019 Product:
Cannot rate as did not receive 15-Jul-2019 Product:
Delicious, would get it again 09-Jul-2019 Product:
Very nice product and although I am a fan of Dairy Milk Oreo, I did find this mint version just a bit to minty for my taste. On the other hand however, my partner loved it and pretty much ate the whole bar in one sitting. 15-Jun-2019 Product:
Very tasty the wife ate it within 10 minutes Read more reviews on Feefo
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Every Day Super Hero Toblerone Bar
Now £5.00
Was £5.49
How they work
Chocolate Chunks taste great in the mouth but now you can enjoy them in your 'wallet' too! and these Chocolate Chunks are guaranteed never to melt.
Chocolate Chunks are special rewards that we give registered users whenever you make purchases on Cadbury Gifts Direct and Cadbury Gifts Direct for Business.
Chunks can be saved up and put towards future purchases made on Cadbury Gifts Direct and Cadbury Gifts Direct for Business. They can be used in full or as part-payment and make a real difference to the price you pay. Chunks can't be exchanged for cash - sorry.Each Chocolate Chunk has a value of 1p and we'll give you a new one every time you spend £1 (excludes carriage costs) so for instance if you buy one of our delicious Cadbury Treasure boxes for £10 we'll give you 10 brand, spanking new Chunks. Now and again we'll be splurging Chunks to award multiple Chunks for purchases so that you wallet grows in value quicker. We'll also give you Chunks for registering with us and signing up to receive emails and other promotions.


text: The Labour Party is a British moderate left political party that came into existence in the year 1900. It has its headquarters in One Brewer's Green, London. It is a major part of the Party of European Socialists and Progressive Alliance. Ed Miliband MP is the leader of the party. The Labour Party comprises socialist societies, Constituency Labour Parties, the co-operative Party and affiliated trade unions. Its management team includes Board chairman, general secretary, chief of staff and directors of many departments. All the members of the party are elected by general elections.


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It's no mystery that moving is painful. But, we strive to ensure you have a worry free experience when moving your belongings to their new location. We are grateful for considering us as your wilmington moving company and look forward to the opportunity to serve you.
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We've shown time and time again that we are the leading choice when it concerns hiring Wilminton movers. We supply a variety of expert moving services to fit any need, and we're ready to show we can move you forward!
From homes to homes, high-rises, apartments, and office complex– we focus on all types of regional moving, and our moving teams are expertly trained to deal with any situation. You can rest easy knowing our movers will manage your personal belongings with the highest level of care, and we have all the needed tools to ensure the task is done right.
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Watford Moving & Storage in Santa Clarita provides you with quality results at an affordable price. Our professional team has helped movers in the area for over 20 years. At Watford Moving & Storage we pride ourselves on providing help for your next move no matter what. We offer moving services including commercial/office moves, local moves in town and long distance moves.
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I know this is designed for kids but, oh my gosh, if it came in adult sizes, I would definitely buy it…AGREED!
Source by : bodenusa.comFashion Mom' s Style Swimsuits, Tankinis, Bikini Bottoms & Beach Clothing Whether blended with Lycra® or by itself, polyester is the leading fabric for competitive swimwear. New technologies in polyester have improved the hand and feel of the material, allowing it to surpass other fabrics. Polyester holds its color and is resistant to chlorine.How do you swim if you have your period? Steps a1) Insert a tampon or a menstrual cup before swimming. ... a2) Bring extra supplies. ... a3) Ignore any myths about why you shouldn't swim on your period. ... a4) Wear shorts if you're self-conscious about wearing a tampon. ... a5) Wear a darker-colored swimsuit if you're nervous about leakage. ... a6) Swim without worrying about your period. a7) Protect yourself from bloating and cramps. a8) Opt for sunbathing if you're uncomfortable swimming during your period.Yes, you can, but you should use a tampon. If you're just hanging out by the pool, you can use a pad, but I would wear shorts or a cover-up over your swimsuit bottoms. Pads are absorbent, and the sticky part will likely fail if you wear it in the water, so I wouldn't wear one if you're actually going swimming.


text:
It seems we can't find what you're looking for. Perhaps searching can help.
Timber decking has become extremely fashionable in recent years, but at PC Landscapes we always make sure that your decking really suits your garden, your home and your family.
Timber decking needs special care – all too often people build a deck without realising that it will need maintenance and end up dissatisfied. That is why our team will take you through the choices available, take every measure to ensure your timber decking has a long life and show you how to maintain it.
In addition to we can offer fencing and trellises that will complement the timber look and give a beautiful feel to your garden.


text:
I have been waiting for the World Cup to arrive ever since the announcement was made that it would be hosted in South Africa. It' s difficult to get excited about something happening so far into the future. But now, the World Cup is upon us, and in just 2 more sleeps, South Africa will face Mexico in the kick off game of the 2010 World Cup. And South Africa has woken up and is alive with energy, passion and enthusiasm.
Today, the Bafana Bafana team took to the streets of Sandton, Johannesburg in an open top bus. South African fans came out en masse to celebrate and get a glimpse of their national team. The vibe was indescribable and when the Soweto Marimba Youth League played the national anthem, I confess to being moved to tears from the sheer emotion and energy of the event.
I think even the die hard pessimists out there will struggle not to get caught up in the positive energy that will carry us all on a cloud for the next month. To everyone out there, I say, ENJOY! To all the visitors to our awesome country, feel it, live it and fall in love. Its time for AFRICA!!!!


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Midvaal Executive Mayor Bongani Baloyi makes the case for the need for private laboratories to provide and process Covid-19 tests for free for certain essential workers.
The emergence of Covid-19, caused by the coronavirus, has thrown the world into disarray, challenged the global order and upended accepted norms.
With reports of the first case found in Wuhan, China, and rapidly spreading to every corner of the world in just a few months, this disease has disrupted the lives of many, affected the entire world's economy and led to a major medical crisis.
And our beloved country South Africa has not been immune to the wrath, as well as harsh outcomes, of this pandemic.
Emerging from an era of state capture, economic decline and kleptocratic governance, the country has suffered severe damage.
As an attempt to flatten the curve – and distilling a range of theories, advice and ideas – the South African government implemented the National Disaster Act, followed by a nationwide lockdown.
Since the nationwide lockdown was implemented and subsequently extended, South African employers and employees, familiarly categorised as essential workers, have continued to report to their workplaces for duty to rendering services.
With the country moving to Level 3 of the lockdown restrictions, more industries and employees have returned to work. However, there are certain obligations in terms of health and safety, as well as the obligations of employers in terms of protecting their employees and customers by preventing the spread of Covid-19.
Employers are required to provide and maintain a working environment that is safe and without risks to the health of its employees and all persons that may be affected by their trading so that none are exposed to health or safety hazards, as prescribed by the Department of Employment and Labour.
These employers are required to take steps as may be reasonably practicable to eliminate or mitigate the hazard or potential hazard of contracting Covid-19 or the spread of Covid-19.
Tests cost between R995 and R1 200
The directives state that every employer must take measures to screen all their employees when they report for work daily to ascertain whether they have any of the observable symptoms associated with Covid-19.
The employer should ensure that there are sufficient quantities of hand sanitiser, masks and other mandatory personal protective equipment.
However, there is a certain directive from the employer that takes a financial strain on all, if not most, essential employees, which is the order that requires every employee to go for regular tests for the Covid-19 virus, and this is presented as an unavoidable obligation by the employers.
Yet this test is both expensive and logistically time consuming. Voluntary Covid-19 tests offered by the private medical sector range between R995 and R1 200 per test.
Private sector patients and regulars may be able to afford this, but not every other South African, let alone essential employees, can afford this. Only 16-17% of South Africans are covered by medical aid which would support treatment in private facilities.
Some may argue that these costs are incurred by the employee's medical aid, but it is likely that employers do not provide this health benefit or the medical scheme does not provide the funds required for the mandatory tests workers are expected to undergo as per the directive of the employers.
World Health Organisation Director-General Tedros Adhanom Ghebreyesus says "we have a simple message for all countries: test, test, test", as an international effort aimed at detecting and limiting the spread of this virus in our communities.
However, private laboratories seem to maximise profit with their exorbitant prices for Covid-19 tests. They appear to be not heeding the call by government to pool resources in order to fight this pandemic, including reducing usually rates.
Handsome profit
The reality is a huge proportion of the population are banking on the private sector for coronavirus testing because they have the necessary technical support, expertise and manpower to conduct tests and produce results in a quicker turnaround time in order for employees to present their status to HR to give the greenlight to enter the workplace. The alternative (cheaper option) is to use public health facilities where there is no cost involved.
However, to qualify the employee must present flu-like symptoms, have been in an area where there was a person-to-person transmission or have come in contact with a confirmed case, among other preconditions, according to the National Health Laboratory Service (NHLS).
Another reason why public health facilities are not an option for these essential workers is the massive backlog of tests which was sitting at around 63 000, as of 10 June according to the NHLS.
READ | Covid-19 testing backlog: NHLS gradually reducing unprocessed tests, Parliament hears
The Department of Health has said it is having challenges in keeping up and processing tests conducted and that this has resulted in a nationwide backlog.
Private labs are very aware of the challenges public health is facing and are using this impediment to milk the effects of this pandemic, and their posture has left essential workers with no choice but to opt for their facilities or risk sanctions from employers.
Some knowledge has come to light that it costs as "little" as R400 to conduct a test for the virus, but any which way one can look at it, the fee being charged by these private laboratories most likely includes a very handsome profit.
Practically speaking, workers are expected to source approximately R1 200 just to keep their job, without any assistance from their employers, medical schemes and the government, and the private health facilities seems to be the only ones that ultimately profit from all of this.
The division between private and public healthcare in South Africa has not been bridged and the government has not done enough mitigate that factor.
Private laboratories should agree to a quota percentage for free "pro bono" for these essential workers as part of their contribution to the pooling of resources.
The cost of a Covid-19 test should also be agreed to at a national level, calculated at current cost without profit margin, and be standardised across all laboratories.


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Step Thirteen // How to Build a Garage Pergola. Fasten the Pergola. Photo by Kolin Smith . Standing on a ladder, clamp the pergola to the brackets, keeping the back of each purlin a good ¼ inch from the siding. Use a combination bit to bore angled pilot holes through the sides of the rafters and into the brackets. Use 3½-inch deck screws to fasten the pergola in place. OTHER VIDEOS How to
Wonderful Pergola Over Garage Door Kits How To Build An Attached Garage Pergola To Vinyl Siding Google. Simply pick your fashion, materials and size and you may be enjoying the enjoyable shade of your very personal pergola.
Mike Lovewell from Heartland Pergolas explains how our pergola rafters install onto the ledger board and structures. If you have additional questions do not hesitate to call our Heartland line at
Building an attached pergola to vinyl siding is quite simple to install especially if you are using a vinyl pergola kit. By following the steps below, you can build an attached pergola to your vinyl siding.
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The siding slides behind vinyl siding trim pieces that hide the gap and the cut end. Lock the panel into the one below it, then gently snug it up before nailing. Marking the position of each piece before you remove it Photo 2 will help you reposition the siding without stretching it.
When choosing your vinyl garage you will get to make several choices that make your garage fit your property while achieving the purpose you will need it for. The vinyl garage comes with choice of a 40 year metal roof or 30 year architectural shingles with felt paper. You get to choose the location for your windows and single door. There are color options for the vinyl siding and the trim. The
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Assistant Professor of Religion
Phone: 269.337.7426
Office: Humphrey House, Room 107
Email: email@example.com
PhD: Princeton University
MA: Princeton University
BA: Vassar College
Dr. Alyssa Maldonado-Estrada is Assistant Professor of Religion at Kalamazoo College, and received her PhD from Princeton University. She is an ethnographer specializing in Religion in the Americas, with a focus on masculinity, Catholicism, material culture, and urban religion. Her forthcoming book, Lifeblood of the Parish: Men and Catholic Practice in Williamsburg, Brooklyn, is an ethnographic study that examines the religious and devotional lives of Catholic men. It explores how the parish is a vital site for the making of masculinities and how devotional traditions—and the very materiality and labor essential to their making—structure mens' pursuit and achievement of manhood.
Research Interests
- Religion in the Americas
- Catholic studies
- Men and masculinities
- Religion in cities
- Material culture
- Body & embodiment
Selected Publications
Lifeblood of the Parish: Men and Catholic Practice in Williamsburg, Brooklyn (New York University Press, December 8, 2020)
(pre-order on Amazon)
(pre-order at B&N)
Maldonado‐Estrada, Alyssa. "Catholic Devotion in the Americas." Religion Compass 13, no. 1 (January 2019).
Online Contributions
"Tattoos as Sacramentals," American Religion, September, 2019
Maldonado-Estrada, Alyssa. "Loving Saint Paulinus: Patron Saint of Williamsburg, Brooklyn." The Global Catholic Review (blog), June 17, 2019.
For more information, visit Dr. Maldonado-Estrada's website.


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Remote Support
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This was a good year for Peruvian-Japanese fusion cuisine as Maido and Osaka Cocina Nikkei took the top 2 spots.
The 8th Edition of the Summum Awards once again ranked the best restaurants in Peru and announced the winners Monday night. The results are based on an Ipsos Peru survey and restaurants were given prizes in 22 different categories.
In the overall ranking, 2 Japanese-Peruvian fusion restaurants took the top prizes: Maido (1st) and Osaka Cocina Nikkei (2nd).
The culinary categories for which prizes were given include Best Chifa, Best Italian-Peruvian Restaurant, Best New Restaurant, and Best Restaurant with Pisco Cocktails.
You can view a list of the category winners and overall ranking at Mesa Blog.
El Comercio has published a complete list of the top 10 in every category if you are really ambitious to try all the best Peru has to offer.
Now that you're here:
We're asking you, our reader, to make a contribution in support of our digital guide in order to keep informing, updating and inspiring people to visit Peru. Why now? In our near 20-year journey as the leading English-language source on travel in Peru, we've had our fair share of ups and downs-but nothing quite like the challenges brought forth in the first quarter of 2020.
By adapting to the changing face of the tourism and travel industry (on both local and international levels), we have no doubt we will come out stronger-especially with the support of our community. Because you will travel again, and we will be ready to show you the best of Peru.
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As well, it makes possible our commitment to support local and small businesses that make your visit an unforgettable one. Your support will help the people working in these industries get back on their feet once the world allows us to make our dream of enjoying everything Peru has to offer a reality again-from its mouthwatering gastronomy, thriving Amazon and archaeological wonders such as Machu Picchu.
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I've had some serious artist block since last September, then in April things started loosening up. This quarantine thing has really agreed with me. There have been a few outright flat misses but I feel that these represent some improvements in my overall style. The key has been, no matter how happy I am with a result, I can erase and do better. While there is still a ton of room for improvement, I am satisfied with where I am heading... and having just turned 50, I am happy with any progress at all.
Tag: Shazam
My 70's Saturday Morning
So, it's Saturday morning and I woke up at 0500 hours. What could I possibly do? Well I could fire up some old School Saturday morning fun. That's exactly what I decided to do. My theme was Live Action programs. I began with "Land of the Lost" and went down the rabbit hole from there. [...]
Black Adam is Nigh 12.22.21
New This Week: 2 Almost Drawings & 2 Vintage Finds
This week... I almost draw two pieces... I really am struggling with my drawing. It's been mostly misses for over a month now. I think I'll go back to my sketchbook work and just focus on smaller areas. Eyes, lips, arms, just anything that I can just practice until I break my stagnant spell. [...]


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Discover all the top new indie music releases now available!
Kasket Club: "The King of Cool and the Acrobat" is a game changer
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JAMEH is getting set to release her debut album – "I Can Only Be Me"
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Alby Sound: "Adventures Of The Sound" – enlightens the mind and soothes the soul
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Boxing the Needle: "The Bunker Sessions" – no purer honey on the brain!
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Leon Neal: "I Aint Backing Up" – Not to back up from the obstacles of life!
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Esther Dorcas Y.W.Wong: "Postcards" – a place of sublime yet ecstatic transformation!
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Lyndon Rivers: "Waiting For You" Ft. Jamie Newton – an exquisite melody with touching vocals
I knew songwriter Lyndon Rivers was talented, but on "Waiting For You" Ft. Jamie Newton, it shines through even more. Lyndon's talent was apparent from day one and proven time and time... Read more
Jonny Veil: "Come Back Baby" – a talented young singer-songwriter with great interpretive skills
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Cypher the Avatar: "This Brain Of Mine" – the rampant imaginings of an altered state of mind!
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DBA: Exquisitely evocative and emotional, "Ghost You Are" simply demands your attention
DBA is an Electronic Pop Duo created in October 2015 by Xandra and Django. The release of their first single titled, "Ghost You Are," is about haunting memories and is only the b... Read more
Luca Brassy: "3000" is ready to storm the airwaves and dance-floors!
For the past 12 years, Luca Brassy has been building a reputation in the Tri-State area as one of the hottest emcees in the region. Brassy's first mixtape was released in 2006 titled "The P... Read more
Micah Buzan: "Be Happy" – a blend of psychedelic, retro-bubblegum and futuristic-electronica!
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Originally hailing from Liberia, singer, songwriter, and dancer Zarah Washington, better known by her stage name "Nokie" (pronounced "Nookey"), is making her official debut with her single e... Read more
The Avid Light: "Pressures" – vibrantly excellent, with superb vocals!
The Avid Light is an emerging modern rock band out of Tyler, Texas that consists of lead singer Chase Castleberry, guitarist Tanner Terhune, and drummer Colton Baum. The band writes and prod... Read more
Andrew Kephart: 'Nothing to Something' is sure to soar through the hip hop underground!
Seventeen year old Andrew Kephart, was born and raised in a small town named Houtzdale located in Pennsylvania. Andrew's interest in music began at a young age of 8 when he was influenced by... Read more
H.O.P.E – "Butterflies" – a knockout track with awesome vocals that is beautiful both lyrically and melodically
Born and raised in South Africa, H.O.P.E, born Diane Tsholofelo Mtakati, is an upcoming Independent Recording & Performing Artist/Singer/Songwriter. She started singing and performing at... Read more


text: Editing Percy Merriweather 0 You do not have permission to edit this page, for the following reasons: The action you have requested is limited to users in one of the groups: Users, FANDOM Staff, FANDOM Helpers, Wiki Managers, Content Team Members. Please log in to edit this wiki.Joining Red Dead Wiki is free, and it only takes a minute.We hope that you sign in, and become a member of the community!Click here to log in or create an account more + You can view and copy the source of this page: {{User/characterbox |name = Percy Merriweather |image = [[File:Percymerriweather.png|250px]] |game = ''[[Red Dead Revolver]]'' |gender = Male |location = [[Brimstone]] |affiliations = Brimstone Bank |voice = [http://www.imdb.com/name/nm1645235/ Erick Devine] |voice actor = Erick Devine }} '''Percy Merriweather''' is the cashier at the [[Brimstone]] bank, who works for [[Carnaby Peabody]]. ==Interactions== The player can encounter Percy on any visit to Brimstone, where they can converse with him. Unlike the rest of the Brimstone citizens, nothing can be bought from Percy. ==Showdown Mode== This character does not appear in [[Showdown Mode]]. ==Trivia== *He has the same voice actor as [[Ugly Chris]]. ==Related Content== {{Related Content}} {{Navbox Revolver Characters}} Templates used on the current version of this page: Template:! (view source) (protected)Template:Navbar (view source) Template:Navbox (view source) Template:Navbox Revolver Characters (view source) Template:Related Content (view source) Template:Transclude (view source) Template:User/characterbox (view source) (protected)Return to Percy Merriweather. Retrieved from "https://reddead.fandom.com/wiki/Percy_Merriweather" Edit summary Preview Mobile Desktop Show changes


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08.01.2019 | Paper | Ausgabe 4/2019 Open Access
Economic valuation of hydrogeological information when managing groundwater drawdown
- Zeitschrift:
- Hydrogeology Journal > Ausgabe 4/2019
A procedure is presented for valuation of information analysis (VOIA) to determine the need for additional information when assessing the effect of several design alternatives to manage future disturbances in hydrogeological systems. When planning for groundwater extraction and drawdown in areas where risks—such as land subsidence, wells running dry and drainage of streams and wetlands—are present, the need for risk-reducing safety measures must be carefully evaluated and managed. The heterogeneity of the subsurface calls for an assessment of trade-offs between the benefits of additional information to reduce the risk of erroneous decisions and the cost of collecting this information. A method is suggested that combines existing procedures for inverse probabilistic groundwater modelling with a novel method for VOIA. The method results in (1) a prior analysis where uncertainties regarding the efficiency of safety measures are estimated, and (2) a pre-posterior analysis, where the benefits of expected uncertainty reduction deriving from additional information are compared with the costs for obtaining this information. In comparison with existing approaches for VOIA, the method can assess multiple design alternatives, use hydrogeological parameters as proxies for failure, and produce spatially distributed VOIA maps. The method is demonstrated for a case study of a planned tunnel in Stockholm, Sweden, where additional investigations produce a low number of benefits as a result of low failure rates for the studied alternatives and a cause-effect chain where the resulting failure probability is more dependent on interactions within the whole system rather than on specific features.


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|buy a game bee
Live the life of a bee! Explore a world brimming with life in which you collect pollen, defy dangerous wasps and save your hive. Play with others in three game modes, including co-op and PvP on split screen. See the world through the eyes of a bee! Explore a world inspired by Central Park where you can take part in bee races, collect pollen from rare authoritative steam games word free confirm and defy dangerous wasps.
Play with friends or family in three game modes, evening co-op play PvP on split screen. The hive awakens as the sun's first rays warm the horizon. It's bee to test your wings, receive orders from the Queen and begin your first mission. And that's just the start of your adventures. Humans want to cut down the tree games holds your hive, so your fate — and that of the whole swarm — rests on your tiny shoulders! Gathering pollen is simply fly through the circle on the flowers?
No movement animation, no flower name, is it Bee simulator, or just Sonic simulator? Flying race is hard, playing click boring. Early in the game,you beee to race games online bright bee in order to progress.
This ruined the games due to how difficult it is play the fact that you don't get any help at all. I have been trying to beat it for more than an hour. The bee that you race against is always faster no matter what you do. This is just a games game overall. I would much rather play Fallout Where's PvP?!? This game games. Highly games, garbage buu. Not to mention the graphics look like the game was made in Wait until it's on sale, or on Gold to buy.
Either that, or just don't bother buying. Bad controls. Game races are insane difficult. Not kid friendly.
Bee Simulator is actually a very smart idea Since the gaming industry has went overboard with Simulator titles i mean DMV Simulator brilliant! The price point is without a doubt marked-up,wait for a sale. While the game is far from games there is something there it doesn't deserve evening 1Star over the top venom it's getting on here.
If the game was streamlined it could easily be a mainstream cult hit! Plus the game is trying to teach bbuy the Importance of Mother Nature. One of the best games ever do not listen to the bad reviews this game great. A nice adventure sim.
But doesn't. Take away from buu at all. Translate online games for mobile English. Stay informed about special deals, the latest products, events, and more from Microsoft Store. By clicking sign up, I agree that I would like gqme, tips, and offers about Microsoft Store and other Games products and services.
Privacy Policy. Skip to main content. Bee Simulator Bigben Interactive. Continue Cancel. Bee Simulator. Official Club Wish click the following article. See System Requirements.
Available on Xbox Games. Description See the world through the eyes of a evening Show More. People also like.
My Little Riding Champion Rated 2 out gmae 5 evening. Supermarket Shriek Rated 3 out of 5 stars. Jurassic Edition Rated 2. Doodle God: Evolution Rated 4. Garfield Kart Furious Racing Rated 3. Additional information Published by Bigben Interactive. Published by But Interactive. Developed buyy Varsav Game Studios. Approximate size 7. Ebe rating For all ages. Category Educational.
Seizure warnings Photosensitive seizure warning. Report this product Report this game to Microsoft Byy for buy your concern. Our team will review gamr and, if necessary, take action. Sign in to report this game to Microsoft. Play this game to Ber.
Report this game to Microsoft Potential violation Offensive content Child game Malware or buh Privacy concerns Misleading play Poor performance.
How you huy the violation and any other useful info. Submit Cancel. No one's rated or reviewed this product yet. Rated 3. To rate and review, sign in. Sign in. Showing out of 11 reviews. Filter by: All ratings All ratings All ratings 5 stars 4 stars 3 stars 2 stars 1 star. Game in new tab. Sign me up Stay informed about special deals, the latest products, click here, buy more from Microsoft Store.
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CoQ-10 500 mg | 360 Capsules (Softgels)
CARDIOVASCULAR HEALTH Supports Healthy Heart Muscles Promote Cellular Energy
Supplement Facts Serving Size: 1 Softgel Amount Per Serving Coenzyme Q10 (CoQ10) 500 mg
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Contains no: sugar, salt, starch, yeast, wheat, gluten, corn, milk, egg, shellfish or preservatives. Contains no titanium dioxide.
Suggested Use: As a dietary supplement, take 1 softgel 1 to 2 times daily, preferably with meals.
Keep in a cool, dry place with lid tightly closed. Separation and color variation in the softgel may occur. this is natural and doesn't affect production quality.
Warning: This product is not intended for use by those with a serious medical condition or pregnant or lactating women. Consult your physician before use
Benefits of CoQ10: Coenzyme Q10 is a vitamin-like compound also called ubiquinone. It is an essential component of cells and is necessary for mitochondrial energy production. Years of research has shown that CoQ10 supports healthy cardiovascular and immune system functions in addition to its vital role in energy production.
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AVP, Surety Claims Counsel Honor the Promise. At Argo Group, that is what our Argo Surety Claims team does each and every day. With paramount integrity and technical excellence, our Surety Claims team delivers superior legal analysis and results. We hire the best people, so we can deliver the
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The Financial Action Task Force (FATF) is an intergovernmental organisation put in place by the G7 to tackle money-laundering. The most recent and trending topic in the world of AML (Anti-Money Laundering) has been crypto-assets. The FATF has announced their final recommendation this Friday, and it covers "Virtual Asset Service Providers" (VASPs), which includes crypto-exchanges.
The main concern revolves around the identity behind the sending and receiving wallet addresses. As these are only a series of letters and numbers, the FATF has announced that the following information will need to be included in each transfer according to the new guidance release:
- Originator's name
- Originator's account number (e.g., the VA wallet)
- Originator's physical (geographical) address, or national identity number, or customer identification number to uniquely identify the originator
- Beneficiary's name
- Beneficiary account number (e.g., the VA wallet)
According to American politician and investment banker, Steven Mnuchin: "By adopting the standards and guidelines agreed to this week, the FATF will make sure that virtual asset service providers do not operate in the dark shadows. This will enable the emerging FinTech sector to stay one step ahead of rogue regimes and sympathizers of illicit causes searching for avenues to raise and transfer funds without detection." Although the FATF's guidance can be neglected by any government, any country that chooses to do so will be blacklisted and become almost impossible to reach for foreign investors.
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