text:
- Research article
- Open Access
Validation of reference genes for normalization of qPCR gene expression data from Coffea spp. hypocotyls inoculated with Colletotrichum kahawae
- Andreia Figueiredo†1Email author,
- Andreia Loureiro†2,
- Dora Batista2,
- Filipa Monteiro1,
- Vítor Várzea2,
- Maria Salomé Pais1,
- Elijah K Gichuru3 and
- Maria Céu Silva2
© Figueiredo et al.; licensee BioMed Central Ltd. 2013
- Received: 21 May 2013
- Accepted: 25 September 2013
- Published: 28 September 2013
Coffee production in Africa represents a significant share of the total export revenues and influences the lives of millions of people, yet severe socio-economic repercussions are annually felt in result of the overall losses caused by the coffee berry disease (CBD). This quarantine disease is caused by the fungus Colletotrichum kahawae Waller and Bridge, which remains one of the most devastating threats to Coffea arabica production in Africa at high altitude, and its dispersal to Latin America and Asia represents a serious concern. Understanding the molecular genetic basis of coffee resistance to this disease is of high priority to support breeding strategies. Selection and validation of suitable reference genes presenting stable expression in the system studied is the first step to engage studies of gene expression profiling.
In this study, a set of ten genes (S24, 14-3-3, RPL7, GAPDH, UBQ9, VATP16, SAND, UQCC, IDE and β-Tub9) was evaluated to identify reference genes during the first hours of interaction (12, 48 and 72 hpi) between resistant and susceptible coffee genotypes and C. kahawae. Three analyses were done for the selection of these genes considering the entire dataset and the two genotypes (resistant and susceptible), separately. The three statistical methods applied GeNorm, NormFinder, and BestKeeper, allowed identifying IDE as one of the most stable genes for all datasets analysed, and in contrast GADPH and UBQ9 as the least stable ones. In addition, the expression of two defense-related transcripts, encoding for a receptor like kinase and a pathogenesis related protein 10, were used to validate the reference genes selected.
Taken together, our results provide guidelines for reference gene(s) selection towards a more accurate and widespread use of qPCR to study the interaction between Coffea spp. and C. kahawae.
- Reference Gene
- Common Bean
- Normalization Strategy
- Candidate Reference Gene
- Receptor Like Kinase
Coffea arabica L. production in Africa can be seriously affected by coffee berry disease (CBD), caused by the hemibiotrophic fungus Colletotrichum kahawae JM [1]. Despite the fact that several plant organs can be affected, maximum production losses occur when infection takes place in expanding green berries, leading to their premature dropping and mummification [2, 3]. Crop damages due to CBD, along with chemical control costs, accounts annually for a loss of US$ 300–500 millions in Arabica coffee production [4].
Since its first report in 1922 in Kenya [5], CBD rapid outbreak throughout Eastern Africa urged the development of breeding programmes in several countries (such as Kenya, Ethiopia and Tanzania) giving rise to the release of several resistant coffee varieties for coffee growers [2, 4]. In Kenya, the most relevant example is the hybrid commercial variety Ruiru 11, which was bred for resistance to CBD and coffee leaf rust (Hemileia vastatrix) using lines of the coffee cultivar Catimor as resistance sources. Breeding programmes remain the most economical and environmentally sound method for a sustainable coffee production. Presently, with the advances in genomic and transcriptomic resources, breeding for resistance can be supported and thus improved by molecular based-information.
Molecular research on the coffee - C. kahawae pathosystem has been lagging behind other plant-Colletotrichum spp. interactions although advances on the mechanisms of pathogen infection and host resistance at cellular level were achieved [2, 3, 6] Deepening the knowledge on the molecular basis governing coffee resistance to C. kahawae is thus fundamental to get some insights on the distinctive processes underlying plant resistance response. Monitoring gene differential expression and validating high-throughput RNA sequencing (RNA-seq) data is ideally achieved through quantitative real-time PCR (qPCR) analysis. Regardless of being an extremely powerful technique relative to sensitivity, specificity and broad quantification range, accurate data normalization with a reference gene(s) is an absolute requirement for qPCR correct measurement of gene expression. In this study, we have tested 10 candidate genes for qPCR normalization of gene expression during the first hours of interaction (12, 48 and 72 hpi) with C. kahawae. Two coffee genotypes, resistant and susceptible to C. kahawae were used. The best combination of reference genes determined for each dataset was used to further assess the expression of a pathogenesis-related protein 10 (PR10) and a receptor like kinase (RLK) known to be induced during coffee – leaf rust interactions [7, 8]. Here we provide, for the first time, a set of reference genes suitable for gene expression studies in both resistant and susceptible coffee genotypes to C. kahawae.
Results and discussion
Normalization is one of the key factors affecting the accuracy and reliability of quantitative gene expression analysis. Here, we describe an assessment of ten candidate reference genes (RGs) for their use as internal controls in gene expression studies of coffee defence responses against C. kahawae in two different genotypes, Caturra as susceptible and Catimor 88 as resistant. Data were analyzed considering the entire dataset and each of the coffee genotypes separately.
Amplification specificity and efficiency
Candidate reference genes and target genes primer sequences, amplicon length and qPCR analysis
Gene (coffee
Primer sequence
Amplicon length (bp)
Ta (°C)
Tm (°C)
PCR efficiency (%)
Regression coefficient (R2)
Average Cq value
source gene)
Gamm et al. [29]
Fw: ATAGAGAGAGAGCCCCCAATTC
Discarded (unspecific amplification, primer dimer)
Rev: CCGAGGAAGCCAAAAAAAG
UQCC (isotig09120)
Fw: CCTCGGGCTTCATCTCTACTC
Rev: TCTCCCCAGCATTTTTTGTC
SAND (isotig05620)
Selim et al. [30]
Fw: GATTTGTCTACGAACCCTGCTT
Rev: GGACCACCAACACCAATAAAC
Ramiro et al. [7]
Fw: AACATTGAGGGTGGTTCTGTTC
24.5 ± 0.95
Rev: GCAGAAAACCAACTAAGACCTAACAA
S24 (SNG-U349723)a
Cruz et al. [28]
Fw: GCCCAAATATCGGCTTATCA
20.61 ± 1.04
Rev: TCTTCTTGGCCCTGTTCTTC
IDE (isotig10635)
Borges et al. [12]
Fw: TGATCTAAGCTGGTGGAAAGC
24.46 ± 1.30
Rev: TCAGGTGCATCAGGATGATT
β- Tub9 (isotig08544)
Fw: ACCCTCCAGCAAACTGATGA
19.40 ± 0.82
Rev: AGGATGCCACTGCTGATGAT
14-3-3 (SGN-U356404)a
Barsalobres-Cavallari et al. [18]
Fw: TGTGCTCTTTAGCTTCCAAACG
22.76 ± 1.47
Rev: CTTCACGAGACATATTGTCTTACTCAAA
GADPH (SGN-U347734)a
Fw: TTGAAGGGCGGTGCAAA
21.42 ± 1.96
Rev: AACATGGGTGCATCCTTGCT
RPL7 (SGN-U351477)a
Barsalobres-Cavallari et al. [18]
Fw: CATTCGAGGTATCAATGCTATGCA
23.76 ± 1.28
Rev: TGTCTCAGGCGCAGAAGCT
Genes of interest (GOIs)
Ramiro et al. [7]
Fw: GCCACCATCCTTGAAGAGAA
19.35 ± 2.28
Rev: CAACTCTCTGCTTGGCAGTCT
Fw: ATGGGAGAAAAGAATGGCAGAAG
24.81 ± 2.02
Rev: GGCCAATTACAGTTTGAAAACACC
PCR efficiency of each primer pair was calculated through the standard curve method using a pool of all cDNA samples in a ten-fold serial dilution. The amplification efficiency (E) of the reactions ranged from 1.886 (89%) to 2.033 (103%) with the correlation coefficients R2 varying from 0.98 to 0.99 (Table 1). To assure that any variation between biological replicates was not related to the treatments but intrinsic to the gene itself, data from the biological replicates was analysed separately by statistical algorithms [9, 10].
Analysis of gene expression stability data
The expression stability of the selected RGs was addressed by three different statistical applets: GeNorm, NormFinder and BestKeeper. The results were analyzed by dividing the data into three different datasets: all samples in the assay (entire dataset), biotic stress applied to the coffee cultivars Catimor 88 and Caturra, separately.
The geNorm algorithm calculates the gene expression stability measure (M value) for each gene based on its average pairwise expression ratio relative to each of the other genes in the analysis. A gene displaying a high M value presents a high variance in its expression. After, GeNorm estimates the normalization factor (NF) using the geometric mean of expression levels of n best reference genes, using a pairwise variation (V) with a cut-off value of 0.15 as threshold [11].
Reference genes ranking for the entire dataset calculated by the GeNorm, NormFinder and BestKeeper
Entire dataset
2.54 ± 0.62(2)
2.61 ± 0.59(4)
1.98 ± 0.47(1)
2.44 ± 0.47(3)
2.79 ± 0.57(5)
2.23 ± 0.54(6)
Reference genes ranking for Catimor 88 and Caturra inoculated with C. kahawae
Biotic stress
Catimor 88 (resistant)
Caturra (susceptible)
2.82 ± 0.69(4)
2.09 ± 0.50(2)
2.59 ± 0.58(3)
2.71 ± 0.61(5)
2.09 ± 0.49(1)
1.86 ± 0.44(1)
2.34 ± 0.45(2)
2.37 ± 0.46(4)
2.87 ± 0.59(5)
2.34 ± 0.48(3)
1.75 ± 0.43(6)
2.61 ± 0.63(6)
2.85 ± 0.58(7)
NormFinder is based on a variance estimation approach, which calculates an expression stability value (SV) for each gene analysed. It enables estimation of the overall variation of the RGs, taking into account intra and intergroup variations of the sample set. According to this algorithm, genes with lowest SV will be top ranked [15]. For the entire dataset, IDE was considered as the most stable gene (Table 2), while for Caturra and Catimor 88 datasets it appeared in the three top ranked genes, showing in general a high stability as an internal control for coffee hypocotyls inoculated with C. kahawae. In addition, the best combination obtained for gene expression analysis of the different genotypes inoculated with C. kahawae was β-Tub9/40S ribosomal protein S24 (S24) for Caturra and β-Tub9/IDE for Catimor 88 (Table 3). GAPDH and UBQ9 appeared as the least stable genes when analysing the entire dataset and the susceptible genotype Caturra. For the resistant genotype Catimor88, GAPDH and S24 were the most unstable genes.
The BestKeeper tool calculates standard deviation (SD) and the coefficient of variation (CV) based on quantification cycle (Cq) values of all candidate RGs [16]. Reference genes with SD values greater than 1 could be considered as inconsistent and should be excluded. Conversely, genes with the lowest SD value are proposed to be the most stable [16]. BestKeeper estimates the intergene correlation of all reference gene pairs, and the Pearson correlation coefficient (r) and the probability (p) value are predicted for each correlation. In this study, BestKeeper analysis considered RPL7 and β-Tub9 as the most stable genes for the entire dataset, with SD values of 0.47 in both cases (Table 2) with p < 0.001. For cultivar Caturra during inoculation time-course β-Tub9 and S24 were considered the most stable with SD values of 0.46 and 0.48, respectively, with the best correlations (r = 0.882 and 0.787, respectively) and a p value of 0.005. For Catimor 88, IDE was selected as the most stable with an SD value of 0.69, high correlation (r = 0.790) and a p value of 0.005 (Table 3).
Interestingly, despite belonging to important classes of cellular functioning, GAPDH and UBQ9 were indicated by BestKeeper analysis as the least stable expressed genes for the entire dataset and for Catimor 88 under biotic stress, which was consistent with the results obtained from geNorm and NormFinder. GAPDH has been widely used as a reference gene in many experimental conditions [17] and is one of the best RGs for measuring gene expression in different C. arabica tissues/organs, namely root, stem, leaf, flower and fruit [18]. However previous examples of this gene leading to wrong results have also been reported due to its lack of stability under specific experimental conditions [17, 19]. In the present analysis, GAPDH was not among the best RGs between experimental groups. One of the possible reasons for those discrepancies may be that GAPDH not only acts as a component of the glycolytic pathway but takes part in other processes as well, thus the expression profile of GAPDH might fluctuate according to the corresponding experimental conditions. UBQ9 was also pointed out as a good RG for C. arabica leaves inoculated with Hemileia vastatrix[7]. Yet, for coffee hypocotyls inoculated with C. kahawae it was considered one of the least stable genes, which is in accordance with Borges et al. [12] whom pointed out Ubiquitin as one of the least stable genes for common bean inoculated with C. lindemuthianum in several tissues including hypocotyls. These variations in the expression profiles of genes normally used as internal controls confirm the need for validation of RGs under each specific condition of interest.
A comprehensive ranking analysis considering the three applets (Additional file 2) revealed that, in the specific pathosystem coffee hypocotyls-C.kahawae, the two most stable genes, were IDE and β-Tub9 for the resistant genotype and the combination IDE + β-Tub9 or S24+ β-Tub9 for the susceptible genotype.
Expression analysis of PR10 and RLK
The coffee RLK gene is predicted to encode a receptor-like kinase [20], being some members of this class of proteins involved in resistance signalling pathways [21], while PR10 seems to be related with the jasmonic acid (JA)-dependent resistance pathway [22] and to accumulate in host cells in incompatible interactions [23, 24]. Both PR10 and RLK genes have been described to be induced during coffee infection with Hemileia vastatrix[8, 20].
The expression of both PR10 and RLK was studied during both coffee compatible and incompatible interactions with C. kahawae. Three normalization strategies were followed in order to validate the results obtained by GeNorm, Normfinder and Bestkeeper: 1) using the two best RGs given by the comprehensive ranking, (2) selecting the optimal number of RGs based on GeNorm pairwise variation value (the 0.15 cut-off value was followed) and (3) using the two least stable RGs. Thus, PR10 and RLK expression was normalized in Catimor 88 with a normalization factor (NF) calculated based on the expression of (1) IDE and β-Tub9, (2) RPL7, IDE, 14-3-3 and β-Tub9 and (3) GADPH and UBQ9. For Caturra, the NF was calculated based on the expression of (1) IDE and β-Tub9 or S24 and β-Tub9, (2) IDE, S24 and β-Tub9 and (3) GADPH and UBQ9.
For Caturra inoculated with C. kahawae, β-Tub9 appeared as the most stable, followed by both IDE and S24. Thus, normalization with the two best RGs was tested using either a combination of IDE + β-Tub9 or S24+ β-Tub9. When analysing RLK and PR10 expression in comparison to control samples, no significant expression differences were obtain at 12 hpi with the three normalization strategies tested (Figure 2B, Additional file 3). Also, at 48 hpi and 72 hpi, using the two best RGs for normalization (two best genes selected by the statistical applets and also by GeNorm V2/3 value), expression levels and SD values did not varied significantly either with IDE + β-Tub9 or S24+ β-Tub9 (Figure 1). However, when using the combination S24+ β-Tub9, lower SD values were obtained and PR10 and RLK expression levels were very similar to those obtained with the three best RGs given by GeNorm V3/4 value (Additional file 3). It may be considered that despite IDE and S24 shared the second position in the comprehensive ranking of the three statistical applets; S24 is more stable than IDE and thus S24+ β-Tub9 combination should be used for normalization. Interestingly, expression of both genes was not significantly altered when normalizing with the two most unstable genes (GADPH and UBQ9). On the contrary, PR10 and RLK expression levels were slightly smaller or presented higher SD values when normalizing with the three best RGs given by geNorm V = 3/4 (Figure 1). For the susceptible coffee cultivar Caturra inoculated with C. kahawae, despite being considered the most unstable RGs by GeNorm, NormFinder and BestKeeper, GADPH and UBQ9 presented a more stable expression than in the resistant genotype Catimor 88 (Figure 2). These results reflect the need to not only validate reference genes for specific experimental conditions, but also for different genotypes.
A non- parametric test was used to account for any significant differences in the expression of PR10 and RLK calculated with the different normalization strategies (Additional file 4). As no statistically differences were found, we may assume that for an accurate normalization, in the present experimental system, the two most stable RGs suggested by the three applets may be used, i.e. IDE + β-Tub9 for Catimor 88 and S24+ β-Tub9 for Caturra.
In the present study, we have evaluated the expression stability of 10 candidate reference genes along C. kahawae inoculation time-course of two coffee genotypes showing a susceptible vs resistant response towards this pathogen, aiming to identify a set of stable reference gene(s) for data normalization of gene expression studies. Analysis of expression stability using GeNorm, NormFinder and Bestkeeper revealed that the expression of β-Tub9 and IDE is most stable across all datasets tested. In addition, several normalization strategies were tested under an additional validation step using RLK and PR10, and statistical analysis revealed that for an accurate normalization the two most stable RGs suggested by the three applets should be used and that a different combination of reference genes should be applied for susceptible and resistant genotypes inoculated with C. kahawae. Thus IDE + β-Tub9 should be used for transcript normalization of Catimor 88 hypocotyls inoculated with C. kahawae and S24+ β-Tub9 for transcript normalization of Caturra hypocotyls inoculated with C. kahawae.
Since, up to our knowledge, no reference genes have yet been established for inoculation studies of coffee hypocotyls, we believe that the information provided by our study will greatly facilitate future coffee research and enable sensitive and accurate quantification of gene expression in coffee genotypes showing different degrees of resistance towards C. kahawae.
Inoculation of coffee hypocotyls
Hypocotyls were used as a model material to study CBD in controlled conditions since previous studies have shown a correlation between the pre-selection test on hypocotyls and mature plant resistance in the field (r = 0.73–0.80) [25]. Coffee seeds were sown in greenhouse conditions at temperatures between 16°C and 28°C (average minimum and maximum temperatures respectively), during 8 weeks. Conidia of C. kahawae isolate Que2 (from Kenya) were produced after 7 days at 22°C on extract malt agar [26]. Hypocotyls of cultivars Catimor 88 (from Kenya) and Caturra (CIFC 19/1), resistant and susceptible to the C. kahawae isolate used, were then inoculated according to the technique described [25] with slight modifications. Briefly, hypocotyls were vertically placed on plastic trays containing a wet nylon sponge and sprayed with a conidia suspension (2×106/ml) or with water (mock-inoculated hypocotyls – control samples). After inoculation, trays were covered with plastic bags and and kept in a Phytotron 750 E at 22°C in the dark for 24 h, and then under a photoperiod of 12 hours during the infection time-course.
Sample collection
According to previous microscopic analysis [27], hypocotyl tissues were sampled at times corresponding to different stages of pathogenesis: i) Conidia germination and appressoria differentiation (in both coffee genotypes) at 12 hpi; ii) fungal penetration and establishment of biotrophic phase (susceptible genotype) or beginning of hypersensitive cell death (HR) and accumulation of phenols (resistant genotype) at 48 hpi; iii) switch to the necrotrophic phase (susceptible genotype) or display of HR and phenols deposition in more that 50% of infection sites (resistant genotype) at 72 hpi. Hypocotyls were thus harvested at 12, 48 and 72 hours post inoculation (hpi). Two independent experiments were conducted and 40 hypocotyls were collected for each coffee genotype (Catimor 88 and Caturra, control and inoculated) at each of the three time-points. Plant material was immediately frozen in liquid nitrogen and stored at 80°C.
RNA extraction and cDNA synthesis
Total RNA was isolated from hypocotyls with the Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, USA) according to the manufacturer's instructions. Residual genomic DNA was digested with DNase I (On-Column DNase I Digestion Set, Sigma-Aldrich, USA). RNA purity and concentration were measured at 260/280 nm and 260/230 nm using a spectrophotometer (NanoDrop-1000, Thermo Scientific), while RNA integrity was verified by agarose gel electrophoresis. Genomic DNA (gDNA) contamination was checked by qPCR analysis of a target on the crude RNA [11]. Complementary DNA (cDNA) was synthesized from 2.5 μg of total RNA using RevertAid®H Minus Reverse Transcriptase (Fermentas, Ontario, Canada) anchored with Oligo(dT)18 primer (Fermentas, Ontario, Canada), according to manufacturer's instructions.
Candidate gene selection and primer design
Ten candidate genes were selected based on their previous reports of suitable qPCR reference genes associated either to Coffea arabica or to biotic stress [7, 12, 18, 28–30]. Five of these genes were previously described as reference genes for C. arabica: S24, 14-3-3, RPL, GAPDH) and UBQ9. The other genes were retrieved from coffee (HDT 832/2) database (unpublished data) as being homologous to grapevine VATP16, SAND, UQCC and homologous to common bean IDE and β-Tub9.
Specific primers (Table 1) were designed with Primer Express software version 3.0 (Applied Biosystems, Sourceforge, USA) using the following parameters: amplicon length between 75 and 250 bp; size: 20 ± 2 bp; annealing temperature (Ta) between 55°C and 60°C; GC content: ± 50%.
Quantitative real time PCR
Quantitative RT-PCR (qPCR) experiments were carried out using Maxima™ SYBR Green qPCR Master Mix (2×) kit (Fermentas, Ontario, Canada) in a StepOne™ Real-Time PCR system (Applied Biosystems, Sourceforge, USA). A final concentration of 2.5 mM MgCl2 and 0.2 μM of each primer were used in 25 μL volume reactions, together with cDNA as template. The amplification efficiency of each candidate/target gene was determined using a pool of identical volumes of all cDNA samples. The pool was diluted and used to generate a five-point standard curve based on a ten-fold dilution series. Each standard curve was amplified in two independent qPCR runs and each dilution was run in triplicate. Amplification efficiency (E) was calculated from the slope of the standard curve (E = 10(−1/a) -1) where a is the slope of the linear regression model (y = a log(x) + b) fitted over log-transformed data of the input cDNA concentration (y) plotted against Cq values (x).
To investigate candidate reference gene stability, cDNA samples were 10-fold diluted. Thermal cycling for all genes started with a denaturation step at 95°C for 10 min followed by 45 cycles of denaturation at 95°C for 15 s and annealing temperatures (Table 1) for 30 s. Each set of reactions included a negative control with no template. Dissociation curves and agarose gel electrophoresis were used to analyze non-specific PCR products. Two biological replicates and three technical replicates were used for each sample.
Determination of gene expression stability
The expression stability of each candidate reference gene and the best combination of reference genes were obtained using a pairwise method by GeNorm [11], a model-based method by NormFinder [15] software and the BestKeeper tool [16]. The analysis was performed considering three groups: resistant hypocotyls (Catimor 88) compared to control (mock-inoculated) samples dataset, susceptible hypocotyls (Caturra) compared to control (mock-inoculated) samples dataset and entire dataset. The definition of the optimal number of genes required for normalization was achieved by GeNorm pairwise variation analysis [31]. A comprehensive ranking was established by calculating the arithmetic mean ranking value of each gene using the three applets [32], and each gene was ranked from 1(most stable) to 7 (least stable).
Finally, RefFinder (http://www.leonxie.com/referencegene.php) was used as a verification tool of our results (Additional file 5). RefFinder is a comprehensive tool that integrates the currently available major computational programs (GeNorm, Normfinder, BestKeeper, and the comparative ΔCt method) and based on the rankings from each program assigns an appropriate weight to an individual gene, and calculates the geometric mean of their weights for the overall final ranking [33].
PR10 and RLK gene expression
The expression of two defense-related genes, a receptor like kinase (RLK, CF589181) and a pathogenesis-related protein 10 (PR10, CF589103), previously described as being differentially expressed in coffee leaves inoculated with Hemileia vastatrix[7, 8] was studied in the coffee-C. kahawae interaction. Three normalization strategies were tested: 1) using the two top genes given by a comprehensive ranking considering the three methods (GeNorm, NormFinder and BestKeeper); 2) using the optimal number of reference genes selected by the GeNorm V value (pairwise variation analysis, Figure 1) for each condition studied, and 3) using the two most unstable genes considering the comprehensive ranking of the three methods (GAPDH and UBQ9).
To assess gene expression, relative quantities (RQ) were calculated for both RGs and genes of interest (GOIs) by the formula RQ = EΔCq, where E represents the amplification efficiency (E) for each gene and ΔCq the difference in the Cq from each target sample and calibrator (ΔCq = Cqcalibrator – Cqtarget) [16, 34]. A normalization factor calculated as the geometric mean of the relative expression of the RGs selected for each normalization strategy was used to obtain the normalized relative quantities (NRQ) [35].
The statistical significance (p < 0.05) between the three normalization strategies used was determined by the Kruskall-Wallis test using IBM® SPSS® Statistics version 20.0.0 (SPSS Inc., USA) software.
This work was funded by Portuguese national funds through Fundação para a Ciência e a Tecnologia (project PTDC/AGR-GPL/112217/2009, grants SFRH/BPD/47008/2008 and SFRH/BPD/63641/2009) and through BIOFIG PEst-OE/BIA/UI4046/2011. We also appreciate the technical support provided by Paula Leandro and Sandra Sousa.
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Book Designers
Book Designers In Minnesota
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Page Designers In Minnesota
Nader Youssef
Freelance Book Designer & Page Designer
Saint Paul, Minnesota, United States
2 Skills
To establish a long-term career in computer support and software training with a large reputable organization
• Certified Quality Manager Diploma, The American University in Cairo - [From: Sept. 2001 To: May 2002]
• Professional Diploma in TQM, The American University in Cairo - [From: Sept. 2000 To: July 2001]
• Professional Program in Quality & ISO -9000, The American University in Cairo - [From: Sept. 1996 To: May 1997]
• (2596) Managing Microsoft systems management server 2003, Microsoft Egypt - [Jan. 2005]
• Implement MS Win2003 professional & Server, IBM Egypt - [From: Oct. 2003 To: Nov. 2003]
• B.Sc. Social Work, Higher Institute of Social Work in Cairo, Egypt.- [From: 1984 To: 1988]
Computer Support, Metro Social Services – [Nov 2010 till present]
• Windows server 2003 and ISA server Installation and Management
• Software and hardware support
• Network installation and support
• Analyze and develop a customized software solution for most of the organization's activities
Computer Academic Manager, Michigan Academy Egypt (MAE) - [Jun 2006 till May 2010]
Microsoft gold certified partner, Microsoft CPLS and Michigan Academy franchisee, MAE provides Computer, Soft skills and English courses.
Academic Manager
• Oversaw all academic operations and management of a center and outsourced educational services to six universities.
• Prepared, supervised and led workshops to orient new teachers
• Developed and maintained productive relationships with all key stakeholders.
System Architect
• Defined and design software system architectures for the Academy management computer systems.
• Determined systems specifications and identified input/output processes.
System Analyst
• Analyzed and examined client existing business models and flow of data.
• Discussed findings with the client, and design an appropriate improved solution.
• Translated client requirements into highly specified project briefs.
• Identified options for potential solutions and assessed them for both technical and business suitability.
• Created logical and innovative solutions to complex problems.
• Oversaw developers to ensure technical compatibility.
• Oversaw the implementation plan of the system.
Project Manager
• Managed projects from meeting with clients to determine their requirements, till having the system go live with all related activities including test plan and end-user training.
Trainer and Computer Specialist, the American University in Cairo (AUC) – [Apr 1996 till Jun 2006]
Principal Responsibilities:
• Prepared and taught classes and led workshops for new software for students, staff and faculties.
• Developed a user guide for CALL software for instructors and students.
• Installed, maintained and administered
• Developed and maintained designated computer security systems, passwords, and file protection protocols
• Planned, organized, directed and controlled the information system and electronic data processing.
• Managed annual operating budget
• Designed and administered ELI audio listening final exams.
• Overseeing and prioritizing the maintenance of computer software; diagnosing and repairing network software problems; working with outside vendors for major repairs.
• Searching and evaluating new CALL "Computer Assisted Language Learning" software.
• Provided technical assistance on vendor issues, services, programs, software, etc.
• Worked efficiently and effectively with various software, operating systems "both IBM and Apple, and databases systems to include installation, upgrades, maintenance and troubleshooting.
• Presented to the university faculty and staff IT technological alternatives to streamline office functions and improving productivity.
Volunteer Center Director, Saint Mary Language Center (SMLC) – [May 1999 till Jan 2006]
A not for profit organization provides Computer and English Classes
• Oversaw the establishment of the center and the development of curriculum of courses in language and computer software training (Microsoft office, …………….).
Administration and Marketing Analyst, ELS Egypt – [Aug 2001 till May 2002]
• Designed and implemented information systems (databases) for class schedule control, students' database, etc.
• Developed and implemented the marketing plan
• Developed a quality assurance measures to ensure the quality of the services provided at the center.
Software and Hardware Support, Arab Consulting Engineers - [Nov 1992 till Mar 1996]
• Researching and reviewing new technology of computer sets and accessories at local and international markets.
• MICS "Management Information & Control System" Program Using MS ACCESS Data Base
• Free Lance IT consultant, The American Embassy in Cairo
In charge of Systems analysis, development and maintenance of the AECWA membership database program.
From: Sep. 2001 till: May, 2010
* Software analysis, development and maintain the commission's mailing system, Delegation of the European commission in Egypt
In 2005
* MS Access instructor at one of the AID development projects
From: April 2000 till: July 2000
* Publisher for the Payroll System manual, technical editor assist and help file builder at Intercom
From: Jan. 1996 till: July 1996
* Publisher at Saudi Soft (MS Access Project).
From: Feb. 1994 till: June 1994
* Book Setting on MAC "Structure Mechanics and Engineering".
From: Mar. 1992 till: Aug. 1993
* Free Lance Computer Instructor
* Trainer & Support for a Computerized AV Language Lab at Arab Banking Institute
From: May 1995 till: Aug. 1995
Skills (
Book Design
Page Design
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subset: commoncrawl


text: Hi there! I was diagnosed last Friday after a really difficult couple of years. I had my uterus removed in Nov 2012 as I had adenomyosis. I won't go into all the details as its so long but in brief had a perforated gall bladder, kidney stones of unusual composition and all last year chronic bladder and pelvic pain and urinary frequency day and night. I have been batted between gynae and urology the entire year. I was beginning to accept the prospect that this was Interstitial Cystitis as my gynae diagnosed, the laparoscopy only occurred to discount the urologists theory of endo as I was still bleeding substantially every month, so thankful now that I know what I'm dealing with. It's all over my bladder, cervix and right pelvis I have been excised and cauterised. I'm waiting for my follow up and crammed this week as I knew very little about endo. My gynae said it will be a drug treatment to put me in a menopausal state then he will remove my cervix. My question is, is that enough I'm bothered by what I've read that my ovaries are being left although I won't have a cervix or uterus? Grateful for any advice!! Very confused! Lx


text: "Isha and Rohan on their journey to find a perfect match for their client, encounter a very difficult client. Will they be able to find a match for her considering her behavior? Interestingly she has a past to understand too, Watch further to find out? Opposites attract? Really? Think again. Rishta.com is the story of these extremely opposite but compassionate souls who claim to be living life the right way. Rishta.com is the story of two contrasting individuals namely Isha and Rahul who open a matrimonial agency and try to unite the matches made in heaven, on earth. But while accomplishing the most challenging job, they encounter extreme cases, and illogical requests of their clients. As two strikingly different individuals, the way they deal with these circumstances proves the line that men are from mars and women are from Venus. What follows is a series of hilarity and the perfect sweet-salty recipe for a Happily Ever After.This show is a story of a couple who reiterates the fact that opposites attract and make a better life despite of the differences in their lifestyles and nature"


text:
Your furry family member loves watching Flames games with you. Sometimes your four-legged friend even beats you to the chair and you have to establish your place as top dog to get it back. Reward that Calgary spirit and keep tabs on your four-legged fan with this team pet ID tag. It features a Flames logo on one side, and space to write in your pet's name and contact information on the reverse!
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UNICO 20˚87˚ Hotel Riviera Maya
Why We Love It
- Adults-only all-inclusive resort opened in March 2017
- One of Travel + Leisure's "Hotel Openings to Watch Out for in 2017"
- Top Chef Mexico contestant runs one of five on-site restaurants
- All stays include fitness classes, water sports and select spa treatments
"Unusual take on custom itineraries brings you beyond the predictable all-inclusive experience" -- Brit & Co.
Map & Directions
Rooms & Rates
Opened in March 2017, the UNICO 20˚87˚ Hotel Riviera Maya is an all-inclusive resort catering to the needs of adult guests and curating personalized itineraries beyond the typical packaged vacation experience. Visitors are paired with butlers to book Mayan cooking lessons, tours of Tulum's ancient ruins, canoe trips to the nearby cenotes or zip-lining excursions through Cancun's jungles.
All rooms are decorated with works from local artists, are furnished with regional woods and have private patios with plunge pools or furnished balconies with spa tubs. Standard accommodations face the pool and gardens, while upgraded rooms have partial or full ocean views. Families and groups traveling together can spread out comfortably in villas with living rooms, kitchens, dining tables, sweeping terraces and additional bedrooms and bathrooms to accommodate up to six adults.
All-inclusive rates include unlimited meals and drinks at the property's five restaurants and six bars. Cueva Siete is run by Chef Christian Bravo, once a competitor on Top Chef Mexico, who brings his experience cooking across Mexico and Spain to Riviera Maya. Guests can also dine on coastal Italian favorites at Mi Carisa or sample three different kinds of Japanese cuisine at Mura House. The beachfront buffet at Restaurant 20.87 is open for breakfast and dinner daily as well as brunch on the weekends.
Day and night, traditional cocktails with a Mayan twist are served at Bar Balam, or guests can sip frozen concoctions by the pool at Bar Unica. For a nightcap with a taste of nostalgia, the Palmera Lounge is decorated like a Prohibition-era Cuban speakeasy.
The resort helps guests stay on track of fitness goals at the gym offering personal training sessions and daily group classes with options like Pilates, cross-training and spinning. Active visitors can borrow tennis and basketball gear to play on the illuminated courts, and enjoy unlimited use of the catamarans, kayaks, pedal boats and stand-up paddleboards in the ocean.
To unwind after a tough workout, all guests are treated to complimentary spa treatments and can purchase additional services. The property features three pools with swim-up bars, and the staff delivers food and drinks to sunbathers on the beach.
- "Eschewing a one-style-fits-all design, this new all-inclusive adults-only resort has gone the custom-designed route" -- Frommer's
- "An all-inclusive that encourages guests to get out" -- Travel + Leisure
- Bar
- Gym / fitness center
- Hot Tub / Jacuzzi
- Outdoor pool
- Pool bar
- Private beach
- Restaurant
- No pets allowed
- Spa
- 100% smoke-free
- Sauna
- Steam Room
- 24-hour front desk
- 24-hour room service
- 24-hour security
- Airport transfer (fees apply)
- Beauty treatments / massages (fees apply)
- Concierge service
- Hair salon (fees apply)
- Laundry / dry cleaning
- Pillow menu
- Room service (limited hours)
- Safe in reception (fees apply)
- Shuttle service (fees apply)
- Business center
- Safe in reception
Room amenities
- Air-conditioning
- Central heating
- Coffee / tea-making facilities
- Fridge
- Iron and ironing board
- Kitchenette in some rooms
- Mini bar
- Jacuzzi in some rooms
- Outdoor space for some rooms
- Safe
- Sitting area
- DVD / CD player & library
- Flatscreen TV
- iPad on request
- iPod/iPhone docking station
- Complimentary Internet access
- Complimentary Wi-Fi throughout
- Bathrobes and slippers
- Hair-dryer
- Luxury toiletries
- Bathtub or shower
- Complimentary parking
- Complimentary valet service
- Public car park
- Accessible bathroom in some rooms
- Accessible meeting room
- Accessible parking
- Accessible path of travel
- Accessible pool area
- Accessible restaurant
- Accessible spa
- ADA compliant rooms
- Facilities for guests with special needs
- Roll-in shower
- Wheelchair accessible
Standard Time is 3pm. Minimum age required to check-in: 18.
Minimum age required to check-in: 18.
Checkout: Standard Time is 12pm.
Children and additional guests:
This is an Adults Only resort.
Extra beds are not available.
No pets allowed.
Room price is based on two adults. For each additional guest (age 18 and older) an additional per person, per night fee will apply.
This fee varies by room and season. Please call 888-680-7625 with your confirmation number for specific pricing based on your arrival dates if you wish to add extra guests to your room.
The additional person fees are NOT included in the total price shown.
Charges will be applied by the hotel.
Cancellation: Cancellation policy & booking conditions vary by 'rate plan'. These will be displayed on the payment page after you have searched for your dates and selected a rate.
Have a Question? Contact Travelzoo.
- 1-877-966-0000 U.S. and Canada (toll-free)
- +44 (0) 20 3564 9000 U.K. and Europe
Customer Service available Mon-Sun, 9am-9pm ET
Rates may vary by date, are subject to availability and are not guaranteed until your purchase is complete.
The room rates listed are for double occupancy per room unless otherwise stated and exclude tax recovery charges and service fees.
It is the responsibility of the hotel chain and/or the individual property to ensure the accuracy of the photos displayed. Travelzoo is not responsible for any inaccuracies in the photos.
*Unless otherwise stated, Travelzoo-exclusive extras are valid once per stay (based on two adults sharing). Value is an estimate based on double occupancy. Extras are only included with Member Rates and may not be available on all dates. Extras are non-transferable and have no cash value. Total value may vary depending on time of booking and availability. When included, breakfast may be limited to 2 guests per stay subject to hotel's discretion. Other restrictions may apply; see our T&Cs for details.
Please note: Some amenities and services may not be available in all rooms or may incur an additional charge. All prices stated have been verified at the time of publication and may vary at the discretion of the hotel.
(5 Member Ratings)


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Justin Bieber Joins Cody Simpson On Stage [Video]
Two titans of teen pop -- Justin Bieber and Cody Simpson -- collided in Simpson's latest tour diary.
The new episode, which is about 12 minutes in length, shows the routine and procedures during a regular day of production on Simpson's Paradise tour. If you were ever curious about the nuts and bolts and the inner workings of a tour, this video will give you that insider's, keyhole view.
You see soundcheck and more, as it's a guided tour provided by the Aussie blond.
However, it's not just a regular day, as The Biebs joins Simpson at about nine minutes in. Wearing a wife beater and a cap, The Biebs partners with Simpson for a little rendition of his hit 'Pretty Brown Eyes,' grooving and making the girls go nuts, like bats. Cray cray!
Backstage, The Biebs jokes that Simpson used to be his little bro, but now he's his big bro, since Simpson has had a growth spurt and is now taller.
Check out the Bieber bling.
The Biebs also pimps out Simpson's new music, saying that the singer is working hard in the studio and improving those dance moves!
This video is TO. DIE. FOR. We almost can't handle this much awesomeness in one video.


text:
This web site contains about The World of the Hunger Games (Hunger Games Trilogy) 6-8 thousand ebooks from countless authors. The number of these publications are usually in the following groups: fictions/novels, short testimonies, poems, essays, takes on, nonfictions. Many of those books are traditional works of National Literature, English Literary works, and Irish Literature from well-known authors including William Shakespeare, Draw Twain, Charles Dickens, Oscar Wilde, Robert Ice, Edgar Allan Poe, Alexander Pope, Arthur Conan Doyle, Helen Austen. and more authors' works will probably be added to the web collection. Inside these special webpages, you will find The World of the Hunger Games (Hunger Games Trilogy) many give winning publications intended for your online reading. Get pleasure from and please tell a friend, thanks.
Description : Title: The World of the Hunger Games Binding: Hardcover Author: Inc.Scholastic Publisher: ScholasticInc.


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Vagabond Players presents Over the River and Through the Woods
12:00 am - 10:00 pm
Queen's Park
New Westminster, BC
Vagabond Players presents Over the River and Through the Woods, running February 4 – 27, 2016, Thursday – Saturday at 8pm and Sunday at 2pm, at The Bernie Legge Theatre, Queen's Park, New Westminster.
Nick is a single, Italian-American guy from New Jersey. His parents retired and moved to Florida. That doesn't mean his family isn't still in Jersey. In fact, he sees both sets of his grandparents every Sunday for dinner. This is routine until he has to tell them that he's been offered a dream job. The job he's been waiting for – marketing executive – would take him away from his beloved, but annoying, grandparents. He tells them. The news doesn't sit so well. Thus begins a series of schemes to keep Nick around. How could he betray his family's love to move to Seattle, for a job, wonder his grandparents? Well, Frank, Aida, Nunzio, and Emma do their level best, and that includes bringing to dinner the lovely – and single – Caitlin O'Hare as bait…


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This essay has been submitted by a law student. This is not an example of the work written by our professional essay writers.
Published: Fri, 02 Feb 2018
Art is product of human creativity
Art is subjective. Art is a product of human creativity and is nearly impossible to intrinsically define. So, it is unfortunate that to protect art from being copied, and therefore devalued, in the UK one must pigeon-hole it into narrow fields. Subject matter which is protected under UK copyright law are called "works". One of these are "artistic works", to narrow down this category subcategories have been introduced which are defined for the most part by the materials used in creation. For example a photograph is protected and defined by the method in which the artist captures the "art", by way of a camera. This method of defining art does not take into account artistic input on any level. The Copyright Design and Patents Act (CDPA) 1988 in Section 4(1)(a) it is laid out that graphic works, photographs, sculpture or collage must be protected "irrespective of artistic quality". The subcategory of "artistic craftsmanship" outwardly acknowledges that the aforementioned categories are inherently artistic in nature and that some arts and crafts may not be artistic, but those that are should be protected. The courts limited this category by highlighting the legislative inclusion of "artistic" but each judge in the leading case on the subject had a different opinion on why a prototype piece of ordinary commercial furniture was not a "work of artistic craftsmanship". The discrepancies amongst the judges have commentators reiterating that judges should not have to or need to decide a case based on artistic merit, but Booton uses the judgements as an example of why the definitions of the categories should be based instead on "artistic character" so they would not be forced to look at merit. These criticisms aside, the fact that none of these judges could come to the same conclusion demonstrates that there needs to be a change in the law and that it cannot come from the courts as it will only lead to more confusion. When dealing specifically with three dimensional art the CDPA 1988 is limiting in itself for what can fall into the category of "artistic work". The courts interpretation of what falls into the category of "sculpture" or "work of artistic craftsmanship" is confused and confusing in its short evolution to where artists stand today in their quest to protect their artistic work.
The most debated issue surrounding the UK's approach to copyright is the limiting reality of the CDPA. Artistic works must fit into a prescribed category to receive protection, due to the exhaustive list provided by the act it is difficult for any new form of subject matter to find an appropriate area to fit itself into. In Creation Records v News Group the assembling of objects to set a scene for a photograph to be taken around a pool could not be protect by copyright. It did not fit into the three dimensional categories as a sculpture because "[t]he scene was not a sculpture since no element in the composition had been carved, modelled or made in any of the other ways in which sculpture was made" it was also not a work or artistic craftsmanship "since it was merely an assemblage of objets trouvés and not the subject or result of the exercise of any craftsmanship". The scene set for the cover of the Oasis album could not amount to an artistic work because the courts were unable or unwilling to read the section as a living piece of legislation that is to evolve with the artistic community. The courts fear the idea of having to judge art, defining it by an aesthetic standard, this has led the courts to allow blatantly non-artistic works to fit into this category based on the composition or technique of creation. In Wham-O v Lincoln Industries the New Zealand Court of Appeal held that a wooden model to create frisbees was a sculpture under the act. This is an example of how the courts have expanded to protect industrial models under the umbrella of artistic works, but were not able to protect the actual artistic endeavour found in Creation Records.
The court's expansion of the definition has been criticized as protecting non-artistic works and excluding actual artistic works. The courts have attempted to limit the amount of non-artistic works that are protected by keeping the technique definition of sculpture narrow. This can be seen in J&S Davis (Holdings) v Wright Health Group where it was held that a dental cast was not made for the purposes of sculpture because it was made out of plasticine and therefore was not meant to be permanent. This approach to interpretation does not deal with the underlying problem of limiting the copyright protection on actual works of art. Booton is under the impression that if works were not pigeon-holed into techniques for development and instead required to display "artistic character" the courts would be able to protect works which are artistic and could exclude those which are not. "Artistic character" is established by the creator being aware of the "art world" being in the background. This definition does have it's own problems, for instance the ambiguous solution outlined in Booton's thesis to how the courts are to use this vague definition of "artistic character" to define works of art is not entirely clear. He leaves the reader to infer that because Laddie J seems to be using a similar approach to his that it is the way of the future. This assumption is suspect, but it his points are all relevant, the fact that copyright does not define three dimensional object as art where the art world would is a problem. The solution of incorporating expert opinion into this definition is not a new concept for the law, and should be utilized in this instance.
In the case of Metix (UK) Ltd v GH Maughan (Plastics) Ltd a Booton sympathetic approach was used by the court, which changes the way three dimensional objects are interpreted by the court. Laddie J said that 'sculpture' should be construed narrowly, that they should be defined in a way an ordinary person would agree with. He defined sculptures to be three dimensional objects made by an artists hand. This inclusion, that the creator must either believe he is an artist or be consider himself to be an artist, brings in an element of limiting the category of artistic works to only extend to protecting art whether good or bad. This in turn should be able to extend the definition to include new and experimental three dimensional works of art into the protection of the CDPA, but Laddie J only used his definition in the negative sense in excluding the functional moulds for making cartridges. If this definition were to be expanded positively to include any three dimensional object made by an artist which has more than just a functional aspect to it then most installation, conceptual art should also be protected. Cheng fears that this logical extension would lead to ordinary household items being capable of being protected, if placed in a museum and given a symbolic meaning. She complains that if a case was to come to court regarding Damien Hirst's unpreserved cow there would be difficulty because the cow itself is not the important part of the art, it is what it is symbolizing. Therefore if the idea behind the symbol can not be copyrighted, then Damien Hirst will own a copyright on all unpreseved cows, which seems like a stretch of the law. Cheng is at odds with Booton's ideal definition, she is under the impression that do decide if something has "artistic character" then the judiciary will be placed in a position of deciding artistic intention and that the intention of many conceptual artists cannot be found in the art itself, but in the museums description card. Cheng's states that if intention cannot be found in the object then the artistic merit will actually be evaluated, but when it comes to conceptual art she is not opposed to judgement. In this area Cheng is, simply put, wrong. Copyright is supposed to protect artists from being copied without compensation so that income can be derived from artistic endeavours. If successful artists, recognized by galleries and critics cannot protect their work through copyright, how is a struggling artist trying to begin their career supposed to hope to protect their works? It is obvious that art is subjective and not all pieces will be loved or appreciated by all patrons, but discrimination is not the solution.
The courts had developed definitions to each subcategory of artistic work based upon the techniques used to create the art. This approach did not work for sculptures or artistic craftsmanship because they are a product of a technique. The courts used the technique definition here until it proved impossible and then turned to the idea of requiring artistic intention, as seen in the Upholstery case and in Metix. The courts have not come to a consensus of what the definition of artistic is and we are left with Laddie J's interpretation which is only applied to purely functional objects, and does not extend to other forms of art that are not currently protected by the CDPA.
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The 2013 FIFA Confederations Cup kicks off in Brasilia on Saturday night, with host nations Brazil entertaining Asian champions Japan. The match, to be played at the Estadio Nacional in the country's capital, is a Group A fixture. Italy and Mexico are the other sides in the group. Spain, Tahiti, Uruguay and Nigeria are the other sides involved in the tournament and are in Group B.
Where to Watch Live
Kick-off is set for 8pm BST. Live coverage will be on BBC Three. Catch highlights of the game on Match of the Day, on BBC One and BBC One HD at 10.30pm BST.
Brazil will kick off in front of demanding home fans after a comfortable 3-0 win over fellow former World Cup champions France in an international friendly. Chelsea midfielder Oscar opened the scoring in the second half, Lazio's Hernanes doubled the lead and Paris St Germain (PSG) attacker Lucas Moura completed the rout from the penalty spot.
The win was only the Seleção's second since the start of 2013; the other was a 4-0 defeat of Bolivia, also in a friendly match. Other results this calendar year include draws against Italy, Chile and England in March, April and earlier this month.
Brazil have played England twice this year, losing 2-1 at Wembley before forcing a draw at the newly renovated Maracana. The former world champions may be unbeaten since that defeat at Wembley but Luiz Felipe Scolari, in his second stint at the helm of the national team, will know the performances have not been as impressive as they ought to be, given the talent he has at his disposal.
Brazilian fans tend to be passionate and very demanding and with home field advantage, they will expect their stars to shine.
Meanwhile, Japan make it to South America by virtue of winning the AFC Asian Cup in 2011. The Asian powerhouse beat Australia 1-0 in the final but may count themselves a trifle unlucky after having drawn Brazil, Italy and Mexico in their group.
Managed by former Inter, AC Milan and Juventus coach Alberto Zaccheroni, the Japanese come into this fixture on the back of a narrow 1-0 win over Iraq in a 2014 FIFA World Cup qualifier earlier this month. Stuttgart striker Shinji Okazaki scored late in the game to qualify for the competition next year.
The Japanese have lost only once in the last five games - a 2-0 friendly defeat to Bulgaria.
Team Form
- Last Five (all competitions, most recent first): W D D W D
- Last Result: Brazil 3 - 0 France
- Last Five (all competitions, most recent first): W D L W W
- Last Result: Iraq 0 - 1 Japan
Team News
Scolari is likely to field an attacking line-up, with Oscar, Hulk and Barcelona's latest signing Neymar likely to play behind Fred. David Luiz will pair up with PSG's Thiago Silva in the heart of the side's defence.
Probable Line-Up: Julio Cesar; Alves, David Luiz, Thiago Silva, Marcelo; Paulinho, Luiz Gustavo; Hulk, Oscar, Neymar; Fred
Japan have been handed a major boost after news midfielder Keisuke Honda and full-back Yuto Nagatomo are likely to feature. The duo were thought to be ruled out after picking up knocks.
Probable Line-Up: Kawashima; Uchida, Yoshida, Konno, Nagatomo; Endo, Hasebe; Okazaki, Honda, Kagawa; Maeda


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Soccer is an incredibly typical game globally. There are plenty of football crazy fans all over the world. Lionel Messi had been also one reasons why youth had been addicted to soccer game. Soccer provides individuals an effective way to learn how to achieve in life.
Argentina soccer players are among the most effective soccer athletes inside whole world. A number of the most useful young players on earth play inside this league.


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Past Event
This event featured a presentation of the EIB's 2018 Investment Report.
Past Event
What is the place of civil society in the digital age as well as the role of technology in society?
Past Event
How to measure China's digital economy?
Past Event
The 2018 Annual Meetings will be held on 3-4 September and will feature sessions on European and global economic governance, as well as finance, energy and innovation.
External Publication
Technological development, and in particular digitalisation, has major implications for labour markets. Assessing its impact will be crucial for developing policies that promote efficient labour markets for the benefit of workers, employers and societies as a whole.
Parliamentary Testimony
Testimony before the European Parliament's Committee on Industry, Research and Energy (ITRE).
Past Event
On 6 June Bruegel is organising a closed-door brainstorming workshop on: The European automotive sector: future challenges and opportunities.
Working Paper
The rise of influential Chinese digital giants, including Baidu, Alibaba, Tencent and Xiaomi has shown the world that China is a global leader in digital innovation and it is not surprising that China has started to influence the global digital market. But is China exploiting its full potential in this area? To answer this question, the authors assess how big China's digital economy is relative to the rest of its economy, and how China performs compared to the rest of the world.
Blog Post
The development of e-commerce has affected both demand and supply fundamentals of markets, changing the way competition works. In the effort to develop a frictionless and welfare maximizing digital single market across the EU, it is necessary to carefully review the disruptive forces on e-commerce on markets and firms' strategies.
Past Event
At this event, we looked into the progress made towards achieving the main priorities for strengthening the digital single market, the opportunities and the challenges at EU level.
Policy Contribution
The author looks at how concentrated corporate R&D is in Europe, compared with sales and employment. The US and China are more likely to produce new R&D leaders that take over some of the top positions from incumbent R&D leaders. How is the EU coping with technology shifts and creating the next generation of new leading firms?
Past Event
How can we address digital taxation in the EU? Is the proposed "equalisation tax" on turnover the best policy to tackle the challenges posed by digital taxation?


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I recently became interested in tsum when i walked into hallmark and saw these adorable things. I noticed on the tag their was a website i think a game for them and i downloaded I haven't stop playing the game in 2 days(gosh). I think its a rare daisy valentine edition.I name this baby Esmeralda I love this name so much for some reason! In the future I would totally name one of my adoptive kids this name (lol if its a boy too).
Happy Thanksgiving!!
xoxo lily


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Welcome to Connect Charter School
Anne Tingle Library & Learning Commons
Introducing Library Staff and Roles
It's never just an ordinary day!
The Library Collection
Young Adult Fiction- Grades 7-9
Parent Resources and Policies
Online Reference Centre @ Learn Alberta
ibrary and learning commons
supports and enhances the learning within our unique inquiry-based learning community, with the collection, the space and our services. Students and staff work within a setting characterized by collaboration, authentic learning experiences, and ubiquitous access to computer technology. Inquiry-based learning teaches our students to be information literate, prepared for lifelong learning in the 21st Century, with a disposition of inquiry de
y embedded into who we are as learners and citizens.
"A school library has never meant, "just books"- it has always been a gathering of resources, services and spaces for people, with a focus on teaching and learning (and I don't think it has meant "shushing" for a very long time!). A school library reflects and supports the mission and vision of the learning community in which it lives." Donna Alden, Teacher-Librarian
Observe students working with other students, students with with teachers, teachers working with other teachers, small groups and classes. Sometimes teachers are offering instruction, other times a class may be interacting with a guest expert as a class. Students and teachers may be incorporating a range of multimedia and using laptops, viewing a keynote projected from the LCD projector, or interacting with the SmartBoard in our collaboration centre. We learn within a collaborative, active culture of mutual respect. Everyday there will be students in the library reading books, digital screens, magazines, and sometimes, even textbooks! We are a school library which has evolved purposely into an authentic
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Wondering about the effects of the BP oil spill on the fishing and seafood industries in the Gulf?
Click here to read Dan Barry's great piece from The New York Times this week, which detailed the interconnectedness of those involved in the water-to-plate journey of the Gulf's oysters.
Friday, July 16, 2010
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text: This might sound silly, but I just can't seem to figure it out. I've had trouble learning to copy & paste text on my Pro since I've had it. I LOVED the option on my BBerry because I could just hold down a key and roll over the text I wanted to select and it would highlight it easily for me. Not so much luck with the Droid Pro. Since the recent software update, I noticed a new feature where a little "bar" shows up in the area where I'm texting that I can move around. I noticed that it also sometimes shows up in text from emails or websites and when I accidentally moved it around, it highlighted various text! Surprise surprise! But I have no idea how to GET that feature to work when I want/need it. It looks like exactly what I need, but I just have to figure out how to make it work. *LOL* Hope that made sense. Thanks in advance if you can help.


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We treat large and small customers with equal care, partnering so that we all can be successful. No order is too small for us to give it full attention and care.
We pride ourselves on serving the robust Twin Cities Printing industry. We provide rapid turnaround on converting long and short runs, special sizes and special window envelopes from preprinted stock. We provide regular seal gum, resin gum, latex seal and peel and seal.
We provide a wide range of customers with unique services ranging from plain stock envelopes to custom imprinting of special envelopes with customized inside tints to maintain corporate identity for small branch offices.
We can produce sizes from small coin envelopes and credit card protectors to 15"x21" open end envelopes with a full range of sealing options. We can do two color printing on the inside and outside of most envelopes. Our collection of envelope and window dies is as extensive as any in the industry.
Whatever your envelope needs we are confident we can provide a solutions that will satisfy your requirements and provide and economical solution. Give us an opportunity to show what we can do for you and your business.


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Plan to boost GP mental health role fails to win Govt support
Health Minister Sussan Ley has ruled out a recommendation of a Government-commissioned review of the mental health system to boost the role of GPs and Primary Health Networks in providing mental health care by redirecting $1 billion of Commonwealth funding from public hospitals to primary health providers and community-based mental health services.
A leaked copy of the long-awaited National Mental Health Commission review of mental health care, obtained by the ABC's 7.30 Report, has urged a shift in funding priorities away from 'downstream' services like acute care and the Disability Support Pension income support to prevention and early intervention care provided by GPs and other primary health providers.
"It is clear the mental health system has fundamental structural shortcomings," the review said. "The overall impact of a poorly planned and badly integrated system is a massive drain on peoples' wellbeing and participation in the community."
The Commission has argued that changing to a "stepped care approach" with a major focus on prevention and early intervention would reduce the severity and duration of mental health issues, ultimately slowing demand for expensive acute hospital care and lowering the incidence of long-term disability.
"The Commission believes one of the most fundamental elements of the stepped care approach lies in prioritising delivery of care through general practice and the primary health care sector," the report said, citing modelling from consultancy KPMG suggesting this would "slow the rate of increase in Disability Support Pension and Carer Payment costs and the costs of acute care and crisis management".
The Commission report said international evidence showed that strong primary health care made for healthier people and less costly health care than a focus on specialist and acute care, and suggested the creation of Primary Health Networks was an opportunity to "better target mental health resources".
Commission Chair Professor Alan Fels told ABC's AM program that, "we believe that over the next couple of years the Commonwealth should get ready to somewhat rebalance its spending away from it all going into hospitals when it would be better spent on services that keep people out of hospitals".
The Commission found that mental health problems are inflicting enormous costs on the country despite massive Commonwealth spending.
It said that, despite the expenditure of $9.6 billion on mental health care in 2012-13, around 3.6 million Australians experience mental ill-health every year – including 9000 who die prematurely – and the problem costs the country up to $40 billion a year.
"Our 'mental health system' is instead a collection of often uncoordinated services introduced on an often ad hoc basis, with no clarity of roles and responsibilities or strategic approach," the Commission's report said. "We need system reform to rebalance expenditure away from services which indicate system failure and invest in evidence-based services like prevention and early intervention."
Professor Fels said the Commission was not arguing for money to be taken out of mental health care, but that the focus of effort be redirected, suggesting the reallocation of "a minimum" of $1 billion of Commonwealth funding from acute hospitals to community-based mental health services from 2017-18.
"There's a great deal of Commonwealth spending on people, for example, going into the disability support payment scheme because they're not well and haven't been treated well. So, we believe there should be better prevention mechanisms, and system needs to be rebalanced in that direction."
But Ms Ley has poured cold water on the idea, saying the Commonwealth will not make such a change without the full support and co-operation of the states and territories.
"My strong view is we need to be partners with the states if we are to address serious mental health issues in this country long-term," the Minister told the West Australian. "Our preference, therefore, is to work with the states to deliver better co-ordination of existing roles and funding arrangements, rather than reduce the part they play."
The Commission's report was delivered to the Government on 30 November, but it is yet to make a formal response, causing frustration in the mental health sector.
Adrian Rollins


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13 December 2010
A new way of peering inside biological cells using carbon nanotubes as tiny multifunctional endoscopes has been developed by US researchers. The team says its nanotube endoscopes allow many different cellular responses in a single cell to be studied in parallel without damaging the cell, and could be important for drug discovery and testing applications.
Existing methods for interrogating cells such as glass micropipettes or nanoneedles often cannot handle fluids well and run the risk of damaging cells. Fluorescent marker molecules are commonly used to probe cells, but cannot follow different cellular responses simultaneously.
Now, a team at Drexel University in Philadelphia, Pennsylvania, US, has come up with an all-in- one solution in the form of nano-sized endoscopes made from carbon nanotubes attached to standard glass micropipettes. 'In many ways it's similar to the kind of endoscopic tools physicians employ to probe the inside of a human body in a minimally invasive fashion. Ours is just many orders of magnitude smaller,' says Gary Friedman, a co-author of the study.
The team made the endoscopes by using a fluid flow technique to place multiwalled carbon nanotubes at the tip of a glass pipette which were then sealed with epoxy. By filling the CNT with magnetic magnetite (Fe3O4 nanoparticles, magnets could then be used to create tiny deflections in the CNT tip to precisely control it.
A multiwalled carbon nanotube is attached to the end of a glass pipette, which is coated with a non-conducting epoxy on the outside and a conducting epoxy on the inside. Inset: Scanning electron micrograph of an endoscope with 100nm carbon nanotube
© Nature Nano.
Experiments with the endoscopes revealed that optical signals observed via Surface Enhanced Raman Spectroscopy (SERS) from different regions within a cell matched existing observations on intracellular SERS. The team was also able to record electrochemical signals and transfer fluids and nanoparticles to and from a cell.
To determine if the endoscopes caused any damage the team looked at cellular calcium signals which increase when a cell becomes stressed. In addition, they made microscopic observations of cellular membranes and intracellular filamentary networks. They found minimal cell damage occurred compared to that caused by standard glass micropipettes.
'This presents an extremely powerful multifunctional intracellular probing technology that can penetrate deep into cells, manipulate intracellular components remotely with high resolution, and perform optical and electrochemical diagnosis at the single organelle level,' says Dejian Zhou, who develops functional nanomaterials at the University of Leeds, UK. 'Its main advantage over other cell interrogation technologies is the cylindrical shape of the probe, which allows for deep cell penetration without causing too much cell destruction,' he adds.
'The CNT endoscopes can be used whenever someone wants to follow in parallel different cellular responses to extracellular stimuli or injections of various agents through the endoscope,' says Friedman. 'This could be important in decoding various cellular signals, genetic and protein interaction networks useful for drug discovery.'
James Urquhart


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Brick Assassin "Bite the Hand that Feeds" 12" EP
BRICK ASSASSIN out of Chicago have presented you with a few stellar EPs to date, so it should come as no surprise that this newest release on Foreign Legion Records maintains the same intensity on these 6 tracks. With Kyle taking the reins on vocals, the gravely vocals compliment the the music of a similar ilk. Great Art, great tracks, limited release. Get it. BRICK ASSASSIN.
A1 Let Me Bleed 3:01
A2 Ruled By You 2:59
A3 Bonehead Dicks 2:06
B1 Poison 1:50
B2 Piles on Piles 1:47
B3 Scabs (King Of The Rats) 2:44
Pressing Information
300 Black Vinyl on Foreign Legion Records


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We got a note from the nice people at Maryhill Winery asking us if we'd like to try their wines before we visit Washington this summer. Something about the name was familiar.
Then Friday night after we sampled two of their whites, it hit me and promptly stuck in my head! It was a song from Billy Joe Royal. I didn't quite understand the lyrics when I first heard them — because I was 7 years old. But I was already watching American Bandstand and would generally pick up on anything I could sing. While not as racy as some of the tunes on the radio today, the song made it clear that Mary Hill did what she wanted without worrying about what other people thought. I guess at that age, since I had more fun playing football outside with the boys rather than inside playing Barbies with the girls, I identified with these lyrics:
Mary Hill used to hang out in Cherry Hill Park
The game she played lasted all day to way after dark
Now all the girls they criticized her but all the guys just idolized her
cause Mary Hill was such a thrill after dark, in Cherry Hill Park
The 10th largest winery in Washington state, family-owned Maryhill Winery is located at the Southern tip of the Columbia Valley appellation in Goldendale, Washington. Named "2009 Washington Winery of the Year" by Wine Press Northwest, Maryhill produces 23 varietals and 28 wines. In addition to the winery, Maryhill hosts weddings and special events, with an adjacent 4,000-seat outdoor amphitheatre that hosts a summer concert series with artists like Lyle Lovett, Jackson Browne, Natalie Merchant and Earth, Wind and Fire (and that's just this summer!)
Last weekend we tasted two of Maryhill Winery's white wines and found them very enjoyable; the 2008 Viognier from Columbia Valley; and the 2007 Winemaker's White.
Maryhill Winery 2007 Winemaker's White – Columbia Valley
Reminiscent of a white Bordeaux with a bit of a twist, the Maryhill Winemaker's White combines 49% Chardonnay, 36% Sauvignon Blanc, 11% Sémillon and 4% Viognier for a well-balanced semi-dry white table wine perfect for seafood, or Mediterranean fare. We tried it with stuffed grape leaves; hummus; feta and Greek olives that we had left over from our Greek wine tasting the previous evening. Plenty of citrus on the nose, with a bit of zesty lemon, peach and apricot. With notes of melon and tropical fruit on the palate, it is both creamy and refreshing, the Sémillon providing the perfect balance to the acidity of the Sauvignon Blanc. 13.5% Alcohol by Volume. A nice value at just $12.00.
Joe teases me and says I taste honey in every bottle of white. Not true, I say, sometimes I taste banana – not regular banana, but saltwater taffy banana. But that's only a particular type of Chardonnay whose maker has decided that there never can be too much malolactic fermentation. Believe me, there can be. And it's the one wine I will take one sip of and then give the rest of the glass or bottle to whomever happens to be in my vicinity. Cannot stand the stuff – at all! Thank goodness we tasted none of those!
Maryhill Winery 2008 Viognier – Columbia Valley
The 2008 Viognier from Maryhill Winery is more honeysuckle and star jasmine than Sue Bee on the nose, with delightful aromatics of sweet wild florals. Once in the mouth, we tasted Granny Smith apples and Anjou pear, with just a hint of vanilla bean, a bit of honey and a smidgen of toasty French oak. Light acidity and balanced sweetness cut the flash of spice and garlic in our leftover Baba Ghanoush. 13% Alcohol by Volume, down from the 14.3% of the 2007 vintage. Also a nice value at just $15.00.
The Winemaker
Richard Batchelor joined Maryhill Winery in July 2009 and is responsible for directing all production activities at the winery including winemaking, bottling and harvest coordination. Prior to joining Maryhill Winery, Batchelor was head winemaker at Hall Winery in the Napa Valley, and was responsible for some of the fabulous 2006 vintages we enjoyed during our lunch and visit to Hall Winery last summer. A native of New Zealand, he holds a postgraduate degree in Viticulture and Oenology, as well as a Bachelor of Science degree from Lincoln University in Christchurch.
Established in 1999 by Craig and Vicki Leuthold, Maryhill Winery is a 90-minute drive from Portland, Oregon and a three-hour drive from Seattle, Washington. Located at 9774 Highway 14 in Goldendale, they are open daily from 10 a.m. to 6 p.m.
For those of you not familiar with the song; take a listen to Billy Joe Royal's Cherry Hill Park, which peaked at number 15 on the Billboard Hot 100 in the year 1969. I apologize in advance if it sticks in your head!
The WineWonkette


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BANGKOK — An anti-coup activist in Thailand called Friday for a weekend rally to defy the military government's ban on demonstrations, urging those opposed to the takeover to wear masks and be ready for cat-and-mouse chases with soldiers in the capital.
The call to rally on Sunday raised concerns of a showdown, as the military reiterated its ban on political gatherings and warned it will not tolerate protests against the coup it staged May 22.
"The authorities will take legal measures against those who come out to oppose" the coup, deputy army spokesman Col. Winthai Suvaree told a news conference. "We cannot tolerate this situation happening."
The warning came as the military sealed off a major Bangkok intersection for a second day Friday to prevent a possible protest. The massive show of force — involving hundreds of troops during the evening rush hour— came in response to small but near-daily demonstrations that have raised tension and concerns the army will crack down on protesters.
The country's powerful army chief, Gen. Prayuth Chan-ocha, defended the coup as a means to restore order after seven months of increasingly violent political turbulence.
In the past week, the junta has moved to silence its critics and warned that it will not tolerate dissent.
It has summoned more than 250 people, including members of the government it ousted and other leading political figures, journalists, scholars and activists seen as critical of the regime. Roughly 70 people are still in custody.
Among those summoned was veteran social activist Sombat Boonngam-anong, who is allied with "Red Shirts" who backed the ousted government and has played a role in organizing anti-coup protests.
Sombat has gone into hiding but taunted the military by posting the call to protest on his Facebook page, summoning 10,000 people to come in disguise for a "mask party to celebrate the coup." Protesters have started wearing masks with the faces of political personalities, including the army chief, Prayuth.
"There is no need to be aggressive in opposing the coup. Smile, please, and take it easy," Sombat said. "The masks you wear ... will be enough to make the dictators in the military lose face."
"The goal is to tell the world what we think about the coup," he said, setting the meeting place at a McDonald's restaurant in central Bangkok. Anticipating that soldiers might seal the area, he urged protesters to "go play" elsewhere and listed different neighborhoods in the capital where they could regroup.
One of the McDonald's branches in Bangkok has become a gathering place for protests because of its convenient location. Some protesters have used the McDonald's logo in their anti-coup signs, replacing the "m'' in democracy with the yellow golden arches.
Earlier this week, McDonald's in Thailand stated its political neutrality and warned it will take "appropriate measures" to protect its trademark.
"We wish to clarify that McThai maintains and will continue maintaining a neutral position in the current political situation in Thailand," the company said in a statement.
The army coup overthrew a government that won a landslide election victory three years ago.
At the center of Thailand's deep political divide is Thaksin Shinawatra, a former prime minister supported by many rural Thais for his populist programs but despised by others — particularly Bangkok's elite and middle classes — over allegations of corruption, abuse of power and disrespect for the monarchy. He was ousted in a 2006 coup and lives abroad in self-imposed exile, but held great influence over the overthrown government, which had been led by his sister until a court ousted her earlier this month.
Despite the latest political upheaval, life has continued largely as normal in most of the country, with tourists still relaxing at beach resorts and strolling through Buddhist temples in Bangkok and elsewhere.
A curfew remains in effect from midnight to 4 a.m. but has not affected critical travel, including that of tourists arriving at airports.
Associated Press writer Jocelyn Gecker contributed to this report.


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Published Oct 9, 2014
Who pours the best Guiness and serves the best fish and chips?
"They have Magners. What more do you need? It's easy to get to, plenty of parking, and various forms of entertainment..."
via Saryn C.
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"…Tom Bergin's rocks my socks off. Down to earth, neighborhood bar that welcomes all. Be a part of history and work towards getting a shamrock, you'll…"
"…best pub every luv it. you'll have a great time…"
"…Best place to go for any Boston game. Pizza used to be the best in town, but not sure they still serve. Karaoke nights too!…"
"…Best Guiness outside of Dublin…"
"…Love Casey's, especially their summer concert series! Live music on the patio with a drink and BBQ = fabulous way to spend a Sunday.…"
"…On a sunday afternoon if you close your eyes you could very easily be sitting in Shanleys pub in Clonakilty, County West Cork! they weren't voted best…"
"…it's not only the best irish pub in l.a. it is the best irish pub in southern california.
the owner and staff are amazing and the people are very fr…"
"…Also best happy hour ;) @tylerrass - right?!…"
"…Makes me proud to be Irish when I take a pint at Gallaghers,...R. Sullivan…"
"…This is the best pub in California! Great pints of Guinness and they show soccer and rugby. Live music, and fantastic food make this pub a must stop i…"
"…This is close to the real deal. They've got great beers on tap and their shepherd's pie rocks my socks.…"
"…I once saw a leprechaun there! No seriously it was Wee Man! this is a cool industry bar and you never know what star will be drinking there.the owner …"
"…The best Irish pub ever! Its a great place to catch a game also the staff is fun, very helpful and good looking. The general manager is intimidating b…"
The 2014 LA HOT LIST awards are based on more than 19,000 votes from local Los Angeles-area experts.
See the full results →
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© CityVoter, Inc.


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NUTLEY, NJ - 1872 applications were received to be a part of the first class at Hackensack Meridian School of Medicine at Seton Hall University where the first class of 55 students will start July 9.
Those students will be a part of the plan to address the shortage of physicians in New Jersey and forge a new path in medical education that addresses profound changes in health care which have resulted in more community-based care and an unprecedented focus on wellness and population health according to founding dean Dr. Bonita Stanton. Research shows that physicians often practice where they train which would help the state ease a shortage of an estimated 3,000 doctors in New Jersey by 2020.
"Our goal is to maximize health in all of the communities we serve, a goal best achieved through an interdisciplinary approach based on an understanding that health and wellness, as well as disease and sickness, occur where people live, work and play,'' said Dr. Bonita Stanton. "We are humanizing health care.''
Interviews of candidates for the first class started last week, according to Dr. Jeffrey R. Boscamp, Associate Dean of Medical Education Continuum. Acceptances to the new school of medicine will being to go out in the next few weeks, Dr. Boscamp said.
Along with the opening of the School of Medicine, Seton Hall University will relocate its College of Nursing and School of Health and Medical Sciences to create an Interprofessional Health Sciences (IHS) campus in Nutley and Clifton this month, said Mary Meehan, Ph.D., interim president of Seton Hall University.
"We have created a rigorous academic curriculum that combines traditional science with a focus on the new frontiers in medicine – prevention, population health, genetics and team-based care delivered in the community setting,'' Dr. Meehan said.
Additionally, the Hackensack Meridian Health Board of Trustees recently voted to establish a $100 million endowment fund for scholarships to the school, fulfilling a high priority to ensure top students can afford a medical education, Garrett said.
"Dynamic changes in health care require a new approach to medical education and we are thrilled to announce that we are accepting applications from so many talented and high achieving students,'' said Robert C. Garrett, co-CEO of Hackensack Meridian Health. Students will train in several of Hackensack Meridian Health's 16 hospitals.
Additionally, the innovative curriculum will help future physicians navigate major changes in health care that are underway in the U.S. including the transition to value-based care in which physicians and hospitals are paid to keep people well. It's a major shift from fee-for-service medicine in which providers are paid for each treatment and procedure.
The strategy is essential to improve outcomes and lower the cost of care as the U.S. faces an epidemic of diabetes and other chronic disease, which is costly and in many cases preventable. Even though the U.S. spends far more than virtually all nations, we lag behind other peer nations in all major areas of health including maternal and infant health and life expectancy. This new approach aims to eliminate disparities in health outcome by closely coordinating care and intervening earlier when problems develop.
Students will develop partnerships with families living in stressed communities and work with them to jointly understand and overcome factors that can impede or contribute to well-being, ranging from access to grocery stores to taking advantage of new developments in telemedicine
The new school is also unique in that it offers a three-year program, one of only a dozen or so in the nation to take this approach which can lower the cost of a medical education.
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