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Home > Journals > Biomedical & Life Sciences > AJPS
American Journal of Plant Sciences > Vol.9 No.10, September 2018
Cloning and Expression Analysis of RrGT2 Gene Related to Anthocyanin Biosynthesis in Rosa rugosa ()
Xiaoming Sui, Yang Wang, Mingyuan Zhao, Xu Han, Lanyong Zhao*, Zongda Xu*
College of Forestry, Shandong Agricultural University, Taian, China.
DOI: 10.4236/ajps.2018.910146 PDF HTML XML 415 Downloads 645 Views Citations
At present, the research about flower color of Rosa rugosa is a very inno-vative and practical study. Glycosylation modification fulfills an important role in increasing the stability and solubility of anthocyanin in plants. In this study, based on the transcriptional database of R. rugosa, a gene with full length cDNA of 1422bp, encoding 473 amino acids, designated as RrGT2, were isolated from flowers of R. rugosa 'Zizhi' and then functionally characterized. According to online software prediction, the molecular formula of the protein encoded by the RrGT2 gene is C2334H3628N602O711S18, the relative molecular mass is 52,075.17 Da, and the theoretical isoelectric point is pI = 4.76. The result of the RrGT2 protein 3D model construction showed that it had the highest homology with the UDP-glycosyltransferase 74F2 protein model in the database (39.53%). Sequence alignments with the NCBI database showed that the RrGT2 protein is a member of the GTB superfamily. Homology analysis revealed that the coding regions of RrGT2 was highly specific among different species, but still had typical conserved amino acid residues called PSPG that are crucial for RrGT2 enzyme activity. RrGT2 transcripts were detected in five flowering stages and seven tissues of R. rugosa 'Zizhi', R. rugosa 'Fenzizhi' and R. rugosa 'Baizizhi', and their expression patterns corresponded with the accumulation of antho-cyanins. Therefore, we speculated that glycosylation of RrGT2 plays a crucial role in anthocyanin biosynthesis in R. rugosa.
Rosa rugosa, RrGT2 Gene, Clone, Gene Expression, Anthocyanin
Sui, X. , Wang, Y. , Zhao, M. , Han, X. , Zhao, L. and Xu, Z. (2018) Cloning and Expression Analysis of RrGT2 Gene Related to Anthocyanin Biosynthesis in Rosa rugosa. American Journal of Plant Sciences, 9, 2008-2019. doi: 10.4236/ajps.2018.910146.
Rosa rugosa is an important ornamental plant which belongs to the genus Rosa in the family Rosaceae. It is native to China and is widely distributed in the world. Because of its unique fragrance, color, cold resistance and drought resistance, it has great development potential in garden application [1] . There are many varieties of roses, but most of them are traditional colors such as pink, purple, etc. A few varieties are white, lacking yellow, bright red, orange and compound color, etc. [2] . Therefore, how to innovate rose color has become the main goal of breeders. The analysis of the pigment composition of rose and the study of the expression characteristics of the key enzymes encoding genes that catalyze the synthesis of rose pigment are the important prerequisite for rose color oriented molecular breeding [3] . Anthocyanin determines the color of higher plant organs. Its biosynthesis pathway related structural genes (CHS, CHI, F3H, F3'H, DFR, ANS, 3GT etc.) and regulatory genes (MYB, mostly R2R3 MYB, BHLH and WD40 classes) have been cloned, sequenced and protein function studies in many plants, such as petunia, maize, snapdragon and so on. But less research has been done on R. rugosa.
Anthocyanins, derived from the anthocyanin biosynthesis pathway, are the largest group of water-soluble plant flavonoids found in organs of plants and crops [4] [5] . Anthocyanins are unstable in plants, mainly in the form of glycosides in the vacuole [6] . Anthocyanins play an important role in insect pollination, auxin transport, protection of leaves from ultraviolet radiation, inhibition of diseases and insect pests, etc. [7] . In addition, as a safe, non-toxic natural food pigment, anthocyanins also have anti-oxidation, anti-cancer and anti-arteriosclerosis functions [8] .
Anthocyanin biosynthesis pathway is one of the secondary metabolic pathways in plants. At present, the research on it has been more clear. Firstly, naringin was formed by the catalysis of CHS and CHI with coumaryl coenzyme A and malonyl Co A. Then flavonols were formed under the action of F3H. The next step is to form colored anthocyanins under the action of DFR and ANS. Finally, through glycosylation, methylation, acylation, hydroxylation and other modifications, a variety of anthocyanins with a stable structure were formed [9] . Flavonoid 3-0-glycosyltransferase (3GT) gene is a downstream gene in anthocyanin synthesis pathway. It can catalyze the glycosylation of UDP glucose to replace the 3 hydroxyl groups of anthocyanin and make anthocyanin glycosylation to produce colored and stable anthocyanins. And move the maximum absorption spectrum to the ultraviolet end, thus increasing the blue tone of anthocyanins [10] . Glycosylation can change the hydrophilicity, biochemical activity and subcellular localization of anthocyanins, which is beneficial to the transport and storage of anthocyanins in cells and organisms [11] .
Some studies have shown that the anthocyanin content of plants lacking 3GT also decreased significantly [12] . 3GT gene belongs to a glycosyltransferase (GTs) family 1, whose enzyme protein has a conserved domain of about 44 amino acids at its C-terminal, known as plant secondary product glycosyltransferase (PSPG) box. At present, 3GT has been cloned and analyzed in many plants such as Zea mays [13] , Vitis vinifera [14] , Gentiana trflora [15] , Petunia hybrida [16] and so on, which has laid a foundation for understanding the metabolic regulation of anthocyanin synthesis pathway.
At present, the studies on R. rugosa are mainly focused on morphological classification, geographical distribution, essential oil extraction and food quality evaluation, and there are few reports on the anthocyanin biosynthesis mechanism, so we don't know exactly how it works. In this study, based on the R. rugosa transcriptome data, we cloned and identified RrGT2 gene from the petals of R. rugosa 'Zizhi' for the first time. We carried out detailed bioinformatics analysis, homology analysis and the temporal and spatial expression pattern analysis of the RrGT2 gene in order to provide some useful informations for the subsequent color improvement project in R. rugosa.
2.1. Plant Materials
For R. rugosa, the plant materials were the same as the varieties used for transcriptome data determination. Three varieties (R. rugosa 'Zizhi', R. rugosa 'Fenzizhi' and R. rugosa 'Baizizhi') cultivated in Rose germplasm nursery of Shandong Agricultural University was used as the test material. All three varieties were crossed with Rosa davurica as female parent and Rosa rugosa Thunb. as male parent. We collected the petals at the budding stage, initial opening stage, half opening stage, full opening stage and wilting stage in the forenoon On sunny days from 20 April to 10 May 2017. Seven tissues (roots, stems, leaves, petals at the budding stage, sepals, stamens and pistil) of R. rugosa 'Zizhi' were collected at the same time. After quick freezing of liquid nitrogen, all samples collected with three replicates were put into −80˚C refrigerator for storage.
2.2. Extraction of Total RNA and Synthesis of the First-Strand cDNA
The extraction of total RNA is based on the specification of EASY spin plant RNA rapid extraction kit (Aidlab Biotech, Beijing, China). The integrity of RNA was detected by gel electrophoresis with 1.0% nondenatured agarose, the purity and concentration of RNA were detected by Nanodrop2000C ultramicro spectrophotometer (Thermo Fisher Scientific, Wilmington, Delaware, USA), and the qualified RNA was preserved at −80˚C. The first-strand cDNA was synthesized by referring to the steps of 5 × All-In-One RT MasterMix reverse transcription kit (ABM Company, Vancouver, Canada) and synthesized according to the requirements of RT-PCR and qRT-PCR.
2.3. Full-Length CDS Cloning of the RrGT2 Gene
3'RACE specific primers were designed based on the sequence information provided by the Rosa transcriptome data. The cDNA 3'terminal sequence of the target gene was amplified by 3'RACE technology. The known target gene sequence in transcriptome data and the 3'terminal sequence obtained by RACE technique were spliced with DNAMAN software to obtain the full-length cDNA sequence of the RrGT2 gene. According to the sequence obtained by splicing, the upstream primer RrGT2-F containing the starting codon of the RrGT2 gene and the downstream primer RrGT2-R containing the terminating codon (Table 1) were designed and amplified. The estimated length of amplification was 1422bp.
2.4. Bioinformatics Analysis of the RrGT2 Gene
Bioinformatics analysis softwares and tools were used to predict the physicochemical properties and structural functions of the protein encoded by the RrGT2 gene, which provided a reference for the future research and application of the gene. The basic physical and chemical properties of the protein encoded by the RrGT2 gene were analyzed by using Prot-Param tools in ExPasy (http://web.expasy.org/protparam/). The CD-Search function (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) on NCBI was used to predict the conserved domain in the protein encoded by the RrGT2 gene. The 3D structure of the RrGT2 protein was predicted by online tool SWISS-MODEL. DNAMAN software and Blast tool in NCBI were used to analyze the amino acid sequence of the RrGT2 gene. The phylogenetic tree is constructed by neighbor-joining method using MEGA5.0 software.
2.5. qRT-PCR Detection
The gene expression was analyzed by qRT-PCR on a Bio-Rad CFX96TM Real-Time PCR instrument (Bio-Rad, Inc., USA). The qRT-PCR mixture (20 μL total volume) contained 10 μL of SYBR® Premix Ex Taq™ (TaKaRa, Inc., Japan), 8.2 μL of ddH2O, 0.4 μL of each primer and 1 μL of cDNA. The PCR program was carried out with an initial step of 95˚C for 30 s; 40 cycles of 95˚C for 5 s, 60˚C for 30 s; and then, 95˚C for 10 s, 65˚C for 5 s and 95˚C for 5 s for the dissociation stage. Each gene was assessed with three biological replications. The relative expression levels of the genes were calculated by the 2−ΔΔCt method [17] .
3.1. Cloning of RrGT2 and Sequence Analysis
We obtained the RrGT2 gene 3' terminal sequence of 236 bp length by nested PCR (Figure 1(a)). The full length CDS sequence (Figure 1(b)) of the RrGT2 gene was cloned by full-length primers (Table 1) and confirmed by sequencing. The RrGT2 gene has a complete ORF from the starting codon ATG to the termination codon TAA, encodes a 473-amino acids protein and has a polyA tail. Sequence alignments with the NCBI database showed the RrGT2 protein is a member of the GTB superfamily and has a typical plant secondary product glycosyltransferases conserved domain consisting of 44 amino acid residues at the
Table 1. Primers used in the present study.
Figure 1. The results of 3'RACE amplification and full-length CDS amplification of the RrGT2 gene. (a) 3'RACE amplification product of the RrGT2 gene. (b) Full-length CDS amplification product of the RrGT2 gene.
C-terminal. According to online software prediction, the molecular formula of the RrGT2 protein is C2334H3628N602O711S18, the relative molecular mass is 52075.17 Da, and the theoretical isoelectric point is pI = 4.76.
3.2. Protein 3D Model Construction
During the construction of the RrGT2 protein 3D model, templates search with BLAST and HHBlits has been performed against the SWISS-MODEL template library. It was found that it had the highest homology with the UDP- glycosyltransferase 74F2 protein model in the database (39.53%), so the RrGT2 protein 3D model (Figure 2) was constructed on the basis of its model.
3.3. Homology Analysis
Sequence alignment of multiple species amino acids (Figure 3) showed that the RrGT2 protein has strong species specificity in the N-terminal region and PSPG conserved domain in the C-terminal region. Using MEGA5.0 software,
Figure 2. The protein modelling results and quality estimation of RrGT2. (a) Visualizable 3D model of RrGT2. (b) Local quality estimation of the protein. (c) As a statistical value, Z-score can be used to represent the matching degree between template proteins and proteins to be tested. QMEAN: A comprehensive scoring function for model quality assessment. All Atom: Normalized all-atom potential energy of the residue calculated from the short-range statistical potentials. CBeta: Normalized cbeta potential energy of the residue calculated from the short-range statistical potentials. Solvation: Normalized solvation potential energy of the residue calculated from the short-range statistical potentials. Torsion: Torsion energy of the residue exposed relative solvent accessibility, calculated by dividing the maximally accessible surface area (ASA) of a residue by the observed value. (d) Comparison with Non-redundant Set of PDB (Protein Data Bank) structures. (e) Alignment between the model and the template.
phylogenetic tree (Figure 4) was constructed from 21 plant amino acids sequences including R. rugosa RrGT2. The results showed that the RrGT2 gene had the closest genetic relationship with FaUGT and FvGT, and its homology reached 89%.
3.4. Temporal and Spatial Expression Patterns of the RrGT2 Gene
The expression levels of the RrGT2 gene, which significantly differed, were assessed during five flowering stages. For R. rugosa 'Zizhi' (Figure 5(a)), the highest expression level was observed during the full opening stage, and the lowest was observed during the budding stage. For R. rugosa 'Fenzizhi' (Figure 5(b)), the highest expression level was observed during the wilting stage, and the lowest was observed during the budding stage. And for R. rugosa 'Baizizhi' (Figure 5(c)), the expression level was highest during the initial opening stage but lowest
Figure 3. Amino acid sequences homologous analysis of the RrGT2 gene and other species related genes. Alignments were performed using DNAMAN. The relative genes of 21 species including the RrGT2 gene were selected for comparative analysis. The blue box shows the PSPG domains. Black triangles (from left to right) indicate 22, 23 and 44 amino acids in the PSPG box.
also during the budding stage. The expression patterns of the RrGT2 gene in R. rugosa 'Zizhi', R. rugosa 'Fenzizhi' and R. rugosa 'Baizizhi' showed different trends.
Then the expression levels of the RrGT2 gene in three varieties was compared in five flowering stages (Figure 5(d)). In the budding stage, full opening stage and wilting stage, R. rugosa 'Fenzizhi' > R. rugosa 'Baizizhi' > R. rugosa Zizhi', while in the initial opening stage and half opening stage, the trends of gene expression in the three varieties were consistent: R. rugosa 'Baizizhi' > R. rugosa 'Fenzizhi' > R. rugosa 'Zizhi'.
The expression levels of the RrGT2 gene, which also significantly differed, were assessed in seven different tissue types of R. rugosa 'Zizhi' (Figure 5(e)). The order of relative expression levels of the RrGT2 gene in seven tissues was: sepal > leaf > stem > root > flower at the budding stage > pistil > stamen.
Although many genes have been reported to regulate the formation of flower color, there are few reports of downstream structural genes such as GTs. The final formation of anthocyanins depends on the glycosylation of GTs, so it is very important to elucidate the function and influence of the RrGT2 gene in R. rugosa color formation. In this study, we successfully cloned RrGT2 gene with full length cDNA of 1422bp, encoding 473 amino acids from the petals of R. rugosa 'Zizhi'. It was predicted that the molecular formula of the protein encoded by the RrGT2 gene is C2334H3628N602O711S18, the relative molecular mass is 52,075.17 Da, the theoretical isoelectric point pI = 4.76, which belongs to GTB superfamily. During the construction of the RrGT2 protein 3D model, it was found that the RrGT2 protein had the highest homology with the existing UDP-glycosyltransferase 74F2 protein model protein model (39.53%), which confirmed the function of the RrGT2 gene to some extent.
The evolutionary analysis of flavonoids GTs by Sawada et al. [18] showed that GTs, which catalyze the glycosylation of flavonoids in different positions
Figure 4. Phylogenetic tree of RrGT2 and GT members from other plant species. The tree was constructed by neighbor-joining method using MEGA 5.0 software. Branch numbers represent as percentage of bootstrap values in 1000 sampling replicates and scale indicates branch lengths.
(3-O, 5-O, 7-O), were formed into different evolutionary branches (F3Gly T, F5Gly T, F7Gly T) without restriction of species. It suggested that the region-specific of flavonoids GTs to glycosyl receptors (catalytic site specificity) was formed before species differentiation. In the course of evolution, the ability to utilize UDP-sugar (UDP-glucose, UDP-rhamnose, UDP-galactose) was obtained. Phylogenetic tree analysis showed that RrGT2 was linked to FaUGT and
Figure 5. The temporal and spatial expression patterns of RrGT2. The relative expressions of the RrGT2 gene in five flowering stages of R. rugosa 'Zizhi' (a), R. rugosa 'Fenzizhi' (b) and R. rugosa 'Baizizhi' (c). S1, budding stage; S2, initial opening stage; S3, half opening stage; S4, full opening stage; S5, wilting stage. (d) Comparison of relative expressions of RrGT2 in five flowering stages of three varieties above. (e) The relative expression of RrGT2 in seven different tissues of R. rugosa 'Zizhi'. RrGAPDH was used as the internal control. Error bars represent the SDs of triplicate reactions. The experiment was repeated three times with similar results. Different lower case letters represent significant difference which is calculated using LSD analysis at the level of P < 0.05.
FvGT of 3-O-glycosylation of flavonoids, suggesting that RrGT2 may be involved in the glycosylation process of 3-O positions of flavonoids.
The alignment of amino acid sequences between RrGT2 and glycosyltransferases from the other 21 species indicated that RrGT2 possessed a common PSPG motif of the glycosyltransferases superfamily (Figure 3). Previous studies have shown that the conserved region of PSPG is related to substrate recognition and catalytic activity of enzyme proteins [19] [20] [21] [22] . If the 44 amino acids of the PSPG domain are numbered, the amino acids at position 22, 23 and 44 play an important role in the selection of enzyme proteoglycan donors. The twenty-second position of tryptophan (Trp, W) can correctly locate UDP-glucose, while arginine (Arg, R) can make UDP-glucuronic acid locate correctly; the twenty-third position of serine (Ser, S) is highly conserved in UDP-glucurono- syltransferase [23] [24] [25] ; the forty-fourth glutamine (Gln, Q) and histidine (His, H) have strong conservatism in glucosyltransferase and galactotransferase respectively [22] . In the PSPG domain of the RrGT2 gene, the amino acids at position 22, 23 and 44 are tryptophan (Trp, W), asparagine (Asn , N) and glutamine (Gln, Q), respectively. Therefore, we speculated that the RrGT2 gene is using UDP-glucose as the main glycosyl donor, but has no glucuronyltransferase activity.
The expression levels of the RrGT2 gene during flower development and in different tissues were investigated. It was found that the expression of the RrGT2 gene showed a different trend during different flowering periods in three R. rugosa varieties, indicating that the expression of the RrGT2 gene was developmentally regulated in the process of anthocyanin biosynthesis. Studies have shown that the accumulation of anthocyanin in red skinned sand pear, strawberries and litchi is positively correlated with the activity of UF3GT. Boss et al. [26] also detected the expression of UF3GT in the peels of red grape which accumulated anthocyanin, but not in other tissues of red grape and white grape without anthocyanin accumulation. Gong et al's [27] studies showed that the partial structural genes of Perilla frutescens anthocyanin metabolic pathway were only expressed in the leaves of red varieties, but not in green varieties or the expression in the green leaves was very low. About the tissue-specific expression in R. rugosa 'Zizhi', it is worth mentioning that the stems of R. rugosa 'Zizhi' are purple, which is consistent with the relatively high level of expression of the RrGT2 gene. But for the relatively low level of expression in flowers, this did not mean that the RrGT2 gene had no effect on flower color formation. We believe that this was the result of comparing flowers at the budding stage as tissue types. Because in the budding stage, the expression of the RrGT2 gene was lowest, but in other stages was very high. In addition, RrGT2 was also highly expressed in the sepals and leaves, so we infer that RrGT2 is also involved in the glycosylation of secondary metabolites in sepals and leaves and plays an important role.
In conclusion, the cloning and expression analysis of the RrGT2 gene was beneficial to analyzing the molecular synthesis and regulation mechanism of anthocyanins, and also provided some important informations for the improvement of R. rugosa flower color in the future.
This project was supported by the Agricultural Seed Project of Shandong Province ([2014] No. 96).
*These authors contribute equally.
#Corresponding authors.
[1] Li, M. (2006) Survey and Quality Evaluation of Shandong Rose Varieties. Shandong University of Traditional Chinese Medicine.
[2] Feng, L.G., Shao, D.W., Sheng, L.X., et al. (2009) Study on Investigation and Morphological Variation of Wild Rosa rugosa in China. Journal of Shandong Agricultural University (Natural Science Edition), 40, 484-488.
[3] Chen, S.-M., Zhu, X.-R., Chen, F.D., et al. (2010) Expression Characteristics of Anthocyanin Structural Genes in Different Flower Color Chrysanthemum Cultivars. Journal of Northwest plants, No. 3, 453-458.
[4] Kim, S.H., Lee, J.R., Hong, S.T., et al. (2003) Molecular Cloning and Analysis of Anthocyanin Biosynthesis Genes Preferentially Expressed in Apple Skin. Plant Science, 165, 403-413.
[5] Martens, S., Preuß, A. and Matern, U. (2010) Multifunctional Flavonoid Dioxygenases: Flavonol and Anthocyanin Biosynthesis in Arabidopsis thaliana L. Phytochemistry, 71, 1040-1049.
https://doi.org/10.1016/j.phytochem.2010.04.016
[6] Ge, C.L., Huang, C.H. and Xu, X.B. (2012) Research on Anthocyanins Biosynthesis in Fruit. Acta Horticulturae Sinica, 39, 1655-1664.
[7] Forkmann, G. (2010) Flavonoids as flower Pigments: The Formation of the Natural Spectrum and Its Extension by Genetic Engineering. Plant Breeding, 106, 1-26.
[8] Springob, K., Nakajima, J.I., Yamazaki, M., et al. (2003) Recent Advances in the Biosynthesis and Accumutation of Anthocyanins. Natural Product Reports, 20, 288-303.
https://doi.org/10.1039/b109542k
[9] Yonekura-Sakakibara, K., Nakayama, T., Yamazaki, M., et al. (2009) Modification and Stabilization of Anthocyanins. Springer, New York, 169-190.
[10] Zheng, Z.L. (1994) Flower Color Gene Engineering of Flower Crops. Northern Horticulture, 3, 37-38.
[11] Vogt, T. and Jones, P. (2000) Glycosyltransferases in Plant Natural Product Synthesis: Characterization of a Supergene Family. Trends in Plant Science, 5, 380-386.
[12] Tohge, T., Nishiyama, Y., Hirai, M.Y., et al. (2005) Functional Genomics by Integrated Analysis of Metabolome and Transcriptome of Arabidopsis Plants Over-Expressing an MYB Transcription Factor. Plant Journal, 42, 218-235.
[13] Goto, T., Kondo, T., Tamura, H., et al. (1982) Structure of Gentiodelphin, an Acylated Anthocyanin Isolated from Gentiana makinoi, that Is Stable in Dilute Aqueous Solution. Tetrahedron Letters, 23, 3695-3698.
[14] Hall, D., Yuan, X.X., Murata, J., et al. (2012) Molecular Cloning and Biochemical Characterization of the UDP-Glucose: Flavonoid 3-0-Glucosyltransferase from Concord Grape (Vitis labrusca). Phytochemistry, 74, 90-99.
[15] Tanaka, Y., Yoshikazu, K., Fukuchi-Mlizutani, M., et al. (1996) Molecular and Biochemical Characterization of Three Anthocyanin Synthetic Enzymes from Gentiana triflora. Plant & Cell Physiology, 37, 711-716.
https://doi.org/10.1093/oxfordjournals.pcp.a029004
[16] Yamazaki, M., Yamagishi, E., Gong, Z.Z., et al. (2002) Two Flavonoid Glucosyltransferases from Petunia hybrida: Molecular Cloning, Biochemical Properties and Developmentally Regulated Expression. Plant Molecular Biology, 48, 401-411.
https://doi.org/10.1023/A:1014043214943
[17] Schmittgen, T.D. and Livak, K.J. (2008) Analyzing Real-Time PCR Data by the Comparative C(T) Method. Nature Protocols, 3, 1101-1108.
https://doi.org/10.1038/nprot.2008.73
[18] Sawada, S., Suzuki, H., Ichimaida, F., et al. (2005) UDP-Glucuronic Acid: Anthocyanin Glucuronosyltransferase from Red Daisy (Bellis perennis) Flowers. The Journal of Biological Chemistry, 280, 899-906.
[19] Gachon, C.M., Meurinne, M.L. and Saindrenan, P. (2005) Plant Secondary Metabolism Glycosyltransferases: The Emerging Functional Analysis. Trends in Plant Science, 10, 542-549.
https://doi.org/10.1016/j.tplants.2005.09.007
[20] Sakakibara, K.Y. and Hanada, K. (2011) An Evolutionary View of Functional Diversity in Family 1 Glycosyltransferases. The Plant Journal, 66, 182-193.
[21] Wang, X.Q. (2009) Structure, Mechanism and Engineering of Plant Natural Product Glycosyltransferases. FEBS Letters, 583, 3303-3309.
https://doi.org/10.1016/j.febslet.2009.09.042
[22] Kubo, A., Arai, Y., Nagashima, S., et al. (2004) Alteration of Sugar Donor Specificities of Plant Glycosyltransferases by a Single Point Mutation. Archives of Biochemistry and Biophysics, 429, 198-203.
https://doi.org/10.1016/j.abb.2004.06.021
[23] Shao, H., He, X.Z., Achnine, L., et al. (2005) Crystal Structures of a Multifunctional Triterpene/Flavonoid Glycosyltransferase from Medicago truncatula. Plant Cell, 17, 3141-3154.
https://doi.org/10.1105/tpc.105.035055
[24] Li, L.N., Modolo, L.V., Trevino, L.E., et al. (2007) Crystal Structure of Medicago truncatula UGT85H2-Insights into the Trusctural Basis of a Multifunctional (iso) Flavonoid Glycosyltransferase. Journal of Molecular Biology, 370, 951-963.
https://doi.org/10.1016/j.jmb.2007.05.036
[25] Modolo, L.V., Li, L.N., Pan, H.Y., et al. (2009) Crystal Structures of Glycosyltransferase UGT/8G1 Reveal the Molecular Basis for Glycosylation and Deglycosylation of (iso) Flavonoids. Journal of Molecular Biology, 392, 1292-1302.
[26] Boss, P.K., Davies, C. and Robinson, S.P. (1996) Expression of Anthocyanin Biosynthesis Pathway Genes in Red and White Grapes. Plant Molecular Biology, 32, 565-569.
[27] Gong, Z.Z., Yamazaki, M., Sugiyama, M., et al. (1997) Cloning and Molecular Analysis of Structural Genes Involved in Anthocyanin Biosynthesis and Expressed in a Forma-Specific Manner in Perilla frutescens. Plant Molecular Biology, 35, 915-927.
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redpajama_set_name: RedPajamaCommonCrawl


N 51° 37' 14" and W -3° 56' 35"
Swansea in City and County of Swansea (Wales) with it's 300,352 citizens is a city in United Kingdom about 164 mi (or 264 km) west of London, the country's capital city.
Local time in Swansea is now 08:40 AM (Sunday). The local timezone is named Europe / London with an UTC offset of one hour. We know of 8 airports in the vicinity of Swansea, of which 3 are larger airports. The closest airport in United Kingdom is Swansea Airport in a distance of 5 mi (or 9 km), West. Besides the airports, there are other travel options available (check left side).
There are several Unesco world heritage sites nearby. The closest heritage site in United Kingdom is Blaenavon Industrial Landscape in a distance of 45 mi (or 73 km), East. Also, if you like golfing, there are a few options in driving distance. Looking for a place to stay? we compiled a list of available hotels close to the map centre further down the page.
When in this area, you might want to pay a visit to some of the following locations: Landore, Morriston, Clydach, Bishopston and Pennard. To further explore this place, just scroll down and browse the available info. Let's start with some photos from the area.
Frankie Fryer checks out all the sights for a Swansea Away Day.
http://www.visitswanseabay.com - get away on a romantic break in our neck of the UK, where moments last forever. Make the weekend last a lifetime, Visit Swansea Bay.
Just a video of this year's parade. Shot from my kitchen overlooking the top of Wind Street and Castle Gardens.
http://www.visitswanseabay.com - start your adventure with an activity holiday in the UK. Make the weekend last a lifetime, Visit Swansea Bay.
Marsch zum Liberty Stadium, 03.10.2013.
Located at 51.6203, -3.9412 (Lat. / Lng.), about 0 miles away.
Located at 51.6253, -3.9409 (Lat. / Lng.), about 0 miles away.
Located at 51.6164, -3.93861 (Lat. / Lng.), about 0 miles away.
The Maritime Quarter, or Swansea Marina, is a residential area of Swansea, Wales, UK located immediately south of the city centre shopping core. It falls within Swansea's Castle ward. The area is bounded by Brynmill to the west, Swansea Bay to the South, the River Tawe to the east and the A4067 to the north.
Located at 51.6154, -3.9386 (Lat. / Lng.), about 0 miles away.
Swansea Marina is a marina located behind the Swansea barrage at the mouth of the River Tawe in Swansea, south Wales. Swansea Marina was awarded blue flag status in June 2005http://wales. gov. uk/news/archivepress/environmentpress/enviropress2005/707946/?lang=en and has been awarded five gold anchors by the Yacht Harbour Association. http://www. ewpdev. com/ami/news2.
Located at 51.6158, -3.93833 (Lat. / Lng.), about 0 miles away.
The LC is a leisure centre located in the city centre of Swansea, Wales, UK. Originally Swansea Leisure Centre, the rebranded facility re-opened to the public on 1 March 2008 after a £32 million makeover. The LC's exterior has been revitalised by replacing the old concrete panels with clear glass, translucent glazing and timber panelling.
Located at 51.6169, -3.94056 (Lat. / Lng.), about 0 miles away.
The Swansea Museum in Swansea, Wales, UK is the oldest museum in Wales. The building was built for the Royal Institution of South Wales in 1841 in the neo-classical style.
Located at 51.6178, -3.9381 (Lat. / Lng.), about 0 miles away.
The Glynn Vivian Art Gallery is a public art gallery in Britain. It is the public art gallery of the City and County of Swansea, in South Wales. The gallery is situated in Alexandra Road, near Swansea railway station, opposite the old Swansea Central Library.
Located at 51.6239, -3.94444 (Lat. / Lng.), about 0 miles away.
Swansea Market situated in the heart of Swansea city centre is the largest indoor market in Wales. The market is covered by a steel arched portal frame roof clad in steel and glass. The current market was built in 1959-1960 by Percy Edwards. It is the fourth market to be built at the site in 200 years. The previous market on this site had existed since 1894 and was destroyed during bombing raids during World War II. The site has been used as a market since the Middle Ages.
Located at 51.618, -3.95 (Lat. / Lng.), about 0 miles away.
The Quadrant Shopping Centre is the principal under-cover shopping centre in Swansea, Wales. The centre opened in 1979. Since the 1980s it has been home to the Swansea Devil, a controversial carved wooden statue of the Devil. The centre and surrounding areas are owned by the City and County of Swansea council.
Located at 51.618, -3.945 (Lat. / Lng.), about 0 miles away.
Swansea bus station, or Swansea city bus station, is a bus station serving Swansea, Wales. It lies immediately to the west of the Quadrant Shopping Centre. The bus station also has a taxi rank to the south. The station has 20 stands for local bus services with 3 stands for national coach services.
Located at 51.6168, -3.94586 (Lat. / Lng.), about 0 miles away.
St Mary's Collegiate and Parish Church is an Anglican Church in the centre of Swansea, Wales, UK. There was a church on the site of St Mary's since circa 1328, erected by Henry de Gower, Bishop of Saint David's. One Sunday morning, in 1739, the roof of the nave collapsed into the church. Fortunately this was before the waiting congregation entered the building. The whole structure was re-built apart from the tower. 1822 saw the church being lit by gas for the first time with thirty six lamps.
Located at 51.619, -3.943 (Lat. / Lng.), about 0 miles away.
The old Swansea Central Library in Swansea in Britain was a public lending and reference library located in Alexandra Road, in the Swansea city centre. The old library building is now occupied by Swansea Metropolitan University's Welsh School of Architectural Glass.
Located at 51.6236, -3.94389 (Lat. / Lng.), about 0 miles away.
The Tower, Meridian Quay is the tallest building in Wales standing at 107m (351ft). The tower has 29 storeys, double the number of the previous tallest building in Swansea, the BT Tower. Most of the tower houses residential apartments. The ground floor features a concierge desk which is manned 24 hours a day, whilst the top three floors consist of the Grape and Olive restaurant run by the Cardiff brewery SA Brains. This was opened following the unsuccessful 290 cover Pentouse restaurant.
Located at 51.6139, -3.94318 (Lat. / Lng.), about 0 miles away.
The Plantasia is a large public hothouse, located in the Parc Tawe retail park, Swansea, Wales that opened in 1990.
Located at 51.6217, -3.93861 (Lat. / Lng.), about 0 miles away.
This article is about the shopping centre in Swansea. For the shopping centre of the same name in Cardiff, see St. David's (Cardiff) St. David's Shopping Centre is a shopping complex in Swansea, south Wales. The complex is located adjacent to the Quadrant Shopping Centre and opposite St Mary's Church. Completed in 1982, the complex has thus far been fairly unsuccessful, in terms of filling its outlet spaces and retaining customers.
Located at 51.618, -3.943 (Lat. / Lng.), about 0 miles away.
Parc Tawe is a retail park and leisure area in Swansea, Wales. It is located in the eastern area of the city centre on the west bank of the River Tawe in the Lower Swansea valley. The area includes "out-of-town" style stores with car parks located outside the shopping area. A highly visible building in the area is the Plantasia - a large triangular tropical plant house. Entertainment in the area include a 10 screen Odeon cinema and a ten pin bowling alley.
Located at 51.6217, -3.93917 (Lat. / Lng.), about 0 miles away.
The Palace Theatre is a building located at the northern end of High Street, Swansea, Wales, recognisable for its distinctive wedge shape. Originally built in 1888 as a traditional music hall, the building's original name was 'The Pavilion'. During its lifetime, the building has been used as a bingo hall as well as a nightclub. The Grade II Listed building is one of just two purpose-built music halls left standing in the whole of the UK.
Located at 51.6275, -3.94111 (Lat. / Lng.), about 0 miles away.
The Castle Cinema is a former cinema building located adjacent to the grounds of Swansea Castle in Swansea, south Wales. According to Kinematograph year book of 1914 the Castle opened in October 1913. It was the only building left standing in Swansea's Castle Street vicinity after the Nazi German Luftwaffe bombing raids during the Second World War. It is a grade II listed structure. In 1963 the Castle Cinema changed its manager (Mr.
Located at 51.6208, -3.94139 (Lat. / Lng.), about 0 miles away.
Swansea Central police station is a 21st-century-designed building, located on the former Swansea Fire Station site in Alexandra Road, near Swansea Magistrates Court, Swansea. The police station is the area headquarters for the Western Division of South Wales Police.
Located at 51.622, -3.946 (Lat. / Lng.), about 0 miles away.
The Weaver building was a flour mill and corn storage building in the North Dock of Swansea, Wales. The structure was built by French engineer Francois Hennebique in 1897 and was the first reinforced concrete building in Europe. The Weaver building survived World War II bombings in 1941, but was demolished in 1984 to make way for a new Sainsbury's superstore that now stands on the site.
Located at 51.62, -3.938 (Lat. / Lng.), about 0 miles away.
The BT Tower is located near Swansea Castle and is the second tallest building in Swansea after The Tower, Meridian Quay. It has 13 floors and is 63m/206.64 ft high. Completed in 1970, it is used as an office building for BT Group plc, its original developer.
Located at 51.6206, -3.94028 (Lat. / Lng.), about 0 miles away.
The Environment Centre in Swansea, Wales, is an independent charity organisation for environmental information, education and activity. Environment Centre may also refer to the building the charity is located in. The Environment Centre's aims are to raise people's awareness of environmental issues, to increase the participation in environmental projects and to work towards a more sustainable future. It promotes, for example, the concept of the three Rs (reduce, reuse, recycle).
Located at 51.6185, -3.93637 (Lat. / Lng.), about 0 miles away.
The Dylan Thomas Theatre is a theatre based in the Maritime Quarter, in the centre of the city of Swansea in Wales. The theatre officially opened under its present name in 1983, but was home to the Swansea Little Theatre from 1979.
Located at 51.6169, -3.93722 (Lat. / Lng.), about 0 miles away.
Swansea Victoria is a former railway station in Swansea, south Wales. It closed in 1964 under the Beeching Axe.
Located at 51.6174, -3.9397 (Lat. / Lng.), about 0 miles away.
These are some bigger and more relevant cities in the wider vivinity of Swansea.


Patients often remember a family member who suffered from blood clots. Blood clots can occur both in arteries and veins. Arteries carry blood from the heart to the tissues and veins return the blood from the tissues back to the heart. Both artery and vein clots are potentially dangerous but are different from each other in many respects. Only venous blood clots commonly referred to as DVT or DVT clot are explained below. DVT is abbreviation for deep vein thrombosis. Thrombosis is the medical term for blood clot.
Clotting of the blood is a necessary safety mechanism. It prevents a person from bleeding to death from a minor cut or every day injury. The injured tissue produces a substance that promotes blood clotting at the site of injury.
Nature has provided several mechanisms to prevent blood from clotting inside the blood vessels. The movement of blood inside the blood vessels, itself is a protective mechanism. Blood clot does not form unless there is stagnation of blood ("stasis"). However, stasis within veins can occur in patients who are inactive or bed ridden. Muscle activity in the legs such as in walking is necessary to keep the circulation going in veins. A form of this type of DVT clot sometimes occurs when patients travel long distances in a sitting position.
Most DVT blood clots occur in leg veins. Arms are less affected as arm movements are maintained even in sedentary patients. The incidence of DVT blood clot is very high in patients who are very sick such as those who are admitted to the intensive care unit in the hospital. Stasis can also occur in patients who are immobilized by plaster casts on the legs or restricted to bed by back problems, joint replacement, multiple fractures following motor vehicle accident or other forms of injury. Often, tissue factors that promote blood clots following these types of injury add to the stasis problem to increase the risk even further. Stroke often confines the patient to bed for weeks or months and leg muscle action may be impossible from paralysis. The occurrence of DVT clots are high in these patients.
Several substances are present in the circulating blood that act against formation of a blood clot. Many of these molecules have been identified but some are not identified yet. Some patients are deficient in one or more of these substances as a hereditary defect. The medical term for this condition is Thrombophilia (Greek word for 'prone to blood clot') These patients are 'hypercoagulable'– medical jargon to mean 'prone to blood clots'. Some Thrombophilia defects are more serious than others, so the risk of blood clot varies according to the type of defect. Factor five deficiency, also known as Leiden gene defect is one such mild defect. It is present in one in five individuals. Prothrombin gene deficiency is another such mild deficiency which is also present in many individuals. The blood clot risk in these mild defects is only slightly increased over the general population. The risk of DVT clots is much higher in certain other forms of molecular deficiency.
There are several natural conditions such as pregnancy that may increase clotting tendency. The risk is highest in late stages of pregnancy and during several weeks after child birth. The risk of blood clots gradually increases with age. Some hormones and medications can also increase clotting tendency. The risk of DVT clots from birth control pills and estrogen replacement pills have greatly decreased because of better formulations. But some mild risk is still present. Some patients on these medications may develop DVT clots when a mild form of Thrombophilia such as Leiden gene or prothrombin gene deficiency is also present increasing the risk.
Some diseases particularly cancer is a strong promoter of DVT clots. Vein problems such as varicose veins or leaking valves in the deep veins can also increase the risk of DVT clot formation. A DVT clot causes damage and scarring of the veins. This scarring produces narrowing of the veins. This in turn slows the movement of blood in veins causing stasis. Thus a vicious cycle is produced where a previous DVT clot increases the risk of future DVT clots.
When a blood clot travels to the lung, it is called "pulmonary embolism". A piece of DVT clot that breaks away and travels with the circulation to another part of the body is known as an embolus. Most often this produces no symptoms at all or only mild to moderate symptoms such as shortness of the breath. In a small percentage, perhaps 1 in 100 sudden death may occur. These are usually sick patients prone to blood clots for a variety of reasons described above. The travelling blood clot has to be large in size to produce death. Not all blood clots travel to the lung. Blood clots in the superficial veins such as varicose veins do not break easy to travel. Therefore, the risk of pulmonary embolism is very low. But they may cause intense pain and inflammation over the clot for two to three weeks. Blood clots from the arms also do not often travel to the lung. DVT clots in the deep veins of the leg are often silent—there is no pain or other indication that a DVT clot has formed. Thy are the ones that tend to travel to the lung. They are also large enough to cause death. It is estimated that 90% of blood clots that travel to lung originate from deep veins of the leg. The treatment for blood clots in the deep veins of the leg is administration of blood thinners. This is also the main treatment for pulmonary embolism—clots that have already travelled to the lung. The lung has great ability to dissolve clots that have travelled there if blood thinners are given for 3 to four months. In contrast, blood thinners do not dissolve DVT clots that are still in place in the deep veins of the leg. They do help to prevent the clot from growing larger and may help them shrink in size. Eventually, most DVT clots in the deep veins of the leg turn into scar tissue that narrows the vein.
In cases where blood thinners cannot be safely given because of bleeding risk from stomach ulcers for instance, filters (known as IVC filters) can be inserted above the clot to trap them if they try to move. They are needed only temporarily, say for 2 months or so. By then the clot has turned into solid scar tissue within the vein and is not able to move. IVC filters should be removed as soon as they have served their purpose. This is because, the IVC filters themselves can scar the deep vein after they have been in place for 3 months or more.
Scar tissue that is a result of a DVT clot in the deep veins of the leg generally cause no serious health problems. However, annoying symptoms such as leg pain, heaviness and swelling and occasionally even leg ulcers can develop from narrowing of the vein. This prevents healthy circulation of the legs. The is known as Post thrombotic syndrome– which literally means a symptom condition that develops after a deep vein DVT clot. Medical grade support stockings can be a great help in controlling these symptoms. In selected cases where compression stockings fail to provide relief, stents may be placed to open up the narrowed vein. Stents look like a large coil spring made of metal. They can be inserted into the vein through needle sticks.


Being a human, our mind generates hundreds of questions when we try to make any decision. But when we are up to invest money to buy a product, thousands of questions are randomly generated. Today in this article I am going to answer all those common questions that are produced in our mind related to Athletic Greens. For this section, I have trawled the internet and tried to gather all the common questions that are asked on the web associated with Athletic Greens. So let's start our Athletic Greens faq.
Q – What is/are Athletic Greens?
Q – Who owns Athletic Greens superfood powder?
Q – What is Athletic Greens made of OR what ingredients are in Athletic Greens?
Q – Do/Does Athletic Greens powder really work?
Q – What/How does Athletic Greens taste like?
Q – How to make Athletic Greens taste better?
Q – How to use/take Athletic Greens?
Q – When to drink/take Athletic Greens?
Q – What to mix with Athletic Greens?
Q – What do you mix Athletic Greens with?
Q – How to mix or prepare Athletic Greens superfood powder?
Q – How many calories in Athletic Greens powder?
Q – How many carbs in Athletic Greens?
Q – Is there caffeine in Athletic Greens superfood powder?
Q – Is Athletic Greens keto friendly?
Q – Are Athletic Greens good for you?
Q – Are Athletic Greens any good?
Q – Are Athletic Greens healthy?
Q – Are Athletic Greens safe?
Q – How much is/does Athletic Greens cost?
Q – Is/Are Athletic Greens worth the money or price?
Q – Where can you/I buy or purchase Athletic Greens powder?
Q – Who sells/carries Athletic Greens?
Q – Where to/can I buy or purchase Athletic Greens in US/Canada/South Africa/Australia?
Q – Can you buy Athletic Greens in stores?
Q – What stores sell/carry Athletic Greens?
Q – Does GNC sell/carry Athletic Greens?
Q – What does Athletic Greens do?
Q – Why are Athletic Greens so expensive?
Q – What is comparable to Athletic Greens?
Q – How to cancel Athletic Greens?
Every query starts in our mind with the introduction of the product, and we get eager to know about the product we come across. Here are some common questions related to Athletic Greens introduction.
Ans – Athletic Greens is a premium superfood and cocktail of green smoothie powder. It is marketed as the best alternative to your daily nutritional deficit. It is perfectly blended and combined with all essential vitamins, minerals and other nutrients that are important for our body's daily requirement that most of us fail to get from our regular diet.
It is essential to know who is manufacturing or producing the supplement as the credibility of the manufacturer creates reliability and assurance in the buyer's mind.
What we first check in any product or supplement is the list of ingredients used in the formulation. It is essential as a buyer should be aware that isn't the product includes any component that does not suit his body.
Ans – The official website claims that the Athletic Greens is formulated with 100% natural ingredients and includes nutrition in pure extract form. The supplement consists of the total of 75 vital elements that is equivalent to consuming 15 types of fruits and vegetables. Some of the prominent ingredients are Ashwagandha root extract, Rhodiola Rosea extract, Green tea extract, Cocoa bean extract, and CoQ10.
Every product or supplement officially claims in the market that it helps in boost immunity or treating various conditions from the root, but it is hard to believe as we cannot judge the product from the cover and therefore it obvious that question arises in mind related to efficiency.
Ans – As I crawled throughout the official website, I found many big claims made regarding the efficiency of this green drink powder. It is claimed that no other superfood supplement in the market can match the ability of Athletic Greens. Consumption of this supplement is the equivalent of consuming five different types of vitamins and mineral pills. But talking about the facts, though the product is not as efficient as claimed, overall it can be said that it is an excellent green drink supplement in the budget.
As we all know that any green drink, whether it's a homemade or packed supplement, doesn't taste well and that is the reason people hesitate in consuming such products. Therefore it is essential to know whether Athletic Greens taste similar to its league or it is different.
Ans – In this factor, I must say that Athletic Greens is much better, or we could say best in the market. I have gone through various customer reviews and found that almost everyone appreciates the taste of this green drink. It is much delicious, sweet and smooth than any other green drink.
Ans – Although you may not need to add anything to make it taste better because it usually tastes sweet and delicious but if you want you can add some artificial sweetener or fruits in it.
To drive out maximum result out of any supplement, it is essential to use it as recommended by the manufacturer. This raises some common questions related to the usage of this green drink powder.
Ans – It is recommended to take this green drink powder twice a day in the form of shake by mixing it in water.
Ans – The manufacturer suggests taking it twice a day once with breakfast and another with dinner.
Ans – Though the official website states to mix the powder with water, but you can also use milk, only if you are not diabetic or overweight or any other condition which restricts protein intake.
Ans – To prepare a green shake, you should mix two tablespoons of AG powder in 200 ml water.
When we talk about a superfood supplement, it gets essential to know how that supplement will help in proving essential nutrients. Here are some common questions related to the nutritional quality of this superfood green drink.
Ans – There are 40 calories in one tablespoon serving of Athletic Greens of which 42% is carb, and 58% is protein.
Ans – There is net 4.8 gram of carb in each serving of powder.
Ans – No, the ingredient list doesn't include caffeine, and therefore it could be said that it is caffeine free drink.
Ans – No, because in the ketogenic diet you are dependent on healthy fat and avoid carbs but in Athletic Greens, you don't take fat and use carbs as the primary fuel. Therefore, Athletic Greens and Ketogenic diet cannot be continued parallelly.
It is crucial for anyone to know whether the supplement or product he or she is going to use, produces any side effects or any harmful effect on the health. Find out answers to such questions related to Athletic Greens.
Ans – These are the essential questions. Therefore, it needed some in-depth research. I have scanned various review websites and forums along with an official website to get the most excellent answer for this question. Being a natural ingredient supplement, there are no such possible side effects, but if you are suffering from any chronic health condition, then it is recommended to consult with your physician before consuming it. If you are on a low protein diet, then you must avoid taking Athletic Greens because it provides 58% protein that would be not good for your diet.
Every buyer's primary focus is on the price as they are restricted to their budget. Investing more than the pocket sometimes leads to complete dissatisfaction when you don't get the desired results. Here are some common questions related to cost & best price of Athletic Greens.
Ans – The cost of Athletic Greens is quite high as the pack of 30 Servings costs you $77 per month whereas the package of 60 servings costs you $147 per month. There is a trial offer pack that costs you $97, but you can avail it one time only.
When you pay a high amount for any product or service, you expect better results from it, and if the product delivers you prominent results, you don't hesitate in investing in the future. But what about Athletic Greens, it is cost worthy?
Ans – Although we could say that this supplement is quite costly compared to other alternatives available in the market, if you go through the ingredients and results delivered, the price looks worthy. As per the official website, no such supplement matches the ingredient quality of Athletic Greens. If you individually buy those supplement, it will cost you around $445 per month and therefore in such case you are saving about $365 per month, and that's a considerable amount.
When you make your decision of buying a supplement, the next thing that comes is the best place to make your order. This is because there are lots of scams prevailing on the internet and millions of people are getting trapped and wasting money on it. Here are some common queries related to purchase of Athletic Greens.
Ans – Well, there are various options where you can buy this superfood green drink such as official website, good affiliate sites and online stores. But it is highly recommended to place your order through official website as it ensures that you are getting the genuine product and not the fake or name resembling.
Ans – Athletic Greens is a US-based superfood green drink that is very popular in the US and Canada. You can easily buy it from the official website and online stores in US and Canada. Although the official website does not ship this product outside the US and Canada therefore if you live outside the US and Canada, you can make your order through online stores.
Ans – You cannot find this superfood green drink in local or nearby stores or supermarket. Other than the official website you can only get it through Amazon.
Ans – Although many review websites claim that Athletic Greens is duly available for purchase on GNC but I checked the GNC website to find out Athletic Greens, but there was no such product available on the store.
Ans – This superfood green drink works in 3 step formula, Nourishment – Absorption – Synthesis. It nourishes your body with all vital nutrients, improves your digestive system and synthesis your cells to allow your body to utilize all the nutrients delivered.
Ans – The manufacturer's response for this question is that the ingredients used in the formulation are real extracts that are expensive in processing and also 75 types of nutrients increase the cost of this supplement.
Ans – When comparing Athletic Greens with other such supplements, two names are prominent in the market, Total Living Drink Green and Shakeology. But these two are even much costlier. The closest competitor to AG is Green Vibrance. It is also an excellent green drink, but I will still stay with Athletic Greens because it is useful, affordable and safe.
Ans – Cancellation process is available on the monthly subscription option. If you have subscribed for regular monthly supply, then you can easily cancel or pause your subscription by logging into your account or by calling out the customer service team on 1-888-390-4029.
Hopefully, I have covered all the questions regarding Athletic Greens that are arising in your mind. If I have missed some or if you have some other questions related to this product, you can email us. Also checkout unbiased review on Athletic Greens for more details. Search for more superfood supplements on MGD that can be alternative to Athletic Greens.


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RenaissanceRe Holdings announced that Cythnia Trudell, 65, has been nominated to serve as an independent director of the Company, and that Edward J. Zore, 73, will retire from the Board at the end of his term, each to occur in conjunction with the Company's Annual General Meeting of Shareholders in May 2019.
Ms. Trudell most recently served as Executive Vice President and Chief Human Resources Officer of PepsiCo, Inc., and previously served in a number of executive and management positions with General Motors Corporationand Brunswick Corporation, including as chairwoman and president of Saturn Corporation, president of IBC Vehicles and president of Sea Ray Group.
Kevin J. O'Donnell, President and Chief Executive Officer of RenaissanceRe, said, "The entire RenaissanceRe executive team and my fellow directors are delighted by the nomination of Cynthia. Her distinguished background as a chief executive and as a leader of organizational talent across industries confronting change, as well as her experience as a director in financial services, is ideal for our Company at this time.
James L. Gibbons, non-Executive Chair of RenaissanceRe added, "The entire RenaissanceRe Board is pleased to nominate Cynthia for election by our shareholders.
"We are confident that Cynthia's broad experience as a chief executive, as an operating manager, and as a chief human resources officer of a leading global enterprise will support the Board's oversight at a time when the Company's organic and inorganic growth is expanding its operational footprint and breadth of client services significantly.
From 2011 until her retirement in September 2017, Ms. Trudell served as Executive Vice President and Chief Human Resources Officer of PepsiCo, Inc. having served as Senior Vice President and Chief Personnel Officer of PepsiCo from 2007 to 2011.
Prior to her tenure at PepsiCo, Ms. Trudell held a number of executive operating and general management positions with General Motors Corporation from 1981 to 2001, and with Brunswick Corporation from 2001 to 2006, including chairwoman and president of Saturn Corporation, president of IBC Vehicles and president of Sea Ray Group.
Since 2015, Ms. Trudell has served on the board of ISS A/S, a global facility services provider based in Denmarkand publicly traded on the NASDAQ OMX Copenhagen, where she currently serves as a member of the Remuneration Committee and the Nomination Committee.
Since 2013, she has served as a member of the Defense Business Board, which provides business advice to the U.S. Department of Defense. She also served on the boards of PepsiCo from 2000 to 2007,Canadian Imperial Bank of Commerce from 2005 to 2008 and The Pepsi Bottling Group, Inc. from 2008 to 2010.


"Peter was just unbelievably comprehensive in his envisioning of this world. He was the encyclopedia that I read, in order to play my part. There was wonderful leadership, done with this infectious, joyous, childlike passion for the elements of the story and for the film."
Sam Elliott should just voice all the animated movies forever.
He also talks about taking over the project from previous director Bob Peterson, making the protagonist younger, the frontier setting, and more.
Originally scheduled for release in May 2014, the buddy comedy has been completely retooled.
'Toy Story 4' won't be a continuation of the past films, but rather a brand new chapter in the 'Toy Story' world.


Colorful गुलबहार, डेज़ी. A dasiy of many रंग. Wallpaper and background images in the रंग club tagged: colors colorful flower daisy.
This रंग photo contains एडलवाइस, leontopodium alpinum, एडेलवीस, लेओटोपोडियम एल्पाइनम, एडेलवेइस, रुए एनीमोन, anemonella thalictroides, र्यू एनेमोन, एनेमोनेला थैलेिक्टाइरेड्स, rue एनेमोन, hippeastrum, hippeastrum puniceum, हिपपेस्ट्रम, and हिपपेस्ट्राम पाइनिसियम. There might also be matilija अफीम, कैलिफोर्निया पेड़ अफीम, romneya coulteri, मैटीलीज पोस्ता, कैलिफ़ोर्निया पेड़ पेप्सी, रोमेनेय कल्टेरी, सूरजमुखी, हेलियनथस, हेलिएन्थस, पहाड़ लिली, लिलियम auratum, लिलिअम अरातुम, and तटीय auratum.
An नारंगी, ऑरेंज फल called "Orange"


text:
Story launches 'owned' channel consultancy in thriving city of Leeds
MISSION brand, Story, increases its presence in the North of England with a new office opening in Leeds.
Headed by experienced CEO and entrepreneur Mark Reardon, Story Leeds will work collaboratively with organisations large and small to help them unlock the brand and commercial value of their 'owned' channels. This will involve a holistic approach to channels and assets as varied as identity, product innovation, experience, content, technology and employee engagement.
Story Leeds will also provide Clients with access knowledge, expertise and talent from across MISSION Group, which has 1,150 staff in 31 offices across the UK, US, Europe and Asia, with an annual turnover of nearly £80m.
With the office located in The Calls in the heart of the city, MISSION is the latest national Group to move into this growing creative hub, at the centre of one of the fastest growing regional economies for financial and business services.
Mark Reardon, CEO of Story Leeds, said: "Modern day brands are built in non-traditional ways, via consumers engagement with creativity, experience and technology increasingly in owned channels. That's our focus and we're looking forward to bringing this approach, and our expert consultancy, to one of the most exciting and creative cities in the UK."
James Clifton, MISSION Group Chief Executive said "We're excited to be growing our successful Story brand in Leeds, building on its Edinburgh home and newly opened Newcastle office. Like all MISSION Agencies, it will have its own specialisms, expertise and creative culture. Story Leeds will fit perfectly within our operating model – a strong and unique local proposition, with the ability to draw on talent and experience from across the Global Group."


Michael Melluish, Chairman of the Arundel Castle Cricket Foundation from 2003–2011, died on February 8th aged 81. His contribution to cricket at Arundel was both inspiring and selfless. He worked tirelessly behind the scenes, superbly supported by his wife, Anna, to strengthen the Board of Trustees and so in turn to improve the financial impact of the Foundation. Thousands of young people have benefited, without knowing it, from Mike's energy and commitment.
He loved Arundel dearly and visited the ground with Anna whenever he could during the summer. His kindness towards people, as well as his great affection for the game, shone from a man of impressive intellect, wide-ranging interests and a mischievous sense of fun. Of course he will be very much missed at Arundel and elsewhere but those who knew him will remember him with great joy and a broad smile on their faces.
St John's Wood Church, London, with a reception afterwards in The Long Room at Lord's Cricket Ground.
Michael Edward Lovelace (MEL), OBE, of Bradford on Avon, formerly of Mayflower Cottage, Broadham Green, died peacefully at home on Saturday 8th February 2014 after a wonderful innings of 81. Much loved husband of Anna, brother of Bat, father of Sarah, Louise and Sam, father-in-law of David and Martin and adored Grandpa of Gina, Susanna, Eliza, Jamie and Max. Private funeral. Celebration of Mike's life to be held in due course. No flowers please, donations if desired to Arundel Castle Cricket Foundation (Charity No: 1150351), Arundel, West Sussex,.— BN18 9LH.


text:
We Treat Our Clients Like Family · Hablamos Español
Conspiracy Charges
Pandering & Sex Trafficking
Status Crimes
Las Vegas Criminal Lawyer > Las Vegas Criminal Defense > Las Vegas Possession of Stolen Vehicle Lawyer
Las Vegas Possession of Stolen Vehicle Lawyer
Possession of a stolen vehicle can carry a lengthy prison sentence if a person is convicted. Read the following information below to learn more information about possession of a stolen vehicle, its penalties, and its defenses.
What constitutes possession of a stolen vehicle?
NRS 205.273 states that any person commits an offense involving a stolen vehicle if the person:
Intends to produce or pass a title to a motor vehicle in which he/she knows or has any reason to believe that the vehicle was stolen; or
Has in his/her possession a motor vehicle which the person knows or has any reason to believe that the vehicle was stolen.
It is important to note that this law also applies to motorcycles and trucks as well.
Penalties and punishments of possession of a stolen vehicle:
The penalties and punishments for possession of a stolen vehicle vary depending on the cost of the vehicle stolen.
If the vehicle is worth $3,500 or less, you are guilty of a category C felony and will be punished:
A minimum term of 1 year and a maximum term of 5 years in a state prison; and
A possible fine of no more than $10,000; and
Required to pay restitution
If the vehicle is worth $3,500 or more, you are guilty of a category B felony and will be punished:
A minimum term of 1 year and a maximum term of 10 years in a state prison; and
Defenses to possession of a stolen vehicle charges in Las Vegas:
There are several defenses to this charge that can either reduce the charge or even dismiss it.
You had no reason to believe the vehicle was stolen.
You were never in possession of the vehicle.
In order to properly evaluate your Possession of a Stolen Vehicle charges, it is important that you contact an experienced Las Vegas stolen vehicle lawyer who can get you the results you deserve. Adrian Lobo has dealt with several Possession of a Stolen Vehicle cases and has worked tirelessly to get her clients the help they need. Contact Adrian Lobo today to schedule a consultation.
Acquittals, Dismissals,
& Reductions
State v. Individual, 2018
Charge: Driving Under the Influence while also being undocumented.
Received a reduced charge sentence by the Superior Court of California
Charge: Attempt Murder with the Use of a Deadly Weapon and Possession of a Firearm by a Prohibited Person.
Attempt murder charges were dismissed and case negotiated to possession of a firearm by a prohibited person.
Charge: Murder with the Use of a Deadly Weapon and Possession of a Firearm by a Prohibited Person.
State dismissed the charges.
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text:
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Do you see what I am doing wrong here?

redpajama_set_name: RedPajamaStackExchange


text:
Report: Around the World, a Christian Is Martyred Every Six Minutes
3 Signs of an Unhealthy Relationship You Can't Ignore
This year, almost 90,000 Christians died for their faith, according to an Italian research group.
The Center for Studies on New Religions (Censur) will publish its annual report next month, and it's devastating. That 90,000 number means in 2016 one Christian was martyred every six minutes.
Earlier this week, the center's director, Massimo Introvigne, told Vatican Radio that this number makes Christians the most persecuted religious group in the world. (It's important to note that while Christians represent the most persecuted religious group, both according to this and other studies, they are also the most populous in the world.)
Introvigne explained that most of these killings (around 70 percent) resulted from "tribal conflicts" in Africa, often because Christians refused to take part in violence.
Michael Lang, an 18-Year-Old Freshman in Ohio, Has Died of COVID-19
Another part of the problem in Africa, and particularly in North Nigeria, is the terrorist group Boko Haram.
Introvigne said the other 30 percent of Christian martyrs happened in terrorist attacks and as the result of government persecution.
Aaron Cline Hanbury is a contributing editor for RELEVANT. You can follow him on Twitter at @achanbury
Aaron Cline Hanbury December 30, 2016
Research: 74 Percent of Americans Still Identify as Christians
DAPL Protestors Hung a Giant Banner From an NFL Stadium During a Game Yesterday


On Thursday,we had our corporate Xmas event.
Fun was had by all. And the food was tasty (we had 3 tutors to help and show us how to).
We are enjoying a nice sun today. Osiris tells you to enjoy life as much as she does.
Oh, what a fabulous party! We don't get anything close to that nice where I work.
De quoi vous faire regretter de ne plus aller au taf, lol!
Bon début pour la saison des fêtes, en tout cas.
MMmmmm tasty treats for everyone. FUN.
What a unique way to pique the interest of people at the event! Fun!


Accept chip cards and NFC payments with Square's most advanced reader yet. Plug it into Square Stand or connect wirelessly to your mobile device.
Swipe magstripe cards with the included magstripe reader.
Accept EMV chip cards, Apple Pay, Google Pay, and other contactless payments.
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The world is our RUNway and we believe in looking good for victory! Life will show up for you when you show up for life!
We believe that it's your story of triumph and victory that create the DNA and narrative of Mission I'mPOSSIBLE.
The RUNway is where dreams become a reality, ideas turn into action, life turns into living and tests turn into testimonies.
We believe in using the RUNway to serve our community.
From local to global activations, Mission I'mPOSSIBLE's mission is to bring light to the darkest places!
Through fashion, fitness and community activations, we lift the heads of the lost and bring hope to the hopeless.


text:
El hotel Paysant es una mansión privada situada en el 4 Distrito de París construido en el siglo XVII.

Historia
Fue adquirido en subasta en 1644 por el notario Etienne Paysant que lo arrendó a los marqueses y marquesas de Sévigné que se instalaron allí en diciembre de 1644. La marquesa, cansada de las infidelidades de su marido, en particular de un romance con Ninon de Lenclos, pidió la separación de bienes y dio permiso a su propietario en septiembre de 1650 para instalarse en el Château des Rochers. En 1660, Marie Paysant, hija de Etienne Paysant, heredó la casa e hizo reconstruir el edificio principal del patio. Jean-Baptiste du Féron, Gran Maestre de las Aguas y los Bosques de Île-de-France compró el hotel que permaneció en manos de la misma familia hasta 1808. La residencia fue adquirida por Jacques Happey, propietario del cercano Hôtel de Vieuville, y siguió siendo propiedad de sus descendientes, los Barbiers d'Aucour hasta la década de 1920.

Descripción
La fachada de la calle ha conservado las buhardillas clave rematadas por frontones alternativamente curvos y rectangulares. Las rejas son de la época Luis XV con remates y espirales. El patio está bordeado a la derecha por un pabellón que incluye una escalera siglo XVII. en hierro forjado, a la izquierda de un pabellón que contiene una escalera Luis XIII con balaustres de madera. El edificio principal al final del patio XVIIsiglo XVII . siglo está perforado con arcadas de carpintería, decorado con mesas y rematado por un frontón triangular.

Referencias

Le Marais
Wikiproyecto:Francia/Artículos

redpajama_set_name: RedPajamaWikipedia


On Imzy. It'll be an extension of sorts to this blog, in case you want to follow my half-formed and probably sleep-deprived thoughts on there.
Fair warning, I'm really bad at doing any kind of social media. This one might be a little easier for me to keep up with though, since I can have multiple profiles without having to create multiple accounts.
If you need an invite, I have a few 200 or so. Or, you can try getting one from the asexuality community.


text:
Plaatsen
Arak (Iran), een stad in Iran
Arak (Algerije), een dorp in de Algerijnse gemeente In Amguel, in de provincie Tamanrasset
Dranken
Arak (Nabije Oosten), een anijslikeur
Arak (Verre Oosten), een alcoholische drank uit onder meer Indonesië
Arak (Mongolië), een Mongoolse alcoholische drank van merriemelk
Overige
Salvadora persica, de tandenborstelboom of mosterdboom
NGC 3 (ARAK 1), een lensvormig sterrenstelsel in het sterrenbeeld Vissen


Saying goodbye to Turkey and hello to England..
On Sunday I packed up my life into a suitcase once again, boarded the plane alone and arrived back in the UK, this time for longer.
I'd been putting it off for weeks, but a month ago I booked my flight and tried to make the most of everyday I had left there in Turkey. Each day was marred by the constant thought of 'this is the last time I'll do this' or 'this is the last time I'll eat this', 'this is the last time I'll go here'. Those thoughts filled my head and everything just felt different, I could no longer enjoy my time there, once I'd booked my flight the countdown began and it was awful.
While everyone else was excitedly opening their advent calendars and counting down the days til Christmas, I was wishing time to go slower, hoping that by some miracle the 14th December would never arrive. Inevitably it did, of course.
Berkay had been able to get the day off work so we could spend the day together, we done a lot of our favourite things, had a lovely Turkish breakfast outside on the balcony, played backgammon with a cup of coffee, walked Boncuk along the seafront and had a gorgeous BBQ outside. The hours ticked by and once I was all packed and ready we went to the beach to watch one 'last' sunset. (See what I mean? Always 'lasts' on my mind!) It reached 5.30 pm, I took my case and walked out of my front door for the 'last' time (again…). "Bye house".
We went and waited in the hotel garden for Berkay's friend to arrive, he was taking us to the airport in his car. I played with Boncuk and explained to her what was going on, although of course she's clueless and is probably still waiting for me to go back and play ball with her and wondering where I am now. We saw our friends car drive around the corner and then it hit me, it was time to go.
Berkay took my suitcase to the car and I sat on the steps cuddling Boncuk, telling her to be good and that I'd see her again soon. I gave her a little treat and she ran off with it, bouncing around all happy and none-the-wiser. She was so happy with her little treat that when I climbed over the fence and walked away she didn't even come to say bye. She usually stands with her paws up on the fence, crying or howling as we walk away, but not this time. At least one of us wasn't!
A 45 minute minute car journey later and we were in Dalaman. Our friend hadn't had dinner so we stopped at a Çiğ köfte place to kill some time and eat and then carried on to the airport.
My flight was the only one flying out that evening so the airport wasn't very busy but the queues to check in were very long as the flight was one of the last ones direct from Dalaman before Christmas and it was full of expats flying back to the UK for the holidays. We queued for around an hour, but I didn't mind as that meant delaying the 'goodbye' further. Eventually the inevitable happened, I got to the front of the queue and after trying to reduce the weight of my case from 25kg down to just 20, I was all checked in and ready to go.
After a tearful goodbye and a cuddle, I walked through security and passport control where they stamped my passport with an exit date stamp, that was it, the point of no return. I looked back and waved at Berkay and he blew me a kiss, cheesy! Then that was that, I walked around the corner and found my gate, which was full of people already waiting. I spotted a Facebook friend who had previously told me she was on the same flight with her husband and daughter, she too has a Turkish husband and has faced the same teary goodbyes. I went over to her and she asked if I was ok and gave me a big hug, at which point I burst into tears. Poor woman! Still, I felt better after that and I didn't have to wait very long until we started boarding the plane. The plane was full of young children and before take off there were alot of screaming babies, they looked exactly how I felt on the inside!
On take off I said 'bye Turkey, bye Berkay, bye Boncuk' and had one last look out of the window, I even gave a little wave.
Before I knew it, the plane was landing back in Gatwick. "Welcome home" the pilot said. Not really home for me though. I got through passport control quickly and retrieved my baggage. I saw my friend again and apologised for crying on her, "it's ok I know how it feels" she said, which was reassuring. Then I walked out through the doors and saw my mum and dad waiting for me with more hugs, along with a cheddar cheese and HP sauce sandwich and packets of pickled onion monster munch, yum!
I can't tell you how strange it is to be back here. The moment I walked into my room again it just hit me, but it was like nothing at all had changed. My calendar is still stuck on the page of June, the month I left. Everything is the same here, yet different. It really does feel like I've never been away, like the past 6 months were all just a dream, like I wasn't really there. I have to keep looking at photos of our house to remind myself that it wasn't all a dream.
It upsets me that I'll probably never step foot in my house again, a week ago I was there, sleeping next to Berkay, waking up next to him, eating breakfast together on the balcony, making dinner in the kitchen, watching films in the living room… now all traces of us are removed from that house, and someone else is living there.
Berkay is now living under the hotel (which is still closed for the winter) in a concrete room which is used for staff accomodation during the summer. Boncuk is staying there with him for now and will go somewhere else once he's in the army, we'll get her back again once he's finished in February 2016, which seems like a lifetime away.
Forget about the arrival of 2015 in 2 weeks time, roll on 2016 when we can finally settle and live together without the worry of these goodbyes again, that's what I say!
Oh Danni I really feel for you,I hope you will soon settle and while your waiting you can look forward to spending the rest of your life together,time soon passes hope to see you at mums charity do, take care and have a lovely christmas lots of love.
Danni you know what,you have some beautiful memories to look back on,it won't stop you missing Berkay but the things you have shared with us will keep you strong,the next year may seem like a lifetime away but just think how fab it will be when Berkay has done his service and you will be back together. Use the time apart to work hard n save hard,maybe go to college part time and get some qualifications that you could possibly use in turkey or here in the uk.
Try not to be sad,you have a family who love you and want the best for you. Never give up.
I was just reading about J.R.R. Tolkien's parents, who got engaged back in the 1880s – and then had to wait to get married until the man was "in a position to support a wife and children" – he went off to work in a bank in South Africa and she stayed in England. Imagine? No emails or telephones, only letters! They had to wait three years! I can't believe people used to think that was a good/proper thing to do – nowadays she'd have gone out there with him. Who cares if you have to eat bread and cheese (or whatever) as long as you're with the one you love!
I hope Berkay gets some leave soon and you two can meet up somewhere!
Amazing! Thanks.Wish to see more articles.
I just bought a property in Kalkan , I am an IT expert, wish to find job also in this amazing country.
← Winter Sunsets in Calis..


The Dutch statures were taken from data kindly supplied by Molenbroek, of Delft University of Technology (ref 52). 265 male students and 89 female were measured at 4 different times between 1985 to 1986. In 1986, 152 males and 457 females aged 65-74 were measured.


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Our View: The best things about our city
We all have favourite aspects of our communities that enhance our enjoyment of living in the Capital Region.
Whether it's the Dallas Road walkway, the beaches at Witty's Lagoon or Willows, the view from atop Mount Douglas or other gems and jewels, Greater Victoria has plenty of characteristics to love that keep our minds and bodies active.
In this year's Best of the City, we present what we think are interesting, sometimes behind-the-scenes stories on some of the things readers enjoy most, top-10 lists of the best places or activities, and of course, your top three selections in more than 100 activity, retail and culinary categories.
Displaying a Best of the City plaque or certificate has become akin to wearing a badge of honour, one that encourages people to stop in and have a look because there's something special going on.
Our communities have many special things worth checking out. We encourage you to give Best of the City a read. You'll no doubt find some new things to love about your city.
Team forges royal link to past
The name for Victoria's new Western Hockey League franchise, the Royals, is appropriate.
Victoria has long been considered, by those in the tourist trade at least, as "a little bit of old England." And we are, after all, named after a British monarch.
But the nickname has been used by other teams, including the Royals Athletic Association, whose field lacrosse team competed from 1908 to 1913 at what would become Royal Athletic Park.
The name is more romantic than the other frontrunners, Capitals, Eagles or Destroyers. But it makes sense given our links to all things royal – not to mention the surge in popularity of royals with the wedding of Prince William and Kate Middleton.
-Vic News
No sympathy for Savoie's budget 'complaining'
Better recycling bylaw enforcement needed


"Our product development process starts with researching customer needs," says Rick Gambaccini, Sr. Product Manager-Cordless of Milwaukee Electric Tool Corporation. "Users want more power in a smaller and lighter size. With this request in mind, we have delivered new impact wrenches that will provide the power they need for heavy-duty applications and the size they want for more control and less fatigue."
With its Milwaukee 4-pole frameless motor it delivers a staggering 610 Nm of torque ideal for powering through the toughest of applications.
Ideal for heavy applications such as steel fabrication mechanical fastening industrial construction and automotive work.


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Proton X50 – 100,000 Bookings Received, Best-Selling SUV in Malaysia
1 min read March 4, 2022
Just last month, Proton revealed that it has sold over 100,000 SUVs locally, securing its position as the number one SUV brand in Malaysia. Well, the national carmaker seems to be maintaining this momentum: it just revealed that the exceedingly popular Proton X50 has received over 100,000 bookings.
The X50 managed to achieve this impressive figure in only 16 months, making it the best-selling SUV in Malaysia. Just last month in February 2021, as many as 3,002 units of the B-segment SUV were delivered to customers.
Deliveries took a dip in January 2022 due to the floods, where 2,601 bookings of the X50 were fulfilled. As for the previous two months, as many as 4,310 and 4,010 units were registered in December and November 2021 respectively. In 2021 itself, the SUV found 28,774 homes.
Given the sheer value for money the Proton X50 offers, it's certainly proving to be a success for the national carmaker. If you'd like to learn more about this highly sought-after SUV, watch our full review right here.
automotiveprotonproton x50


DISC is one of the leading institutions in North America for the study of Islam and Muslims. Its comparative, cross-cultural approach to Islamic studies will foster fresh interpretations of Islam and encourage creative solutions to the economic, political and social challenges involving Muslims.
We are committed to working with partners at home and abroad to provide undergraduate and graduate students, professionals and policy makers with the knowledge about Muslims and Islamic cultures, beliefs and practices that will enable them to operate effectively in a multicultural world.