text:
Identification and characterization of new Δ-17 fatty acid desaturases
- 1.4k Downloads
- Citations
ω-3 fatty acid desaturase is a key enzyme for the biosynthesis of ω-3 polyunsaturated fatty acids via the oxidative desaturase/elongase pathways. Here we report the identification of three ω-3 desaturases from oomycetes, Pythium aphanidermatum, Phytophthora sojae, and Phytophthora ramorum. These new ω-3 desaturases share 55 % identity at the amino acid level with the known Δ-17 desaturase of Saprolegnia diclina, and about 31 % identity with the bifunctional Δ-12/Δ-15 desaturase of Fusarium monoliforme. The three enzymes were expressed in either wild-type or codon optimized form in an engineered arachidonic acid producing strain of Yarrowia lipolytica to study their activity and substrate specificity. All three were able to convert the ω-6 arachidonic acid to the ω-3 eicosapentanoic acid, with a substrate conversion efficiency of 54–65 %. These enzymes have a broad ω-6 fatty acid substrate spectrum, including both C18 and C20 ω-6 fatty acids although they prefer the C20 substrates, and have strong Δ-17 desaturase activity but weaker Δ-15 desaturase activity. Thus, they belong to the Δ-17 desaturase class. Unlike the previously identified bifunctional Δ-12/Δ-15 desaturase from F. monoliforme, they lack Δ-12 desaturase activity. The newly identified Δ-17 desaturases could use fatty acids in both acyl-CoA and phospholipid fraction as substrates. The identification of these Δ-17 desaturases provides a set of powerful new tools for genetic engineering of microbes and plants to produce ω-3 fatty acids, such as eicosapentanoic acid and docosahexanoic acid, at high levels.
Keywordsω-3 Desaturase Δ-17 Desaturase Long-chain polyunsaturated fatty acids ω-3 Fatty acids Yarrowia lipolytica Metabolic engineering
Long-chain polyunsaturated fatty acids (LCPUFAs), especially ω-3 LCPUFAs eicosapentanoic acid (EPA) and docosahexanoic acid (DHA), are essential nutrients critical to human nutrition and health. These fatty acids cannot be synthesized de novo in mammals and so must be obtained either directly through diet or indirectly through further desaturation and elongation of other polyunsaturated fatty acids (PUFAs) widely available in the diet, such as linoleic acid (LA) or α-linolenic acid (ALA) (Holman 1986; Lands 1992; Bézard et al. 1994; Moghadasian 2008). LCPUFAs are part of the cellular membrane system and play essential roles in determining the structure and function of the membrane (Rabinovich 1991; Stillwell and Wassall 2003; Ma et al. 2004). Physiologically, they are necessary for proper development in mammals, particularly in the developing infant brain, and for tissue formation and repair. They are also precursors to several important eicosanoids in mammals, e.g., prostacyclins, eicosanoids, leukotrienes, and prostaglandins (Funk 2001; Smith and Murphy 2002). Additionally, a high intake of long-chain ω-3 PUFAs produces cardiovascular protective effects (Dyerberg 1978; Shimokawa 2001; Kris-Etherton et al. 2002; von Schacky 2003; Calder 2004; Diniz et al. 2004; von Schacky and Harris 2007; Tavazzi et al. 2008). Numerous clinical studies have documented wide-ranging health benefits conferred by administration of ω-3 LCPUFAs against a variety of symptoms and diseases such as asthma, cancer, depression, diabetes, immune disorder, and skin conditions (Mickleborough et al. 2006; Colomer et al. 2007; Damsgaard et al. 2007; Amminger et al. 2010; Wall et al. 2010; Djoussé et al. 2011).
The Δ-6 desaturase/Δ-6 elongase pathway (called Δ-6 pathway from hereon) is predominantly found in algae, mosses, fungi, nematodes, etc., and is characterized by the production of γ-linolenic acid (GLA; 18:3 ω-6) and/or stearidonic acid (STA; 18:4 ω-3) as intermediates. This pathway can also produce EPA with the help of a Δ-15 desaturase, which converts LA to ALA followed by Δ-6 desaturase converting ALA to stearidonic acid (STA, 18:4 ω-3), then C18/20 elongase converting STA to eicosatetraenoic acid (ETA), and finally Δ-15 desaturase converting ETA to EPA.
The Δ-9 elongase/Δ-8 desaturase pathway (called the Δ8 pathway from hereon) operates in organisms such as euglenoid species (Wallis and Browse 1999). It is characterized by the production of eicosadienoic acid (EDA; 20:2 ω-6) and/or eicosatrienoic acid (ETrA; 20:3 ω-3) as intermediates. The Δ-8 pathway can also use ALA as an intermediate to produce EPA in combination with a Δ-15 desaturase, in which the Δ-15 desaturase converts LA to ALA, followed by Δ-9 elongase converting ALA to ETrA, then Δ-8 desaturase converting ETrA to ETA, and finally Δ-5 desaturase converting ETA to EPA. EPA can be further elongated by C20/22 elongase and desaturated by Δ-4 desaturase to form DHA.
As the world population expands, the ocean fishery resource is under increased pressure to supply food enriched in ω-3 fatty acids. It is generally recognized that the current practice of wild fish harvest and commercial fish farming are not sustainable over the long term. For example, to raise a kilogram of farmed salmon, oil from 4 kg of wild fish has to be consumed to produce the feed. Furthermore, pollution remains an issue with fish-derived ω-3 fatty acid oil. A sustainable supply of clean EPA and/or DHA could reduce the pressure on ocean resources and protect both the integrity of ω-3 fatty acid products and the environment. Toward this goal, a variety of different organisms including plants, algae, fungi, and yeast are being investigated as hosts for sustainable commercial production of EPA and DHA. Genetic engineering has demonstrated that the natural abilities of some hosts (even those natively limited to LA and ALA fatty acid production) can be substantially improved to produce important fatty acid molecules such as arachidonic acid (ARA), EPA, and DHA (Napier and Sayanova 2005; Graham et al. 2007; Zhu et al. 2010; Petriea et al. 2010; Tavares et al. 2011).
Genetic engineering of plants or microorganisms to produce EPA and DHA through the desaturase/elongase pathway requires the heterologous expression of various pathway enzymes. Regardless of whether the Δ-6 or Δ-8 pathway is used, a ω-3 desaturase is necessary for conversion of ω-6 PUFAs into their ω-3 counterparts. Because of the essential role Δ-17 desaturase enzymes play in the synthesis of long-chain ω-3 PUFAs, there has been considerable effort to identify and characterize these enzymes from various sources. However, so far only two Δ-17 desaturases have been isolated, one from Saprolegnia diclina (Pereira et al. 2004) and the other from Phytophthora infestans (GenBank accession no. CAJ30870). Identification of additional Δ-17 desaturases would be of great value to the effort of genetically engineering an organism to produce ω-3 fatty acids in a sustainable way. Here we report the identification and characterization of three genes encoding Δ-17 desaturases from the oomycetes, Pythium aphanidermatum, Phytophthora sojae, and Phytophthora ramorum. The enzymes were expressed in Yarrowia lipolytica to demonstrate that they are Δ-17 desaturases with high activity. We further compared their substrate specificity with the bifunctional Δ-12/Δ-15 desaturase from Fusarium monoliforme, another type of ω-3 desaturase (Damude et al. 2006).
Materials and methods
Genetic elements introduced in Y4070U
Enzyme activity
F. monoliforme
Δ-12/Δ15 Desaturase
E. gracilis Δ-9 elongase
E. gracilis Δ-9 elongase
M. alpina C16 elongase
E. gracilis Δ-8 desaturase
E. gracilis Δ-8 desaturase
F. monoliforme
Δ-12/Δ15 Desaturase
E. gracilis Δ-9 elongase
E. gracilis Δ-5 desaturase
Peridinuim sp.
Δ-5 Desaturase
E. gracilis Δ-5 desaturase
Media and chemicals
Yeast extract, yeast nitrogen base, and malt extract medium were from Difco Laboratories (Detroit, MI, USA). Trizol reagent was from Invitrogen (Carlsbad, CA, USA). Restriction enzymes were from Promega (Madison, WI, USA). Ex Taq polymerase was from TaKaRa Bio Inc. (Otsu, Shiga 520-2193, Japan). Qiagen miniprep DNA preparation kit and PCR purification kit were from Qiagen (Valencia, CA, USA). Universal Genome Walking kit was from BD Clontech (Mississauga, ON, Canada). Other chemicals were from Sigma Aldrich (St Louis, MO, USA). Yeast culture media were prepared according to standard recipe (Sherman 1991).
DNA and RNA isolation
Total RNA and genomic DNA were isolated from P. aphanidermatum cells scraped off a malt extract agar plate, using the Trizol reagent (Invitrogen), according to the manufacturer's protocol. Specifically, scraped cells were resuspended in 1 ml water and centrifuged for 30 s in an Eppendorf microfuge at 14,000 rpm. The cell pellet was resuspended in 0.75 ml Trizol reagent, mixed with 0.75 ml of 0.5 mm glass beads, and homogenized in a Biospec mini beadbeater (Bartlesville, OK, USA) at the highest setting for 3 min. The mixture was centrifuged in an Eppendorf centrifuge for 30 s at 14,000 rpm to remove debris and glass beads. The supernatant was extracted with 150 μl of 24:1 chloroform/isoamyl alcohol (Invitrogen). The upper aqueous phase was used for RNA isolation and the lower organic phase for DNA isolation, according to manufacturer's protocol.
cDNA synthesis
Double-stranded cDNA was synthesized directly from the P. aphanidermatum total RNA. Three microliters of total RNA sample (0.9 μg) was used as template for first strand cDNA synthesis using the BD-Clontech CreatorTM SmartTM cDNA library kit following the manufacturer's protocol. The resulting first strand cDNA synthesis mixture was then used as template for PCR amplification to generate double stranded cDNA, using the manufacturer's condition. The PCR primers were 5′ PCR primer and CDSIII/3′ PCR primer from the kit. Amplification product was purified with a Qiagen PCR purification kit following the manufacturer's protocol exactly. Purified cDNA product was eluted with 50 μl of water.
Cloning of genomic and cDNA fragments containing the Δ-17 desaturase
Sequences of primers used to amplify potential Δ-17 desaturase
Nucleotide sequence
Amino acid sequence
GC analysis of fatty acid profile of P. aphanidermatum
Fatty acid profiles of P. aphanidermatum were obtained by GC analysis of extracted lipids from cells grown on malt extract agar plate (Difco Laboratories). Cells were scraped off the plate and resuspended in 600 μl of sodium methoxide dissolved in methanol. The sample was shaken for 20 min, and 50 μl of 1 M NaCl was added. After mixing, 600 μl of heptane was added. The sample was vortexed and centrifuged in an Eppendorf microfuge for 1 min. The upper layer was carefully separated from the lower layer and placed in a glass vial for GC analysis as previously described (Damude et al. 2006).
Synthesis of the codon optimized P. sojae and P. ramorum Δ-17 desaturase ORFs
P. sojae and P. ramorum Δ-17 desaturases were identified by BLAST analysis using sequence of P. aphanidermatum Δ-17 as a query, against the P. sojae and P. ramorum databases of Department of Energy's Joint Genome Program (http://genome.jgi-psf.org/Physo3/Physo3.home.html). The coding region of the Δ-17 enzymes were optimized based on the general rule of RNA stability, the codon preference, and the consensus sequence around AUG of genes from Y. lipolytica. DNA fragments encoding the codon optimized versions of candidate Δ-17 desaturase ORF were synthesized de novo by Genscript (Piscataway, NJ 08854, USA). A NcoI site was incorporated at the start codon and a NotI site after the stop codon to facilitate cloning of the synthetic ORFs into expression vectors. Internal NcoI sites were removed.
Extraction and separation of lipid fractions
Acyl-CoAs and phospholipids from approximately 0.5 g of wet yeast pellets were extracted using a modified version of the method of Schjerling et al. and Domergue and Heinz et al. (Domergue et al. 2005; Schjerling et al. 1996). Yeast cultures were centrifuged to collect the cells. Wet cells were weighed into a 13-ml glass tube and resuspended in 800 μl of ice-cold water containing 1 ppm of 17:0 acyl-CoA as a standard. Three milliliters of 2:1 chloroform/methanol was then added and the cells vortexed five times for 1 min each, with intermittent cooling in an ice bath. One milliliter each of chloroform and water were added to each sample, and the mixtures were vortexed for one additional minute. After centrifuging for 5 min at 3,000 rpm, the upper phase was removed and discarded. The interphase containing the acyl-CoAs was treated with 400 μl of extraction buffer consisting of 2 ml 50 mM potassium phosphate buffer (pH 7.2, 2 ml IPA, 50 μl acetic acid, and 80 μl of 50 mg/ml BSA), 10 μl saturated ammonium sulfate, and 1.2 ml of 1:2 chloroform/methanol solution. After vortexing for 3 min, the samples were incubated fro 20 min at RT before being centrifuged at 3,000 rpm for 5 min. The supernatant was analyzed by HPLC for acyl-CoA, as described by Schjerling et al. (1996). The lower phase was transferred into a second 13-ml glass tube and dried using a turbovap. The dried down fraction was re-suspended in 6:1 chloroform/methanol and plated onto TLC plates. After developing with a solvent of 70:20:1 hexane/ethyl ether/acetic acid, the bands were scraped into 13-ml glass vials and esterified using 5 % methanolic HCl. They were then extracted with hexane and analyzed by GC as described below.
GC analysis of fatty acid profile of Y. lipolytica transformants
Twenty-five-milliliter cultures of Y. lipolytica transformants in synthetic minimal medium (MM) (Sherman 1991) containing 2 % glucose were grown for 2 days at 30 °C and 250 rpm in 125-ml flasks. Cells in 10 ml of the culture were collected by centrifugation, resuspended in 25 ml of a medium containing 8 % glucose and 0.2 M sodium phosphate at pH 7.0 (HG medium), and allowed to grow for five more days at 30 °C and 250 rpm in 125 ml flasks. Cells were then harvested by centrifugation. Lipid extraction and fatty acid analysis were done as described previously (Damude et al. 2006). Substrate conversion efficiency for ω-3 desaturase is calculated as \( {C_{\text{product}}}/\left( {{C_{\text{product}}} + {C_{\text{substrate}}}} \right).{C_{\text{product}}} = \% \) of product ω-3 fatty acid and Csubstrate = % of substrate ω-6 fatty acid.
ω-6 Fatty acid substrate feeding
Strain ATCC #76982 and L38 carrying various plasmids were grown in MM medium individually. Each overnight culture was diluted to an OD600 of 0.5 before aliquoting into three 3-ml cultures. After growth for another 6 h, the cultures were harvested by centrifugation and resuspended in fresh 3 ml MM medium containing 1 % Tergitol and 0.5 mM each of GLA, EDA, and ARA, and allowed to grow for 24 h, at which time they were harvested, washed once with 12 ml 0.5 % Triton X-100, and once with 12 ml distilled water. The pellets were analyzed for fatty acid composition (% of total fatty acids) by GC as described above.
Sequence comparison and structure prediction
Sequence alignment was done with Vector NTI (Invitrogen) using the Clustal W software. Sequence homology was analyzed using the BLAST tools at NCBI. Initial prediction of transmembrane (TM) domain was carried out using program TMHMM ("Prediction of transmembrane helices in proteins"; TMHMM Server v. 2.0, Center for Biological Sequence Analysis, BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby, Denmark). Multiple sequence alignment was generated for a number of Δ-17 desaturases and the three conserved His-rich motifs were identified. The topology model was adjusted to bring the three His-rich motifs to the cytoplasmic side (Diaz et al. 2002).
GenBank accession numbers
P. aphanidermatum Δ-17 desaturase: FW362186.1
P. ramorum Δ-17 desaturase: FW362214.1
P. sojae Δ-17 desaturase: FW362213.1
Fatty acid profile analysis of P. aphanidermatum
Cloning of the Δ-17 desaturase cDNA from P. aphanidermatum
Based on the sequence of the two known Δ-17 desaturases from S. diclina and P. infestans in the public databases, we designed a set of 14 degenerated primers for amplification of the potential Δ-17 cDNA (see Table 2). The primers correspond to the highly conserved regions of the known Δ-17 desaturases. Genomic DNA and cDNA samples were prepared from P. aphanidermatum cells and used as template for degenerated PCR amplification of the Δ-17 gene. Three pairs of primers, PD17-F1/PD17-R5, PD17-F4/PD17-R2, and PD17-F6/PD17-R2, produced DNA fragments of the expected size, and these fragments were cloned and sequenced (see "Materials and methods" for details). Assembly of the sequences generated a 614-bp contig containing part of a putative ORF with 76 % identity to Δ-17 desaturase from P. infestans and 69 % identity to Δ-17 desaturase from S. diclina. DNA fragments corresponding to the 5′ and 3′ ends of the putative ORF were obtained by genome walking, and sequenced (see "Materials and methods"). Assembly of various sequences yielded a 1,533-bp contig that contained the entire ORF plus 5′ and 3′ untranslated regions. The coding region of the ORF from P. aphanidermatum (PaΔ17) is 1,080 bp long and encodes a peptide of 359 amino acids (GenBank accession number FW362186.1).
Identification of genes encoding Δ-17 desaturase from P. sojae and P. ramorum
Confirmation of the activity of P. aphanidermatum Δ-17 desaturase
To confirm that the cloned PaD17 indeed has Δ-17 desaturase activity, we constructed an expression vector pFM-PAD17 containing the PaD17 ORF under the control of the strong constitutive Y. lipolytica promoter FBAIn (Hong et al. manuscript in preparation) from Y. lipolytica (see "Materials and methods" for details of the plasmid). The pFM-PAD17 construct was used to transform Y. lipolytica strain Y4070. This strain contained three copies of Δ-9 elongase genes, two copies of Δ-8 desaturase genes, and three copies of Δ-5 desaturase genes ("Materials and methods"), enabling it to produce ARA at approximately 10 % of total fatty acids. Introduction of a functional Δ-17 desaturase is expected to lead to production of EPA from ARA.
Improved expression and conversion efficiency of Δ-17 desaturase by codon optimization
Codon bias of an organism often leads to sub-optimal expression of a heterologous gene. We thus synthesized codon optimized versions of the Δ-17 desaturases from P. aphanidermatum (PaD17S), P. sojae (PsD17S), and P. ramorum (PrD17S) for enhanced expression in Y. lipolytica ("Materials and methods"). Comparison of the amino acid sequences of P. sojae Δ-17 and P. ramorum Δ-17 revealed that the N terminus of the PsD17 has two extra residues Q and E at position 5 and 6. To make a synthetic version of PsD17 with optimized codons (PsD17S), we designed the new gene without these two residues so that the N terminus region of PsD17S was similar to the PrD17S. In all, for PaD17S, 17.4 % of the 1,080-bp coding region were changed, which led to 48.6 % codons being optimized. The GC content was reduced from 61.8 % to 54.5 %. For PrD17S, 15.5 % of the 1,086-bp coding region was changed, leading to 44.2 % codons being optimized. The GC content was reduced from 64.4 % to 54.5 %. For PsD17S, 16.0 % of the 1,092-bp coding region was changed, leading to 46.2 % codons being optimized. The GC content was reduced from 65.1 % to 54.5 %.
Conversion of ARA to EPA by the three synthetic versions of ω-3 desaturase
ARA (% of total)
EPA (% of total)
Y4070 + pFM-MOD
Y4070 + pFM-PaD17s
Y4070 + pFM-PrD17s
Y4070 + pFM-PsD17s
Determination of omega-6 fatty acid substrate spectrum of different omega-3 desaturases
Average fatty acid composition of different transformants of wild-type (WT, ATCC #76932) and Δ-12-desaturase disrupted (d12KO) Y. lipolytica cells grown in glucose medium
Fatty acid composition (% total fatty acid)
The FmΔ-12/Δ-15 desaturase is a bi-functional desaturase with high level of ω-6 desaturase activity as well. To test if the Δ-17 desaturases were also bi-functional desaturases, the Δ-12 desaturase activity of the three Δ-17 desaturases was determined by transforming their expression constructs into a Δ-12 desaturase-disrupted Yarrowia lipolytica strain L38, which was derived from ATCC #76982. The FmΔ-12/Δ-15 desaturase was used as a control. If there is any Δ-12 desaturase activity present, we expect to see the production of LA and ALA in these transformants. The fatty acid composition of L38 with different constructs (Table 4) revealed that cells carrying the three Δ-17 desaturases did not produce detectable levels of LA or ALA, indicating a lack of any Δ-12 desaturase activity. In contrast, cells carrying the F. monoliforme bifunctional Δ-12/Δ-15 desaturase produced a significant level of ALA (due to the high Δ-15 activity) and residue level of LA, as expected. Thus, unlike the Δ-12/Δ-15 desaturase, the newly identified Δ-17 desaturases are not dual function desaturases, but strictly ω-3 desaturases.
Fatty acid profile in various lipid fractions
ARA and EPA content in PL, CoA, and total lipid fractions
ARA (% of total)
EPA (% of total)
Oomycetes such as Pythium and Phytophtora are known to produce LCPUFAs, such as ARA and EPA (Stinson et al. 1991). Fatty acid profiles of these organisms suggest that they produce these LCPUFAs through the desaturase/elongase pathway, rather than the polyketide pathway used by marine bacteria and thraustochytrids (Stredansky et al. 2000). These organisms thus are excellent sources for various desaturase and elongase genes of the LCPUFA pathway. We have identified and isolated three new ω-3 desaturases from these organisms that convert ω-6 fatty acids to the ω-3 unsaturated form. The newly identified ω-3 desaturases belong to the so-called type II desaturase that introduces double bond near the methyl end of an unsaturated fatty acid (Uttaro 2006). They are highly similar in amino acid sequences, with >73 % identity. Each contained the signature desaturase motifs, six membrane domains, and share extensive similarity in secondary structure arrangement. However, they share less identity to other ω-3 desaturases such as the Δ-17 desaturase from S. diclina (55 % identity) and the Δ-15 desaturase from F. monoliforme (31 % identity). Nevertheless, the secondary structure arrangements were similar among these ω-3 desaturases, based on structure predictions. All three desaturases also contained the consensus ER membrane protein retention signal at the C terminus, suggesting that they are localized to the ER membrane like that of the S. diclina Δ-17 desaturase (Pereira et al. 2004). Similar to the S. diclina Δ-17 and other ω-3 desaturases, they lack the cytochrome b5 domain and need to interact with independent cytochrome b5 to be active (Pereira et al. 2004).
The activity and substrate spectrum of these enzymes are analyzed by expression of the genes in both wild-type and genetically engineered Y. lipolytica strains. The wild-type P. aphanidermatum enzyme, when introduced into the ARA producing strain Y4070, converted 54 % of the endogenous ARA to EPA. The codon optimized version converted 10 % more ARA than the wild-type version (64 % versus 54 %). The codon optimized version of P. sojae and P. ramorum enzymes also converted >60 % of ARA to EPA. This result showed that these enzymes are highly active when expressed in Yarrowia. It suggests that they retained the correct conformation and intracellular location. The enhanced activity of the codon optimized version of P. aphanidermatum enzyme, compared to the wild type, indicated a further improvement in expression level resulting from codon optimization.
Like Δ-17 desaturases from S. diclina, the three new Δ-17 desaturases could also convert different types of ω-6 fatty acids to ω-3 fatty acids. Fatty acid feeding experiments showed that these three ω-3 desaturases had the strongest substrate preference for ARA, relatively lower preference for EDA and DGLA, and least preference for GLA (Fig. 7). The activity of the three enzymes toward C18 substrate GLA is in contrast to that of the S. diclina Δ-17 desaturase, which was reported to be an exclusive C20 desaturase with no activity on the C18 substrates (Pereira et al 2004). These three ω-3 desaturases had also significant levels of Δ-15 desaturase activity, as shown by their ability to convert LA to ALA. The conversion efficiency of the Δ-15 activity of these enzymes is about the same as the Δ-17 conversion efficiency with substrates other than ARA, such as EDA and DGLA. Thus, it appears that the enzymes are optimized for the production of EPA but have broad substrate specificity at lower activity levels. Furthermore, the substrate specificity analysis showed that these enzymes are the v + z-type desaturases that require a pre-existing double bond in the substrate molecule, and generate a new double bond three carbon away towards the methyl end of the fatty acids (Shanklin and Cahoon 1998). Expression of these three ω-3 desaturases in a Δ-12 deletion strain, L138, showed that, unlike the previously reported bi-functional Fm Δ-12/Δ-15 desaturase, none of them have any detectable Δ-12 desaturase activity. Therefore, the enzymes are not dual function desaturases like the Fusarium Δ-12/Δ-15 desaturase (Damude et al. 2006). The sequence divergence between the Pythium enzymes, the S. diclina enzyme and the F. monoliforme enzyme probably contributed to the observed difference in substrate specificity. As shown in Fig. 5, the F. monoliforme enzyme in particular has two stretches of extra amino acids (a.a. 165–184 and 214–230) that are not present in other Δ-17 desaturases. This could be the key factor determining the substrate specificity of the Δ-15 enzyme. More detailed mutagenic study will have to be done to fully understand the factors contributing to the substrate specificity.
Our analysis on the substrate preference of Pr Δ-17 showed that a small amount of activity on acyl-CoA substrate can be detected when cells are fed with ARA supplied in the culture medium. However, compare with the total activity, this acyl-CoA based activity is insignificant. The S. diclina Δ-17 desaturase was reported to desaturate fatty acids not connected to PL (Pereira et al. 2004). It was not established whether Sd Δ-17 also acts on PL because ARA supplied in the medium is not incorporated into PL (Pereira et al. 2004). Taking advantage of the availability of strains that produce ARA de novo, we were able to show that in an engineered ARA producing strain carrying Pr Δ-17, the PL fraction contained higher percentage of EPA than either the CoA fraction, or the total lipid fraction under oleaginous condition. This strongly suggests that these Δ-17 desaturases could use, and likely prefer, fatty acids in PL fraction as substrates. It also suggests that like the Sd Δ-17 desaturase, these enzymes do have activity toward CoA substrates as well.
The high activity of these newly identified ω-3 desaturases render them excellent choices for genetic engineering of organisms for the production of EPA. One of the major issues in engineering an organism for efficient production of omega-3 fatty acids is the conversion efficiency of the enzymes used for engineering. With the elongase/desaturase pathway, the precursors of the ω-3 fatty acids are ω-6 fatty acids. The physiological effects of the ω-3 and ω-6 fatty acids are quite different (Simopoulos 2011; Ramsden 2010). It is thus important to control the ω-6 fatty acid at a low level in the final product. To do this, the ω-3 desaturase activity level in the host needs to be high. The previously characterized S. diclina Δ-17 desaturase, when expressed in yeast, has a conversion efficiency of only 26 % in a feeding experiment. The Δ-17 enzymes described in this study have conversion efficiencies ranging from 36 % to 56 % in feeding study, significantly higher than that of the S. diclina enzyme. The activity is even higher toward endogenous ω-6 fatty acids. We have demonstrated that in a genetically engineered ARA producing Y. lipolytica strain, the expression of any of the PaD17, PrD17, and PsD17 from a low copy plasmid led to the conversion of up to 64 % of the ARA to EPA. Importantly the codon optimized version produced more EPA, suggesting that it is possible to increase the conversion efficiency via enhanced expression of the enzyme. By constructing a stable transformant with three copies of Δ-17 desaturase gene integrated into the genome, the conversion efficiency from ARA to EPA could be as high as 95 % (Zhu et al., unpublished result). Thus, by using multiple copies of the highly active Δ-17 desaturase, it is possible to lower the ω-6 fatty acid concentration to less than 5 %. Having multiple Δ-17 desaturases allowed us to avoid repeated use of a single ORF, thereby reducing the chance of unwanted recombination events and increasing the stability of the engineered strain. This paves the way for the construction of production organisms that are capable of producing high levels of ω-3 fatty acids EPA and DHA.
We would like to thank Lisa Hoffman of DuPont Agriculture Products for providing the Pythium apanidermatum strain used for cloning the Δ-17 desaturase and Kelley Norton for critical reading of the manuscript.
Open Access
This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.
- Amminger GP, Schäfer M, Papageorgiou K, Klier CM, Cotton SM, Harrigan SM, Mackinnon A, McGorry PD, Berger GE (2010) Long-chain omega-3 fatty acids for indicated prevention of psychotic disorders: a randomized, placebo-controlled trial. Arch Gen Psychiatry 67:146–154. doi:10.1176/appi.ajp.163.5.790 CrossRefGoogle Scholar
- Andersson H, Kappeler F, Hauri H (1999) Protein targeting to endoplasmic reticulum by dilysine signals involves direct retention in addition to retrieval. J Biol Chem 274:15080–15084. doi:10.1074/jbc.274.21.15080 CrossRefGoogle Scholar
- Baik I, Abbott RD, Curb JD, Shin C (2010) Intake of fish and n-3 fatty acids and future risk of metabolic syndrome. J Am Diet Assoc 110:1018–26. doi:10.1016/j.jada.2010.04.013 CrossRefGoogle Scholar
- Bézard J, Blond JP, Bernard A, Clouet P (1994) The metabolism and availability of essential fatty acids in animal and human tissues. Reprod Nutr Dev 34:539–68. doi:10.1051/rnd:19940603 CrossRefGoogle Scholar
- Calder PC (2004) N-3 fatty acids and cardiovascular disease: evidence explained and mechanisms explored. Clin Sci (Lond) 107:1–11. doi:10.1042/CS20040119 CrossRefGoogle Scholar
- Chung H, Nettleton JA, Lemaitre RN, Barr RG, Tsai MY, Tracy RP, Siscovick DS (2008) Frequency and type of seafood consumed influence plasma (n-3) fatty acid concentrations. J Nutr 138:2422–2427. doi:10.3945/jn.108.089631 CrossRefGoogle Scholar
- Colomer R, Moreno-Nogueira JM, García-Luna PP, García-Peris P, García-de-Lorenzo A, Zarazaga A, Quecedo L, del Llano J, Usán L, Casimiro C (2007) N-3 fatty acids, cancer and cachexia: a systematic review of the literature. Br J Nutr 97:823–31. doi:10.1017/S000711450765795X CrossRefGoogle Scholar
- Damsgaard CT, Lauritzen L, Kjær TMR, Holm PMI, Fruekilde M, Michaelsen KF, Frøkiær H (2007) Fish oil supplementation modulates immune function in healthy infants. J Nutr 137:1031–1036Google Scholar
- Damude HG, Zhang H, Farrall L, Ripp KG, Tomb J-F, Hollerbach D, Yadav NS (2006) Identification of bifunctional Δ12/ω3 fatty acid desaturases for improving the ratio of ω3 to ω6 fatty acids in microbes and plants. Proc Natl Acad Sci U S A 103:9446–9451. doi:10.1073/pnas.0511079103 CrossRefGoogle Scholar
- Diaz AR, Mansilla MC, Vila AJ, de Mendoza D (2002) Membrane topology of the acyl-lipid desaturase from Bacillus subtilis. J Biol Chem 277:48099–106. doi:10.1074/jbc.M208960200 CrossRefGoogle Scholar
- Diniz YS, Cicogna AC, Padovani CR, Santana LS, Faine LA, Novelli EL (2004) Diets rich in saturated and polyunsaturated fatty acids: metabolic shifting and cardiac health. Nutrition 20:230–4. doi:10.1016/j.nut.2003.10.012 CrossRefGoogle Scholar
- Djoussé L, Biggs ML, Lemaitre RN, King IB, Song X, Ix JH, Mukamal KJ, Siscovick DS, Mozaffarian D (2011) Plasma omega-3 fatty acids and incident diabetes in older adults. Am J Clin Nutr 94:527–533. doi:10.3945/ajcn.111.013334 CrossRefGoogle Scholar
- Domergue F, Abbadi A, Zahringer U, Moreau H, Heinz E (2005) In vivo characterization of the first acyl-CoA Δ6-desaturase from a member of the plant kingdom, the microalga Ostreococcus tauri. Biochem J 389:483–490. doi:10.1042/BJ20050111 CrossRefGoogle Scholar
- Dyerberga J, Banga HO, Stoffersena E, Moncadab S, Vane JR (1978) Eicosapentaenoic acid and prevention of thrombosis and atherosclerosis. Lancet 312:117–119. doi:10.1016/S0140-6736(78)91505-2 CrossRefGoogle Scholar
- Fournier P, Abbas A, Chasles M, Kudla B, Ogrydziak DM, Yaver D, Xuan J-W, Peito A, Ribet A-M, Feynerol C, He F, Gaillardin C (1993) Colocalization of centromeric and replicative functions on autonomously replicating sequences isolated from the yeast Yarrowia lipolytica. Proc Natl Acad Sci USA 90:4912–4916CrossRefGoogle Scholar
- Funk CD (2001) Prostaglandins and leukotrienes: advances in eicosanoid biology. Science 294:1871–1875. doi:10.1126/science.294.5548.1871 CrossRefGoogle Scholar
- Graham IA, Larson T, Napier JA (2007) Rational metabolic engineering of transgenic plants for biosynthesis of omega-3 polyunsaturates. Curr Opin Biotechnol 18:142–147. doi:10.1016/j.copbio.2007.01.014 CrossRefGoogle Scholar
- Holman RT (1986) Nutritional and functional requirements for essential fatty acids. In: Ip C (ed) Dietary fat and cancer. Liss, New York, pp 211–228Google Scholar
- Hong SP, Seip J, Walters-Pollak D, Rupert R, Jackson R, Xue Z, Zhu Q (2012) Engineering Yarrowia lipolytica to express secretory invertase with strong FBA1(IN) promoter. Yeast 29:59–72. doi:10.1002/yea.1917 Google Scholar
- Jackson MR, Nilsson T, Peterson PA (1990) Identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum. EMBO J 9:3153–3162Google Scholar
- Kris-Etherton PM, Harris WS, Appel LJ (2002) Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease. Circulation 106:2747–2757. doi:10.1161/01.CIR.0000038493.65177.94 CrossRefGoogle Scholar
- Lands WEM (1992) Biochemistry and physiology of n-3 fatty acids. FASEB J 6:2530–2536Google Scholar
- Ma DWL, Seo J, Switzer KC, Fan Y-Y, McMurray DN, Lupton JR, Chapkin RS (2004) n-3 PUFA and membrane microdomains: a new frontier in bioactive lipid research. J Nutr Biochem 15:700–706. doi:10.1016/j.jnutbio.2004.08.002 CrossRefGoogle Scholar
- Metz JG, Roessler P, Facciotti D, Levering C, Dittrich F, Lassner M, Valentine R, Lardizabal K, Domergue F, Yamada A, Yazawa K, Knauf V, Browse J (2001) Production of Polyunsaturated Fatty Acids by Polyketide Synthases in Both Prokaryotes and Eukaryotes. Science 293:290–293. doi:10.1126/science.1059593 Google Scholar
- Mickleborough TD, Lindley MR, Ionescu AA, Fly AD (2006) Protective effect of fish oil supplementation on exercise-induced bronchoconstriction in asthma. Chest 129:39–49. doi:10.1378/chest.129.1.39 CrossRefGoogle Scholar
- Moghadasian MH (2008) Advances in dietary enrichment with n-3 fatty acids. Crit Rev Food Sci Nutr 48:402–10. doi:10.1080/10408390701424303 CrossRefGoogle Scholar
- Napier JA, Sayanova O (2005) The production of very-long-chain PUFA biosynthesis in transgenic plants: towards a sustainable source of fish oils. Proc Nutr Soc 64:387–93. doi:10.1079/PNS2005447 CrossRefGoogle Scholar
- Pereira SL, Huang Y-S, Bobik EG, Kinney AJ, Stecca KL, Packer JCL, Mukerji P (2004) A novel ω3-fatty acid desaturase involved in the biosynthesis of eicosapentaenoic acid. Biochem J 378:665–671. doi:10.1042/BJ20031319 CrossRefGoogle Scholar
- Petrie JR, Shrestha P, Mansour MP, Nichols PD, Liu Q, Singh SP (2010) Metabolic engineering of omega-3 long-chain polyunsaturated fatty acids in plants using an acyl-CoA Δ6-desaturase with ω3-preference from the marine microalga Micromonas pusilla. Metab Eng 12:233–240. doi:10.1016/j.ymben.2009.12.001 CrossRefGoogle Scholar
- Rabinovich AL (1991) Ripatti PO (1991) On the conformational, physical properties and functions of polyunsaturated acyl chains. Biochim Biophys Acta 1085:53–62. doi:10.1016/0005-2760(91)90231-6 CrossRefGoogle Scholar
- Ramsden CE, Hibbeln JR, Majchrzak SF, Davis JM (2010) n-6 Fatty acid-specific and mixed polyunsaturate dietary interventions have different effects on CHD risk: a meta-analysis of randomised controlled trials. Br J Nutr 104:1586–1600. doi:10.1017/S0007114510004010 CrossRefGoogle Scholar
- Sayanova O, Napier JA (2004) Eicosapentaenoic acid: biosynthetic routes and the potential for synthesis in transgenic plants. Phytochemistry 65:147–158. doi:10.1016/j.phytochem.2003.10.017 CrossRefGoogle Scholar
- Schjerling CK, Hummel R, Hansen JK, Borsting C, Mikkelsen JM, Kristiansen K, Knudsen J (1996) Disruption of the gene encoding the acyl-CoA-binding protein (ACB1) perturbs acyl-CoA metabolism in Saccharomyces cerevisiae. J Biol Chem 271:22514–22521. doi:10.1074/jbc.271.37.22514 CrossRefGoogle Scholar
- Shanklin J, Cahoon EB (1998) Desaturation and related modifications of fatty acids. Ann Rev Plant Physiol Plant Mol Biol 49:611–641. doi:10.1146/annurev.arplant.49.1.611 CrossRefGoogle Scholar
- Sherman F (1991) Getting started with yeast. In: Guthrie C, Fink GR (eds) Methods Enzymol 194. Academic, New York, pp 3–20Google Scholar
- Shimokawa H (2001) Beneficial effects of eicosapentaenoic acid on endothelial vasodilator functions in animals and humans. World Rev Nutr Diet 88:100–108. doi:10.1159/000059773 CrossRefGoogle Scholar
- Simopoulos AP (2011) Evolutionary aspects of diet: the omega-6/omega-3 ratio and the brain. Mol Neurobiol 44:203–215. doi:10.1007/s12035-010-8162-0 CrossRefGoogle Scholar
- Smith ML, Murphy RC (2002) The eicosanoids: cyclooxygenase, lipoxygenase and epoxygenase pathways. In: Vance DE, Vance J (eds) Biochemistry of lipids, lipoproteins and membranes, 4th edn. Elsevier, Amsterdam, pp 341–371CrossRefGoogle Scholar
- Stillwell W, Wassall SR (2003) Docosahexaenoic acid: membrane properties of a unique fatty acid. Chem Phys Lipids 126:1–27. doi:10.1016/S0009-3084(03)00101-4 CrossRefGoogle Scholar
- Stinson EE, Kwoczak R, Kurantz MJ (1991) Effect of cultural conditions on production of eicosapentaenoic acid by Pythium irregulare. J Ind Microbiol 8:171–8. doi:10.1007/BF01575850 CrossRefGoogle Scholar
- Stredansky M, Contia E, Salaris A (2000) Production of polyunsaturated fatty acids by Pythium ultimum in solid-state cultivation. Enzyme Microb Technol 26:304–307. doi:10.1016/S0141-0229(99)00146-5 CrossRefGoogle Scholar
- Tavares S, Grotkjær T, Obsen T, Haslam RP, Napier JA, Gunnarsson N (2011) Metabolic engineering of Saccharomyces cerevisiae for production of eicosapentaenoic acid, using a novel Δ5-desaturase from Paramecium tetraurelia. Appl Environ Microbiol 77(5):1854–61. doi:10.1128/AEM.01935-10 CrossRefGoogle Scholar
- Tavazzi L, Maggioni AP, Marchioli R, Barlera S, Franzosi MG, Latini R, Lucci D, Nicolosi GL, Porcu M, Tognoni G (2008) Effect of n-3 polyunsaturated fatty acids in patients with chronic heart failure (the GISSI-HF trial): a randomised, double-blind, placebo-controlled trial. Lancet 372:1223–30. doi:10.1016/S0140-6736(08)61239-8 CrossRefGoogle Scholar
- Uttaro AD (2006) Biosynthesis of polyunsaturated fatty acids in lower eukaryotes. IUBMB Life 58:563–571. doi:10.1080/15216540600920899 CrossRefGoogle Scholar
- von Schacky C (2003) The role of omega-3 fatty acids in cardiovascular disease. Curr Atheroscler Rep 5:139–45. doi:10.1007/s11883-003-0086-y CrossRefGoogle Scholar
- von Schacky C (2007) Harris WS (2007) Cardiovascular benefits of omega-3 fatty acids. Cardiovasc Res 73(2):310–315. doi:10.1016/j.cardiores.2006.08.019 CrossRefGoogle Scholar
- Wall R, Ross RP, Fitzgerald GF, Stanton C (2010) Fatty acids from fish: the anti-inflammatory potential of long-chain omega-3 fatty acids. Nutr Rev 68:280–289. doi:10.1111/j.1753-4887.2010.00287.x CrossRefGoogle Scholar
- Wallis JG, Browse J (1999) The Delta8-desaturase of Euglena gracilis: an alternate pathway for synthesis of 20-carbon polyunsaturated fatty acids. Arch Biochem Biophys 365:307–16. doi:10.1006/abbi.1999.1167 CrossRefGoogle Scholar
- Wallis JG, Watts JL, Browse J (2002) Polyunsaturated fatty acid synthesis: what will they think of next? Trends Biochem Sci 27:467–473. doi:10.1016/S0968-0004(02)02168-0 CrossRefGoogle Scholar
- Zhu Q, Xue Z, Yadav NS, Damude H, Pollak DW, Ruppert R, Seip J, Hollerbach D, Macool D, Zhang H, Bledsoe S, Short D, Tyreus B, Kinney A, Picataggio S (2010) Metabolic engineering of an oleaginous yeast for the production of omega-3 fatty acids. In: Cohen Z, Ratledge C (eds) Single cell oils: microbial and algal oils. AOCS, Urbana, pp 51–73Google Scholar


text:
Lucy Pinder :: David Lodge/Getty Images
I'll be honest with you, I know nothing about NASCAR, Indy Car, F1 or any other racing leagues. In fact, I know nothing about cars. I consider it a success each morning when I put my key in the ignition, turn it and the car starts. That's the extent of my car knowledge. But, I do that the Indianapolis 500 is synonymous with Memorial Day weekend. Now, since I can't possibly say anything half intelligent about the race, I'll just link to this gallery of 56 Insanely Hot Race Car Grid Girls, which includes Lucy Pinder. Another big sporting event that kicks off this weekend is the French Open. Cuzoogle has posted the odds (and some photos) for the ladies side of the draw.
With Terminator Salvation and Star Trek now in theaters, Bleacher Report is re-imagining sci-fi classics by putting athletes in key roles.
Home Run Derby shows us that names aren't the only thing fans put on the back of their baseball jerseys.
John Cena, Shane McMahon
Have you ever wondered why Chuck Taylors are called Chuck Taylors? If so, you're in luck. Mental Floss looks at how nine famous sneakers got their name.
New York Giants offensive lineman Dave Diehl recently performed at a comedy club in New York. I don't know much about his act, except for the details provided by Sports Rubbish. But all anyone needs to know is that Diehl mocked Brett Favre.
As I wrap up the links section, it would be a travesty for me not to properly honor the unofficial beginning of summer, Memorial Day weekend. So here is a list of The 101 Sweetest Things About Summer. Lots of sports-related activities are featured, such as the MLB All-Star Game, badminton, playing Frisbee, softball, surfing and Wimbledon. "Bikinis" also made the cut, which gives me an excuse to link to this in-depth piece on bikini competitions.
For more links, follow me on Twitter and join the Hot Clicks Facebook group.
Daniel Williams, of Nicholasville, Kent., said, "Jimmy, I love Hot Clicks and read it everyday. Thanks for all the great work you do! A couple months ago I sent you an e-mail about Courtney Lee from my beloved Western Kentucky University. Well, Wednesday night he dunked on THE LeBron James. Here is the link. Of course, ESPN didn't show the highlight at all because anything that makes the King look bad can't be shown. Could you please post this video and show James getting shown up by a Hilltopper? Go Tops!" Since it's a holiday weekend -- and Marv Albert does the play-by-play, I'll honor this request.
Memo to the New York Yankees: Get rid of the lame and tired YMCA bit, kill the Cotton Eyed-Joe nonsense and stop with the stupid "which subway will finish first" game. Just hire this kid to entertain the crowd in between each inning. Now! (Thanks to Stefan, of Toronto, for the link.)
Did you know all the things paper towels can do? (Thanks to Patrick, of Melfort, Saskatchewan, for the link.)
I would love to interview any person who actually bought a car from these two people. (Thanks to Hillis Waymer, of Decatur, Ga., for the link.)

subset: commoncrawl


text:
Welcome! I would like to thank you for stopping by my website. This website is designed to be a valuable resource for you, providing helpful information and tools to stay informed on happenings in state government, at the State Capitol, and in the 10th Senate District. In addition to press releases, events, and blogs, you can track legislation and contact me to share your views on issues of interest.
I hope you find the website helpful. Please take time to "like" my Facebook page.
Thanks for visiting!


text:
It's an exciting time for our youngest learners here in the district, as we begin our second year in our 4K Learning Academies at G.D. Jones, Hawthorn Hills, Riverview and Thomas Jefferson elementary schools. Whether your child is enrolled in one of the district's 4K Learning Academies or one of the community outreach sites, you and your child will be warmly welcomed and come to know and love the 4K program mascot, Tucker Turtle!
Step one in the registration process is to complete this online form, which in part is to indicate your interest in our 4K program and tell us where you would like your child to attend. After you submit this form, you will receive an email confirmation that includes directions for completing step two, which is a paper registration packet.
If you have questions, contact Sarah Fisher at 715-261-0265 for assistance.


text:
Elise M. 'Sheila' Leather Belt
My Review
This women belt is popular on market, you can buy Elise M. 'Sheila' Leather Belt at very low prices. If you would like to order Today. Please check the product prices before deciding to order the women belt. Thank you so much for visit us.
The Elise M. 'Sheila' Leather Belt is a product that is selling well right now. We recommend that you should rush to order the women belt before it was out of stock. We think you go to this page via a Search Engine by your smart keywords. We are not sure what you're looking for the price of this women belt or not. However, if you are searching for discount rates for this women belt and we hope that the information in this page is useful to you. We know that everyone would likes to order at the cheapest price. But sometimes a little more expensive but it's faster shipping, it's a great alternative to shopping.
Be careful, These product may out of stock soon! are you searching for Elise M. 'Sheila' Leather Belt. I wish this is the great times for your shopping on this month.
Elise M. 'Sheila' Leather Belt Shopping Tips
No online shopping store/website needs your social security number or your birthday to do business Then you don't give this information to store/website. AND Don't wait for your bill to come at the end of the month. Go online regularly during the holiday season and look at electronic statements for your credit card, debit card, and checking accounts. Make sure you don't see any fraudulent charges. If you found fraudulent charges. If you would like to buy Elise M. 'Sheila' Leather Belt. Please buy it from trusted store.
Other Reviews
Review 1
Before we dive into the secret here is the most important rule to live by the CEO or other housewives enhance your natural beauty! We live in an era of improved artificial: the false eyelashes, false hair implants and many more are false, the add-on can help you change the way you look. But do you really want to do this?
Review 2
Pin Up Elise M. 'Sheila' Leather Belt skirt these enhance the image of the person wearing it. The rooms are tight and make-up looks curvaceous Generally these kinds including Elise M. 'Sheila' Leather Belt, skirts, pie, lemon chiffon and banning novelty to print. Shoes worn PIN to complete the operation to be less than the vintage style rockabilly. To make the hair and make use DOS for a look at the SDAs and reflect modern and vintage styles.
Review 3
The current strategy of visual culture in the luxury retailers that are integrated heavily into several reigned mind our society are satisfied immediately recognized through platforms like Instagram, which helps. users can upload and share photos within seconds Perhaps it is social media, which is now forced to brands. re-Strategise And prioritize the heritage of the brand and philosophy rather than creating visual culture. Of luxury has always been heavily dependent on the idea of ??'exclusivity' and the other as platforms like Instagram allows users to form communities, which may be perceived as a brand with the idea of ??'exclusivity' elegant interior. should not be confused With the idea of ??'the cost.
5 Shopping Tips when Buying Women Clothing Online
This Video will assist you with the tips which must be applied while ordering customized clothing for women. Moreover, the same will not just facilitate you in receiving the right measurement, but anticipated fabric and design as well.


text:
MOVIE SYNOPSIS [ General Audiences ] With the actor direction of Joan Capdevila, José Mota leads the cast of this movie of Spain, with dialogues in Spanish (with subtitles), whose official premiere was scheduled for 2015. You can watch No Se, No Se online on TV cable or theaters with original audio in Spanish at different dates. This film premiered in theaters in 2015.
Rating: 5 by 10 users./
Runtime: 100 minutes
José Mota, Lolita Flores, Estopa, Justo Molinero, Bernard Fougeres and Javier Romero.
100-minute film (full movie) directed by Joan Capdevila.
The original audio of No Se, No Se has dialogues in or Spanish, it was produced in Spain.
It premiered in cinemas or television in 2015.
Christian Moyès.
Joan Capdevila.
Composed by Rumba Tres.
Without commercial breaks, the full movie No Se, No Se has a duration of 100 minutes; the official trailer can be watched in streaming on the Internet. You can watch this full movie free in English or Spanish in movie TV channels, renting the DVD or with VoD services (Video On Demand player, Hulu) and PPV (Pay Per View, Netflix).
See the page of this full movie, No Se, No Se in FULLTV Movies.


text:
Newegg.com - A great place to buy computers, computer parts, electronics, software, accessories, and DVDs online. With great prices, fast shipping, and top-rated customer service - once you know, you Newegg.
If you are reading this message, Please click this link to reload this page.(Do not use your browser's "Refresh" button). Please email us if you're running the latest version of your browser and you still see this message.
Newegg.com - Computer Parts, Laptops, Electronics, HDTVs, Digital Cameras and More!
Sold and Shipped by Newegg
The Radeon™ Performance Series is optimized for the latest generation of AMD and Intel processors. This series is built using high-quality memory chips designed to offer unrivaled performance. The low profile heat spreader provides additional protection with the added assurance of high compatibility testing, all backed by a lifetime warranty. Running frequencies up to 1866MHz, the Performance Series comes equipped with AMD Memory Profile Technology, enabling an easy overclocking experience through AMD OverDrive™ on compatible AMD-based platforms. The Performance Series is the perfect solution for PC users seeking extreme reliability and high performance for gaming and memory-intensive multimedia applications.
All AMD Radeon™ memory modules are compatible with AMD and Intel platforms. They are designed to deliver the best quality and reliability, and are tested to the highest standards to help ensure stability and performance.
Designed to take advantage of your system memory to create a virtual drive enabling additional performance enhancements to your PC.
Designed to give new or experienced overclockers an easy way to tune their memory's performance, enhancing their PC with AMP-enabled memory and platforms. Mainstream users can easily choose from a preset memory optimization from the AMD OverDrive™ software while PC enthusiasts can manually set the preset optimization during the boot process.
AMD Radeon™ memory is the only memory that comes equipped with both AMD Memory profiles and Intel XMP profiles - making it easier for you to overclock and switch your memory modules between AMD and Intel platforms without breaking a sweat.
Your request will be processed within the next 24 hours.
Please add "email@example.com" to your address book to ensure delivery.See Today's E-Blast Promo
Some manufacturers place restrictions on how details of their products may be communicated.
Some manufacturers place restrictions on how details of their products may be communicated.
Some manufacturers place restrictions on how details of their products may be communicated.


text:
'Sansa Stark' Kicks Steve - O in the Balls
Description:Sophie Turner, who plays Sansa Stark in Game of Thrones has a the privilege of meeting Steve-O recently. When Steve-O meets someone he admires, what does he do? Have them kick him in the groin. Never change, Steve.
Added:3 years ago
0 likes, 0 dislikes
Embed / Iframe code:
Make your selection, copy and paste the embed code above.
Standart sizes: Custom size:
What is the problem?
Please do not abuse this section. The videos ,which are on investigate list, are manually checked by editor.
Security code:(is not case-sensitive) can't read
Write a comment
at least 10 characters, up to 400 characters
400 characters remaining
Security code:
(is not case-sensitive) can't read


text:
If you love decorative birdhouses, come explore this gallery to grab ideas for your garden.
Also see, how to make a stone birdhouse.
I may receive a commission if you purchase something mentioned in a link on this post for sites including Amazon.com. Other links may go to websites where I have been paid to write a blog or article. See the entire disclosure here.
What's Your Favourite Birdhouse Style?
I think you can tell a lot about a gardener's style by their choice of birdhouse. This gallery has everything from stone to wood, rustic to modern, traditional to eclectic. See which ones are your favourites.
If you love bold colours, also see 12 Colourful Garden Ideas. I've got some freshly-painted birdhouses in my garden too!
Most birdhouses are suitable simply as garden art decor and not really appropriate as nesting boxes (see this post for more information on making choices that are safe for birds).
Rusty Birdhouses
Love rusty garden art? Welcome to Team Rust.
Stone Birdhouses
The first birdhouse I ever made for my garden was a stone birdhouse. I had seen a rather magnificent one for sale on a garden tour and, after seeing the sticker price ($275), I thought it might be a good idea to try making my own instead.
I have provided free instructions for making a stone birdhouse here.
Colourful Birdhouses
Birdhouse Green Roof
Get the DIY green roof instructions here
Rustic & Wood Birdhouses
Colourful Birdhouses
Multiplex Martin-Style Birdhouses
Birdhouse Villages
White Birdhouses
Tall Tower Birdhouses
Tall tower birdhouses are often decorative or ornamental because they are made from a wood log and not intended for nesting birds. The log is embellished with windows, doors, and a roof to give the impresson of a house.
Modern Architecture Birdhouses
Garden Junk / Art / Repurposed Birdhouses
These cowboy boot birdhouses were hand-painted by Empress of Dirt Facebook friend Gaye.
Gazebo Birdhouses
Birdhouse Furniture
I love this one.
~Melissa the Empress of Dirt ♛
|Find Your Plant Hardiness Zone
* United States
* Canada


text:
Free Shipping Every Day
Estimated 6-9 business days for delivery.
Available in fun prints to cheer up a rainy day, these leakproof rubber rain boots for toddlers will stand up to all the mud and puddles your kids can find – and trust us, they'll find plenty.
Questions? Chat with an Expert


text:
The front of this insert is dedicated to all the 'typical' Valentine's day memories. Since this insert will be sitting within my Week 7, I included some of the yellows and blues present in that layout, so it would coordinate nicely. I did, however, add in some more colors, like pink… because Valentine's Day, duh.
The card on the left was a free printable from a Valentine's Day set, that I cannot for the life of me remember where I got it. So if you recognize the "Heart You" card, let me know in the comments where it's from so I can share the love! I kept this card super simple with just my planer picture and some journaling.
The card on the right showcases a cookie bouquet my parents had sent to Melissa and I earlier in the week. The "Especially For" embellishment is actually the card that came with the basket, I just trimmed it down to stick in the pocket. I then transcribed the note that was written inside the card onto a yellow label. I added some washi tape, and a cute little chipboard embellishment that represents both the year and the date (February 14, 2014).
The card on the left shows all the goodies my Valentine showered me with. I simply stuck it onto a 3×4 freebie printable. I then added the "lucky" transparent sticker (fromt an SC kit) right onto my photo, and added in "girl" using SC Color Theory alphas.
The card on the right was made by adhering a 3×4 card from Sweet Shoppe Designs onto a 4×4 piece of cardstock. I added my photo into the blank journaling area. Then I cut out the "Love" letters from another freebie card to make my title. I stuck each letter onto a foam adhesive to give the card a little more dimension.
This card is a close up of the flowers Melissa got me stuck onto a plain 3×4 piece of cardstock. I then fussy cut around the banner embellishment, which was originally a 3×4 card from Sweet Shoppe Designs. In order to not cover up too much of the pretty flowers, I let the banner overflow out of it's pocket, so it sort of overlaps with the card above it as well.
The card on the right was again using a printable freebie 3×4 attached to a 4×4 piece of cardstock. The heart arrows were also cut out from a 3×4 card from Sweet Shoppe Designs, to turn them into embellishments. I finished things off with a 3D glittery heart sticker.
Going back over the process of making a layout really helps to highlight the patterns you used to create it, and which techniques you relied on a little more heavily than you may have realized. For the front of this layout, I pretty much made the entire thing using free, printable cards, with the exception of about 3 embellishments. This proves that you don't have to break the bank to make a fun and beautiful Project Life layout!
I also noticed that I was doing a lot of cutting up cards. Since they were printable, and I didn't feel bad about "ruining" them (since I could always print out more), I cut out a lot of the designs on cards and used them as embellishments rather than actual cards. I love discovering new and creative ways to use all the fun products that come with this hobby!
For the back of my insert, I focused on another aspect of our Valentine's Day. We just so happened to put a hold on our current apartment on that day. Some people might hate doing errands like this on such a romantic holiday, but we were so excited about moving into our dream apartment, making it official was the best way we could have spent our Valentine's Day.
I made a mini title card for this side of the insert highlighting the entryway of our new apartment. For the 'bones' of this pocket, I used a 4×4 piece of cardstock, and a map 3×4 card that came in a SC kit. I hand lettered the word 'welcome' going up the side, and used Thickers to complete the title. I also added in a tiny pink banner behind the H, so my white alphas didn't blend in to the white background.
The card on the right holds a super cool selfie we took before going in to sign all the papers. I embellished with a few printable embellishments (found here), and added the journaling "best Valentine's Day" to a little label.
The next 4×4 pocket shows off the business card of the leasing agent who was so incredibly helpful while we were figuring everything out. She dealt with my million and one questions, and did not judge the crazy lady who wanted to take a ton of pictures of an empty apartment #lifeofablogger. This entire card was again embellished using nothing but free printables (found here, here and here).
Of course, a layout about our new apartment would not be complete without a walk-in closet shot. I have moved apartments many times in the last 5 years, and my number one requirement is always a walk-in closet. The fact that this closet was even bigger than most others, and had TWO rows of shelves, only solidified it's place as my favorite closet ever. The picture is attached to a free printable card, and embellished with a printable label and a 'my fave' tid-bit.
However awesome the closet was, what we were most excited for with this apartment was having two floors. Living within a single floor for the last 5 years really made me desperate for some stairs. They are really what closed the deal on this place. So obviously, those epic stairs made it into my album. The card is cut down to 4×4 from a 4×6 free printable. I embellished with a shipping tag and a cluster of colorful free printables (found here, here and here), and journaled some details about the apartment.
The last card is definitely my favorite of this layout. I had started out wanting to include about 15 apartment photos in this little six pocket insert, which obviously wasn't going to work. I then had a 'duh' moment, and realized I could just link to my blog post about this day right in my layout! The blog post has a ton more pictures and details, so I could include all that information without wasting that much space in my album. So smitten.
I sound like a broken record, but I again embellished with a bunch of different free printables (found here, here and here), and then added in the link using wood veneer letters from SC. I then realized Bitly is case-sensitive, so I included a little hint to myself on the bottom of the card for typing in the link.
And that is my mini insert for Valentine's Day 2014! Does anyone else not realize they're forming a creative pattern until you're all finished and discover you really favored a certain technique in your layout? I didn't realize that I pretty much made this entire layout using free printables until I was writing this post. Who else has fun, unexpected ways they like to use Project Life supplies? I was really digging cutting up cards to use as embellishments with this insert. I'm always looking for new techniques, so shout out your favorites in the comments, I'm all ears!


text:
This promotion is for Rewards members only. To take advantage of exclusive offers like these, join Office Depot® OfficeMax® Rewards Everything now!
- Home /
- Product Details
3 Breaker Maintenance Bypass Panel enables maintenance bypass of Tripp Lite SU40K UPS. Wallmount 3 breaker manual bypass panel with Kirk Key interlock system isolates the UPS from connected loads for maintenance with no loss of network availability.
Product Details
|Item #
|Manufacturer #
|brand name
|Tripp Lite
|gray powder coat
|Tripp Lite SU40K UPS
|11.7 in.
|form factor
|42.4 in.
|Tripp Lite
|Weight (Approximate)
|220 lb
|30.1 in.
Keep High-Output UPS Applications Operational During Maintenance Periods
|Tripp Lite's SU40KMBPK 3-Breaker Maintenance Bypass Panel is used in conjunction with select Tripp Lite 3-Phase UPS systems to maintain total continuity of power to connected load circuits when bypass of the UPS equipment is required for performance of regular service and maintenance. The SU40KMBPK works by using 3 built-in CH-type circuit breakers to isolate 40kVA 3-Phase UPS systems from connected loads with no loss of network availability. The Kirk Key interlock system enhances system reliability. For enhanced safety, the unit also includes a 150A breaker with a KAIC rating of 65. The SU40KMBPK supports wall-mount installations.
When a UPS needs maintenance, the SU40KMBPK keeps power supplied to components via its mechanical interlock system by switching power to the external bypass breaker. Simply unscrew the interlock switch, rotate the switch from the UPS AC input breaker (SW1) to the bypass breaker (SW3), then turn on the bypass breaker and turn off the UPS and isolation breakers to perform maintenance without service interruption.
Compatible with Select 40kVA 3-Phase UPS Systems
The SU40KMBPK is compatible with Tripp Lite's SU40K 40kVA Modular 3-Phase UPS system.
- Wall-mount manual bypass panel isolates UPS from connected loads for maintenance with no loss of network availability
- Kirk Key interlock system enhances system reliability
- 150A breaker with KAIC rating of 65
- 3 built-in CH Type circuit breakers minimize sequencing errors
- Compatible with select Tripp Lite 40kVA 3-Phase UPS systems
- 2-year warranty
- SU40KMBPK 3-Breaker Maintenance Bypass Panel
Founded in 1922 in Chicago, Illinois, Tripp Lite is a leading manufacturer of products to power, protect, manage, house, cool, connect and mount your equipment. With a large selection of connectivity products, Tripp Lite has everything from USB hubs and reversible USB cables to fiber-optic cables and over Cat5e/Cat6 signal extenders. Whether you need to connect high-speed networks to switches, routers and servers in your data center, or send high-resolution video signals to displays and digital signs, Tripp Lite has the complete solution. Plus, before and after your purchase, our Chicago-based, in-house technical support specialists are available to assist you by phone, email and live chat. With over 90 years of quality products and service, Tripp Lite is a brand you can trust.
was removed.
Thank you, you will now be redirected to comparisons.
Just a moment while we prepare the page
Check In-Store Availability


text:
The Spinitron advantage
Spinitron is an online playlist-management solution for non-commercial community and educational radio stations. It handles playlist data capture, archiving, reporting, and dynamic online publishing among many other features.
We provide the database, application, servers, technical and customer service, and protection of your data. There is no software to install – all you need is web access.
Sophisticated features for data entry and manipulation offer many benefits for DJs and stations. But when playlists are online in real-time, benefits flow also to listeners and others.
For radio stations
- Log and publish playlists online
- Track all music played in a structured database
- Prepare reports of use for ASCAP, BMI, SESAC and SoundExchange
- Generate statistical reports such as Top-30-type charts
- Manage shows, program schedule and DJs
- Integrate Spinitron into your station's web site
For DJs
- Put your playlist online in real-time
- Connect with listeners interested in the music you play
- Fast data entry reusing previously logged data to save typing
- Enter playlists before, during or after your show from anywhere on the internet
- Record the artistry of your programming for posterity
- Help bands and artists by publishing your spins online
For listeners
- Find out about music you hear on the radio
- Discover music by exploring playlists by your favorite DJs, disks by the same artist or on the same label
- Browse all the artists, disks, songs and labels logged by a station
- Find a station's program schedule
- RSS feeds of spins on a station
For artists and record labels
- Browse your disks logged on Spinitron
- Find who spun your music and when
- Spinitron helps listeners find details of you and your music online
- When you ask them, stations can find out if your music has been played and how often, by whom, etc.
- NOTE: please do not send Spinitron promotional material. We are not a radio station!


text: I don't know if JonZ was going in this direction, but another potentially interesting storyline would be to have the Avengers go after Hulk as agents of SHIELD. Somewhere in there throw in a storyline about them being manipulated leading to them becoming independent. That would also be a throwback to the comics.


text:
I Have Had a Mind Control Experience
Personal Stories, Advice, and Support 27 People
I let a girlfriend mind control me once,or so she says, and she says I'm still mind controlled, or hypnotized or whatever to be obsessed with girls feet..wierd, cause I kinda am. :D
jbaxter06 26-30, F 1 Response 4 Jun 16, 2014
My Symbolic Life.I see you there, over there. Dancing on the black and white stones. Inbetween the twin pillars, Whispering in your hear. All awaiting the ritual. Just Say the Devilish words now. And we will go, far away. and dissasociate. The Endless maze, only to find out that were going no...
TokyoKid 18-21, M 1 Jun 24, 2012
My Mind Control ExperienceIntroduction: As far as I can ascertain, I have experienced this phenomenon my entire memorable life. To be sure, it hasn't been constant, and recently has become more and more frequent and each occurance has lasted longer. Until the night of Friday, September 6, 2002, I had...
JonBobElder 36-40, M 10 Responses 2 Oct 25, 2008


text:
Another election? Quebeckers are rolling their eyes. If Canadians are indeed once more called to the polls, this will be the sixth time in five years for Quebeckers. Since 2004, on top of three federal elections, there have been two provincial elections, the latest in December, 2008. Not to mention the fact that a federal campaign will likely interfere with municipal elections scheduled throughout the province.
The Liberals are pinning high hopes on Quebec. Insiders believe they can win as many as a dozen new seats (they now have 14 of 75 Quebec ridings). Of course, compared to the golden era of Pierre Elliott Trudeau, this is a modest goal. The Bloc Québécois remains an unbeatable adversary, and right now one can't see what could make the Bloc lose its grip on the province. The latest polls indicate the Liberals are more or less neck and neck with the Bloc, but such numbers are deceptive since a great deal of the Liberal vote is concentrated in the Montreal area and the Bloc remains by far the favourite among the francophone electorate that controls the majority of ridings.
Outside political and intellectual circles, Michael Ignatieff is even less known in Quebec than elsewhere in the country, and nobody knows why exactly he wants to topple the government. Strangely, Mr. Ignatieff didn't take advantage of the summer to make himself more visible. At the end of August, he did spend several days in the Eastern Townships, but provoked a wave of fury when he said Canada should stop exporting asbestos. (The industry supports 700 jobs and is the lifeline of the area.)
Most Quebeckers will be left cold by Mr. Ignatieff's lyrical view of Canada as a work in progress and a land in need of a collective dream. Francophone Quebeckers might have relinquished the ideal of sovereignty, but they don't think of Canada in romantic terms.
At best, it's a country where they feel secure, a good place to raise a family, a tolerant space that allows French to flourish, but certainly not the stuff of emotions. Mr. Ignatieff's passionate brand of patriotism will fall flat in Quebec. In any case, the Liberal leader knows and understands this reality - he often acknowledges Quebeckers have varied identities. So the Liberal campaign in Quebec will focus on other themes, including demonizing Stephen Harper, who has become a rather unpopular figure in the province.
For years now, the Quebec wing of the Liberal Party has been an empty shell. It's in dire need of new blood. But it's a bad omen that in the eight months since he became leader, Mr. Ignatieff has managed to recruit only three prominent Quebeckers, of whom only one will actually run for office.
Steven MacKinnon was an adviser to former New Brunswick premier Frank McKenna, and is said to be "cabinet material." He will run in Gatineau. Meanwhile, Jean-Marc Fournier, a former provincial Liberal minister, is working as a strategist for Mr. Ignatieff, and Marc-André Blanchard, a former president of the provincial Liberal Party, will co-chair the Liberal campaign in Quebec.
But for the public, the only real household name among federal Liberals is the ubiquitous Denis Coderre, a populist MP of the old Chrétien school. Mr. Ignatieff must attract more new faces if he wants to renovate the party's image.
At least, as is shown by the appointments of Messrs. Fournier and Blanchard, Mr. Ignatieff has managed to re-establish some informal links between his party and the Jean Charest government. If some seasoned provincial organizers follow suit, the alliance should help counterbalance the formidable one of the Bloc and the Parti Québécois.
But the question remains why, exactly, Mr. Ignatieff wants to replace Mr. Harper. Will he be able to provide a credible answer in the weeks to come, after having spent the past eight months avoiding the serious issues?Report Typo/Error


text:
- Please select a date to see the proper prices!
- Meal can be requested on the spot
Best offer
102 € / person / night, no meal
Description of the accommodation:
Rooms, services Metropole Brussels is welcoming all guests in City of Brussels with 286 comfortable rooms to stay in. You are also welcome at the hotel for a fine local meal in the restaurant. Parking possibility for your vehicle is available nearby. Car rental service and organized city tours are also available to help you discover all key highlights of the area. In case you like doing exercises while you are on a trip, you should definitely try the gym as well. The café and the bar are an ideal place to host a personal meeting or a pleasant evening talk. You can secure your valuables in the safe. The hotel is offering wireless Internet connection (WiFi) without charging extra fees, so you may go online anytime with your laptop during your stay. To learn more about the exact range of services of Metropole Brussels (including services for an extra fee) and the exact equipment of the rooms, please check the information sheet below.
Payment For the reservation you may need to provide advance payment by credit card or credit card guarantee, depending on the exact date, room and price.
Arrival, opening hours The earliest arrival time is 15:00, the latest time to leave the rooms is 12:00. The personnel can communicate with you in the following languages: Dutch and English.
Metropole Brussels rooms Rooms
- Please select a date to see the proper prices!
Accommodation services
Parking (25 €/night)
Free WiFi in shared places
Relaxation: Fitness
Organized sightseeing nearby, Car rental nearby
- Hairdresser's parlour
Pet friendly (50 €/night), Bar, Cafe, Elevator, Safe deposit box
Own restaurant
Bathrobe, Hairdryer, Desk in the room, Mini-bar


text:
1/144 CGD Mini Stronghold with Flak Pantherturm Summer
Sold for:
Start Free Trial
or Sign In to see what it's worth.
- Sold Date:
- Source: eBay
Flat Rate Shipping Promotion!!Flat rate shipping cost of USD 9 for any number of items you buy!
And Free Shipping for multiple purchases totaling over USD 100.00. Call me stupid or what, I have a hard time figuring out the eBay shipping rule settings. If you find that you are going to be overcharged with shipping, ask me to send you an invoice , and I'll make sure you get the shipping promotion!!
1:144 Mini Diorama
Special Edition
Mini Stronghold with 55mm Flak Pantherturm
- Summer
Base on the propular CGD Mini Diorama "Pillbox", this is specially modified to make use of the CGD 3D Printed 55mm Flak Panther turret and Pantherturm.The Pantherturm may be the deadliest defense weapon introduced by the Germans in WWII, where a Panther tank turret was put into a concrete/wood/steel sub-structure as a gun emplacement. The 55mm Flak Panther however, was a "Paper Project". That means it never got past the drawing board phase. But it is still an exciting model to have and an excellent chance to boost up you Anti Air capability if you are making "What if" dioramas. This diorama features a modified "Pillbox" diorama base, 1 trees, and a 3 parts 55mm Flak Panther Turret and a Camo Net. (Soldiers, vehicles NOT INCLUDED.)
Each of the diorama base is individually hand modified... read more


text:
Per Schefter - more info coming
Lots of weapons for the Eagles
Edit - for a 5th rounder
Per Schefter - more info coming
Lots of weapons for the Eagles
Edit - for a 5th rounder
Last edited by Jetsguy; 03-13-2014 at 09:28 AM.
|Sponsored Links
damn. thats a good move for the eagles
30 Team Stadium Checklist: 10 to go
1) Yankees 2) Orioles 3) Rays 4) Red Sox 5) Mets 6) Braves 7) Phillies 8) Nationals 9) Marlins 10) Pirates 11) Padres 12) Astros 13) Mariners 14) Twins 15) Cubs 16) White Sox 17) Cardinals 18) Indians 19) Tigers 20) Royals 21) Reds
Jets traded their 4th for Ivory last year, and that was an awesome move.
This is better, great move by Chip
Go Knicks!
All time
This kills shady fantasy value!
|Sponsored Links
Eagles will trade for Chris Johnson and will have the fastest pair of RB's in the league
Looks like it's gonna take 2+ years to rebuild this defense
wasting shady's prime years
IMO, Brown is odd man out. Eagles have struggled since Brian Mitchell (since Desean was sooooo underused) for the return game. Solid move.
Carson, trust me, even if you turn out to be a really bad QB, BDawk4Prez will always defend you on PSD.
I don't think so. 1 it gives another back to spell him on passing situations with Brown who can spell him on running situations. Most importantly, besides Jenkins, all of our signing have been on beast special teams players, which we were horrible at last year. This will give us shorter fields and also pin teams back and not set our D up for failure like the playoff game against the Saints.
sproles will be a big upgrade in the return game, johnson was awful last year, and smith was meh
|Sponsored Links


text:
Sordid tales about vampire is a word out of literary imagination than fact of any significance. Vampire is by itself a sordid tale of ghosts, werewolves, tales of horror and unsolved mystery etc. Vampire assumes the form of a woman or a big bat or a man with a penchant for human blood as in the case of count of Dracula. Dracula is the fictional character out of imagination depicted as an example of a human vampire. He was a creation by Bram Stoker, a horror story writer and become popular for the work. The narration of the story in movie makes women faint.
Now it is proved by the scientists that these dangerous creatures do exist in the form of bats and humans but with a twist. A case of a woman Anastasie Dieudonne in Haiti, confessed to sucking the blood of her niece driven by an urge absolutely uncontrollable with a mental imbalance. This is the case of a human vampire. There have been many cases reported similar to this from different walks of life more dramatic and horrifying.
With reference to bat vampires, Dr. August Kronheit of the German Academy of Science, and member of a number of leading American societies, has made an elaborate study of them in South America. Dr. August Kronheit of the German Academy of Science has done a in-depth research on the existence of bat vampires in south America. He identifies the true vampire as a monster natured black-brown bat with a large wing spread and razor-sharp teeth with easily unrecognizable nose resembling a pig. As a bat, it is nocturnal and sucks blood by attacking animals and humans at sleep. A case of a young girl in Bolivia proves this case when the girl's dad saw a bat crouching on his daughter's shoulder at night in the porch. He crushed it to death and later found that the vampire was sucking blood from the girl.
This is not to frighten the readers as most of the bats are harmless. They live by eating insects or by sucking juices from fruits. Vampire bats are known to exist in tropical countries. The puzzling fact is that the victims are not awakened during sucking of blood by the vampire. Reason is attributed to the flat razor-sharp teeth that shave the upper portion of the skin. Blood is sucked through the abrasion by suction. It is neither biting nor stinging and has no depth and so the wound created doesn't create any pain for the victim. Human vampires are psychologically affected people with a passion for sucking human blood and investigations do point out that such people existed earlier and exist now also.


text:
Weather Stock Photos and Illustrations
the products of the Weather category are presented here.
PIXTA's images are high quality and low price. Prices are based on the size of the image, starting at $5 per image.
All images are free of royalty charges, thus, they can be used multiple times, indefinitely.
Popular Images of Weather
This section introduces popular stock photos and illustrations in Weather category that have sold most.
PhotosMore Weather -themed photos
IllustrationsMore Weather -themed illustrations
Search items from popular categories of Weather
This section introduces popular categories and seasonal ones of Weather.


text:
The upcoming sequel Angry Birds Rio won't be available from the Android Market. Rather, you'll have to download it from the Amazon Appstore.
Rovio has been offering free, ad-supported versions of Angry Birds and Angry Birds Seasons through the Google Android Market for months, but has yet to offer ad-free paid apps for Android. In today's announcement, the company also promised that ad-free versions of both games will be available soon. They'll debut in the Amazon Appstore, but it's likely that they'll eventually be available fromt the Android Market.
Angry Birds is one of the most popular games for any mobile platform, with millions of users on Android, iOS, and webOS devices. Now it looks like if you want the next version of the game for Android, you're going to have to install Amazon's store on your device.
PC Magazine says Angry Birds Rio will be available on Mach 22nd, which means the Amazon Appstore could be open in the next wek or so.
Download From: Amazon Appstore


text:
Australian bishops appeal on behalf of detained refugees
As concern over the treatment of asylum seekers in Australia grows - the Australian Catholic bishops have issued an official statement calling on the government to reassess the way they are handling the crisis. Their statement follows: The President of the Australian Catholic Bishops' Conference, Archbishop Francis P Carroll, has called on the Australian Government to re-assess procedures dealing with the asylum seekers in the detention centres. He said that while we can sympathise with the Government in dealing with the problems of asylum seekers arriving in unpredictable numbers without following proper procedures, and the difficulties of processing applications, there is widespread unease in the Australian Community about the current horrific situation. "I urge the Government to respect the human dignity and rights of the asylum seekers, hear their cries for help, and to heed the disquiet of the Community', said Archbishop Carroll. "I also urge the Government to avail itself of the offers of practical assistance from the many groups that wish to assist in the care of the asylum seekers. The Catholic Community and its agencies are more than ready to work with Government and other bodies to bring relief and support". While destructive behaviour and inflicting harm on children are repugnant, said Archbishop Carroll, they must be recognised as the response of deeply traumatized people brought to a point of desperation. Children should not be in such centres in the first place and the causes of desperation much be addressed. Archbishop Carroll welcomed the Government's decision to resume the processing of applications for residence, but pointed out the need for the process to be streamlined and expedited. After the overthrow of the Taliban regime, the Afghan asylum seekers are in a special category. "Should not the Government consider giving them "safe haven" temporary visa arrangements similar to those extended to the East Timorese and Kosovars", asked the Archbishop. "The tragic plight of the asylum seekers, especially the women and children calls for an immediate and compassionate response", concluded Archbishop Carroll. source: Australian Bishops Conference


text:
GraphicRiver Year Calendar 2015 9433219
Print Template \ Stationery \ Calendars
Calendar \ Template \ Company \ Business \ University \ School \ Vector \ Builders
Print Template - Year Calendar 2015 9433219 by XpertgraphicD @ GraphicRiver
Print Template \ Stationery \ Calendars
Item Description .
Very stylish, clean and modern calendar template. This calendar template is fully vector graphic. You can use this professional design to promote your business. This calendar template can be used in office, school, college, university, firm, clinic, bank, insurance, builders company or any other business company.
Item Features .
- Standard A3 sizes with 0.125inch bleed
- Fully editable and resizable
- Fully vector files
- CMYK color mode
- Print ready
- EPS and AI files
- High 300 dpi
- Free font used
- Month start with monday
- Total 14 EPS and 14 Ai files
- Preview image are not included in main file
Font used : Roboto Font & Open Sans Condensed Font(bold)
Layered: No; Graphics Files Included: Vector EPS, AI Illustrator; Minimum Adobe CS Version: CS5; Print Dimensions: 11.69x16.54.
Travel Point Logo; Home Services Logo; Market Arrow Logo; Mountain M Letter Logo; Star Design Logo; Mountain Studio Logo; Power Author Logo.
Keywords: annual, bank, builders, business, calendar, calender, clinic, college, company, corporate, creative, date, firm, insurance, latest, monthly, new, office, photography, school, university, wall, year.


text:
Spouse, children, and grandchildren of CBS Alumni can take advantage of courses offered at College of Biblical Studies. Some courses are offered with a special alumni discount.
College of Biblical Studies 7000 Regency Square Blvd., Houston, TX 77036
The College of Biblical Studies-Houston provides biblically based education for the Body of Christ, with primary focus on African-American and other ethnic minority groups, and equips its students with a biblical worldview for Christian service to the church and the world.
© 2017 College of Biblical Studies | Truth. Training. Transformation. | 7000 Regency Square Blvd., Houston, TX 77036


text:
How deep does his tool drawer run? On the same day he didn't drive in a run for the first time in 12 games, he stole two runs, potentially three, from the Dodgers on Sunday at Dodger Stadium in a game the Rockies
won 7-6.
Three words: Do the math.
Technically, Rafael Betancourt got the save in a game that enabled the Rockies to avoid a three-game sweep. But everyone who saw Gonzalez's catch to end the fourth inning knew better.
"That catch," said Rockies manager Jim Tracy, "was the play of the day."
If Gonzalez didn't make a diving catch on Justin Sellers' drive down the right-field line, two runs would have scored to pull the Dodgers within one. And Sellers would have been standing on second or third, representing the tying run.
Right. Like CarGo wasn't going to catch the ball.
"I thought when the ball came off the bat, there was no chance he was catching it," said Ty Wigginton, who had a good view from first base. "He actually made it pretty easy. He's special. As the year goes along, he's starting to get into that form that people in Colorado have seen."
Said Gonzalez, when asked about the catch, "Which one?"
Exactly. There's no such thing as a circus catch by CarGo. Because he makes the spectacular seem routine.
"Just put it on the list," Tracy said.
As for Gonzalez's hitting, his franchise-record 11-game RBI streak was snapped. He had to settle for three hits, including a double, that extended his hitting streak to 15, during which he has hit .417 (25-for-60) with eight homers and 26 RBIs.
"It's fun," Gonzalez said. "It's the baseball that I play. Right now, I'm just going to enjoy it and take advantage of whatever opportunity is in front of me. When you're hot, everything coming off your bat is hits.
"I wasn't looking for any record. The only record I care about is the record of the team."
The Rockies have a lot of work to do on that front, even after Kevin Kouzmanoff's four RBIs — three on a bases-loaded bloop double in the first — helped the Rockies win this one. At 64-70, they're 11 games behind the Diamondbacks in the National League West.
Even a sweep of Arizona this week probably wouldn't get them back in the race, but it would have been nice to arrive in Phoenix with a rested bullpen.
Instead, Jhoulys Chacin's command issues reappeared after the Rockies gave him a 5-0 lead in the first.
Chacin (11-10, 3.60 ERA) escaped the fourth thanks to Gonzalez's catch. Two innings later, he walked the first three batters to load the bases, beginning a procession of five relievers.
"I just lost my focus a little bit," Chacin said. "It's just the walks. You've got to throw strikes and give the defense a chance to make plays."
Said Tracy: "Jhoulys got the win, but that's not the way you'd like to see it be done. You have an opportunity there, with what we did early, to leave a lot of (relievers) sitting down there, but ball four continues to haunt him. He's just got to understand that. He's got to clear that hurdle."


text:
Ding ding ding SQUAWK, you know what that means, time for an Angry Birds update – today's update is to the movie tie-in game Angry Birds RIO, the expansion another chapter from the movie in what they're calling Carnival Upheaval. What you'll be finding here is a new bird, (one half of the two blue bird shooter you were using before,) this new bird floating normally through the sky with a several hit combo when activated. An oddball bit of fodder to be sure. Also look for this set of levels to be the most epileptic of all the levels, more seizure inducing than any level before!
This set of levels includes the first half of Angry Birds RIO Carnival Upheaval, the second part coming whenever the fine folks at Rovio decide that we're worthy of it. This first half contains 15 levels, each of them taking place on a series of carnival floats, the entirety of the street carnival on either sides of the street you're going down. No beads allowed, no drinks either, because you'll need to use everything in your arsenal just to pay attention to the game because there is a LOT of confetti coming down and you WILL get a headache if you look too long at the screen.
You'll find a couple new bouncy balloon blocks on the ground in several stages here, the main challenge in these levels being the surprising amount of stone blocks to break down or push for your benefit. Behold a series of levels where it's the non-interactive environment that makes the game the hardest to play. Rovio, if you're listening, feel free to release an update that allows you to turn OFF confetti.
This expansion of the Angry Birds RIO game is available right this second in the Amazon Appstore and will eventually be available for everyone who does not like to use the Amazon Appstore, this availability being through the Android Market. Have at it! And if you're looking for golden secrets, be sure to check the labeled boxes for the most part, and in at least one part a labeled beachball!
Lots of fun awaits! If you'd like to see more Angry Birds spoilers, just head to our big fat [Angry Birds Portal O' Fun] for everything you could hope to seek!


text:
Wessex FM News
Spitfire Lands In Dorset
12:04am 10th November 2013
One of the worlds most loved fighter planes that dominated the skies over Britain during the Second World War has come to Dorset.
The one and only hand built Spitfire replica is on display in Wimborne Square to mark Remembrance Day.
It was put together by Terry Arlow and it took over 20 years to build. His arduous task involved trawling through loads of original pictures of the aircraft which were only on microfilm in a museum archive.
After 8 years of close observation and drawings Terry could start building his iconic Spitfire replica using many refurbished genuine parts from the original planes.
You can learn more about Terry's fantastic project here http://www.simplyspitfire.com/
Sign up for the latest local news direct to your inbox


text:
|04 Nov 2012
Computer does not start, LED light on for motherboard...
With Sandy visit in my area, I've lost power couple of times with the computer being on. It had no issues starting it up, other than Windows complaining about not being shut down gracefully.
While I have had power for the last 4-5 days, yesterday the utility power went out with a big boom. I have the generator running and now the computer does not start at all. The LED light is on for the motherboard, indicating that the power is on for the board. Pushing the power on button results in nothing, the system does not start up.
The generator powers the whole house and this computer did work just fine previously when using generator power. The system also had some issues with not maintaining the BIOS settings, as described in a previous thread:
BIOS battery backup issue...
There had been no changes made to the system hardware recently, other than replacing the BIOS battery end of this September.
The LED light goes out in 3-5 seconds, when the PS power switch is shut off and comes on instantly when turned on.
The chances are that either the power supply and/or the motherboard crapped out. Is there a way to positively identify which component is faulty? It does make a difference. If it's the motherboard, I am building a new computer. Conversely, replacing the power supply would save this computer, at least for awhile.
I am planning to get a new power supply today:
Thermaltake SMART Series 850Watt ATX Power Supply SP-850M - Best Buy
It this one doesn't resolve the issue, I'll use it in my soon to build new machine.
I'd appreciate any other suggestion for troubleshooting this issue.
|My System Specs
|04 Nov 2012
Windows 8 64-bit (6.2, Build 9200) (9200.win8_rtm.120725-1247)
If the mobo light comes on it possibly isn't the psu. Could be your cpu or another part of the mobo is fried.
|My System Specs
|04 Nov 2012
Wouldn't the fans kick-in even if the motherboard and/or the CPU fried? There are fans that are directly running from the PSU.
|My System Specs
|Thread Tools
|Similar help and support threads
My Computer Freezes randomly while hdd Light turns solid red.
Hello, SevenForums Community My Computer is giving me a problem for 1 month! The problem is that, my computer Freezes randomly, while that is happening my HDD red light Turns Solid RED! This is totally random, but it happens more times when i'm playing games (ex: Skyrim , GTA San Andreas ,...
|General Discussion
Hdd light on my computer keep's shutting off than my computer freezes
Hi, there is a red light next to my power button that blink's alot & i believe it's called the hdd light. No matter what i'm doing it shut's off after awhile & than my computer freeze's & i don't know why. Everything work's fine into that happen's & it's alway's random. I only had this computer for...
|Hardware & Devices
cpu light stays on alot and slows the computer
Hi i am new to this and need some help with my laptop ... The cpu light stays on a lot and often flickers .... when it flickers it seems to work faster but when the light stays on it really slows down the computer :mad: please help Thank you Lee
|General Discussion
BSOD when computer is bassicaly on light use
Hello,the other day i was ussing my computer with teamspeak,youtube,and some other stuff running in the background,the performance was alright but suddenly i got a BSOD,i ignored it,then the other day when the PC was left idling the BSOD repeated, i wrote don't remember the code but i will ad both...
|BSOD Help and Support
Computer light shows; but no beeps
Hello. Today, my computer decided to not work. I am the thread maker of this thread: https://www.sevenforums.com/system-security/153948-virus-gone-what-3.html Reading that may give some other, extra, and/or some useless information. Anyways, I will give a longish summary. Within this past...
|BSOD Help and Support
Computer turns itself off and power light stays on
Hey guys. The PC I'm on right now has been turning itself off randomly when I am using it. When it does this the power light is still showing, mouse is still lit up usb sytick still lit up etc. What could be the cause of this and how can I fix this?? Thanks:)
|General Discussion
|Our Sites
|Site Links
|About Us
|Find Us
Windows 7 Forums is an independent web site and has not been authorized, sponsored, or otherwise approved by Microsoft Corporation. "Windows 7" and related materials are trademarks of Microsoft Corp.
© Designer Media Ltd
All times are GMT -5. The time now is 00:32.


text:
Well, I went and saw The Order of the Phoenix today. It's been getting a lot of press about not measuring up to the previous movies. I'm not so sure that this is a fair position to take. The director has changed and it's the shortest movie made from the longest book, so it was always going to be a challenge. However, I think they did a good job of telling as much of the story as they needed to.
As is always the case with movies made from novels, it felt a bit rushed. Lots of important bits and pieces were wedged into the film while enormous chunks of the book were left out. Then again, I think the book was overly long. As usual, I won't go so far as to write an actual review of the film. There's plenty of those posted across the net already. It is worth a look if you're a fan
But I came away with one big question and it's still bugging me. Never mind all the speculation as to whether Severus Snape is a good guy or a bad guy, nor the nail-biting anticipation of whether or not Harry will die at the end of book seven. I have a bigger question.
Remember the Quidditch match injuries and the marvellous ministrations of the Hogwarts nurse (her name escapes me)? Remember the incredible things they can do at St Mungo's hospital? These wizards and witches are all pretty handy at mending a broken magic user.
Why, therefore, does Harry Potter still need to wear glasses?


text: I'm trying to get this latest swype beta since i've never used swype before. I've got my email and when I click the link it says that I'm not running on an android phone... Could this be an issue with 2.1v3. I noticed another poster had the same issue and he's also on the leak. Is anyone else having the same problem or am I just SOL?