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The p.N478D missense mutation in human mitochondrial poly(A) polymerase (mtPAP) has previously been implicated in a form of spastic ataxia with optic atrophy. In this study, we have investigated fibroblast cell lines established from family members. The homozygous mutation resulted in the loss of polyadenylation of all mitochondrial transcripts assessed; however, oligoadenylation was retained. Interestingly, this had differential effects on transcript stability that were dependent on the particular species of transcript. These changes were accompanied by a severe loss of oxidative phosphorylation complexes I and IV, and perturbation of de novo mitochondrial protein synthesis. Decreases in transcript polyadenylation and in respiratory chain complexes were effectively rescued by overexpression of wild-type mtPAP. Both mutated and wild-type mtPAP localized to the mitochondrial RNA-processing granules thereby eliminating mislocalization as a cause of defective polyadenylation. In vitro polyadenylation assays revealed severely compromised activity by the mutated protein, which generated only short oligo(A) extensions on RNA substrates, irrespective of RNA secondary structure. The addition of LRPPRC/SLIRP, a mitochondrial RNA-binding complex, enhanced activity of the wild-type mtPAP resulting in increased overall tail length. The LRPPRC/SLIRP effect although present was less marked with mutated mtPAP, independent of RNA secondary structure. We conclude that (i) the polymerase activity of mtPAP can be modulated by the presence of LRPPRC/SLIRP, (ii) N478D mtPAP mutation decreases polymerase activity and (iii) the alteration in poly(A) length is sufficient to cause dysregulation of post-transcriptional expression and the pathogenic lack of respiratory chain complexes.
The human mitochondrial genome, mtDNA, encodes 13 polypeptides, all of which are essential for the coupling of cell respiration to ATP production (1). These proteins arise from the intra-mitochondrial translation of mt-mRNAs processed from large polycistronic precursors, which are mostly matured by the addition of short (∼50 nt) poly(A) tails (2,3). Adenylation is necessary to complete the termination codon of seven open-reading frames, but the rationale for why human mt-mRNAs are poly- rather than oligo-adenylated remains unclear. Although polyadenylation of transcripts has been almost universally maintained in cells and organelles, its function varies dramatically (4,5). For example, in the eukaryotic cytosol, the polyadenylation of mRNA permits association with poly(A)-binding protein (PABP) family members, leading to an increase in mRNA stability (6,7). Further, the presence of a poly(A) tail and PABPs allows interactions with factors that bind to the 5′ methylguanylate cap structure of mRNAs thereby stimulating translation initiation (8). Conversely, polyadenylation of RNA in various eubacteria can promote degradation and regulate quality control of more stable tRNA or rRNA (9). Furthermore, variations in polyadenylate function are also apparent between organelles. Yeast lack a mitochondrial poly(A) polymerase (mtPAP), and no mitochondrial mRNA carries a 3′ post-transcriptional extension (4,10), whereas polyadenylation of plant and algal organellar RNA follows the eubacterial pattern and stimulates decay (11,12). Trypanosome mitochondria are yet more complicated. Short (15–20 nt) oligo(A) tails are added to pre- or fully edited RNA and are necessary to promote stability, whereas longer U/A extensions of 200–300 nt designate the mRNA as ready for translation (13). It has long been known that human mt-mRNA can be polyadenylated (3), but even after numerous attempts to determine its exact role, the function of this modification is still unclear. A number of different approaches by various groups have been used to address this question. These include siRNA-mediated depletion of transcripts encoded by the PAPD1 gene, now termed MTPAP in the new nomenclature (14–16), occlusion or degradation of poly(A) tails by targeting cytosolic PABP or poly(A) nuclease to the mitochondrion (17), and overexpression of the mitochondrial deadenylase PDE12 (18). In each case, mt-mRNAs were analysed and different effects on stability were observed, dependent on the transcript analysed.
All these methods, however, introduced some level of artificial genetic manipulation, each of which can be prone to artefact and consequently difficulties in interpretation of resulting data. In contrast, we were invited by an Old Order Amish family to investigate the cause of a profound form of spastic ataxia with optic atrophy in a subset of family members. Samples from these patients indicated that their clinical condition resulted from a homozygous p.N478D missense mutation in the MTPAP gene (19). Our initial studies of mRNA transcripts obtained from family blood samples were able to confirm that the mutation caused a loss of polyadenylation in the mt-mRNA species analysed. More recently, using fibroblast cell lines from affected as well as unaffected family members, we have been able to undertake more in-depth analysis into the consequences of non-polyadenylation of mt-mRNAs.
The p.N478D mtPAP mutation causes a defect in oxidative phosphorylation
Our initial investigations were limited to the analysis of blood samples from members of a large Old Order Amish family with multiple children suffering from a progressive and autosomal recessive neurodegenerative disorder. Genomic analysis revealed a candidate pathogenic mutation in MTPAP, the gene encoding mtPAP. We therefore performed mitochondrial poly(A) tail assays of representative mitochondrially encoded transcripts from whole blood. These confirmed an mt-mRNA polyadenylation defect associated with the c.1432A>G (p.N478D) mutation in clinically affected members, but no further studies were possible at that time (19). Here, we present investigations on skin fibroblast cell lines from three family members, two affected individuals homozygous for the mutation (P1 and P2) and one unaffected heterozygote sibling (Het). Initially, it was important to confirm that the polyadenylation defect was recapitulated in the cultured fibroblasts. RNA was isolated, and four mt-mRNAs were subjected to the mitochondrial poly(A) tail length assays as described previously (20). For each mt-mRNA analysed, a lack of polyadenylation was apparent in the pathogenic homozygote lines (P1 and P2), concomitant with an increase in oligoadenylated species (Fig. 1A lanes 2 and 3). The heterozygote behaved similarly to the control line, although there was evidence of a mild increase in oligoadenylated mRNA (Fig. 1A lane 1 cf lane 4). Homopolymeric oligoadenylation was confirmed by sequence analysis of MPAT-derived species for MTCO1 (data not shown). We then assessed whether the marked loss of polyadenylation caused any phenotypic consequences. Cell growth rate was examined first, using either glucose- or galactose-based media. Galactose is a carbon source that forces cells to use oxidative phosphorylation (OXPHOS). The growth defect seen in the homozygote mutant lines was more pronounced on this substrate than on glucose (Supplementary Material, Fig. S1). Cell lysates from the two patient cell lines, the unaffected sibling (Het) and one unrelated control (C) were then prepared and subjected to western blot (Fig. 1B). No observable difference in the steady-state levels of mtPAP could be detected, showing the p.N478D mutation did not affect the stability of the protein. In contrast, in the homozygote p.N478D lines, a substantial decrease was apparent in the mitochondrially encoded proteins of the respiratory chain, ND1, a component of complex I and COX1–3, members of complex IV. NDUFB8, although nuclear encoded, was also present at decreased steady-state levels as it is sensitive to complex I assembly. As expected, no decrease in complex II (SDHA) was observed consistent with all its components being nuclear encoded. Analysis of the fully assembled OXPHOS complexes by blue native gels (Fig. 1C) reflected the severe decrease seen in components of complexes I and IV, and this was further mirrored in the enzymatic activities (Fig. 1D). Intriguingly, complexes III and V appeared unaffected. Taken together, these data are consistent with a selective defect of mitochondrial gene expression.
To confirm that the mtPAP p.N478D mutation was responsible both for the loss of mt-mRNA polyadenylation and the consequent reduction in complexes I and IV, we designed a lentiviral vector to express wild-type mtPAP constitutively. Following transduction and selection of the four cell lines (P1, P2, Het and C), lysates and RNA were prepared. Expression of wild-type mtPAP (+LVMTPAP) in the homozygote mutant cell lines was sufficient to restore steady-state levels of mtDNA gene products at both the mRNA and protein level (Supplementary Material, Fig. S2A and B). Further, analysis of various mt-mRNAs showed a restoration of poly(A) tail for lines P1 and P2 (RNA14, Fig. 1 lanes 2, 3 cf 6, 7; MTND3, Supplementary Material, Fig. S2C lanes 2, 3 cf 6, 7). In all four cell lines transduced with wild-type mtPAP, the final tail length exhibited a slightly increased median length compared with the untransduced controls (lanes 1, 4 cf 5–8 for both RNA14 in Fig. 1A and MTND3 in Supplementary Material, Fig. S2C).
Aberrant mtPAP induces significant effects on the steady-state levels and translation of mitochondrial mRNA species
Depletion of mtPAP, mediated by siRNAs targeting the MTPAP transcript, has been shown by several groups to result in transcript-specific effects on the steady-state levels of mt-mRNAs in cultured human cell lines (14–16). In the homozygote patient lines (Fig. 2A lanes 2 and 3), we also saw transcript-specific effects, where steady-state levels of RNA14 and all MTCO were decreased, MTND1 increased and MTND3 unaffected. A similar trend had been noted when mt-transcripts lacked polyadenylated termini as a consequence of the poly(A) tails being removed by a cytosolic poly(A)-specific ribonuclease that had been targeted to mitochondria (17). To determine what effect these altered steady-state levels, caused by the mutant mtPAP, had on the translation of these transcripts, de novo metabolic labelling was performed. Autoradiographic data analysis of the homozygote compared with the control indicated that there were varied but reproducible differences in the amount of translation products from different transcripts (Fig. 2B). There was an evident decrease in de novo synthesis of the components of the COX2/3 ATP6 triplet, which was in contrast to the increase in ND5 and ND2. Those transcripts that showed decreased stability, such as MTCO3, showed a concomitant decrease in translation product. However, there was no consistent correlation between transcript level and translation as the dramatic increase in stability of MTND1 (Fig. 2A) did not result in a concomitant increase in ND1 (Fig. 2B).
Mutant and wild-type mtPAP are found in mitochondrial RNA granules
It has been shown previously that mitochondrial RNA is processed in distinct foci referred to as mitochondrial RNA granules (MRGs) (22). We posited that the maturation of mitochondrial mRNA may also occur in MRGs. Consequently, we performed immunocytochemistry to detect mtPAP (endogenous, Fig. 3A) and a marker for the MRGs, namely GRSF-1. As shown in Figure 3A, both antibodies revealed discrete co-localizing foci within the mitochondrial network, demonstrating that mtPAP is indeed present in MRGs. Repeat experiments with FLAG-tagged wild-type mtPAP (Fig. 3B) also showed this punctate distribution. We then wished to determine whether the p.N478D mutation led to any loss of mtPAP localization. As seen in Figure 3C, the p.N478D mtPAP also showed a similar pattern of co-localization, consistent with the mutant being present in MRGs.
Activity of the p.N478D-mutant mtPAP is severely compromised
The p.N478D mtPAP mutation did not affect either the stability or the location of the enzyme necessitating further investigations to characterize the effect of this mutation on mtPAP. The crystal structure of this non-canonical poly(A) polymerase has been solved (23) revealing that the protein dimerises but lacks a classic RNA-binding domain. The region encompassing p.N478, however, was disordered in the crystal. To determine whether the p.N478 mutation influences the dimer formation, we overexpressed and purified both the wild-type and mutant enzyme for use in in vitro studies. Both wild-type and mutant mtPAP were isolated to electrophoretic purity and subjected to gel filtration analysis. As shown in Supplementary Material, Fig. S3, both mutant and wild-type mtPAP form dimers indicating that the p.N478D mutation does not affect the overall fold or the oligomeric state of the protein. The mutation, therefore, must affect mtPAP function directly, because the N478 region does not contribute to the dimer interface (23). We subsequently compared polyadenylation activities of the two enzymes in vitro. Initially, we generated long RNA substrates [terminal 277 nt of MTND3 or 248 nt of RNA14, with or without oligo(A8)]. The wild-type enzyme extended from both templates irrespective of the oligoadenylation status. The mutant protein, however, was unable to discernibly extend unadenylated MTND3 substrate (Fig. 4A). Using such a large substrate under these electrophoresis conditions, however, meant that it was not possible to determine whether p.N478D was entirely inactive or only able to oligoadenylate, as could be seen for mt-mRNA in the mutant cell lines (Fig. 1A). The absence of any activity would suggest that another poly(A) polymerase would have to be present to add the oligo(A) modification. We therefore designed a number of short (40 nt) RNA substrates to mimic either internal sections of MTCO1 and MTND3 or the native 3′ end of RNA14 post-processing. The latter substrate was synthesized with or without an oligo(A8) 3′ terminal extension to establish whether mtPAP had a different preference for (i) correctly processed 3′ termini compared with any free 3′ end and (ii) to see whether mtPAP only used pre-oligoadenylated transcripts as a template. Saturating levels of wild-type mtPAP were able to extend ∼40 nt from the 3′ terminus of both the internal MTND3 sequences (Supplementary Material, Fig. S4) and also correctly processed RNA14 with or without oligo(A8) ends (Fig. 4B lanes 2 and 6). Thus, the wild-type mtPAP demonstrated no preference for 3′ terminal sequence of the RNA substrate. In addition to the main population of extended products, there was a second, much longer population. The ratio of this product (Fig. 4C lane 6, *) to the main product varied between experiments. In contrast, the mutant mtPAP showed severely compromised activity, where the extension achieved on either template, with or without oligo(A8), was only 5–10 nt (RNA14 shown in Fig. 4B lanes 3 and 7). The mutant protein was not able to generate longer extensions even in prolonged -h incubations. To determine whether the p.N478D mutation affected polymerase processivity, it was first necessary to identify whether mtPAP was distributive or processive under these in vitro conditions. The extension assay was therefore performed under conditions where the enzyme/RNA substrate ratio fell below 1 : 1. Figure 4C shows that the wild-type enzyme does not fully extend the subset of RNA substrate to which it has bound, indicating that under these in vitro conditions, it is distributive, thus precluding any effect of the mutation on enzyme processivity.
LRPPRC and LRPPRC/SLIRP enhance poly(A) tail length extension
Leucine-rich pentatricopeptide repeat-containing protein (LRPPRC) binds mature or unprocessed mitochondrial mRNA in vivo (24,25) and is often found associated with another much smaller RNA-binding protein, SLIRP (SRA stem loop interacting RNA-binding protein) (26). When the gene encoding LRPPRC or its homologue was deleted in mice or Drosophila, the resulting mt-mRNAs were found at low steady-state level, with reduced poly(A) tail lengths, and in many cases were aberrantly translated (27,28). Further, a recent study concluded that LRPPRC promoted mtPAP-mediated polyadenylation, possibly by resolving secondary structures at the 3′ termini of RNA substrates, facilitating access of mtPAP to unstructured 3′ ends (25). We therefore examined whether the presence of LRPPRC or the LRPPRC/SLIRP complex affected poly(A) extension by wild-type or mutant mtPAP. In the presence of wild-type mtPAP and LRPPRC (0.5 or 1.25 µm) poly(A) tail length was increased by a mean value of ∼12 nt (Fig. 5A cf lanes 2 and 3, 5 and 6). However, the extension was more pronounced on addition of the LRPPRC/SLIRP complex (Fig. 5A lane 4 cf lane 2), whereas SLIRP alone had only a minimal effect on mtPAP activity (data not shown). Assays were performed for 1 h, but increasing extension time to 2 h had only a minimal effect on maximal tail length (Supplementary Material, Fig. S4). Interestingly, a similar modulation of extension by LRPPRC/SLIRP was noted on an RNA substrate carrying eight adenylates (A8) residues at the 3′ terminus (Fig. 5B) precluding the increase in extension being due to LRPPRC/SLIRP resolving 3′ terminal structure. Assays performed with the p.N478D-mutant mtPAP together with the LRPPRC/SLIRP complex also resulted in a further, minor, extension of only 15–20 nt (Fig. 5C lanes 4 and 5), in comparison with an additional extension of ∼50 nt with wild-type mtPAP in the presence of complex (Fig. 5C cf lanes 2 and 3). Intriguingly, the longer population illustrated in Figure 4C lane 6 was in all cases resolved upon the addition of LRPPRC/SLIRP (Fig. 4C lane 12).
The p.N478D mutation in mtPAP causes a severe progressive neurodegenerative disorder in all homozygous patients identified. The resultant cellular defect was identical in both of the patient cell lines analysed and manifested as a profound loss of mitochondrial OXPHOS complexes I and IV. Why such selectivity ? The mtPAP mutation caused selective variability in the stability of mt-mRNAs presumably due to their lack of polyadenylation. There have been several previous studies into the effects of deadenylation on mt-mRNA stability in human cultured cell lines, by using siRNA depletion of mtPAP in human cell lines (14–16), overexpression of a mitochondrial deadenylase PDE12 (18), or by the removal of poly(A) tails by targeting the poly(A)-specific ribonuclease PARN to mitochondria (17). A consensus pattern of stability now appears to be emerging. The lack of a poly(A) tail appears to result in significant and transcript-specific effects on mt-mRNA stability—MTND1, MTND2 increased; MTCO transcripts decreased; MTND3, MTND5, MTCYB mildly or unaffected. A similar trend was noted for the patient homozygote cell lines, albeit that RNA14 was less stable than controls. These effects on transcript stability are clear, and yet, there is no credible hypothesis to explain the transcript-specific variability. What is the effect of this variability on translation? Several recent publications have suggested a link between the polyadenylation status of mt-mRNA and translation (8,17,18). It has been known for many years that translation in the eukaryotic cytosol can be stimulated by the presence of a poly(A) tail by a mechanism promoted through interaction between protein complexes at the 5′ methylguanylate cap and the 3′ tail of the transcript (8). However, mitochondrial mRNA species do not possess a 5′ cap structure or any known 5′ associating complexes (29,30). Artificial deadenylation studies reported dramatic effects on mitochondrial protein synthesis, but these data were complicated by the forced deadenylation causing either the loss of termination codons in open-reading frames (17) or loss of the small mitoribosomal subunit itself (18). Both of these effects would be expected to decrease de novo protein synthesis.
None of the earlier studies assessed the effects of siRNA-induced mtPAP depletion on mitochondrial protein synthesis, although Nagaike et al. (15) did report marked effects on respiration, consistent with a lack of OXPHOS complexes. Our current studies show that de novo mitochondrial protein synthesis was perturbed in the p.N478D mtPAP homozygote cell lines, with certain species being increased (ND5), some decreased (COX1–3) and many unaffected (e.g. ND1–3), and OXPHOS was severely compromised. There was a degree of correlation between effects on mt-mRNA stability and translation, as seen for complex IV subunits, but others did not correlate well (e.g. ND1 and ND5). In other relevant studies, knockout of LRPPRC in mouse cardiac tissue and skeletal muscle resulted in the decreased stability of all mitochondrial transcripts except for the sole L-strand encoded product, MTND6 (28). The remaining pool of transcripts retained only oligo(A) tails, and translation was markedly affected. Although de novo synthesis of most species was decreased as predicted from the effect on transcript stability, one translation product was in substantial excess. Following deletion in the fly of BSF, the homologue of LRPPRC, the initial pattern of severely compromised polyadenylation and steady-state levels of mt-mRNA was apparent and similar to the mouse model (27). However, the fly deletion differed as the transcript showed an additional problem of aberrant processing, and a marked increase in translation was noted, along with the production of novel translation products. This would argue that at least in flies, polyadenylation of mt-mRNA is not a prerequisite for translation. Finally, in human cultured cell lines under conditions where poly(A) tails were masked by mislocalization to the mitochondrion of the major cellular poly(A)-binding protein, PABPC1, a decrease in translation of all mt-mRNAs was seen (17). So, how does the loss of the poly(A) tail on human mt-mRNA result in such profound defects in assembly and stability of OXPHOS complexes? Taken together with several of the studies referenced earlier, it is clear that even relatively mild perturbations in de novo synthesis of mitochondrially encoded proteins can have major consequences for the assembly of OXPHOS complexes, possibly reflecting the intricate process of integrating the various subunits that are synthesized in different subcellular locations. However, this still does not explain the molecular basis of why the absence of the 3′ maturation event of polyadenylation could cause a modulation in protein synthesis that initiates at the other end of the molecule. Many groups contributed to resolving how polyadenylation functioned in the cytosol of eukaryotes to stimulate translation. Research in human mitochondria has the disadvantages of not being able to transfect the organelle with reporter constructs or to have a faithful in vitro system for analysing protein synthesis (31,32). It has therefore taken numerous groups, many years and many experimental approaches to try and understand the exact role of polyadenylation in the mammalian mitochondrion, and despite these efforts, we are not yet in a position to fully explain this observation.
What is the consequence of the p.N478D mutation in human mtPAP? We have shown that the mutated enzyme was correctly dimerized and folded and could function in extending 3′ termini, although the extension lengths were severely curtailed, indicating that the mutation caused a defect in but not a total loss of polyadenylation activity. This may explain the retention of short oligo(A) tails on mt-mRNAs in the homozygous patient cell lines. The mutation is located in the highly conserved fingers domain of mtPAP (23). In the absence of any classical RNA-binding domain in this non-canonical polymerase, it has been suggested that the interface of this fingers domain with the palm region may function in substrate binding, although the exact RNA-binding domain is unclear (5). Alternatively, another enzyme/complex may act in tandem with mtPAP to facilitate either 3′ end binding or processivity as both wild-type and mutant mtPAP act distributively in our in vitro assays, irrespective of LRPPRC/SLIRP addition. The hypothesis that there are more, as yet unidentified, binding partners is particularly appealing, given the poor activity of even the wild-type mtPAP when assayed, in vitro. We have been exploring the possibility that a co-factor is necessary for optimal mt-PAP activity, but our preliminary co-immunoprecipitation assays were unable to identify any candidates to date. Recently, however, a similar co-IP approach was used to identify two associating components, the RNA helicase Suv3p and the RNA-degrading enzyme, PNPase (33). The authors of this intriguing paper supply further evidence to suggest that a transient complex of these three proteins function in vivo to regulate the poly(A) tail lengths of human mt-mRNAs dependent on the ratio of inorganic phosphate to ATP in the mitochondrion.
Finally, how does the interplay between mtPAP and LRPPRC/SLIRP impinge on polyadenylation? It has previously been reported that on its own in vitro, mtPAP was unable to extend 3′ termini beyond short oligo(A) additions and required the presence of LRPPRC to promote extension (25). The authors of that study performed several experiments to show that LRPPRC promoted polyadenylation in vitro, suggesting that the complex acted by resolving double-stranded regions to reveal single-stranded RNA 3′ termini. We found some similarities to this impressive work, but our data differed in several respects. First, we found that mtPAP alone was able to extend from 3′ termini to produce poly(A) tails. Second, although we were able to show a consistent increase on the length of 3′ extension by the addition of the LRPPRC, the extension was markedly enhanced when LRPPRC was complexed with SLIRP. Third, we observed a consistent LRPPRC/SLIRP-mediated increase in poly(A) tail length even in the absence of 3′ secondary structure, arguing that the role of the complex cannot only be to resolve secondary structure. Absolute extension varied mildly with the different buffer conditions tested, but in all cases, the trends remained identical. This promotion of extension is intriguing. In addition to the extension of the major poly(A) species, a longer population of varying proportion (Fig. 4C lane 6) was lost on addition of the LRPPRC alone or complexed with SLIRP (Fig. 4C lane 12). The presence of LRPPRC/SLIRP therefore appeared to act as a molecular ruler. This interplay is reminiscent of a tripartite relationship that regulates poly(A) extension in the eukaryotic nucleus. The cleavage and polyadenylation-specific factor CPSF binds to RNA elements close to mRNA 3′ termini and stimulates poly(A) polymerase-mediated polyadenylation by interacting with the nuclear poly(A)-binding protein PABPN1 that coats the nascent poly(A) tail (34). When the tail reaches ∼250 nt, PABPN1 then acts to disrupt the interaction with CPSF, curtailing further extension. Although this similarity is interesting, it cannot be an exact model for mtPAP. PABPN1 breaks the processivity exhibited by nuclear PAP, and we have been unable to demonstrate processivity of mtPAP in vitro.
In summary, we have shown that the p.N478D mutation in mtPAP is pathogenic. The defect prevents the enzyme from efficiently polyadenylating mt-mRNA. The resultant loss of polyadenylation causes a differential modulation of steady-state levels of specific mt-mRNAs and perturbs mitochondrial protein synthesis leading to profound depletion of complexes I and IV of the respiratory chain, resulting in a form of spastic ataxia and optic atrophy.
Cell culture and viral vectors
Primary human dermal fibroblasts were established from individuals either heterozygous or homozygous for the 1432A>G mutation in the MTPAP gene. Cell lines were immortalized by retroviral expression of the HPV-16 E6E7 gene. Cells were cultured in reagents purchased from Gibco Life Technologies; DMEM supplemented with 10% heat-inactivated foetal bovine serum, 50 U/ml penicillin, 50 µg/ml streptomycin and 50 µg/ml uridine (5% CO2 at 37°C). Wild-type expression of MTPAP was achieved by transduction with lentiviral particles (Genecopoeia) conferring puromycin resistance.
Mitochondrial poly(A) tail assay
The (20) technique was modified to use AlexaFluor® 647-labelled gene-specific nested forward primers instead of radiolabelled primers in the second round of PCR. The resulting PCR products were separated on a 10% polyacrylamide/8.3 m urea gel and directly imaged using a Typhoon FLA 9500 instrument (GE/Fujifilm).
Metabolic labelling of mitochondrial translation
Essentially as described in (35) with a modification of a 1-h incubation with radiolabel, protein aliquots (50 µg) were separated by 15–20% (w/v) gradient SDS–PAGE. Radiochemical signal was detected (Typhoon FLA 9500 instrument GE/Fujifilm) and proteins identified by comparing their migration patterns against established data (21).
RNA extraction and northern blotting
RNA was prepared by TRIZOL (Invitrogen) extraction following manufacturer's recommendations. Northerns were performed as described (36) and signals quantified by scanning with Typhoon FLA 9500 instrument (GE/Fujifilm).
MTPAP cloning, and purification of wild type and p.N478D mtPAP
Human MTPAP (corresponding to residues 44–538) was sub-cloned into the pET28a vector (Novagen) generating an N-terminal 6× His fusion tag. The p.N478D mutant was created with the QuikChange kit (Stratagene). Overexpression of wild-type and p.N478D mtPAP in Escherichia coli BL21 Rosetta (DE3) cells (Novagen) was induced by overnight incubation with 0.5 mm IPTG (Melford) at 20°C. The soluble protein was eluted from nickel affinity gel (Sigma) with 50 mm Tris, pH 8.5, 300 mm NaCl and 250 mm imidazole, further purified by size exclusion chromatography and then concentrated in a buffer containing 50 mm Tris, pH 8.5, 300 mm NaCl and 5% (v/v) glycerol.
Cloning and purification of LRPPRC and LRPPRC/SLIRP
Codon-optimized (DNA 2.0) DNA constructs corresponding to the mature form of human LRPPRC and amino acid 18–109 of SLIRP were cloned in a pCDFDuet-1 vector (Novagen) for co-expression. In addition, LRPPRC was cloned in pJexpress 401 (DNA 2.0). In both constructs, LRPPRC contains a TEV-cleavable 6× His fusion tag at the N-terminus. LRPPRC and LRPPRC/SLIRP were expressed and purified as previously described for the MTERF4-NSUN4 complex (37).
Low resolution polyadenylation assay
RNA substrates were in vitro transcribed using the MAXIscript® SP6 Kit (Ambion) to generate 248- and 277-nt transcripts mapping to the 3′ ends of MTATP6 and MTND3, respectively. Polyadenylation reactions, product separation and visualization were performed as described for high-resolution assays.
High-resolution polyadenylation assay
Single-stranded RNA mapping to the 3′ 40 nt of RNA14, with and without oligo(A8) tails, were purchased from Dharmacon. Polyadenylation reactions were carried out in 15 µl volume composed of 50-ng RNA substrate, 50 mm Tris, pH 8.0, 40 mm KCl, 1 mm DTT, 0.1 mm ATP, 10 mm MgCl2, 0.1 mm MnCl2 and 0.5 U/μl SUPERaseIn RNase inhibitor (Invitrogen). Wild-type mtPAP, p.N478D mtPAP, LRPPRC/SLIRP complex or LRPPRC was added at 0.5 µm each and incubated at 37°C for 1 h unless stated. RNA products were fractionated on 15% polyacrylamide/8.3 m urea gels, stained with SYBR Gold (Invitrogen) and imaged with a Typhoon FLA 9500 instrument (GE/Fujifilm).
Analysis of proteins by immunoblot and BN-PAGE
Proteins were separated by standard SDS–PAGE and then immobilized on PVDF membrane (Immobilon-P, Millipore) by wet transfer (100 V, 1 h at 4°C) in Towbin buffer. Membranes were blotted with primary antibodies followed by HRP-conjugated secondary antibodies (Dako) and visualized by ECL Prime (GE Healthcare). One-dimension blue native gel electrophoresis (4.5–16% w:v) was performed as described in (38) with 40 µg of protein loaded per lane. Primary antibodies were from MitoSciences COX1 (MS404), COX2 (MS405), COX3 (MS406), SDHA (MS204), NDUFA9 (MS111), NDUFB8 (MS105), Complex V α-subunit (MS502), Complex III core 2 (MS304); Santa Cruz ATP8 (sc-84231), TOM20 (sc-17764), LRPPRC (sc-66844); mtPAP (GeneTex GTX70156); COX holoenzyme (gift from L. Nijtmans), ND1 (gift from A. Lombes).
OXPHOS activity assays
The activities of individual respiratory chain complexes were determined in isolated mitochondria as previously described for complex I and IV (39) and for complex V (40).
Immunofluorescence microscopy
HeLa cells were plated on coverslips and transiently transfected with a HA-tagged version of GRSF1 isoform 1 (GRSF1-HA), and FLAG-tagged wild-type or p.N478D mtPAP. Immunolabelling with polyclonal anti-HA, monoclonal anti-mtPAP or monoclonal anti-FLAG was as described (22). Imaging was performed using a Zeiss LSM700 confocal microscope.
Supplementary Material is available at HMG online.
This work was supported by The Wellcome Trust (096919/Z/11/Z) to R.N.L. and Z.C.L.; Pathological Society of Great Britain and Ireland (PhD 2010/04) to Z.C.L. as a PhD Scholarship funding for W.C.W.; Deutsche Forschungsgemeinschaft postdoctoral Fellowship (GZ: HO 3326/2-1 AOBJ:584841) to H.T.H.D. and US NIH grant (R01GM077175) to L.T. Funding to pay the Open Access publication charges for this article was provided by the Wellcome Trust.
Conflict of Interest statement. None declared.


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|"Howard M. Lewis Ship" <hls...@comcast.net>
|[HiveMind] What next?
|Tue, 14 Oct 2003 21:35:47 GMT
I've been touching up the framework and docs and I really think HiveMind is ready to go into beta. My definition of beta is "code complete, no changes outside of focused bug fixes". In other words, by going to beta, we're saying that HiveMind is feature complete for a 1.0 release. We have lots of cool ideas for a 1.1 release ... but despite the surprising amount of interest in HiveMind, my preference is to keep it as small and focused as possible, without omitting any features that will be generally useful. I've lost track of all the cool ideas that have popped up. We need a HiveMind wiki somewhere. -- Howard M. Lewis Ship Creator, Tapestry: Java Web Components http://jakarta.apache.org/tapestry http://jakarta.apache.org/commons/sandbox/hivemind/ http://javatapestry.blogspot.com --------------------------------------------------------------------- To unsubscribe, e-mail: email@example.com For additional commands, e-mail: firstname.lastname@example.com
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System.NullReferenceException: Object reference not set to an instance of an object.
at DotNetNuke.Framework.DefaultPage.OnLoad(EventArgs e) in e:\websites\tvtechnology.com\public_html\Default.aspx.cs:line 791
No matter where you live, natural disasters happen. In California, we live with earthquakes. The biggest one I've experienced was the 1989 Loma Prieta quake in the San Francisco Bay Area. The power, and cable TV, was out. But I was still able to follow the news live that night on a small, battery-powered TV. By contrast, the telephone lines were so jammed that it took about half an hour to get through to my worried mother on the other side of the country.
From the radio news, though, which I relied on until I got home, it wasn't entirely clear to me what parts of local highways had collapsed, given that I wasn't familiar with Oakland and its stretch of Interstate 880 known as the "cypress structure." I suppose if a road I regularly traveled had collapsed, I would have known its local nickname.
However, as someone who took a full month to figure out that "the Bayshore" referred to California Route 101, I'm not at all confident I would figure it out before I found myself trapped overnight in a mammoth traffic jam (as happened to one of my colleagues) or, worse, driving off the jagged end of a collapsed highway.
This is precisely the scenario that members of the Open Mobile Video Coalition (OMVC) have in mind when they insist that broadcast — over-the-air (OTA) — mobile TV is an essential piece of the United States' national communications technology strategy. Last month, research firm IDC released a new report sponsored by the OMVC, "Assessing the Mobile DTV Opportunity and its Role in the United States' Communications Ecosystem," by Danielle Levitas, highlighting live broadcasting's role in the mobile TV ecosystem.
The United States' broadcasting system delivers emergency information to nearly 100 percent of the public through direct news, weather broadcasts, and the national emergency alert system, IDC reports. Mobile TV is essential for extending emergency information to people on-the-go — a time when other networks are most likely to be overloaded.
Further, leveraging the existing system doesn't require any change in people's habits. Most people in the United States get news primarily from local TV broadcasters already, according to media polling company Nielsen, with more than 1 out of 2 Americans reporting that they tune into local news broadcasts daily. IDC's research backs that up, reporting that live news and emergency information tops the list of content that people are interested in watching on mobile devices.
The IDC paper also reports that, as a group, current netbook owners are most interested in mobile TV (40 percent), while current smartphone owners are most interested in mobile TV as a feature in the next device they purchase.
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Read a Five-Part Primer on HDR Video


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On hematoxylin-eosin staining, microscopic sections showed deep dermal and subcutaneous inflammation composed of a mixed population of neutrophils, eosinophils, lymphocytes, and macrophages. Focal areas of necrosis and a few plasma cells were also present. Periodic acid–Schiff and gram stains were negative for fungi and bacteria. However, a Ziehl-Neelsen acid-fast stain revealed clusters of bacilli within the abscess.
A chest x-ray film showed no evidence of active pulmonary disease. The patient was treated empirically with doxycycline (100 mg twice a day) for atypical mycobacterial infection while awaiting identification of the organism. No improvement was observed over the next 3 weeks; in fact, new lesions continued to develop. Because at this point cultures of the first biopsy specimen demonstrated no growth, a second biopsy specimen was obtained for culture and drug sensitivity testing. The antibiotic regimen was also changed to ciprofloxacin. After 1 month, the bacilli were finally isolated and identified as M chelonae. Drug sensitivity testing indicated resistance to ciprofloxacin but revealed sensitivity to clarithromycin, amikacin, doxycycline, and cefoxitin. The lesions healed after a 3-month course of clarithromycin. Five months after treatment, there was still no sign of recurrent infection.
Nodules on the Arm of a Diabetic Patient—Diagnosis. Arch Dermatol. 2003;139(1):93-98. doi:10.1001/archderm.139.1.93-a
© 2017


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So we all think that Sins is an amazing game. We'd like Sins 2. Post here if you want it.
Why no steam?
Multitude of reasons, some with Steam, some not, but mostly because back in the early days of steam (when Steam Early Access meant that you were beta testing the service itself, not some game), Valve, being the ignorant assholes that they were, couldn't tell the difference between a modder, a hacker/pirate, or an in game cheater and banned everyone all the same, and if you argued and even proved to them they were being overzealous douche bags, rather then reinstating your account or refunding your money, they perma-banned you and kept your money. To this day they give a giant middle finger to their earliest adopters and beta testers who made their service what it has become by refusing to unban those banned on the grounds of things they allowed after releasing to the public.
MPlayer was the best, and not because I was a Sage who routinely abused power for comical reasons with hilarity ensuing, but because when you're drunk at 3am and eating greasy ass tacos from the local 24 hour Taqueria and deciding that a machine gunner in C&C should have a 50 round burst instead of the 5 rounds and doing to a crowd of soldiers what a machine gun would actually do to a crowd of soldiers and finding a match with players whom also were probably drunk and would like to see that exact same thing happen, and no bullshit service telling you that you're banned because everyone playing had the exact same files but they weren't the official ones... To watch mPlayer get absorbed into GameSpy and then see their deformed three way love child with Valve become Steam... It's just very disappointing.
Don't even get me started about their TOS agreement telling you they want and will get full read and write privileges to every file on your hard drive... Because no one has ever exploited a security loop hole like that before...
The question SHOULD be why is the next Sins going to be released 10+ years after the original?
Yes I know.. I own the DLC's, but a sequel is at most a five year turn around, why the incredible time between 1 and 2?
Actually sins came out in 2009.
I only just joined Stardock but I have been playing sins since 08 with the original release. And I have to say that I would absolutely support a second full game. Rebellion is in my opinion, the best RTS currently available and it's a shame that it's as old as it is and that people no longer really play...I've only ever LAN'd and play SP but I would really enjoy being part of any active communities that are still playing, in case anyone see's this . Anyway, I digress, PLEASE FOR THE LOVE OF EVERYTHING MAKE A NEW SINS!
Titans and corvettes ruined a lot of the subtlety in rebellion because they outperform other pure power strategies. Sure it makes the game easier for noobs, but if you want sins 2 to have more replayability, than you really should allow for more subtlety.
The developers of sins (Ironclad) focused on a MOBA, the business sense was that they wanted to diversify(though they should have followed Blizzard's model, IHMO). Also, teams get tired of making the same game over and over, the uninspired atmosphere that can occur really shows in the workmanship and therefore game companies go with different games to mix it up and keep everyone interested.
We will not see a Sins2 until StarControl is released and done selling, Stardock/Ironclad would not want the two of them competing for market space.
So figure late 2018 at the earliest.
In the meantime, I plan to whore myself out to them as a source of free labor.
I'd definitely love to see a new Sins2. I'd like to see a 4th race added to it as well. Specifically I'd love to see the race the Vasari are so terrified of and running from to make their official appearance and a Sins2 game.
Of course I want a Sins 2.
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After lots of ENT drama and one post after another ...
How to teach children about money
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- That's how much they can spend each day.
- If they spend nothing one day, they can spend twice as much the next day.
- The longer they save up, the more they'll be able to spend on something they opcje binarne statystyki really want!
If they spend their pocket money right away, they'll have nothing until the next payday. That's a valuable lesson to learn. It's up to you to be strict about this and resist mid-week calls for a top-up: it's an unfortunate fact of life that real-world employers won't cough up more cash just because you ask them to.
binary option trading does it work Don't underestimate their skills
Nobody knows your kids as well as you do, so don't be put off by a game's 'suitable for ages X and up'. That's just a recommendation. Your kids might be ahead of their peers – or you might find a bit of adult help is all they need to play games aimed at older kids.
Plenty of five-year-olds, for instance, are perfect at home playing Monopoly. It's a great way to teach them about counting, budgeting, haggling, planning ahead – skills they'll need all the time when they're grown up. And you don't have to stick to Classic Monopoly. You'll find Lord of the Rings Monopoly, Star Wars, My Little Pony, Power Rangers, football… Find a subject they're really into and all that learning should come a lot more easily.
come giocare in borsa As they say: they'll thank you when they grow up
When we're young, just about everyone dreams of being rich when they grow up. Learning to budget is no magic formula for success, but it might help your kids go further in life. And it should certainly help them get more out of their income – even if they're not earning millions by the time they're your age.
Disclosure: This is a featured post. For more information, please see my disclosure policy.


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Finland: Latest beverage news & analysis
Latest Insights
Soft Drinks, Beer Set to Lose Out in Finland 21 Jan 2011
Soft drinks companies and brewers are facing the possibility of higher excise duties on their products as a result of tax reforms proposed by the Finnish Ministry of Finance. Ben Cooper reports.
Latest News
Tito's Handmade Vodka secures Finland roll-out 17 Mar 2016
Tito's Handmade Vodka has launched in Finland after owner Fifth Generation secured a distribution deal.
Altia appoints managing director for Cognacs 17 Nov 2015
Altia Group has created a managing director role for its Cognac division. Jérôme Durand has assumed the position at Larsen SAS, following the integration of the Cognac house into the group.
Rum owner blasts Finnish ad laws amid debate 15 Oct 2014
A Finnish rum producer at the centre of a national debate over the country's strict alcohol marketing laws has branded current regulations "ridiculous".
Altia forced to cut jobs to 'secure future' 2 Sep 2014
Altia Corp, the Finland-based wine and spirits firm, has announced plans to cut around a tenth of its workforce as it seeks to to boost its "long-term competitiveness".
Altia overhauls Chill Out wine box 23 May 2014
Altia has upgraded the packaging on its Chill Out wine range to make it easier to recycle.
Altia head calls for funds as officials plan sale 20 May 2014
The acting head of Altia has said the company needs owners that are willing to invest after the Finnish Government drew up plans for the state firm's sale.
Carlsberg fined over tax evasion in Finland 29 Apr 2014
Carlsberg has been fined DKK41m (EUR5.5m) after being found guilty of tax evasion in Finland.
Carlsberg regional head set to lead Altia Corp 11 Mar 2014
Altia Corp has poached its next CEO from Carlsberg to replace the previous incumbent, who left late last year.
Altia Corp CEO severance deal sparks outcry 27 Nov 2013
A political party in Finland's Coalition has demanded that the Government intervene to block the severance package agreed with the recently-released CEO of Altia Corp.
Altia Corporation CEO let go after target miss 21 Nov 2013
The CEO of Altia Corportation has been released with immediate effect after the Finnish group fell behind its financial targets.
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How Does Your Squad Stack Up in Our NBA All-Star Weekend Report Card? | Sports | BET
From the Warriors all the way down to the Nets.
From the Warriors all the way down to the Nets.
Adorable Riley and Ryan have gotten so big.
Will the Clippers' center get to see more of his one-year-old son?
Savannah is A1 perfection in the King's eyes.
We have Melo higher than 15.
Here's what Blake Griffin, CJ McCollum and others had to say.
This couple is setting the bar pretty high.
The NBA star's rhymes may be on the brink of a major move.
Here's the latest shot that he took at the defending champions.


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Greetings Guest!!Dismiss Notice
In order to combat SPAM on the forums, all users are required to have a minimum of 2 posts before they can submit links in any post or thread.
Since I last talked about it...I made 70
Discussion in 'WoW General Discussion' started by Guest, Aug 27, 2007.


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What makes Notion different?
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Set up dashboards, invite your team, and share reports within hours—not months—thanks to our simple integration platform and our data collection workflows.
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Everyone on your team will have the data they need, when they need it. Collect and display custom metrics and team polls with beautiful team and private dashboards.
Capture the story behind your data.
With multiple ways to collaborate on your data, you'll add meaningful context to the numbers. One-click reporting and our Slack integration brings data to any conversation.
Notion is helping teams of all sizes make better decisions.
The end of business intelligence.
Establish a single hub of information so your entire team can track, learn, and grow together.
We call it Team Intelligence.
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Empower a data-driven culture that everyone on your team can use. Enhance your Slack conversations with our charts in one click.
Define Team Goals
Set targets to focus on common goals and iterate more rapidly than ever to make sure you're tracking in the right direction.


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Looking for Website Designer ( 1 yrs)
Lyonzone Technologies
Rourkela, RaurkelaIndustrialship
Web Site Designer Job Duties: - Plans site design by clarifying goals; designing functionality. - Develops site navigation by categorizing content; funneling traffic through content. - Develops site content and graphics by coordinating with copywr...
Key Skills : website designer,html,php web developer,wordpress,joomla,web graphic designer,adobe photoshop,photoshop,css,javascript,flash...
Required Dot Net Developer ( Fresher)
Sharp Skill Technology
RS 1,00,000 To 1,25,000 Rourkela, Sundargarh
As A Developer, You Will Be Responsible For Many Phases Of The Software Development Lifecycle Including Technical Research, Requirements Analysis, High Level And Technical Design, Implementation Of Enhancements To The Product, Unit Testing, Debugging...
Key Skills : dot net developer,dot net programmer,net programmer,software developer


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About this Business
- HoursSun-MonClosedTue-Sat10:00 AM - 7:00 PM
From Our Editors
Rumor has it that Pure Daily Bliss Day Spa's relaxing, full-service salon is great for a visit to Norco by yourself or with your best friends, too. Clear up your clogged pores and blackheads with a replenishing facial. If you're looking to clear up your skin, a skin treatment from this salon is exactly what you need. Keep your energy high and your stress low with a relaxing massage treatment from this salon. This salon requires guests to book appointments in advance, aiming to provide the best services possible. On a budget? Check out this establishment's reasonably-priced spa services and keep your spending to a minimum. Payment is simple and all major credit cards are accepted. When you leave Pure Daily Bliss Day Spa, you will leave with a new style and a relaxed and chipper mood.


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NYCdwellers and New York City Real Estate
NYCdwellers is New York City's largest real estate marketplace specializing in residential no fee apartment rentals in Manhattan. We are dedicated to providing outstanding service to New York apartment hunters from all over the world. Our website is comprised of modern apartment search technology and powered by six, full-time staff providing unsurpassed service to NYCdwellers' customers and listing providers.
What does this mean for you? This means a constantly-updated database offering real New York City apartment listings in real time and direct access to all listing providers. Our mission is to find and list all real estate properties available in Manhattan, by owner or by broker. We screen and hand-select all available NYC rentals. No duplicate or outdated listings, no bait and switch. If you are looking to rent an apartment in Manhattan, this is the place to be.
And if an individual listing is represented as a no fee apartment, there is no broker fee for the apartment. Period.
We present a complete selection of New York City rentals. No one makes shopping for apartments for rent in NYC so effortfree. Hunting for New York City apartment rentals can be tough, if you're going it alone. Let NYC Dwellers be your guide. As you are trying to rent in NYC, we are the complete apartment finder. With us you can virtually preview almost all available Manhattan apartments for rent from your home convenience. The system of NYCDwellers.com provides the easiest way to rent apartments in New York City. In your quest for apartments in New York for rent, you will discover that our information is We are reinventing the way folks rent apartments in NYC. NYC Dwellers is a single destination for everyone looking to rent Manhattan apartments.
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Note : Your Quotation Request will be directed to L-3 Narda-MITEQ.
The PD2-2000/4000-100S from L-3 Narda-MITEQ is a Power Divider with Frequency 2000 to 4000 MHz, Insertion Loss 0.4 dB, Isolation 16 dB, Input Power 100 W, Amplitude Balance 0.5 dB. More details for PD2-2000/4000-100S can be seen below.
10 W, 8 Way Power Divider operating from DC to 6 GHz
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45 Barra Road , 4005
This Comfort Suites is located approximately five miles from Old Orchard Beach and 20 miles from Portland International ...more
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We appreciate your business and wish you a safe and happy trip.


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Well I never thought I would find myself here but I am, I've decided an abortion is the best thing for me to do. I know I will not cope with another child and pregnancy right now. I am worried with hubby away how I will cope after, my mum is taking me and dropping me home but I will have to look after the kids myself, I have friends popping over but for the most of it I will be alone and I am really unsure how I will feel after.
Can anyone tell me what to expect physically?
Also I really can't wait for the morning sickness to go away can anyone tell me how long that will take?


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OurSports Central - INDEPENDENCE, MISSOURI - The Missouri Mavericks 2016 Kelly Cup Playoffs campaign begins this weekend at Silverstein Eye Centers Arena with the Western Conference Quarterfinals, as the #1 Mavs take on the #8 Quad City Mallards on Saturday, Apr. 16 (7:05p.m.) and Sunday, Apr. 17 (4:05p.m.).
Local News Provided in part by:
Tann Electric - Kansas City Electricians
Read the full article link below the following comments:


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"Perhaps the most striking..." Kiev Local Custom Tip by Tolik
Kiev Local Customs: 85 reviews and 116 photos
Perhaps the most striking feature of Scythians was the enormous amount of gold they wore and used. The ancient legend tells the story about 'the one-eyed people, Arimaspians in Scythia who had on-going battle with the griffins who guarded the gold'.
This gold undoubtedly came from the rich fields in the Altai district. It is common that the Scythians wore golden ornaments and belts. Gold plates were sewn to their garments and gold gleamed from their weapons. The archaeologists are consistently amazed by the amount of gold offerings deposited in the great burial- mounds of the Scythian kings. The animal style art of the Scythians is evident in hundreds of artifacts found in 'kurgans'(burial mounds) thousands of which were constructed in burial areas across the steppes of Ukraine and Russia from the Carpathian Mountains into the area of northern Iran and extending all the way to the border of China. According to archaeologists Scythian art burst upon the scene around 700 B.C. Much of what has been excavated is gold jewelry, horse ornaments (often gold or bronze), some weaponry, and some silver and gold vessels. The richness of these artifacts confirms ancient reports that the Scythians who sat on the trade routes of Asia were fabulously wealthy.
The Amazon myth also goes back to Scythians, whose women apparently weren't allowed to marry untill after they had killed in battle.
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Binghamton Senators Play Preseason Game on Saturday
The regular season is almost here. Get a taste of what's ahead for the B-Sens when they play Rochester for the only preseason home game on Saturday.
The regular season is almost here. Get a taste of what's ahead for the B-Sens when they play Rochester for the only preseason home game on Saturday.
10 minutes from start to finish is all that it'll take to make these incredibly delicious Italian steak sandwiches!
Racing in the Southern Tier isn't over yet. It's an American Cup Racer Weekend at Five Mile Point Speedway. Kids 12 and younger admitted free.
Week 5 begins tonight when Arizona travels to San Francisco. It's free to play. Speaking of free, I have a free tip about tonight's game.
It may be the last season for the Senators in Binghamton but it'll be an exciting year. They are bringing back many of our favorite promotions along with some new ones too.
While we are getting the giant inflatable ready for Saturday, here's everything you need to know about the Insane Inflatable 5K.
Week 4 of Pro Football Pick'em begins at 8:25 tonight when Miami travels to Cincinnati. It's free to play and you could be the weekly winner of a $25 or $50 dollar gift card.
We may not know their name but we know when they'll play. The 2017 schedule has been released. They are also asking you to take a fan survey.
2 tied for 1st and a 11 way tie for 3rd. You have until Thursday night to get your pick in. Haven't been playing? That's okay, we have weekly winners and it's free.
Devonta Freeman gained 152 yards on 14 carries and Tevin Coleman ran for three touchdowns, as the Atlanta Falcons routed the Saints, 45-32, in New Orleans on Monday night.
The man was the king of golf and had his own army (Arnie's Army) but he connected with the common man. Arnold Palmer has died at the age of 87.
It's now official. The Senators are leaving Binghamton. Why and what's next. Here's what we know.
Golf lost one of its most iconic figures when Arnold Palmer passed away Sunday at the age of 87.
People may love the NFL, but they sure don't love everyone who plays in it.


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Waist strap accent side incision black long zip-up hoodie
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Highest Rated
hmmm, not that simple... i thought moths killed the man inside the sweatshirt, it looks like it! maybe if they went with a shirt hanging or in a tomb it would have been more obvious
yeah, great. Put the shirt in a tomb. That doesn't seemed forced.
I didn't get this campaign AT ALL until your post.
I was reading the word "eliminate" and wondering to myself "but the clothes are still... there. WHAT?"
The copy isn't strong.
I think the copy is extremely obvious and unoriginal, but it may have lost something in the translation. What I don't get is that it looks like the person is eliminated and the clothes, albeit worse for the wear, are still going strong. Seems like this has been executed backwards.
Yeah this is not very good. I didn't get it until reading the posts. Did, however, like the typography
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text: Seller: thepreservationstation (252) 100%, Location: Nashville, Tennessee, Ships to: US, Item: 282244135489 An understated antique newel post, salvaged from a house in Columbia, Tennessee, and dating from the 19th century. The house was designed by architect Nathan Vaughn and finished in 1823 for Major John Brown II, who named the house "Annoatuck," an Indian word meaning "a windy hill." This turned newel features a slender silhouette with clean lines and a ball newel cap. The newel is in good antique condition with the expected wear (as pictured.) It measures approx. 3-1/2" wide and 62-7/8" tall. There is a coordinating newel available under #NNP164-RW. Item # NNP163 Please email or call us at 615.292.3595 with any questions. This item can be viewed at our showroom in Nashville, Tennessee. Preservation Station 1809 8th Ave S. Nashville, TN 37203 615.292.3595 Normal 0 false false false EN-US X-NONE X-NONE


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Bicycle Pizza Cutter
Date Published: 02/25/2014 5:52 AM
The bicycle pizza cutter is a pizza cutter that is in the shape of a bicycle and would make a great addition to any bicyclist's drawer of utensils that always has too much shit in it which then will always jam when you try to open it. I'm not sure how much pizza a bicyclist eats, but it can't be much, but seeing how you are not a bicyclist and in fact obese, this bike pizza cutter will make a fine addition to your repertoire of pizza cutters that have no significant meaning to your life, much like how you own the circular saw pizza cutter when you have never even used one yourself. The Doiy Bicycle Pizza Cutter is a replica of a fixed-gear bike (or fixie bike), it features two sharp cutting discs (the wheels), and have two different styles to choose from, watermelon (pink and mint), and bumblebee (yellow and black).
Alternate Purchase Links: 1 | 2


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Kids' Electric Ride-On Car£79.99 (from Ebuyer) for a kids' Xenta electric ride-on car - DELIVERY IS INCLUDED!
- Get your kid a Xenta electric ride-on car - suitable for 3-6 year olds.
- Features two 6V motors and stylish red body work.
- Plus forward and reverse functions, an MP3 connection and headlights.
- Can be fully remote controlled.
- Top speed of 3.5kn/h.
- Delivery is included!
The Fine Print
- May buy multiples.
- Voucher valid until 30th May 2014.
- Please allow up to 10 working days for delivery after redeeming.
- Subject to availability.
Full Details
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- Measurements: 118cm x 68cm x 49cm
- Weight: 14.5KG
- Maximum load: 25KG
- Automatic brake
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Hotel Gangi A historic building in the heart of the ancient city, the result of a recent and accurate restoration in the center of Piazza Armerina, near the communal gardens and within walking distance from the typical streets. The hotel has three floors, the rooms comfortable and furnished with a tasteful simplicity and elegance, will make you enjoy your stay in the city, both for your business trip or for your vacation pleasure. Among the areas of 'hotel, located on the ground floor, also furnished with warm, natural tones, you can choose to relax in comfortable lounge, or try the hotels bar. Remark: Additional transfer costs airport to Catania 100,00
- Parking
- 24 hours Front Desk Service
- Bar
- Pets allowed
- Low mobility guests welcome
- Restaurant/cafe
- Laundry service
- Air conditioning
- Hairdryer
- Mini bar
- Safe box
- Shower
- Bathrobes
- Coffee/Tea
Gangi Hotel reviews
Show Gangi Hotel on the map
We suggest that you read Foursquare reviews about Gangi Hotel hotel. Usually, they can include useful information, such as: Wi-Fi prices, staff service quality, room facilities and cuisine quality.
Free WiFi here is supposed to be quite fast. The estimated speed is 5.3 Mbps as of March 16, 2015
- Hotel WiFi Test
- 2015-04-13
- 0 likes


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Fionaalice, thats amazing! Of course you can join us.
My current little miracle is a natural one too after giving up IVF after having no success trying again after Ruby. Truly truly amazing. So happy for you!!
Fionaalice, thats amazing! Of course you can join us.
My current little miracle is a natural one too after giving up IVF after having no success trying again after Ruby. Truly truly amazing. So happy for you!!
fionaalice, what a wonderful story and congratulations! So does that mean you must now give up your luxurious Bali life. Sounds divine.
JFB, LOL re the fundraising box. I remember you being ok in the weight gain department with this pregnancy???... if you're still doing ok, what the heck... enjoy oporto's and boost bars... hardly up there in the hard core junk food stakes.. aaaah, just enjoy them anyway. Swimsuits are for young nubile teenagers. (sorry if I've offended any mum's lucky enough to still have bikini bodies - I envy and admire you!... and secretly hate you... in a nice kinda way)
BK and Nessie, LOL re tops being pulled down.... as if having to expose cellulite isn't frightening enough, but then having to worry about involuntary boob flashing. I'm definately palming swimming off to DH.
MG, how exciting ... I'm getting butterflies just thinking about it. Wow, it's all so close now. Yipeee!
Kismet, sorry, I probably haven't helped you any with my swimming comments. Good on you and I can't wait to hear how the littel fish Evie goes at her first lesson. Just watch your top!
Oh and welcome CCCMum!
AFU, 6 week scan on Monday morning, so will hopefully see my little bubbles heartbeat then. I can cope much better with this wait.
Fionaalice - woooowwwww!! talk about a seachange! A big fat congratulations to you and DH, you so deserve this happiness. And you will fit right in here, I think we all saw your previous journey, I remember you. I love hearing about little unexpected miracles. Are you going to continue to stay in Bali?
JFB - oh I definately get into the boardies in public these days too, as much as I hate them, I don't like exposing my thunder thighs. I spent a fortune on Evie today getting new cossies and hat etc for her from the cancer council, she is SO fair I would hate for her to cop even a tiny bit of sunburn, and particularly after having melonoma myself I'm super paranoid with putting her at risk. I was also getting quite envious looking at all the swimwear around thinking I will never wear anything like that ever again, I'm afraid it will be the old granny looking full piece for me in future, only if my thighs shrink, already have the granny grundies so why not the cossies.
Lol wow I am slow! All I looked at when I first posted here was it's a 35+ thread. Now I see it's in the ivf section. So sorry ladies. I'm going to blame my preggy brain..
No problem cccmum. U r welcome stay, or leave if you feel it necessary!! We are a welcoming bunch!!
Fiona Alice - welcome to this thread. Looking forward to getting to know you. I love natural miracle stories. Just when you have given up hope, the universe throws a curve ball in the loveliest way.
Sent from my iPhone using Bub Hub
MG: i saw u had posted and thought there was action!!!!
fiona: congratulations, what a great story!!! you are definately welcome. i bet it didnt seem real at first! it took a long time to sink in this time for me and that was with 2 attempts, with DS i felt very different when i got UTD...it was pure relief, this time it was a very weird type of "oh right, so what happened to the chick who thought she'd never have a kid...gone??"..weird. i have to ask.....when TTC did u ever get the comment "oh just relax and go on a holiday" ...you and DH must be over the moon . you can never undo the effect that multiple ivf's/ infertility had on you tho i have found....regardless of how "easy" later BFP's arrive.
oh dear...when will i learn..my gutz havent coped well this pg with takeaway/ rich stuff....this is about 3rd time ive ended up with post junk stomach cramps/ semi runs (i dont do dirreohea ole iron gutz here )
Nessie. I'm carrying the same as with ruby but a little lower (old flab hadn't really springed back) and I'm bigger. If that's possible! Still a big round ball though. Had a scan today and baby boy is measuring 2 to 3 weeks bigger!!! No wonder I'm so huge
Thanks ladies!!
It is still sinking in as a reality ... still spinning out a little everyday that it IS happening! Feeling hideously sick most of the time is a pretty solid reminder though Trying not to complain too much about it as it's a good sign I guess!
While we were doing IVF and also when we made the decision to stop soooooooo many people said something along he lines of 'as soon as you stop trying it will happen' etc etc and it used to make me so f'n mad ... you know, when you are trying so hard!! Anyway now I have to say they might have been right!! I put it down to warm weather, no stress and good food (and maybe some good sex )
But yeah the worry is there .... trying to still take it one day at a time ...
I am coming home from Bali in 2 weeks, touch base with work and see docs back home, leaving hubby here in Bali and then will return Xmas eve. We'll both return to Melbourne at the end of Jan ... and I guess start planning for the future!!
Thanks for sharing my excitement
kel: boys are bigger...i too have a spare tyre...i definately buying one of those ab king pro things.........i;ll be spinning from here to texas for months............
JFB: I dont think the best ab king pro in the world is gonna help me this time. do you think the surgeon can snip and bit more off whilst doing the c-section? I cant remember if you've said, are you going to find out sex this time??
Pregnant for the first-time?
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Do you dream of starting your own business but fear giving up a stable paycheck?
Rather than focusing on that fear, consider how your life will change when you are doing something that you truly enjoy.
No matter what you choose to do in life, there will be pros and cons. When you focus on the risks of losing your regular source of income, you distract yourself from the benefits of pursuing your dream.
Here are eight "pros" of becoming an entrepreneur:
The post 8 Reasons To Become An Entrepreneur (Infographic) appeared first on Liberty Investor™.


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Quick Look
Property brokered by
Get more listing details on brokerage site
Presented By:
Asia Pacific Properties
+852 2281 7800
Email Agent
Asia Pacific Properties
+852 2281 7800
Email Agent
Property DetailsQuiet corner house in a private gate security guarded. Spacious living, bedrooms, familiy rooms throughout this split level home.
Area Detailed Information
Area Weather
Area Cost Index
Area Property Prices
Additional Property InformationThis rental listing located at Yue Hei Yuen, Hong Kong offers four bedrooms, three bathrooms and one partial bathroom. The property is presently priced at $38,7M (38K/mo,).
Additional features include house of 4307 sq.ft. with 4 bedrooms, 3 full bathrooms, 1 study, 1 powder room, 1 maids room, 1 balcony, 2 terraces and 2 covered car parks included. house includes garage.the house is partially furnished. .
This property is presented by Asia Pacific Properties of Asia Pacific Properties headquartered in China.


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Spirit Stones. iOS
- Publisher: GAMEVIL Inc.
- Release Date: Oct 21, 2013
Summary: Discover the unique Puzzle RPG with a TCG twist!Download now for free and connect with thousands of players around the world!• EASY TO PLAY. DIFFICULT TO MASTER •- Simply SWIPE & MATCH tiles of the same color to engage in battle!- Unleash devastating attacks by planning strategic combos!• BUILD THE ULTIMATE TEAM •- Collect unique, powerful Heroes & assemble the finest team!- Level-up & evolve your cards for maximum power!- Each card comes equipped with beautifully illustrated artwork!• INTENSE GLOBAL PVP •- Battle the best players around the world each week for generous rewards!- Compete against friends and climb the weekly rankings!• RAID THE HELLGATE WITH FRIENDS •- Put your skills to the test by entering Hellgate!- Play with friends and dive to new depths!Join the community at www.facebook.com/PlaySpiritStonesNEWS & EVENTSWebsite http://www.gamevil.com Facebook http://facebook.com/gamevil Twitter http://twitter.com/gamevil YouTube http://youtube.com/gamevil
|GAMEVIL Inc.
Awards & Rankings
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- Home
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- Play Therapy Credentials
- Why Play Therapy?
|Mariellen Griffith, Mariellen Griffith, EdD, LMFT, RPT-S
6 hours agoPlay therapy helps children cope, grow
2/13/2017Therapists see play as a way for children to overcome trauma
2/9/2017What is Play Therapy?
2/9/2017The Power of Play


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Horizontal concrete mixers
Found: 7 products← Back to category "Concrete and grout mixers"
Concrete mixer of B 125
In stock
Price:133.15 EUR
The concrete mixer (concrete mixer) — one of the most demanded types of construction equipment: no building, irrespective of the sizes and assignment Read more
Verified partner for over 2 years
Premium Business
Delivery fromKazakhstan
This company was at the resource today
Display phones
Kovrolinovy rugs of Harmonia 336564
In stock
Price:1397.31 EUR
Kovrolinovy rugs of Harmonia 336564 Read more
Verified partner for over 8 years
Premium Business
Delivery fromUkraine
This company received an enquiry 4 day ago
Display phones
Site: malvast.com.ua
SBR-500A.1 concrete mixer
In stock
The concrete mixer with the electric drive - SBR-500A.1. It is equipped with the pig-iron wreath allowing to increase as much as possible life cycle. Read more
Verified partner for over 2 years
Premium Business
Delivery fromKazakhstan
Display phones
Site: mekenoil.kz
Betonosmesitel RBP-750, RBP-1000 gorizotalny for rigid mixes
In stock
Betonosmesitel horizontal is intended to RBP-750, RBP-1000 for preparation of rigid and plastic moist construction mixes. Betonosmesitel prepares for Read more
Delivery fromUkraine
Betonosmesiteli horizontal, Бетоносмесители,бетономешалки,бетономешалка
In stock
Are intended to Betonosmesiteli for preparation of mobile concrete mixes, construction solutions, and also their dry components. A linear row consists Read more
Delivery fromKazakhstan
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Suppliers will contact you
For last 30 days 108455 leads received the offers from the companies
Trading platform Allbiz offers to see the catalog Horizontal concrete mixers which contains 7 products offers from 3 companies and enterprises. Don't know what Horizontal concrete mixers to buy? You can clarify the specifications, see photos Horizontal concrete mixers and choose the best seller and supplier. Buy Horizontal concrete mixers trough online ! On Allbiz — You can just place your order right on the site.


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Data Deluge
Researchers Turn to Cloud Computing as Genomic Sequencing Data Threatens to Overwhelm Traditional IT Systems
Seeking Young Scientists for Research in Space
Sounds of Science Podcasts
Infectious Fight
Tackling the Inevitability of Resistance One Genome at a Time
Long Noncoding RNAs: Clarity or Confusion?
Because They Are Such Elusive Prey, lncRNAs Have Yet to Emerge as Therapeutic Targets
Charles River Pays WuXi $30M to Renege on $1.6B Acquisition Deal
CRO Charles River Laboratories is bowing to investor opposition by pulling out of its proposed $1.6 billion acquisition of rival firm WuXi PharmaTech. Severing the deal means Charles River will have to pay WuXi $30 million in break-up fees, but the company said it was not willing to go ahead with the merger without full investor support. The Charles River board has now authorized a $500 million stock repurchase plan.
Pulling out of the deal was the only option given the concerns of major shareholders, states James C. Foster, Charles River chairman, president and CEO. However, he adds, "we believed that this transaction, which would have created the premier early-stage contract research organization, would have resulted in long-term strategic benefits for our business and our shareholders."
Charles River claims that the acquisition of WuXi would have given it an immediate presence in China, bolstered its discovery and early development expertise, and created a combined entity capable of offering fully integrated research and drug development services from molecule creation to first-in-human testing.
The firm maintains ditching the merger has not changed its overall strategy, though. It plans to strengthen its existing capabilities in discovery services and early development . "We will enhance our portfolio of essential products and services, deepen our scientific expertise, and maintain our standards of exceptional client service," Foster stresses. "The board's action to authorize a new stock repurchase plan at this time reflects both its belief that our stock price is substantially undervalued and also its faith in Charles River's future prospects. Repurchasing our stock is another means of enhancing earnings growth and improving stockholder value."
The firm has cancelled a prior $600 million stock repurchase authorization, under which there was a remaining balance of some $145 million.
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An agreement paper between Himachal Pradesh CM Virbhadra Singh and insurance agent Anand Chauhan indicates that Singh may have fudged records to legitimise his illegal income.
The date of the agreement is June 15, 2008, but the stamp paper, on which the agreement was executed, may have been purchased after September 2015 when the CBI filed a case against Singh.
The entries in the stamp vendor's record show that the particular stamp paper was bought by someone called Laik Ram of Kotkhai tehsil in Shimla district but that entry was overwritten with Chauhan's name. Both the papers are entered in the vendor's record as 1284. A copy of the stamp papers and the vendor's record is with HT.
"The Enforcement Directorate (ED) has recovered a copy of this particular stamp paper along with a statement from Laik Ram," sources said. Based on the evidence, the ED attached Singh's assets on March 23. The case against the CM and his family is that he has amassed `6 crore from 2009-12 during his stint as the Union minister of steel.
The memorandum of understanding (MoU) says Chauhan would manage the CM's orchid in Shimla for a commission and also invest the earnings for three years. But this agreement may have been done to create a source for the CM's unaccounted income.
Repeated attempts to reach the CM's office yielded no response.


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Greetings Guest!!Dismiss Notice
In order to combat SPAM on the forums, all users are required to have a minimum of 2 posts before they can submit links in any post or thread.
EA&E - This wednesday after the auction - Scavenger Hunt!
Discussion in 'UO Europa' started by Alcestis, Nov 23, 2008.


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Reiki Animal Healing
Alternative Medicine, Natural Therapy, Animal Health,Pet Health,Pet Care,Pet and Pet Services,Veterinary Services,Pet Clinic
Reiki Animal Healing now offers remote healing so your pet doesn't even have to leave home - no more stressful trips to the vet! If you can supply a good front face image of your pet and a description of its condition or problem, then we can perform a remote Reiki healing on it for you.
To learn more about how remote Reiki Animal healing can help your pet, visit our website at http://www.reikianimalhealing.com or call us on 07 5574 7777.
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Reiki Animal Healing is an effective alternative to vet treatment for animals with chronic conditions, illness or injuries.
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Domestic Energy Applications
Water systems for home & garden
The MAGNATION Next Generation Home System safely and effectively improves the ability of water to dissolve waste and other compounds. This improved dissolvability means your home requires less energy and less chemicals to support your daily activities. You also spend less money restoring your plumbing.
By Magnation Water Technologies - Magnation Corporation based in Oakland, CALIFORNIA (USA).
High brightness LED (HBLED) for energy/environment sector
By Oxford Instruments plc based in Abingdon, UNITED KINGDOM.
Pulsar noise testing equipment for construction industry
The construction sector and its wide range of associated activities are very varied and from a noise monitoring perspective offer many unique challenges. However, Pulsar Instruments have a solution; a construction Pulsar noise meter with the ability to effectively assess the following two key categories; Noise at Work (potential for noise induced hearing loss due to long term exposure to noisy activities). Environmental Noise (the potential to cause annoyance to surrounding areas or buildings during site work).
By Pulsar Instruments plc based in Filey, UNITED KINGDOM.
Need help finding the right suppliers? Try XPRT Sourcing. Let the XPRTs do the work for you


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The Lugger Hotel, Truro
- Free breakfast, free WiFi, and free parking
Main amenities
- 22 guestrooms
- Restaurant
- Spa services
- Terrace
- Daily housekeeping
- Wedding services
- Luggage storage
- Newspapers in lobby (surcharge)
Feel at home
- Refrigerator
- Private bathroom
- Television
- Free toiletries
- Terrace
- Daily housekeeping
What's around
- In Portloe
- Poppy Cottage Garden (4.1 mi / 6.6 km)
- Caerhays Castle (5.5 mi / 8.9 km)
- Porthluney Cove (5.5 mi / 8.9 km)
- Lost Gardens of Heligan (8.1 mi / 13 km)
- Dodman Point (8.4 mi / 13.6 km)
The Lugger Hotel, Trurofrom $123
- Standard Double or Twin Room
- Deluxe Room
- Cottage (Middle)
- Superior
- Deluxe Double Room, 1 King Bed, Terrace
More great choices
At a glance
Key facts
Hotel size
- This hotel has 22 rooms
- This hotel is arranged over 3 floors
- Check-in time 2 PM-11 PM
- Check-out time is 11 AM
Required at check in
- Credit card or cash deposit required
- Government-issued photo ID required
Travelling with others
- No cribs (infant beds)
- Pets not allowed (service animals welcome)
- Free WiFi in public areas
- Free WiFi in rooms
- Free self parking
Nearby Four Star Truro hotels
In the hotel
- Free breakfast
- Restaurant
- Room service (during limited hours)
- Spa services on site
- Luggage storage
- Wedding services
- Number of buildings/towers - 3
- Terrace
In the room
- Bathrobes
- Iron/ironing board
- Premium bedding
- Private bathroom
- Shower/tub combination
- Free toiletries
- Digital TV channels
- Desk
- Free WiFi
- Refrigerator
- Free bottled water
- Daily housekeeping
Top Truro hotels
Recently reviewed hotels in Truro
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"This is a charming hotel with spectacular views over the historical harbor. We had a beautiful view room with an open deck and lovely furnishings. The breakfast was very good and…"Oct 28, 2016
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"I can't say enough good things about my stay at the Tudor Court Hotel. The room was very clean and well equipped. The Wi-Fi worked very well and never dropped a connection. The…"Oct 27, 2016
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"Stayed 2 nights at this B&B while visiting the St Austell area. Owner/Host Ian was friendly and helpful in every way. Wonderful breakfast atrium looking out on beautiful garden,…"Oct 23, 2016
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"A lovely hotel - I have stayed three times and will keep coming back. The small pool is warm and inviting, and the sauna is great after a brisk swim in the sea. English hot…"Oct 18, 2016
Also popular with travelers from the USA
Special features
Awards and affiliations
Green / Sustainable Property
This property participates in Green Tourism Program, a program that measures the property's impact on one or more of the following: environment, community, cultural-heritage, the local economy.
The Lugger Hotel, Truro's small print
Also known as
- Lugger Hotel
- Lugger Hotel Truro
- Lugger Truro
Optional extras
Pets are allowed for an extra charge
We have included all charges provided to us by this hotel. However, charges can vary, for example, based on length of stay or the room you book.
Recent reviews
Outstanding 4.6 /5 from 98 reviews
Room on the website was not the same as the actual room. Then when I complained, I got an upgrade.
Beautiful view if the sea just out our window. Great staff, very secluded area. A challenge getting to it down winding narrow streets but worth the surprise at the end when you reach the hotel.
Wow..when can I move in ?
Totally amazing place - awesome location and we were treated so well with the room being terribly comfy. Delicious food all inclusive. Macaroons and flapjacks left for us in our room for tea. And evening drink of hot chocolate and marshmallows a great touch in our rooms.
The Lugger was totally amazing. Awesome location, delicious Food and good choice for breakfast. Nothing was too much trouble. Our dogs were also very welcome. It was a nice touch to have a fresh milk jug ever day in the fridge in our room. And to have a afternoon macaroons and flap jacks in our room for afternoon tea. And to be given a hot chocolate mix for our evening night cap! Highly recommend.
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text:
Netherlands to Regulate Online Gaming After Historic Vote
- 4
- July 10th, 2016
- By Natalie Stephanopoulos
This particular bill has also taken steps to safeguard its latest amendment, by prohibiting lotteries and land-based operators already in existence from flooding their current player databases with promotions of online contributions.
As a result of this major move, the door will now be opened to online gaming firms, which have now received the green light after spending many years at a halt.
According to Jasper Hoekert, co-founder of Revenue Engineers, "We are absolutely delighted to see the Dutch Remote Gaming Bill finally pass through parliament."
Hoekert added, "While there are some aspects of the legislation which are not ideal, this is still a very workable framework and I am confident that the Netherlands will quickly emerge as one of Europe's most important gaming jurisdictions. For any operators or providers looking to make the most of this new opportunity, it is important to act quickly and seek local expertise."
Revenue Engineers, a company which integrates marketing and business strategy with technology innovation services, is made up of a team of 16 gaming experts, based in Amsterdam. In fact, it has already put into motion business efforts with some of the Netherlands' local and international partners.
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ReadyClickAndGo Private Day Trips
day tours, ReadyClickAndGo, agra day trip,
Meet your car and driver at your hotel for your Agra day trip from Delhi. Transfer to the train station to take the early morning express train at 6am. The train journey takes 2 hours, you have a first-class seat and breakfast is served on board. On arrival in Agra, meet your English-speaking local guide and private air-conditioned vehicle. Admire the world-famous Taj Mahal, the exquisite white marble monument built by a mighty Mughal emperor for his lost wife as a symbol of his enduring love for her. Afterwards, visit the so-called 'baby taj', then drive across the river to Agra's impressive Fort, one of the greatest forts in India with gateways, battlements, pavilions, mosques and halls, and a fascinating, often bloodthirsty history.
Continue to the deserted 'ghost' city of Fatehpur Sikri, around an hour away, then return to Delhi by train at 8.30pm from Agra with a hot meal served on board. Please note the Taj Mahal is closed on Fridays
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Comprehensive tour of St Petersburg highlights with visits to the Fortress and Cathedral of SS Peter and Paul, and the Church on Spilled Blood.
Private day trip to Mostar from Dubrovnik to see its famous bridge and the picturesque village of Pocitelj, both UNESCO World Heritage Sites.
Full-day private day tour from Zadar to Plitvice Lakes National Park, with your own car and driver.
Private one day tour from Beijing to Great Wall of China at Mutianyu, one of the more picturesque and undisturbed parts of the wall, and to the gardens of the Summer Palace.
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R.I.P.D. Image Gallery 285691
R.I.P.D. > Image Gallery
Hugh Jackman says Logan will not appear in a Deadpool movie
Matt Reeves officially directing The Batman
Rob Zombie lending his voice to Guardians 2?
Starz American Gods set to debut in April
Update: Drew Goddard on Deadpool 2? Carnahan on X-Force
Trailer for Michelle Rodriguez's gender swap actioner Tomboy
Top 10 Worst Movie Diseases You Wouldn't Want to Catch (video)
Joe Carnahan says Uncharted has craziest action he's written
See the prologue to Ridley Scott's Alien: Covenant!
TV Review: Legion - Season 1, Episode 3 - "Chapter 3"
TV Review: Arrow - Season 5 Episode 14 "The Sin-Eater"& more
Natalie Dormer is the HOTM for February 2017 (video)
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Lyrics Depot is your source of lyrics to Into Your Maddness by Ikon. Please check back for more Ikon lyrics.
Into Your Maddness Lyrics
Artist: Ikon
Album: Flowers for the Gathering
There is a world of pain and sorrow
You turned away, away from reason
Desperation it settles in
You loose yourself
You loose yourself
I've lost the battle within
Into the shadows of sin
There is a demon you have set it free
It is deep within
You try not to follow
You have witnessed
Now you are lost
Totally you are lost
Totally you are lost
What do you want me to be
From: Flowers For The Gathering
by Chris McCarter & Michael Carrodus
Ikon Lyrics
Ikon Flowers for the Gathering Lyrics
More Ikon Music Lyrics:
Ikon - Beginning of the End Lyrics
Ikon - Camouflage Heart Lyrics
Ikon - Hallowed Ground Lyrics
Ikon - I've Been Lyrics
Ikon - Life Without End Lyrics
Ikon - Stranger I've Become Lyrics
Ikon - Suicide Lyrics
Ikon - Wheels in Motion Lyrics


text:
- Using The Library
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Library Commission
Members of the Camden County Library Commission are appointed by the Board of Chosen Freeholders. The Commission is responsible for determining the policies that govern the operation and programs of the Library.
You can reach Camden County Library Commissioners through the Library's Administrative Office at (856) 772-1636 x7339.
Patrick A. Abusi, President
Robert F. Weil
Joyce W. Ellis
James J. Clarke
Jennie A. Owens