text: Ryan Watson Lighting Design and Effects – a genius for creating just the right visuals A bit about Ryan Ryan has surpassed all expectations in his role as lighting designer. As Kirk's son we happy to let him 'do the lights' in the early days, however, he constantly strives to enhance the show, introducing such aspects as 'live cam', various intros and promo films (see the 6 of the best video, its quite amazing!) and has been a major influence in our ongoing success. The humblest person you'd ever expect to meet, yet a genius for a Genesis light show! Presenting the Firth of FifthThe light show is a rich tapestry of colour and imagery adding a beautify canvas for the intricacies of the music and the drama of the costumes. This drew a wonderfully eloquent comment from an audience member at our Charterhouse 2017 gig clearly moved by his experience: "An assault on all the senses (but in a good way!)"


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Dictionary of National Biography, 1885-1900/Sanderson, William
SANDERSON, Sir WILLIAM (1586?–1676), historian, born about 1586, is said to have been the son of Nicholas Sanderson, first viscount Castleton in the peerage of Ireland (Chester, Westminster Registers, p. 189); but this seems to be an error, as the Sir William Sanderson who was son of Viscount Castleton died in 1648 (Cal. of Compounders, p. 2790). Sanderson was secretary to Henry Rich, earl of Holland [q. v.], when Holland was chancellor of the university of Cambridge (Wood, Athenæ, iii. 565; Autobiography of Sir Simonds D'Ewes, ii. 68). James Howell describes him as being from his youth bred up at court, and 'employed in many negotiations of good consequence both at home and abroad' ('Address' prefixed to Sanderson's Life of Charles I). He suffered in the cause of Charles I, and was made a gentleman of the privy chamber by Charles II and knighted. Holland had made him a grant of the Paddock Walk, Windsor Park, which was confirmed at the Restoration (Cal. State Papers, Dom. 1660–1 p. 242, 1671 pp. 348, 500). On 7 June 1671 a pension of 200l. per annum was granted to Sanderson and his wife jointly (ib. 1671, p. 304). He died 15 July 1676, aged ninety, according to his epitaph, and was buried in Westminster Abbey (Chester, p. 189; Dart, Westmonasterium, ii. 125). Evelyn attended his funeral and describes him as 'author of two large but mean histories and husband to the mother of the maids' (Diary, ii. 320, ed. Wheatley).
Sanderson married, about 1626, Bridget, daughter of Sir Edward Tyrrell, baronet, of Thornton, Buckinghamshire; she was mother of the maids of honour to Catherine of Braganza, died on 17 Jan. 1682, and was buried in Westminster Abbey (Lipscomb, Buckinghamshire, i. 352; Dart, ii. 125; Luttrell, Diary, i. 159).
Sanderson was author of three historical works: 1. 'Aulicus Coquinariæ, or a Vindication in Answer to a Pamphlet entitled "The Court and Character of King James,"' 1650, 12mo. This was an answer to the posthumous book of Sir Anthony Weldon, and has been sometimes attributed to Heylyn. Sanderson claims the authorship in the preface to his 'History of James I.' 2. 'A Compleat History of the Lives and Reigns of Mary, Queen of Scotland, and her son James,' 1656, fol. In the preface to the second part Sanderson observes: 'For myself, having lived long time in court, and employed (till my grey hairs) more in business than in books; far unworthy, I humbly confess, to have any hand to the helm, yet I cabined near the steerage, and so might the more readily run the compass of the ship's way.' A few anecdotes attest his acquaintance with the life of the court. 3. 'A Complete History of the Life and Reign of King Charles from his Cradle to his Grave,' 1658, folio, with a portrait of the author, 'ætat. suæ 68.' This is a compilation quoting freely from newspapers, speeches, manifestos, and the 'Eikon Basilike;' it is frequently inaccurate and of little original value. Sanderson devoted much space to answering L'Estrange's 'History of Charles I' and Heylyn's observations upon it. This involved him in a controversy with Heylyn, who published, early in 1658, 'Respondet Petrus, or the Answer of Peter Heylyn, D.D., to Dr. Bernard's Book entitled "The Judgment of the late Primate of Ireland," to which is added an Appendix in Answer to certain Passages in Mr. Sanderson's "History of the Life and Reign of King Charles."' Pages 139–57 are devoted to disproving Sanderson, and in particular to refuting his account of the passing of the Attainder Bill against Strafford. Sanderson replied in 'Post Haste, a Reply to Dr. Peter Heylyn's Appendix' (25 June 1658). Heylyn rejoined in his 'Examen Historicum,' 8vo, 1659, over two hundred pages of which consist of a searching criticism of Sanderson's historical works. Sanderson's defence, entitled 'Peter Pursued,' closed the controversy (4to, 1658–9).
His references to Ralegh in the 'Life of James I,' involved Sanderson in a controversy with Carew Ralegh [q. v.], who attacked him in 'Observations upon a Book entitled "A Complete History, &c." by a Lover of Truth,' 4to, 1656 [see under Ralegh, Sir Walter]. Sanderson published in reply 'An Answer to a scurrilous Pamphlet entitled "Observations upon a Complete History of Mary Queen of Scotland and her son James," 4to, 1656.
Sanderson's only other published work was 'Graphice: the Use of the Pen and Pencil, or the most excellent Art of Painting,' folio, 1658, which contains a considerable amount of information on the history of that art in England (see Brydges, British Bibliographer, iv. 226–8). A portrait, engraved by W. Faithorne after G. Zoust, is prefixed (Bromley).[Wood's Athenæ Oxonienses, ed. Bliss, iii. 565; authorities cited.]

subset: commoncrawl


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Mammalian Bre1 complexes (BRE1A/B (RNF20/40) in humans and Bre1a/b (Rnf20/40) in mice) function similarly to their yeast homolog Bre1 as ubiquitin ligases in monoubiquitination of histone H2B. This ubiquitination facilitates methylation of histone H3 at K4 and K79, and accounts for the roles of Bre1 and its homologs in transcriptional regulation. Recent studies by others suggested that Bre1 acts as a tumor suppressor, augmenting expression of select tumor suppressor genes and suppressing select oncogenes. In this study, we present an additional mechanism of tumor suppression by Bre1 through maintenance of genomic stability. We track the evolution of genomic instability in Bre1-deficient cells from replication-associated double-strand breaks (DSB) to specific genomic rearrangements that explain a rapid increase in DNA content and trigger breakage–fusion–bridge cycles. We show that aberrant RNA–DNA structures (R-loops) constitute a significant source of DSBs in Bre1-deficient cells. Combined with a previously reported defect in homologous recombination, generation of R-loops is a likely initiator of replication stress and genomic instability in Bre1-deficient cells. We propose that genomic instability triggered by Bre1 deficiency may be an important early step that precedes acquisition of an invasive phenotype, as we find decreased levels of BRE1A/B and dimethylated H3K79 in testicular seminoma and in the premalignant lesion in situ carcinoma. Cancer Res; 72(8); 2111–9. ©2012 AACR.
Histone modifications are central in regulating basic processes including transcription, gene silencing, differentiation, cell-cycle progression, and DNA repair (1–4). Among modifications of specific histone residues, much attention has focused in the last decade on a distinctive pathway, mediated by the E3 ligase Bre1, which monoubiquitinates histone H2B at lysine 123 (H2BK123) in budding yeast and at lysine 120 (H2BK120) in humans. H2B monoubiquitination plays an important role in regulation of transcription, being a prerequisite for normal levels of methylation of histone H3 residues K4 and K79 (5–8). In addition, studies in Saccharomyces showed that mutants deficient in H2B monoubiquitination are radiation sensitive and defective in recombinational repair, cell-cycle checkpoints, gene silencing, and meiosis (see ref. 9 for review). Several recent publications on the mammalian homolog of Bre1 (RNF20/RNF40) suggest that mammalian Bre1 complex plays similar roles. We have reported that loss of Bre1 in mouse cells compromised homologous recombination (HR) repair, resulting in reduced recruitment of Rad51 to sites of radiation-induced double-strand breaks (DSB) and increased sensitivity to ionizing radiation and DNA cross-linking agents (10). A subsequent study by others concluded that human Bre1 (RNF20, or BRE1A) was involved in chromatin reorganization, facilitating HR protein access to damaged DNA (11). This role of Bre1 seems to be important for response to DSBs in general, as a recent study has shown that BRE1 (RNF20/40)-mediated H2B ubiquitination is induced in an ATM-dependent manner and is essential for timely repair of DSBs (12). These findings place Bre1 at the forefront of the DNA damage response, which serves as a barrier against cancer formation (13), and reduction in Bre1 levels would be expected to compromise stability of the genome.
Given that Bre1 affects multiple functions involved in genome maintenance, it is plausible that Bre1 homologs have a major tumor-suppressing role in higher organisms. Consistent with this, the RNF20 promoter has been found to be hypermethylated in breast cancers (14). Shema and colleagues (14) showed that BRE1A restrained transcription of several proto-oncogenes, including c-myc and c-Fos, and augmented expression of tumor suppressor genes, notably p53, and further concluded that selective transcriptional regulation of proto-oncogenes and tumor suppressors constituted the basis for the tumor-suppressing activity of BRE1 (RNF20). According to this hypothesis, one consequence of the loss of Bre1 would be an increased proliferation resulting from upregulation of growth-promoting oncogenes. However, this conclusion seems to be at odds with our current observation of impaired growth upon depletion of Bre1 (RNF20/40). Also, the transcriptional regulation of select oncogenes and tumor suppressors does not seem to be universal across species, as in our study we detect no reduction of p53 mRNA levels after knockdown of Bre1 (Rnf20 and/or Rnf40) in mouse cells. Thus, additional mechanisms seem to be at play in Bre1-depleted cells driving their transition to a malignant phenotype.
Here, we show that Bre1 acts as an important suppressor of genomic instability. We used RNA interference (RNAi) to deplete Bre1 from mouse cells and then followed the evolution of genomic instability in Bre1-deficient cells from early replication stress to specific genomic rearrangements, which in turn triggered breakage–fusion–bridge cycles, known to accelerate genomic instability. We show that R-loops, the RNA–DNA hybrids that result from the extended pairing of nascent mRNA with the DNA template behind the elongating RNA polymerase II, are a significant contributor to the genomic instability phenotype in the Bre1-depleted cells. Although Bre1 homologs are highly expressed in mouse and human testis, we find reduced BRE1A and BRE1B levels in testicular cancer and the nonmalignant lesion in situ carcinoma. Collectively, our data show that Bre1 deficiency promotes genomic instability, which may be an early step in carcinogenesis preceding the acquisition of an invasive phenotype.
Materials and Methods
Cell lines
Mouse fibrosarcoma RIF-1 cells were obtained from Dr. M. J. Dorie (J. M. Brown laboratory, Stanford University, Stanford, CA; ref. 15) and mouse embryonic stem (MES) cells were obtained from Dr. J. P. Murnane (University of California San Francisco, San Francisco, CA; ref. 16) in 2005. As the cell lines were obtained directly from the source of origin, the authenticity of cell lines was not tested.
RNA interference
For downregulation of Bre1a and Bre1b by RNAi, we used a lentiviral RNAi system based on the BLOCK-iT system (Invitrogen) modified by Dr. E. Campeau (17).
Quantitative RT-PCR
Quantitative real-time PCR (qRT-PCR) analysis was done as described in Razorenova and colleagues (18). Human or mouse TBP was used for normalization of cDNA input. The amplification was specific as judged by melting temperature analysis. The experiments were conducted in triplicate and repeated at least twice. Primer sequences are available upon request.
Visualization of micronuclei and anaphase bridges
The assay was done as described in Chernikova and colleagues (19). In some cases (Fig. 2B, Supplementary Fig. S3B and S3C), cytochalasin B was not added to allow better visualization of anaphase bridges.
Rad51 foci detection
The assay was done as described in Chernikova and colleagues (10).
RNase H1 transfection
GFP-RNase H1 plasmid was obtained from Dr. Olivier Sordet (Cancer Research Center of Toulouse, France), and transfection carried out using procedures similar to described in Sordet and colleagues (20) and in Supplementary Information.
More detailed Materials and Methods can be found in Supplementary Information.
Bre1a/Bre1b-depletion impairs cell growth
To investigate the effects of loss of function of the Bre1a/Bre1b complex in mouse cells, we created lentivirus-based short hairpin RNA (shRNA) constructs targeting either of the 2 subunits of the complex. As a control, we used an shRNA construct targeting GFP. Expression of Bre1a and Bre1b shRNAs in mouse fibrosarcoma RIF-1 cells resulted in a significant reduction of corresponding mRNA transcript levels after 7 days of antibiotic selection, as shown by qRT-PCR; Fig. 1A). Protein levels of Bre1a and Bre1b were also reduced (Fig. 1B). Importantly, the levels of Bre1b protein were decreased in the knockdown of Bre1a (Fig. 1B, top panel), whereas the levels of Bre1b mRNA were not affected by the knockdown of Bre1a (Fig. 1A), suggesting that stability of Bre1b depends on the formation of Bre1a/Bre1b heterodimer.
In agreement with previous studies (21), we showed that downregulation of either Bre1a or Bre1b leads to reduction in ubiquitination of H2B (Fig. 1C) and subsequent reduction of uH2B-dependent methylation of H3K79 and H3K4 (Fig. 1B). The reduction in H3K4me3 was evident in all knockdowns but was substantially less pronounced than the reduction in H3K79me2 (Fig. 1B), which closely followed the reduction in Bre1a or Bre1b levels in all experiments.
Knockdown of Bre1a or Bre1b significantly impaired cell growth in different mouse cell types including RIF-1, C3H 10T1/2, and MES cells (Fig. 1D and Supplementary Fig. S1A), whereas cells grew well after knockdown of either GFP, or tumor suppressor Rb (Supplementary Fig. S1A). To monitor the effects of disruption of the Bre1a/Bre1b complex over time, we created RIF-1–derived cell lines, in which the knockdown of Bre1a or Bre1b could be initiated by addition of doxycycline. A doxycycline-inducible GFP shRNA system was used as a control. A detailed description of the Bre1 shRNA-inducible system is presented in the Supplementary Materials and Methods and in Supplementary Fig. S1B and S1C. Note that the Bre1b shRNA–inducible cell line has a slightly reduced level of Bre1b, even in the absence of doxycycline, compared with the control cell line with GFP shRNA (Supplementary Fig. S1C, lanes 1 and 4, respectively), indicating that the doxycycline-inducible system is leaky and a partial knockdown of Bre1b occurs even when doxycycline is not present. Similarly to cells with the constitutive Bre1b knockdown (Fig. 1B), the inducible Bre1b shRNA cell line showed significant reduction in growth rate upon addition of doxycycline (Fig. 1E, panel a). Consistent with the leaky phenotype, the growth rate of the inducible Bre1b shRNA cells was slightly reduced even in the absence of doxycycline, when compared with the control-inducible GFP shRNA cell line (Fig. 1E). We were able to obtain a more tightly regulated cell line by modifying stringencies of antibiotics' selection (shBre1b2 in Fig. 2A), which corrected the problem of reduced cell growth in the absence of doxycycline. The availability of the leaky cell line shBre1b1 with partial knockdown of Bre1b gave us an advantage to investigate weaker and/or long-term effects of Bre1b depletion (shown further).
Apoptosis contributes to growth impairment in Bre1-depleted cells
To explain the reduction in growth rate observed in the Bre1-deficient cells, we investigated the possibility of increased apoptosis. Apoptosis frequencies after Bre1b knockdown were increased by up to 5-fold compared with uninduced cells, and to both induced control and uninduced control cells (Fig. 1E (panel b) and Supplementary Fig. S2A). Consistent with the leaky phenotype, low level of apoptosis was present in the shBre1b1 cells even in the absence of doxycycline (Fig. 1E, panel b). Apoptosis also increased after depletion of Bre1 in MES cells (Supplementary Fig. S2B). We concluded that the function of Bre1 is critical for homeostasis and that apoptosis contributes to the reduced growth phenotype in cell cultures with low levels of Bre1a and Bre1b.
Bre1 loss compromises genomic integrity
Reduction in growth rate and increased apoptosis also closely correlated with increased frequencies of micronuclei (MN; Fig. 1E and F), supporting the conclusion that the function of the Bre1a/Bre1b complex is essential for maintenance of DNA integrity. Importantly, we observed a tight correlation between H2B ubiquitination and MN levels (Supplementary Fig. S3A and S3B).
Progressive genomic instability in Bre1-deficient cells
Although significant loss of Bre1 was catastrophic to the cells and lead to cessation of proliferation and cell death (Fig. 1E), more subtle changes in Bre1b expression due to the partial knockdown in the uninduced shBre1b1 cell line allowed cells to proliferate and manifest dramatic genomic instability associated with Bre1 deficiency after longer culture periods. When cultured for 3 weeks and longer under conditions of partial knockdown (Fig. 2A), the shBre1b1 cells displayed MN frequencies comparable with that observed on day 5 after complete Bre1b knockdown in the shBre1b2 cells (Fig. 2B). In addition, partial knockdown shBre1b1 cells at higher passages were characterized by the appearance of anaphase bridges (Fig. 2C and Supplementary Fig. S3C), many of which contained telomere signal(s) (Fig. 2C), and by an increased DNA content (Supplementary Fig. S4). To differentiate between fusions resulting from loss of telomere end protection (22, 23) and fusions resulting from DSBs (24, 25), we carried out telomere FISH analysis on metaphase chromosomes from the Bre1-deficient and control cell lines. Because no telomere signal was observed interstitially at fusion points, chromosome analysis provided no evidence for the loss of telomere end capping function, suggesting that anaphase bridges resulted from DSBs.
We also found that loss of Bre1 led to the formation of 3 main types of chromosome aberrations, providing insight into mechanisms underlying the formation of telomere-positive anaphase bridges and to the increased DNA content in the Bre1-knockdown cells. The chromosome aberrations predominantly found in the Bre1-knockdown cells were Robertsonian-like translocations with amplified centromeric heterochromatin (RTCH; Fig. 2D), aberrations involving large regions of amplified centromeric heterochromatin at pericentric and interstitial locations (ACH), and chromatid-type aberrations (CA; Fig. 2E and F), aberrations associated with instability. The amplified regions of centromeric heterochromatin observed in the metaphase chromosomes of the Bre1-knockdown cell line were sometimes involved in dicentric chromosomes, which lead to the generation of anaphase bridges. These bridges contained telomere signals not associated with any telomere end protection defect and subsequent chromosomal end fusions, but rather telomeres associated with chromosomes pulled into the nucleoplasmic tube of an anaphase bridge, providing explanation for the presence of telomere signals within the anaphase bridges of the Bre1-knockdown cell line.
The chromosomal instability phenotype initiated by partial Bre1b knockdown (shBre1b1 cells grown without doxycycline) for 3 weeks and longer was more dramatic than the one initiated by complete short-term knockdown of Bre1b at early passage (Fig. 2E and F). In addition, cells at higher passages accumulated chromatid-type aberrations, which were not present in the lower passage cells, as well as very complex aberrations, which seemed to result from disruption/amplification/decompaction of centromeric heterochromatin (Fig. 2E and F).
Bre1 deficiency leads to replication stress
To elucidate how loss of Bre1 contributes to increased DSBs and genomic rearrangements, we measured γH2AX levels after Bre1 knockdown. Consistent with previous reports (26, 27), we found 2 distinct subpopulations of cells with elevated γH2AX. In the first, an increase in γH2AX signal was detected as a shift of the main cell population in the fluorescence-activated cell sorting (FACS) profile (Fig. 3A) that could be accounted for by both an increase in the intensity of γH2AX foci and/or in the number of cells with γH2AX foci at sites of DSBs (Fig. 3B–D panels a and b). The other population of cells had dramatically elevated pan-nuclear staining of γH2AX (Fig. 3D panel c), which identified apoptotic or preapoptotic cells (26, 27). FACS analysis showed that although γH2AX signal (DSBs) was elevated throughout the cell cycle (Fig. 3A), cells with pan-nuclear staining of γH2AX (apoptosis) were restricted to S-phase cells after knockdown of Bre1 (Fig. 3E). To investigate how Bre1-deficient cells dealt with replication stress, we treated cells with 2 mmol/L hydroxyurea (HU), which stalls replication forks by inhibiting ribonucleotide reductase essential for the synthesis of DNA precursors. A prolonged (24 hours) treatment with 2 mmol/L HU leads to replication fork collapse, which requires HR for repair of resulting DSBs (28). We found that prolonged treatment of control cells with 2 mmol/L HU led to a distribution of γH2AX signal reported previously (29), with γH2AX-positive cells in 3 distinct clusters corresponding to G1, S, and G2-M cells (Fig. 3F). Consistent with increased apoptosis during replication, we also found that loss of Bre1 resulted in depletion of cells primarily from the cluster corresponding to the γH2AX-positive S-phase cells (Fig. 3F). Replication stress and occurrence of chromatid aberrations are consistent with defective homologous recombination in Bre1-depleted cells we reported previously, and we showed that spontaneous Rad51 foci formation was significantly affected by loss of Bre1 (Supplementary Fig. S4C).
Cotranscriptional formation of R-loops contributes to replication stress in Bre1-depleted cells
To explore additional factors contributing to DSB formation in Bre1-deficient cells, we carried out gene expression analysis on Bre1a and Bre1b knockdown cells. Because growth-affecting changes were observed after longer times of Bre1 knockdown (Fig. 1D), we carried out microarray analysis after 7 days of RNAi. Consistent with both subunits of Bre1 complex being required for the H2B ubiquitination, we observed a high correlation between expression profiles in the Bre1a and Bre1b knockdowns (Supplementary Fig. S5). Despite the general role of Bre1 in transcriptional regulation, loss of Bre1 downregulated a distinct set of genes and upregulated another without affecting majority of genes (Supplementary Tables S1 and S2). Further analysis revealed that the group of genes upregulated after Bre1 knockdown was dramatically enriched for genes involved in RNA processing/splicing, showing a response typical of the one to cotranscriptional formation of recombinogenic RNA–DNA hybrids (R-loops; Fig. 4A). R-loops often arise during perturbed mRNA processing as a result of the extended pairing of nascent mRNA with the transcribed DNA strand behind the elongating Pol II, creating DSBs and leading to increased recombination and genomic instability (30, 31). In a genome-wide siRNA screen for genes whose deregulation leads to elevated levels of γH2AX (32), the mRNA processing module represented the most significantly enriched category of genes, suggesting that abnormal mRNA processing was the most common and direct source of genomic instability. To determine whether R-loop formation contributed to increased formation of DSBs in Bre1-deficient cells, we tested whether overexpression of RNase H1, which degrades RNA in R-loops, would reduce levels of γ-H2AX. Figure 4B shows that overexpression of RNase H1 decreased the number of cells with γ-H2AX foci in the Bre1-depleted cells, whereas it did not have an effect on cells in which Bre1 RNAi was not induced. In addition, RNase H1 expression reduced the average number of γ-H2AX foci per cell (Fig. 4C and D). These data suggested a mechanism for the generation of DSBs through the R-loop formation in the Bre1-depleted cells.
We also found that the gene subset upregulated after depletion of Bre1 was strongly enriched for histone genes from the compact chromatin cluster on 13qA3.1 (P < 0.0001; Supplementary Table S3). Normally, replication-dependent histone mRNAs do not have poly(A) tails, but as we relied on poly(A)-dependent amplification of mRNA for hybridization to our microarray chip, overrepresentation of histone genes from the 13qA3.1 cluster is a compelling evidence for the presence of mRNA that has been polyadenylated. These data confirmed the previous finding by Pirngruber and colleagues (33) showing that BRE1(RNF20/40)-dependent H2Bub1 acts as a marker for correct recognition of the histone mRNA 3′-end cleavage site. Due to the massive synthesis of histones during replication, defective processing of replication-associated histone mRNA in BRE1(RNF20/40)-deficient cells may be the main contributor to the formation of DSBs and hence replication stress.
BRE1A/B and H3K79me2 levels are lower in human seminoma than in normal testis
We hypothesized that tissues with high expression of Bre1 homologs would depend on the function of the complex for maintenance of genomic integrity. Western blot analysis showed that among various mouse organs including brain, heart, kidney, liver, lung, muscle, skin, testis, spleen, and bladder, expression of Bre1b was highest in testis and spleen (Fig. 5A), suggesting that the Bre1a/Bre1b complex may play essential roles in these organs. We observed strong Bre1b staining in spermatogonia (Fig. 5B, left panel). We also found a strong signal for H3K79me2 and H3K4me3, the 2 modifications affected by ubiquitination of H2B. H3K79me2 was highest in middle meiosis (Fig. 5B), whereas H3K4me3 was strongest at the beginning and in the middle of meiosis (Fig. 5B), consistent with the roles these modifications play during yeast meiosis (34–36). Importantly, the staining for total histone H3 was uniform throughout the sections (Fig. 5B).
Having shown the importance of Bre1 homologs to maintenance of genomic stability, we hypothesized that suboptimal expression of Bre1 homologs in testis might be associated with testicular cancer. To test this supposition, we analyzed BRE1A (RNF20) and BRE1B (RNF40) expression data available from public databases (37, 38) and found significantly lower levels of BRE1A mRNA in human seminoma compared with normal testicular tissue (Supplementary Fig. S6). Seminoma is a type of testicular germ cell cancer that is the most common solid tumor in otherwise healthy men aged 15 to 35 years. It is widely accepted that seminoma arises from the precursor lesion carcinoma in situ (CIS, also known as intratubular germ cell neoplasia) and can grow rapidly and metastasize (39). Seminomas are characterized by high levels of genome instability and gains of chromosome 12p, which are not present in CIS. Although formerly often lethal, seminoma is highly sensitive to ionizing radiation therapy and chemotherapy and most are now cured.
To assess BRE1A/B protein levels, we stained tissue microarrays containing tissue sections from normal testis and seminoma. Levels of both BRE1B protein and BRE1A/B-dependent dimethylation of H3K79 and trimethylation of H3K4 were significantly lower in seminoma compared with normal tissue (Fig. 5C). Further analysis of publicly available gene expression arrays (37, 38) showed that among different testicular cancers seminoma displayed the lowest levels of BRE1A (Supplementary Fig. S6), and by staining the testis tissue microarrays, we found that among testicular cancers, H3K79me2 was also lowest in seminoma (Fig. 5D, Supplementary Fig. S7 and Supplementary Table S4). Importantly, staining of BRE1A and H3K79me2 in CIS was also lower than in normal tissue from the same patients (Fig. 5E), suggesting that deficiency in BRE1A/B and in methylation of H3K4 and H3K79 occurs before acquisition of an invasive phenotype.
We show that Bre1 (human BRE1A/B (RNF20/40) and mouse Bre1a/b (Rnf20/40)) acts as an important suppressor of chromosomal instability (CIN). This finding complements the previously suggested mechanism for Bre1 tumor suppression through transcriptional regulation of select oncogenes and tumor suppressor genes (14). The types of chromosomal aberrations we observed after knockdown of Bre1 indicated that a defect in HR contributes to CIN in the Bre1-deficient cells (40, 41). This conclusion is consistent with our previous observation that reduced monoubiquitination of H2B in Saccharomyces bre1null mutants and in mouse cells leads to defective recombinational repair of DSBs; refs. 10, 42). We show that R-loops, the RNA–DNA hybrid structures usually formed behind elongating RNA polymerase II when mRNA processing is disturbed (30–32), constitute a significant source of DSBs in Bre1-deficient cells. Overall, our data support a model in which reduction in Bre1-dependent ubiquitination of histone H2B increases genomic instability through increased generation of DSBs resulting from a defect in correct processing of canonical histone mRNA and through inhibition of HR needed for DSB resolution (Fig. 6).
It should be noted that although we interpret the observed Bre1 knockdown phenotype as arising from an impact on the well-known role of Bre1 in H2B ubiquitination, it is formally possible that additional targets for the Bre1 ubiquitin ligase exist, which could contribute to the knockdown phenotype. Testing this with an H2B K120 substitution mutant is not straightforward in mammals because their genomes contain at least 17 H2B genes (43). However, studies using overexpressed ectopic H2BK120R mutant gene in cells with wild-type chromosomal copies of H2B have shown that effects of overexpression of H2BK120R mimic those of loss of BRE1, including the effect on the formation of γH2AX foci, which we used in our study to detect DSBs (11, 12). Also, in Saccharomyces, in which chromosomal copies of the H2B gene number only 2 and can be deleted, H2BK123R mutants do mimic the bre1 deletion phenotype (reviewed in ref. 9), implying that the phenotypes are likely to be conferred through effects on the H2B target. Questions remain, however, as to whether the phenotypes we observed after loss of Bre1 are mediated via the well-known impact of H2B ubiquitination on methylation of histone H3, and, specifically, whether H3K4me3 and or H3K79me2 are involved. Detailed investigation of the role that H3K4 methylation plays in different Bre1 phenotypes is hampered by the fact that there are many SET1 homologs in mammals, some of which may not be uH2B-dependent (reviewed in ref. 9). Also, knocking down the H3K79 methyltransferase Dot1 would completely eliminate methylation of H3K79, whereas loss of Bre1-mediated H2B ubiquitination only eliminates the higher states of methylation of H3K79, so additional phenotypes may be conferred by the Dot1 knockdown beyond those mediated by the impact of uH2B on H3K79. Although the effects of Dot1 knockdown in mice (44, 45) paralleled the impaired cell growth, increased ploidy and centromeric abnormalities, we observed that after Bre1 knockdown, specific clarification of these issues requires further study.
A crucial unresolved question in cancer biology is whether CIN represents an early event and is therefore a driving force of carcinogenesis. Our results support models in which CIN drives tumorigenesis rather than being its consequence. We provide a comprehensive demonstration of a stepwise accumulation of chromosomal changes that start with downregulation of Bre1. Consistent with the CIN model, we also show that BRE1A/B deficiency accompanies early steps of testicular cancer development.
Correct processing of replication-associated histone mRNA is particularly relevant in connection to testicular carcinogenesis. In the testis, a massive synthesis of histone variants accompanies dramatic reorganization of the genome, during which the majority of the histones are replaced by transition proteins and protamines. Lack of Bre1 leading to abnormal presence of polyadenylated histone mRNAs, which are not rapidly degraded at the end of S-phase, could interfere with proper incorporation of the variant histones into chromatin, and lead to testicular dysgenesis. We showed that low levels of BRE1A and BRE1B, and low H3K79me2 are found in intratubular cell neoplasia (CIS), as well as in seminomas. Seminomas show chromosomal changes similar to those found in CIS and therefore are considered a default pathway from the CIS precursor lesion to invasive testicular germ cell tumors (TGCT; ref. 39). Like all TGCTs, seminomas are characterized by high levels of CIN and aneuploidy, and a gain of chromosome 12p (46). Gain of 12p is not present in CIS, and so it is believed that overexpression of gene(s) on 12p is pertinent to invasive growth. Thus, we can speculate that genomic instability initiated by the abnormal downregulation of BRE1A/BRE1B function may facilitate gain of 12p and thus constitute one possible route to seminoma. Seminomas recapitulate the undifferentiated and pluripotent primordial germ cell (PGC) phenotype and are thought to arise when a block in maturation of PGCs prevents them from forming spermatogonia. Downregulation of BRE1A/BRE1B may be associated with such a maturation block (39) and thus may lead to infertility. In fact, men from families with fertility problems are known to have an elevated risk of testicular cancers, especially seminoma (47–49). Hence, deficiency in BRE1A/B may be among etiologic factors in common for both infertility and testicular cancer. In addition, mutation of BRE1 (RNF20) has been found among other CIN genes mutated in colorectal cancers (50) suggestive of a more general role of Bre1 in CIN.
In conclusion, we propose that the mammalian homologs of the yeast BRE1 gene serve as tumor suppressors by preventing replication stress and chromosomal instability that arise from DSBs associated with incorrect processing of replication-associated histone mRNAs and inefficient HR. In addition to clarifying basic cellular mechanisms, the identification of Bre1 as a CIN gene may have specific relevance for estimation of risk and diagnosis of testicular cancer.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Grant Support
The work was supported by grant CA67166 awarded to JMB by the National Cancer Institute.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The authors thank Xin Huang (University of Pittsburgh School of Medicine), Elena Seraia (Stanford Functional Genomics Facility), and Janos Demeter (Stanford Microarray Database) for help with image processing and microarray analysis.
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
- Received July 5, 2011.
- Revision received January 18, 2012.
- Accepted February 10, 2012.
- ©2012 American Association for Cancer Research.


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The world-famous biennial International GS Trophy returns in November, with three riders selected from each of several countries around the world to compete on new R1200GS bikes for victory in a vicious series of off-road competitions and challenges in exotic and faraway South Africa.
Two BMW GS owners and one journalist will be selected from the USA for this once-in-a-lifetime, all expenses paid trip. Their selection will be made during the Adventure Riders/GS Challenge at the RawHyde Adventures facility in Southern California from April 29 – May 2. This year's "Double Jeopardy" event at RawHyde is now revamped to include two sequences: the Adventure Riders Challenge, open to all enduro riders, and the GS Challenge qualifier from which two BMW riders will be selected to compete in South Africa.
"The competition finalists will not only need strength and endurance, superior riding skills and guts, but they must also be able to navigate their route ahead, pace themselves and collaborate with teammates," commented Laurence Kuykendall, Community and Communications Manager for BMW Motorrad USA. "It also helps to have a sense of humor under adversity," Kuykendall added, "because, after all, this is all about fun, isn't it?"
Interested BMW GS riders can learn more about the Adventure Riders Challenge and how they can participate in the competition to ride (and win) in South Africa by visiting www.adventureriderchallenge.com or emailing email@example.com. Registration forms can be downloaded from: www.adventureriderchallenge.com/arcXdbljprdy_applctn.pdf.
In 2008, the high-stakes competition between riders representing five countries took place in the burning sands of Tunisia and was won by Team USA members Brad Hendry (Thornton, CO), Jim Stoddard (Middlegrove, NY), and Jason Adams (Rosendale, NY) along with journalists Jimmy Lewis (Dirt Rider), Ryan Dudek (Cycle World), and photojournalist Jonathan Beck. The GS owners and journalists represented the United States in the 1,900-kilometer desert rally, involving 30 participants from five nations, seeking to demonstrate their skills over challenging terrain on identically prepared BMW F800GS motorcycles.


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Archive for February, 2016
How to Conveniently Arrange a Playdate for Your Dog
Have you ever heard of Koko, the gorilla? She was raised separately from other gorillas and taught sign language, among other simple tasks. Her caretakers realized one day that Koko was lonely, and so they began searching zoos and protected animal habitats to find her a friend. To put this into context, a group of scientists devoted quite a lot of time trying to find a gorilla a date. Probably not what they envisioned they would be doing with their degrees, but animals are social beings. They need members of their own species to keep them company.
We like our pets for the companionship they provide, but just as we go out to socialize with other people, so should our pets. Dogs especially are pack animals. Now, of the roughly 55 million dogs in the U.S., only about 4% are actually diagnosed with separation a


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FREE AD Posting! – Sell your gently used, ready for a new home, Airgun!
AirgunsForSale.net, and airgun classifieds, was created to help like minded airgunners network together to recycle their gently used airguns. We hope you like what you see, and most importantly we insist that folks use this service responsibly. Please note that we take NO RESPONSIBILITY for the ads posted, transaction process, or quality of the products represented. Engage your brain people. If it's too good to be true, you can bet it probably is.
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Fundraising Project History
1918-1970 The League was involved in various fundraising activities, including balls, lectures, bazaars, follies, fashion shows and rummage sales.
Ongoing – Bridge Marathon – Duplicate Bridge tournament includes League members and other community members who meet on a regular basis, September through May. Since 1976 this project has generated more than $98,000 for the JLD programs and purpose.
1970-73 The League held Daisy Day at Elitch's, family fun for an all day ticket price. The League raised approximately $60,000 over the three years.
1973 – Second Hand Rose – The League voted to open a Thrift Shop, Second Hand Rose, which welcomed its first customers in April 1974. In late November of 1981, a fire destroyed the shop, by then named The Second Time Shop and located at 3449 S. Broadway. The Shop reopened on March 10, 1982 at 3372 S. Broadway. A move to new and enlarged space at 351 W. Girard occurred Oct. 19, 1991. Since its inception, the shop has generated over $2 million in net income.
1975-76 A calendar, Colorado: Then and Now, in conjunction with the Colorado Centennial year, was published in 1975.
1977 Colorado Rockies – The League participated in selling season tickets to the Colorado Rockies hockey games, raising over $20,000.
1978 Colorado Cache Cookbook was first published in September 1978. This year, it is in its 28th printing, not including a 10th Anniversary Limited Edition, and is distributed nationally. To date, almost 1,000,000 copies have been sold to support the League's purpose and programs in the community.
1979-86 The Monogram Merchants, a stationary and related gift item sales project, contributed over $177,000 to the JLD during its existence.
1980 Christmas Mart – The first annual League-sponsored Christmas Mart was held in September 1980, featuring unusual gift items from specialty shops throughout the country. The name was changed in 1981 to the Holiday Mart. This fundraiser is one of the most successful events of its kind within the Association of Junior Leagues International (AJLI). Since its inception, the JLD Holiday Mart has raised $3.7 million in net income.
1987 Crème de Colorado Cookbook has sold more than 500,000 copies to date. It is currently in its 12th printing and is nationally distributed.
1986-88 United States Figure Skating Championships – After two years of preparation and planning, the League, in cooperation with the Mile High Figure Skating Association, produced the 1988 United States Figure Skating Championships, January 4-10, 1988, in Denver. Profits from the event were shared between the League, Mile High Figure Skating Association, and the United States Figure Skating Association. The League's portion of this very successful event were $193,000.
1988-91 Two Quick Action fundraisers were inaugurated in 1989-90: Wine Tasting and Italian Cooking Demonstration, generating nearly $3,000 and providing a social outlet as well for League members. The Wine Tasting Event was held again 1990-91, contributing additional funds totaling $2,000.
1990 The Lord & Taylor Department Store Opening on August 15, 1991 was coordinated by JLD volunteers, at the new Cherry Creek Shopping Mall, generating $45,000 for the League.
1995 Colorado Collage Cookbook was published in September of 1995 and is a successful addition to the two C&C Publications cookbooks already established, with over 225,000 copies sold to date. The three cookbooks have earned over $5.4 million to support the League's purpose and programs.
1996 Nordstrom at Park Meadows Grand Opening Gala on August 28, 1996, was a resounding success. The 2,500 tickets sold out within 10 days of the invitations being mailed and the fundraiser netted the JLD $250,000 and a successful on-going relationship with Nordstrom.
1998 The Colorado Shopping Card was launched as the JLD's newest fundraiser in the fall of 1998. For one week in November, card purchasers receive a 20% discount from over 100 participating retailers. The fundraiser is an event of the Denver Post Charities and is a collaboration with the Colorado Children's Campaign. To date it has raised over $17,000 to support the League's purpose and programs.


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Lindsey Stirling (born September 21, 1986) is an American violinist, dancer, performance artist, singer and composer. She presents choreographed violin performances, both live and in music videos found on her YouTube channel, Lindsey Stirling, which she introduced in 2007.
Stirling performs a variety of music styles, from classical to pop and hip-hop to electronic dance music. Aside from original work, her discography contains covers of songs by other musicians and various soundtracks. Her music video, "Crystallize" finished as the eighth-most watched video of 2012, and her cover version of "Radioactive" with Pentatonix won Response of the Year in the first YouTube Music Awards in 2013. Stirling achieved one million singles sold worldwide by August of 2014. As of April 2015, her Lindseystomp YouTube channel exceeded 6 million subscribers and over a billion total views.
Stirling has been named in Forbes magazine's, 30 Under 30 In Music: The Class Of 2015. Forbes notes her quarter-finalist position on America's Got Talent season five in 2010, a #2 position on the Billboard 200 for her second album Shatter Me in 2014, and her 6 million subscribers on YouTube.
Stirling's eponymous debut album was a commercial success in Europe, selling 200,000 copies in Germany, winning a platinum certification; three additional certifications were given by Austria, Switzerland and Poland. The debut album was nominated for the 2014 Billboard Music Awards for Top Dance/Electronic Albums. Stirling's second album won the 2015 Billboard Music Awards for Top Dance/Electronic Album.


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Where do you plan to give birth? It seems a strange question with an obvious answer for most women, who would never dream of going anywhere but a hospital. The Business of Being Born addresses the growing numbers of women who are opting for home births attended by midwives. The film is a fascinating look at America's perception of birth, and the misconceptions surrounding midwives and their practice. The inspiration for this documentary was based on co-creator Ricki Lake's own experiences with both hospital and home births. After finding her first experience in the hospital overwhelmingly negative, she began doing more research into birth alternatives and ultimately deciding to have her second child at home with the help of a midwife. Lake enlisted her friend, director Abby Epstein, to help create this film and show women with low risk pregnancies that they have a choice.
The Business of Being Born presents a strong pro home birth perspective. Although it does not vilify hospital births, it does present several aspects as problematic. The overwhelming majority of women in the United States give birth in a hospital under the supervision of doctors, which they perceive to be the safest choice. While obviously this is the case for women with high-risk pregnancies, it is not clear that this is the best route for low-risk pregnant women. For instance, despite nearly all births taking place in hospitals, the US has the second worst newborn death rate in the developed world, with one of the highest maternal mortality rates among all industrialized countries. Once admitted to the hospital, 90% of mothers receive birth augmentations and currently 1 in 3 women will end up having a Caesarian section. The majority of these surgeries only become necessary due to the domino effect of the drugs, such as Pitocin, administered to labouring women. Although C-sections seem commonplace, there is no mistake that they are major surgery, leading to increased rates of infection and possible complications later in life. These statistics seem to fly in the face of the common assumption that births attended to by doctors in hospitals are "safer".
The film features several pieces of footage from successful home births, including Lake's own. Most importantly, several misconceptions regarding midwives are cleared up. There are two doctors interviewed in the film who express their doubts about midwives, particularly that they are not properly equipped to deal with emergencies, should they pop up. However, they admit that they have little first hand knowledge of midwife practices to back up their opinions. The midwives in the film contend that they put all prospective women through intense screening processes to determine if they are good candidates for home birth, are highly trained and well equipped, and keep an eye out for red flags so, if need be, they can decide to send their patient to a hospital for support.
Lake and Epstein have infused this film with their passion, providing both a wellspring of information, as well as equipping their film with an emotional core. The various birth stories were touching, and I often found myself smiling and laughing out loud. Sadly, it can't all be sunshine and roses. The film presents many expert opinions from doctors, midwives and researchers in the medical field, the majority of whom are pro home birth. Many of their arguments are based in fact, though there are a few points that seemed a little off. For example, a portion of the film addresses the 'authentic' experience of all-natural birth. It sets me on edge any time the term 'authentic' is thrown around. Authenticity can mean a great many things to people. What does it mean to have an inauthentic birth? Does it mean that you are not a real mother, or less of one? The question of authenticity seems absurd in this context.
There was also a brief nature/nurture discussion towards the tail end of the film, in which a doctor seems to suggest that because fewer mothers are giving birth naturally, (and are subsequently missing out on the crazy chemical cocktail of love juice their brain would produce) they are somehow less attached to their infants. It's a suggestion rather than a statement of fact, but I found it disturbing nonetheless. To suggest that women who miss out on this chemical boost, such as those who have Caesarian sections, or are adoptive mothers, are less attached to their children, or have to work harder to love them, is bizarre. Although The Business of Being Born stumbles during these rough patches, they are not big enough flaws to overcome the strengths of this documentary. In the end, this film accomplishes what it set out to do: educate its audience about birth alternatives, and clear the air of misconceptions that surround the practice of midwifery.


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Chez Muriel is indeed a smart choice for travelers seeking comfort, charm and convenience. It is only few minutes' walk to bus stops, shops, restaurants and the beach. It offers impeccable services and modern facilities to ensure satisfactions and a pleasant stay for its guests. The guesthouse can be rented as an entire villa by a group of people or separately.
At your own leisure for more fun and excitement, you can rent a bicycle on site to take a small tour round the island or even car hire can be arranged as well as excursions and tours. The property offers free parking and a mobile phone on the reception is available at your disposal.
Experience the service and facilities Chez Muriel Guest house has to offer such as:


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Floyd Williams: "No Hobbies. Just Music."
Boston-born Floyd Williams had a long career in jazz, first as a musician and then as an educator. In his home town, the drummer was known by his nickname, "Floogie." No one around now knows how he got it. Later in his career, people knew him as Floyd Williams.
His Boston story is an intriguing one. As with many artists of past decades who did not achieve great stardom in New York, there are facts about his story we don't know. We do know he attended Boston public schools, started on piano as a boy, switched to the drums, gigged with friends while still at Roxbury High School, and studied briefly at the New England Conservatory. I have read that Johnny Hodges was his godfather, and I am still looking into that.
Legendary godfather or not, Floogie Williams earned his own recognition as a drummer in the late 1940s, at a time when Boston was incubating an exciting brand of modern jazz. The numerous G.I. Bill students at the music schools were mixing with the local musicians in the clubs clustered around Mass Ave and Columbus Ave. Disc jockey Symphony Sid Torin called this intersection "the jazz corner of Boston." Williams was a regular on the bandstand at the Sunnyside Cafe, where he worked with saxophonists Sam Rivers and Gigi Gryce. He also played occasionally in the big band of Jimmie Martin, where he met trumpeters Joe Gordon and Lennie Johnson, and Jaki Byard—who played trombone!
> Continue Reading


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Bankruptcies, bailouts and bad loans have rocked the financial industry. For investors, this bad news may provide good opportunities to buy low. Let's take a look at some companies that look to be in the best shape to survive the current crisis and profit once it's passed.
CITIGROUP INC. (C) A discussion of the current financial crisis must include Citigroup. With over 200 million customer accounts in more than 100 countries, Citigroup provides consumers, corporations, governments, and institutions with a broad range of financial services.
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Citigroup stock has plummeted sharply over the past year, reaching a 15-year low in mid November. The federal government, fearing what a Citi collapse would do to the financial markets, saved the company with a $25 billion dollar bailout soon thereafter. Although Citi stock rose sharply after news of the bailout, it's still trading near its 52-week low. Its low price combined with government assurances makes Citigroup an attractive long-term stock for value investors.
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WELLS FARGO (WFC) Despite being one of the top sub prime lenders in 2006 and having its business highly concentrated in 3 of the 4 areas hardest hit by the housing downturn, Wells Fargo has weathered the storm better than most, having sold away their bad loans quickly.
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In the summer of 2008, Wells Fargo reported record revenues, claiming to have "negligible exposure to many of the problem areas that resulted in significant costs and write-downs at other large financial institutions." A closer look, however, shows they have a considerable amount of risky loans, which may indicate future write-downs. Its acquisition of Wachovia, however, indicates Wells Fargo is confident going forward and positioned for huge profits once the crisis has passed.
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JP MORGAN CHASE (JPM) JP Morgan Chase, another financial institution that has fared better than its competition, has $2.3 trillion dollars in assets and operates in over 60 countries. JP Morgan's acquisition of Washington Mutual sent a positive message of long-term viability to the financial world.
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As with other financial institutions, JP Morgan stock has benefited from the government's willingness to make sure the financial sector survives. Despite a recent surge in share price, JP Morgan stock remains well off its highs of a year ago. With less exposure to risky loans than its competitors Wells Fargo and Citigroup, and an aggressive approach to helping families facing foreclosure, JP Morgan Chase has remained stable, despite unprecedented turmoil in the financial sector.
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BARCLAYS PLC (BCS) For investors who like a lot of risk with their reward may want to look at Barclays. The London based bank remains floundering near its 52-week low, not reaping the benefits of the U.S. Government's Citi bailout. This means one of two things: either investors are getting a reputable institution at a rock bottom price or they're getting the next institution to hit rock bottom.
Barclays's history of innovation and creativity lead some to believe the bank will figure a way out of the current financial mess. Barclays introduced the world's first cash machine in 1967, introduced Internet banking to England, has been a major player in Exchange Traded Funds, and won the Excellence in Emissions Trading at the Energy Business Awards as recently as 2005. If Barclays stock survives the current onslaught, shareholders may be looking at historic gains when the crisis ends.
Investors considering investing in financial companies might consider hedging the positions with stock options. Investors can use stock options as "stock insurance", put options for example can protect a stock investment from a downside move. Investors can also limit risk and participate in price appreciation with a married put stock option strategy.
For more information about how to identify and research great option trades, visit the PowerOptions website. There you will find the data you need to make quick, clear, and informed decisions. You can trade knowing you have found the best investment. Also, PowerOptions will allow you, with a few quick clicks, to quickly and accurately compare trades. PowerOptions' premium customer support is second to none in the industry. They can be easily contacted when you need them at their toll-free number to answer customer questions. Call them now toll free at 877-992-7971.
PowerOptions provides a free 14-day trial of its service. So join PowerOptions today, and you too can start reaping the benefits of the covered call investment strategy.
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[tags] Bankruptcies, bailouts, bad loans, financial industry, investors, bailout, Wachovia, Washington Mutual, Internet banking, Exchange Traded Funds, C, Citigroup Inc., JPM, J.P. Morgan Chase, WFC, Wells Fargo & Co., covered call investment strategy, investment strategy, iron condor, poweroptions, stock insurance, stock option strategy, stock option trading, stock options [/tags]


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Pitbull - Don't Stop The Party ft. TJR
Pitbull's official music video for 'Don't Stop The Party' ft. TJR. Click to listen to Pitbull on Spotify: http://smarturl.it/PBSpot?IQid=PSTP
As featured on Global Warming. Click to buy the track or album via iTunes: http://smarturl.it/PGWiTunes?IQid=PSTP
Google Play: http://smarturl.it/PSTPPlay?IQid=PSTP
Amazon: http://smarturl.it/PGWAm?IQid=PSTP
More from Pitbull
Timber: https://youtu.be/hHUbLv4ThOo
Rain Over Me: https://youtu.be/SmM0653YvXU
Give Me Everything: https://youtu.be/EPo5wWmKEaI
Follow Pitbull
Website: http://www.pitbullmusic.com
Facebook: https://www.facebook.com/pitbull
Twitter: https://twitter.com/pitbull
Subscribe to Pitbull on YouTube: http://smarturl.it/PITSub?IQid=PSTP
More great Global Hits videos here: http://smarturl.it/GHPlaylist?IQid=PSTP
I'm running through the world like I'm running back
Scarface, world's mine, running back
Thirty thousand people in Hanover to see me
Eighty thousand people in London, Wembley
Ninety thousand people in Morocco
And I'm just getting warmed up, papo
Catch me with Red One in Stockholm,
Beirut, cafe getting my drink on,
Where all the pretty women hit the hooka
All of them sweet, azucar, azucar
Dale disfruta
They can't, they won't, they never will stop the party
They can't, they won't, they never will stop the party
I say, y'all having a good time, I'll bet
Yeah, yeah, yeah
Que no pare la fiesta
Don't stop the party
Yeah, yeah, yeah
Que no pare la fiesta
Don't stop the party


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Join our green movement in MWC Los Angeles 2019
There are three important things you can do in the event to help the environment:
Your flight, accommodation, transportation and food choice generates an impact on the environment. We can help the environment by offsetting our footprint. Follow four simple steps HERE to calculate and offset your carbon footprint during MWC19 Los Angeles:
Do you know that if every attendee recycled their badge, the carbon footprint of the event would decrease by the equivalent carbon absorption of about 254 trees? Help us recycle your badge to reduce our environmental impact. You can find the recycle bins located at the exits of the event.
Use the Event App instead of printing
Download relevant content from exhibitors via a QR code
Use public transportation to reach the venue
Share your car with other members of your company
Bring your own water bottle
Request cardboard cups instead of plastic when you eat
Consider using airlines with carbon footprint offset programs
Book a direct flight to Los Angeles. Non-stop flights are exponentially better for the environment.
Review the carbon footprint program in your accommodation
Turn off any lights, TV and heater when you leave your hotel
We are a carbon neutrality program committed to reducing the impact of our business on the environment, both in our offices and at our events. All 14 international GSMA offices and all GSMA events, including the Mobile 360 series have been certified as carbon neutral since 2016. This makes GSMA a carbon neutral company, a certification we intend to renew each year. Our objectives are:
Waste reduction through our 'Donation Room' Initiative. We collected 2 box truck loads of furniture and office supplies in 2018 and donated them to Habitat for Humanity of Los Angeles.
We provide a free carbon footprint study to space only exhibitors to understand their impact on the environment and help them become more sustainable stands for future events.
Los Angeles Convention Center (LACC) is a straw free facility. We encourage participants to bring their own water bottle during the event. We will be using water dispensers and cups instead of distributing bottled water in most of the event programs. We also encourage participants to bring their own water bottles to the event.


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This shining alphabet has Lower cases and numbers only.
Is make in pretty green colour.
Each alpha is saved in png file , with no drop shadows.
Is a good share for making layouts and you can add to your scraps.
600 x 600 px.
Is for commercial and personal use.
All images not shown in the preview.
Don't forget say : Thank you ♥
Click on image to download.
Enjoy !


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10 Token Packet
- Vendor: Metro Bus
- Type: Fixed Route
The 10 token packet is good for 10 rides on the Metro Bus Fixed Route system. Tokens do not have an expiration date. Valid only on Fixed Route service.
Orders placed before 12:00 p.m. Monday – Friday will be available after 4:00 p.m. Orders placed after 12:00 p.m. will be ready by 10:00 a.m. the next business day. All sales are final.


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Personal information leaked from law firm
Last week saw tit for tat DDoS (Distributed Denial of Service) attacks launched against two law firms (attacks like this are illegal in the UK) seemingly in response to attacks on a number of torrent sites. A ramification of these attacks was that when ACS:Law restored its website, for a short time a number of files that should have not been publicly available were available to those who looked slightly harder than usual.
Of course these files have now made their way onto various torrent sites and other locations around the Internet. What is of concern is that the files obtained contain swathes of personal information such as spreadsheets revealing customer details from broadband providers in response to court orders, and the response from individuals to ACS:Law. Plusnet has responded to this leak of information by flagging any accounts that were disclosed to ACS:Law, with an email being sent to users in addition to a ticket being added to the account. Plusnet is not the only other ISP involved, some Sky customers details have also been leaked. The stance of TalkTalk and Virgin Media public position of challenging content of the court orders appears to have meant that ACS:Law has not pursued potential infringers on their networks.
While learning the address of anyone in the UK is not difficult, if this can be tied to potential infringement of copyright, it leaves people open to carefully targeted spam or maybe even blackmail. Of the biggest concern is the suggestion that credit card details may also have been leaked, after being stored as plain text. The emails indicate that guest houses are doing things like changing how their guest wi-fi works, partners of forces personnel overseas are having to deal with letters, and interestingly some people are willing to pay but only in instalments.
ACS:Law has already been referred to a disciplinary tribunal by the Solicitors Regulatory Authority. It is just possible that this leak may result in further questions and maybe action, questions for example of why was greater care not taken of personal information. The most interesting aspect is that this security breach reveals a lot about how ACS:Law handle cases, which is not likely to endear them to the public, broadband industry and even the industries it is alleging to protect from copyright infringement.
This incident reveals that no matter how careful you are with your own personal information, third parties may not always take the same level of care. Therefore if you have reason to believe your details may have been leaked e.g. previously had had a letter from ACS:Law, then be on your guard for perhaps even more spam email, unsolicited phone calls or post. Whilst most spam is easy to spot, with the level of personal detail revealed carefully crafted spam that could appear real may well appear. The rules of spam email is to never ever open any attachment (no matter how good your anti-virus software), and never reply to the email.
The copyright infringement part of the Digital Economy Act (DEA) 2010 is meant to level the field somewhat, and the letter writing component will require broadband providers to pass on warning letters. It is possible that the Act will bring an end to activities such as those run by ACS:Law, but this is far from certain. What this latest incident will do though is sharpen the focus on how the data generated by the Copyright Infringement Notice process is obtained and protected.
you can't pass laws to sort out the ambulance chasers, and the badly thought out deact will only give them more power. They are a bigger blight on the face of the earth than any pirate.
Advice: If you have paid ACS Law any money in such a way that they have your bank account details (or there is any other reason why they may have your bank details)
I've checked these emails, they are very bad. There is an attattchment with 5000+ Sky broadband users IP addresses, actual address, etc.. There are emails saying they are purposely scaring people into paying.
Very very bad...
Now ACS Law are facing legal action themselves: http://tinyurl.com/3x3nd3k
(Link is to Privacy International - URL contains some dodgy characters)
I hope that ACS Law are savaged by other legal sharks, and that there will be a self destructive feeding frenzy of Legal piranha
There is a document in an email stating...
Total Letters sent: 20,323
Money recovered: £936,570.72
The amount of money to each firm totalling: £341,078.92
That's alot of profit....
OK, people really need to ring up their banks and cancel their credit card if they have dealt with ACS:Law. Yes, i'm being serious!
hope they get dragged over the coals for this... no excuses considering the flippant responses they have made about the attacks in the press.
ACS tried to use ignorance as a defence when fined by Westminster Council. Funny how they wouldn't accept the same argument from the victims they blackmailed!
Very amusing reading!
haha Jelv, ironic and i just love karma :D
So the people this law firm are chasing to fine could end up suing the law firm for negligence? Priceless....
Is nobody else wondering what on earth ACS were doing storing this kind of information on their public webserver? It should have been on their 'internal access' only systems. They should be hauled over the coals for this one. And their head of IT sacked immediately.
Will be interesting to see if I'm there, and what they thought of my refusal to pay...
Apparently I'm not - the folder for the email address I sent one of my replies to was empty, and no mention of my name.
Totally, storing this on the dirty net facing side of your set-up is very very bad practice.
"Plusnet has responded to this leak of information by flagging any accounts that were disclosed to ACS:Law, with an email being sent to users in addition to a ticket being added to the account."
Having promised on the Community forums they would notify all users they have gone silent - no emails have been sent.
I hope they get shut down, this may become a problem when the DEA fully kicks in.
Deleted the emails now so can't view them anymore lol.
Announced on the forum at 21.14 and confirmed all emails sent at 21.51 both statements by the Chief Operating Officer
Has this been covered by TV yet?
They have been running nothing short of an extortion racket,protected by a legal loophole allowing them to operate the way in which they have so far, the amount they try to extort from most people is exactly the same set amount £495.00, regardless of the content,when really it was crosley who set the amount not the so called client
PrivacyInternational.org is planning on taking Crossley to court for gross negligance of Data Protection. It wants to hear from everyone to build its case so check it out.
You can find mirrors of the emails and info here: http://acslaw.blogspot.com/2010/09/breaking-news-andrew-crossleys.html
There's no way Crossley can stop this despite thinking he can stop the internet.
What comes around..........
Just to let you know 2 more databases have been found in the emails
One with 8000+ names (SkyB) and one with 400 names (PlusNet) both of which have which shows music albums instead of porn.
Its finally on BBC News, watching it now.
Just had an email with BBC News, Andrew Crossley have confirmed the email about scaring people into paying :D
The file I downloaded contained a lot of empty directories - so I'm not sure it had everything that was available...
Yeah there is a lot of empty directories but all the emails are in the "cur" folders.
Their website is down again :) how did this muppet firm get away with this for so long and how did they manage get hold of customer details from the ISP's? It sounds to me like they were requested on behalf of <insert IP owner here> and they were given up when the IP owner may never have said anything in the first place, this £495 sum for each breach just doesn't stack up. I hope he gets fined the 500k talked about in the press for the data breach and then investigated and sent to jail for his illicit dealings
It is not surprising that Andrew Crossley has, yet again, been referred to the SRA for a disciplinary hearing. Hea has been found guilty twice by the SRA of 'conduct unbefitting a solicitor'.
Nate Anderson, who assembles the huge corpus of emails into a coherent narrative explaining how ACS operates, with emphasis on the unethical behavior that made it the most hated law firm in the UK. If you only read one story about "anti-piracy" operations, read this one.
@GMAN99... OMG i dont think ive ever agreed with you so much (checks outside that the sky isnt falling ;) ). That last post of yours... VERY well said.
What's the problem? Surely this is just a different form of 'file sharing' (by ACS), which a sizable proportion of contributors usually seem to favour!
"You can't have it both ways" springs to mind.


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QR CodesSimple QR Codes is a beautiful QR codes plugin that displays 9 types of qr codes via shortcodes. Add a simple but very useful feature to your website with this stylish plugin. You can use them anywhere in the website, to display nice short information about things that your visitors can save easily on their mobile devices. You can set 9 types of QR codes with any size and any alignment.
- Easy to use
- Call to Action Text!
- 9 QR Types
- Auto QR Code – Generates a QR code with the page URL
- Custom Size, Alignment & Margin
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Take the pledge. Display the decal. Be the change.
The City of Dublin joined several other Central Ohio communities in battling texting and driving by passing a texting ban ordinance that took effect March 2011.
The ban strengthens the existing distracted driving ordinance by making texting a primary offense and a fourth degree misdemeanor. A fourth degree misdemeanor can result in a maximum sentence of 30 days in jail and a $250 fine. The ordinance prohibits "sending, reading or writing a text message or accessing the Internet while driving.
The Pledge
The City of Dublin, Dublin City Schools and Washington Township care about you, your family, your neighbors and your friends. We encourage you to join us in keeping our community safe. Please don't text and drive.


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BY MARC LEVY, The Associated Press
Blowout at Pennsylvania natural gas well
HARRISBURG, Pa. – Natural gas and fracturing fluid from an out-of-control Pennsylvania well shot 75 feet into the air before crews could tame the flow of explosive gas more than half a day later, officials said Friday. See the video
The well was brought under control just after noon Friday, about 16 hours after it started spewing gas and wastewater, said Elizabeth Ivers, a spokeswoman for Houston-based driller EOG Resources. EOG is also a major natural gas and oil producer in the Barnett Shale of North Texas, where the company had 12 rigs drilling Friday.
Ivers said she could not immediately respond to questions about how the accident happened. Public safety and protection of the environment are of the utmost importance, the company said in a statement.
The well never caught fire, and no injuries were reported, but state officials had worried that the gas might explode before the well could be controlled. State environmental authorities promised an aggressive investigation of the circumstances surrounding the blowout.
"The event at the well site could have been a catastrophic incident that endangered life and property," John Hanger, secretary of the Department of Environmental Protection, said in a statement. "This was not a minor accident, but a serious incident that will be fully investigated by this agency with the appropriate and necessary actions taken quickly." If the agency finds that mistakes were made, it will take steps to prevent similar errors, Hanger said. He did not elaborate on any environmental damage.
There are no homes within a mile of the well, and wastewater containing fracturing chemicals and salt was prevented from reaching a waterway, said Dan Spadoni, an agency spokesman.
The well is on the grounds of a hunting club in Clearfield County, about 90 miles northeast of Pittsburgh.EOG was drilling into the Marcellus Shale, a hotly pursued gas formation primarily under Pennsylvania, West Virginia, New York and Ohio that some geologists believe could become the nation's most productive natural gas field. Many energy companies active in the Barnett are also active in the Marcellus.
The crew had just finished hydraulic fracturing – in which millions of gallons of water, sand and chemicals are pumped underground to shatter tightly compacted shale and release trapped natural gas – and were clearing debris from the well when gas shot out, Spadoni said.
Workers evacuated the site and contacted the Pennsylvania Emergency Management Agency, he said. Environmental Protection wasn't notified until 1:30 a.m., more than five hours later.
The polluted water flowing out of the well and into the woods was stopped by a trench and a pump installed by a contractor, Spadoni said. Companies that specialize in securing out-of-control wells were called in, he said.
David Rensink, incoming president of the American Association of Petroleum Geologists, said gas well blowouts are very rare but can be very dangerous to control, as a spark can set off an explosion. Securing one can take days, he said.
Typically, valves called a blowout preventer sit atop a well and allow workers to control the pressure inside, he said. A blowout preventer also figured into the massive oil spill off Louisiana. The device was supposed to shut off the flow of oil in the event of a catastrophic failure but did not.
There are more than 1,000 Marcellus Shale wells in Pennsylvania alone, some of them within view of homes, farmhouses and public roads.
On Friday, a worker blocked a dirt road to the site, while trucks hauling water tanks streamed past him. He said he was not allowed to talk about what had happened.


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No. 1 Small CompanyProvidence Christian School of Texas
Nonprofit formed in 1989 in Dallas 1 D-FW location with 69 employees
Headmaster Jeff Hendricks' favorite ceremony comes at the end of the academic year when each of his school's eighth-grade graduates is called to the stage for a moment of recognition.
"We write a blessing for each of them that speaks to the unique gifts and character qualities that we've seen them express," Hendricks says. "There's also a charge to continue to cultivate those strengths and use them in service going forward."
That kind of communal intimacy is just one reason why Providence Christian School is a three-time winner and our No. 1 small company.
"We have a tight-knit community, and the spiritual faith component is much of what draws people together," says Hendricks, who became headmaster this year after Tony Jeffrey retired.
Hendricks, 35, has been with the school for nearly 13 years, serving as its head of middle school for the last three. So he's well-acquainted with the heartbeat of this classical Christian school, which serves 450 pre-K through eighth-grade students: It emphasizes "biblical teachings with a focus on grammar, logic and effective communication."
Prayer is a constant lifeline.
"We pray for each other regularly, from the board and administration down to the teachers and their classrooms," Hendricks says. "We recognize that employees have lots going on in their lives outside of work. I think people feel cared for and supported."
The school's 69 employees echo that. Providence also won our special award for showing appreciation for its workers.
"I am doing something important and influencing future generations in a positive way," one employee says. "I work with loving, supportive staff and parents."
"I am so glad to have a job where I am appreciated and needed," says another. "I look forward to giving my best each workday."
If you want to feel the love, you might attend Grandparents Day just before Thanksgiving, an event that draws more than 1,000 family members and guests.
Nature studies include a live animal center and flower and vegetable gardens that the students maintain. Fourth-graders begin to learn Latin, and by the time they graduate four years later, they've earned two years of high school credit.
Students and parents volunteer, partner and support Providence's sister school, West Dallas Community School. Middle school students participate in the Brother Bill's Food Pantry and help elderly neighbors by painting homes, repairing roofs and mowing yards.
Employee benefits include a 403(b) company matching retirement plan; funds for professional growth; health care, dental, life insurance and vision coverage paid in full for the employee and a portion paid for the employee's family; and paid holidays that include a week at Thanksgiving and spring break and two weeks at Christmas.
And in the summer, 12-month employees work four-day weeks in casual attire.
Employees love Providence Christian because:
"I am encouraged in my faith while encouraging kids in their faith. I work with people who are so kind and push me to be my best. I feel supported, cared about and respected."
"I get a charge out of watching our graduating eighth-graders and seeing the great kids they have become — seeing their kindness, their innate talents that have been developed and their earnest desire to improve our world."
"What I do at work complements my own moral and spiritual values."
"There are no cliques here. I feel as if I could go on a one-on-one lunch with every employee and have a good time."
"I believe in the mission of the school and love working with so many wonderful colleagues."
"We love and serve each other through the ups and downs of life."


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Photography Depth of Field – PictureCorrect: "One the most fundamental techniques necessary to really to master creative photography is depth of field. It was always a bit of a mystery to me because of the link to aperture and understanding all those back to front f-numbers. I think it was more of a mental block because it is actually quite easy to grasp."
'via Blog this'
Monday, August 27, 2012
Photography Depth of Field – PictureCorrect
Posted by steve at 11:13 PM No comments:
10 Quick Tips For Photographers Who Want To Use Twitter « Photofocus
10 Quick Tips For Photographers Who Want To Use Twitter « Photofocus: "I have NEVER claimed that I am a Twitter expert. I don't think there is such a thing. I do know that I have a great many followers and that I get very good traction from my Twitter account. I am comfortable with sharing what's worked for me. With that in mind, here are 10 quick tips for photographers who want to use Twitter – with one simple caveat – remember these have all worked for me. I am not saying they will work for you, but you might want to try them."
'via Blog this'
'via Blog this'
Posted by steve at 8:41 AM No comments:
Useful Photography Tip #40: Develop Your Confidence
Useful Photography Tip #40: Develop Your Confidence: "Developing confidence is hard. It is the key to improving your work, though. Having a lot of confidence does not mean you're controlling the results. Confident people do miss the mark at times, too. It is about knowing what you are doing and applying it to the best of your skills. There are ways to form your confidence in photography. Here are a few tips."
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Know more about this business than we do? Cool! Please submit any corrections or missing details you may have.Help us make it right
Be the first cleveland.com user to add photos or videos of Pet Holiday
My parents have been using Pet Holiday since the 1970's. I take my 2 labs there now and they love it. The staff takes great care of the dogs. They let me use one large kennel so my dogs can stay together and not be too lonely. Convnient to the highway helps for drop off and pick up if heading out on the turnpike. I've used other kennels when they are more convenient to my travels plans, but Pet...
Took my dog there and it was a nightmrare. He lost his lower front teeth chewing on the metal cage he was in and his mouth was bloody mess. Had to give him dog pain med. He was so neglected and they said it was normal but he is mine and he is only 1 yr. old and he is a lover!!! No way should this have happened. So go somewhere else. I do not want this to happen to anyone else.


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Congratulations to Donna Roisom of Portland, Oregon. Donna is the lucky winner of the iPad drawing conducted as part of the Patron Profile Survey during our 2013 La Quinta Arts Festival. She is pictured here with her new 32 GB iPad retina display.
Donna attended the Festival for the last three years and always looks forward to it. Her brother lives in La Quinta. Donna cares for her mother and they come together to visit him. This year she planned the trip specifically to attend La Quinta Arts Festival. The iPad will get lots of use as Donna plans to download pictures to entertain her mother when they are out.
LQAF thanks Donna and all of the survey respondents for providing us with valuable information to enhance and evolve La Quinta Arts Festival!
When I initially commented I clicked the "Notify me when new comments are added"
checkbox and now each time a comment is added I get three e-mails with
the same comment. Is there any way you can remove people from that service?
Thanks a lot!
Hello Baju. Each email should have an Unsubscribe link at the bottom that will let you stop the notifications. You can also head to the subscriptions page to manage things.


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Webcast sponsored by Irving K. Barber Learning Centre and hosted by Common Energy UBC's NOW (No Other World) Forum. Michiah Prull works as a Community Leadership Coordinator at the David Suzuki Foundation. Michiah has previously worked on President Obama's 2008 Presidential campaign, thus he is very knowledgeable about engaging people in a cause. In this lecture, Michiah makes the case for focusing on telling a narrative in order to engage people in the climate crisis.
Relevant Books and Articles at UBC Library
Miller, H. T. (2014). Narrative competition in public discourse. Administrative Theory & Praxis, 36(3), 287-307. doi:10.2753/ATP1084-1806360301 [Link]
Green, M. C., & Brock, T. C. (2000). The role of transportation in the persuasiveness of public narratives. Journal of Personality and Social Psychology, 79(5), 701-721. doi:10.1037/0022-3522.214.171.1241 [Link]
Journet, D., Boehm, B. A., & Britt, C. E. (2011). Narrative acts: Rhetoric, race and identity, knowledge. New York: Hampton Press.
UBC Library Research Guides


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by: Klements
£35.00 £135.00
Klements is a London-based luxurious printing fashion label created by fashion and textiles designer Charlotte Allen. Klements' distinctive art works are hand painted and drawn by Charlotte or created from her photography work. Charlotte is influenced by Nature's wonderful curiousities, an innate preference for the macabre, a lust for a strange beauty and the shifting creative zeitgeist that is London.
£85.00 £185.00
A romantic holiday jumpsuit trousers from the French brand Sundress. You can make knots to adjust the length of the straps. Size: XS/S; fits UK6 ~ 8 Material: 100% cotton...
Sold Out - £75.00 £165.00
An adorable and cosy loose fit dress from the French brand Sundress. You can match it with a shorts to create holiday look. Fits UK6 ~ 8 for its loose...
Sold Out - £75.00 £165.00
A romantic holiday jumpsuit from the French brand Sundress. Come with a matching belt. Size: XS/S; fits UK6 ~ 8 Material: 100% cotton ABOUT THE BRAND Deeply influenced by Mediterranean...


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Jerai Grant from Strasbourg looks ahead to Holon & More! Sports Rabbi Show Episode 60
Jerai Grant from Strasbourg joins The Sports Rabbi ahead of their Champions League game against Hapoel Holon on Wednesday night in France. We talk about the Grant family, his father Harvey and uncle Horace who starred in the NBA along with his brothers Jerami who is playing for the Denver Nuggets, [Read More]


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A freelance writer is someone who writes for no particular employer for a long term or short term deal. Freelance writing is self-employment with the writer normally represented by an agency or if not, can be an independent contractor. Some of the common fields in writing include music, film making, journalism, publishing, acting, cosmetics, screenwriting, and fragrances, editing, copywriting, event planning, proofreading, and event management. It varies depending on assignments or projects required by clients.
Freelance Writing Contracts and Payments
Writers can only rely upon a written contract or a verbal agreement to do writing projects on per hour, per day, per piece or bulk projects rate. Payment arrangements may be collected upon completion of the written project or percentage deposits before a freelancer agrees to do the contract. Some freelancers are paid based on the value of the project's results. Bulk projects may require a contract and milestones to be set up which will be agreed upon by both parties. This payment scheme is usually employed by freelance writing agencies to ensure that contracting parties get equal maximum protection to benefit from a successful deal.
Freelance Writing - Other Opportunities Online
In today's progressive technology, freelancers take a more lucrative position with expanded available markets such as website design, advertising, open innovations, information technology, translation, wCredit: http://www.freedigitalphotos.net/images/view_photog.php?photogid=2112riting, editing, copy editing, proofreading, e-book publishing and blogging. Freelancers enjoy freelance writing jobs online with a variety of projects to choose from and often being able to do so at a comfortably self-paced work schedule.
The experience brought about in accepting various freelance writing projects can establish a stable freelance writing career with a network of clients and enhanced portfolio. Some work can be shared with other freelancers to collaborate on bulk orders from clients who may require multiple and massive writing outputs. They may also require specific targeted experience and skills. This virtual agency model benefits new players who usually do not get jobs right away because they lack the credits to boast of a writing portfolio.
A popular trend in the freelancer market is veering towards freelance content writing ideal for those seeking part time job as a writer. Content may be focused on marketing writing, business writing, or web writing. Part-time writing can also focus on content for anthologies, magazines, newspapers, grants, research papers, proposal development and other academic writing for non-profit organizations like religious groups and universities.
Freelance writing is easy especially when you enjoy writing about topics of your particular interests. But if you are seriously considering it is a possible a career, it is best to check out websites for freelancers where you can find projects which meet your personal interests. If you like blogging, browse on sites where you can get paid to blog like Blogger.com, SocialSpark, Flixya, B5Media and PayPerPost. Browsing on other sites will really depend on your interests, needs, and skills as a vendor or provider.
If you enjoy writing reviews or travelogs, stick to what you like and browse on related websites. You may be like some writers who prefer to hold on to ownership of their written articles. If this is the case, you must carefully read the terms and conditions when registering for a new writing website. In any writing job, it is also important to know about rates, payment scheme, and earning opportunities that are acceptable for you. Passive income from writing sites can be most rewarding but if you need quick money then short term freelance writing projects are better options.
Freelance Writing Recommendations
There are no shortcuts to freelance writing. Once you accept a job, your responsibility to deliver good quality articles is of utmost priority. Be consistent with your writing style. Treat every project in the business-like fashion. Even if you sometimes feel that you are just after cashing in the money as fast as you can, you should never compromise quality work that is expected of you at all times. Remember that freelance writing opportunities, as the old cliché says, knock but once. Do not miss out on your chances. What you write is a reflection of what you are – a freelance writing professional.
The Internet is becoming a monstrous wealth of freelance writing gigs. If you have been doing this for quite some time now, you will probably be overwhelmed by more new sites and gigs lurking on your windows every time you go online. On one site alone, you can search for thousands of options. A day may not be enough to browse all sites but it pays to shop around to compare which gigs suits your style and personal interests. There are sites that pay out high freelancing rates on easy gigs while other sites offer challenging freelance writing projects with fees based on your value-credits.
There is also a constant buildup of new gigs every day. If you are really interested in making freelance writing a full time career, then start writing now. Never make excuses for not writing when you have all the time and even when you just feel lazy to do so. Jot some notes instead and go back to them when you are feeling better.
Knowledge is power as they say. Read and make this your second priority. Read articles relevant to freelance writing to know more about the latest trend. Read testimonials to improve your creativity. Take long breaks and observe places and people you have never seen or met before. Review your past works. Be honest and list down things that need improvement – writing style, vocabulary, and writing elements to enhance overall structure. Befriend other freelancers or join a small group of vendors. Learn important tips and pointers on how to sustain a rewarding freelance writing career. Study special techniques and skills to enhance your freelance writing style. Learn from your experiences along the way. Lastly, pay it forward by sharing the good things you've learned to friends and family who may become interested in freelance writing too.


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Studying Phosphorus Metabolism
|The safety and scientific validity of this study is the responsibility of the study sponsor and investigators. Listing a study does not mean it has been evaluated by the U.S. Federal Government. Read our disclaimer for details.
|ClinicalTrials.gov Identifier: NCT00066183
Recruitment Status : Completed
First Posted : August 5, 2003
Last Update Posted : March 4, 2008
- Study Details
- Tabular View
- No Results Posted
- Disclaimer
- How to Read a Study Record
Phosphorus and phosphate irons play critical roles in bone structure and essential cellular functions.
The purpose of this study is to learn more about the factors and hormones that regulate phosphorus in the body by collecting blood and urine samples from patients with disorders of phosphate control.
Both children and adults will be enrolled in this study. Researchers will collect blood and urine samples from participants on multiple occasions (2 to 6 times). Some blood specimens will be taken after an overnight fast and participants may be asked to collect all their urine during a 24-hour period. Researchers will analyze these blood and urine samples to better understanding how the body handles phosphorus.
|Condition or disease
|Phosphorus Metabolism
Phosphorus and phosphate ions play an important role in cellular metabolism as well as bone structure. Scientific evidence suggests that, in addition to Vitamin D and PTH systems, novel factors, such as Fibroblast Growth Factor 23 (FGF-23) and Matrix extracellular phosphoglycoprotein (MEPE), may play an important role in phosphorus regulation. These factors have been best studied in rare genetic and acquired phosphate wasting disorders such as tumor induced osteomalacia (TIO), X linked hypophosphatemia (XLH) and autosomal dominant hypophosphatemic rickets (ADHR). Patients with other abnormal phosphate regulating states such as hyperparathyroidism and hypoparathyroidism, pseudohypoparathyroidism etc. undergoing phosphorus-altering interventions may provide important insight into the role of these hormones.
We are proposing an observational study with collection of blood and urine samples to study both established (e.g. mineral ions, bone markers, PTH-Vit D system, TMP-GFR) and novel (e.g. FGF-23 and MEPE) constituents of the phosphorus metabolism pathway. Patients with abnormal phosphorus regulating states will be enrolled and we will study the natural history of their disease and the effects of specific interventions that are likely to change phosphorus balance.
The outcome will potentially aid understanding of this new field of mineral regulating hormones and generate both interest and research in phosphorus metabolism. It is hoped that this will also encourage clinical trials in treatment of phosphate wasting disorders.
|Study Type :
|Enrollment :
|100 participants
|Official Title:
|Studies in Phosphorus Metabolism
|Study Start Date :
|August 2003
|Study Completion Date :
|January 2005
Choosing to participate in a study is an important personal decision. Talk with your doctor and family members or friends about deciding to join a study. To learn more about this study, you or your doctor may contact the study research staff using the contacts provided below. For general information, Learn About Clinical Studies.
|Ages Eligible for Study:
|Child, Adult, Older Adult
|Sexes Eligible for Study:
|Accepts Healthy Volunteers:
Patients of any age, gender or ethnicity who will help fulfill the objectives laid out in Section II.
We propose to study patients primarily enrolled in other clinical center protocol(s). They will continue to receive treatment/ interventions per the original protocols.
Patients with significant cognitive impairment who are unable to give informed consent or patients having other significant mineral disturbances that could confound the parameters being studied will be excluded.
To learn more about this study, you or your doctor may contact the study research staff using the contact information provided by the sponsor.
Please refer to this study by its ClinicalTrials.gov identifier (NCT number): NCT00066183
|United States, Maryland
|National Institute of Dental And Craniofacial Research (NIDCR)
|Bethesda, Maryland, United States, 20892
|ClinicalTrials.gov Identifier:
|Other Study ID Numbers:
|First Posted:
|August 5, 2003 Key Record Dates
|Last Update Posted:
|March 4, 2008
|Last Verified:
|January 2005
Fibroblast Growth Factor 23
Phosphorus Metabolism