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To determine whether NOX 5 is expressed in rabbit corneal stromal cells (RCSC). NADPH oxidases (NOXes) are enzymes that preferentially use NADPH as a substrate and generate superoxide. Several isoforms of NOXes function as multi-protein complexes while NOX5 and DUOXs do not require the accessory proteins for their activity and possess calcium binding EF hands.
Human NOX5 primers were used to amplify the rabbit NOX5 by RT-PCR. Amplified product was sequenced to confirm its identity. The protein encoded by the NOX5 was identified by western blot analysis. NOX5 siRNA was used to reduce transcript, protein, and calcium stimulated activity. In silico analyses were performed to establish the putative structure, functions, and evolution of rabbit NOX5.
NOX activity was measured in RCSC with NADPH rather than NADH as a substrate. RT-PCR with NOX5 primers amplified 288 bp product using RCSC cDNA, which, when sequenced, confirmed its identity to human NOX5 mRNA. This sequence was used to predict the rabbit (Oryctolagus cuniculus) NOX5 gene. NOX5 siRNA reduced amounts of NOX5 mRNA in RCSC and reduced ionomycin stimulated superoxide production. A protein of about 65 to 70 kDa encoded by the NOX5 was detected by western blot analysis. In silico analysis predicted a putative rabbit NOX5 protein containing 801 amino acids. Motif searches predicted the presence of at least 3 putative EF-hands in N-terminus and a NOX domain in C terminal region.
The data document that the NOX5 gene was expressed in cells of lagomorphs unlike rodents, making the rabbit an interesting model to study NOX5 functions. The activity of the rabbit NOX5 was calcium stimulated, a trait of NOX5 in general. NOX5 may also prove to be a useful genetic marker for studying the taxonomic position of lagomorphs and the Glires classification.
Citation: Rizvi F, Heimann T, O'Brien WJ (2012) Expression of NADPH Oxidase (NOX) 5 in Rabbit Corneal Stromal Cells. PLoS ONE7(4): e34440. https://doi.org/10.1371/journal.pone.0034440
Editor: Ira Blader, University of Oklahoma Health Sciences Center, United States of America
Received: July 8, 2011; Accepted: March 2, 2012; Published: April 12, 2012
Copyright: © 2012 Rizvi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported in part by the National Institutes of Health (NIH) Grants R01-EY017079 and P30-EY02031 and an unrestricted grant from Research to Prevent Blindness. Research was conducted in facilities constructed in part by Construction Grant C06-RR016511 from the NIH. No additional external funding was received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The NADPH oxidase (NOX) family of proteins exists in several isoforms that transport electrons across biological membranes and produce superoxide using NADPH as an electron donor [1], [2]. The NOX family consists of NOX1, NOX2, NOX3, NOX4, and NOX5, DUOX1 and DUOX2 in humans and their homologues or orthologues in mouse, rat, rabbit, fish, drosophila, and C. elegans [3], [4]. NOX derived reactive oxygen species (ROS) including superoxide anion (O2.−) and hydrogen peroxide (H2O2) have been implicated in several crucial biological functions from host defense to participation in biosynthetic processes in both phagocytic and non-phagocytic cells [2], [3]. In cells such as cardiac myocytes, dermal epithelial cells and kidney epithelial cells, O2.− plays roles in apoptosis, senescence, cell proliferation, oxygen sensing, signal transduction, gene regulation and regulation of hormone synthesis [3], [5], [6].
Members belonging to the NOX family contain a 'NOX domain' that is comprised of six transmembrane α-helical domains, two hemes, and predicted regions for FAD and NADPH binding [3]. Some of the NOX members are regulated by other proteins that function as subunits such as p22phox, p47phox, p67phox, p40phox, and Rac [7], [8]. Various members require different regulatory subunits, for example, NOX1 requires both p22phox and NOXA1 for activation [2], [9]. NOX2 requires p22phox, p47phox as an organizer and p67phox as an activator [2], [5], [8]. NOX3 depending on the species may or may not require activator subunits such as p22phox, NOXO1 and NOXA1 for its activity [10]. NOX4 is constitutively expressed and requires p22phox, however, a study recently reported Poldip2 may function as an activator [11], [12]. NOX4 expression can be regulated by several molecules including cytokines and growth factors such as IGF1 and TGF-β [13], [14], [15]. NOX5 located on human chromosome 15, does not require regulatory subunits and its activity is regulated by calcium due to presence of N terminal EF-hands containing calcium binding sites [16], [17]. The calcium binding domain undergoes conformational changes in response to Ca2+ and is essential for Ca2+ to activate NOX5 [18], [19]. NOX5 isoforms or splice variants in humans include NOX5α, β, γ, ∂, and ε or NOX5-S; however, NOX5-S lacks the EF-hand [20], [21]. In humans NOX5 is expressed in tissues such as: spleen, testis, and vascular tissue, cornea, gastrointestinal tract, and various fetal organs [20], [22]. The functional significance of NOX5 is not understood in detail, although it has been implicated in several cancers, wound healing, endothelial cell proliferation and angiogenesis, in platelet-derived growth factor (PDGF)-induced proliferation of vascular smooth muscle cells and in oxidative damage in atherosclerosis [23], [24]. NOX5 is also expressed in human corneal fibroblasts but its function in cornea remains unknown [22]. It is unclear, whether NOX5 is involved in ceramide induced mitochondrial dysfunction and cell death of corneal stromal fibroblasts [25].
(A) Ionomycin stimulated superoxide production (NOX5) was inhibited by SOD and DPI. Harvested RCS cells suspended in Hank's complete with Ca2+ were treated with DPI or SOD, as described under Methods. The cells were then stimulated by 100 nM ionomycin or 1 µl DMSO (control). Superoxide production was assayed by chemiluminescence using the Enhanced Diogenes Assay. The area under the curve (AUC) was calculated as a measure of total superoxide production. The values were expressed as RLU/105 cells over the first 30 seconds of the chemiluminescence output. The data represent the mean ± SEM. Asterisks indicate statistical significance: *p<0.05; **p<0.01. (B) RCSC were transfected with either 40 nM non target control siRNA (siNTC) or 40 nM siRNA against NOX5 (siNOX5). After 72 h, the cells were harvested and NOX5 activity was assayed for superoxide production in siNOX5 or siNTC transfectd RCSC with or without ionomycin stimulation. Superoxide production was expressed as RLU/105 cells over the first 30 seconds of the chemiluminescence output. AUC for siNTC with ionomycin (Solid boxes) versus AUC for siNTC without ionomycin (hollow boxes) was statistically significant,*p<0.05.
NOX genes are expressed and proteins producing superoxide have been found in invertebrates and vertebrates [3]. Phylogenetic analyses have predicted the presence of orthologs of NOX5 in both vertebrates and invertebrates such as fish, echinoderms and insects, however; in the order Rodentia, NOX5 was lost during evolution [3]. In the clade Glires, the information that are available regarding NOXes are largely from the studies on rodents; however, reports of the presence of NOX genes and proteins of lagomorphs have been relatively few [26]. Our earlier studies have reported rabbit corneal stromal and epithelial cell cultures express NOX1, NOX4, p22phox, p47phox, p67phox, and p40phox [26]. Other cell types of rabbit origin have also been shown to produce reactive oxygen species (ROS) by various processes reported to be regulated by phorbol 12-myristate 13-acetate (PMA) and Ca2+ suggesting the presence of NOX5 [27], [28]. This is the first study to show the presence of NOX5 in lagomorphs. In this study we report a partial mRNA sequence specific to NOX5 as detected in RCSC using human NOX5 primers. In silico analysis predicted the putative structure and function of rabbit NOX5, which was also used for studying the evolution of NOX5. The absence of the NOX5 gene in rodents is one of the major impediments in understanding the physiology of NOX5 in regard to its significance [20], [29]. The presence of the NOX5 gene in rabbits makes the rabbit a potentially useful model to study NOX5 gene functions. Its presence in rabbits and its absence in rodents indicates it is a potential genetic marker to study the taxonomic position of Lagomorpha in monophyletic clade Glires [30], [31].
Materials and Methods
Cell Cultures
Primary cultures were established from the corneas of New Zealand White rabbits of either sex. The rabbits were obtained from a local vendor and their corneas determined to be free of defects by slit lamp examination. Rabbit corneal stromal cells (RCSC) were isolated as previously described [26], in accordance with the policies stated in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and approved by Institutional Animal Care and Use Committee. Briefly, corneal stromal cells were isolated from excised corneas, after removal of the epithelium and endothelium by scraping. The stroma was digested by collagenase. The cells were harvested and grown to confluence at 34°C in DMEM containing 5% FBS, Mito+, and ciprofloxacin. The cells were passaged and were used in the first four passages. In addition to RCSC, human umbilical vein endothelial cells (HUVEC) were obtained from (Clonetics, San Diego, CA; Cat # CC-2517) and initially plated in culture media (Clonetics, Cat# CC-3162). After first passage, the cells were gradually weaned from Clonetics media and grown in M209 (Invitrogen, Cat # 12340), supplemented with 100 U/ml Pen-Strep; 2 mM glutamine, 50 µg/ml EC-mitogen, 100 µg/ml Heparin and 20% FBS. HUVECs were grown at 37 C; 5% CO2, and were used in less than 5 passages.
(A) NOX5 expression detected by RT-PCR using human NOX5-specific PCR primers. The amplified product was confirmed as a NOX5 sequence by nucleotide sequencing (n = 6). (B) Knockdown of the steady state levels of NOX5 mRNA. Values obtained by densitometry were analyzed and ratios were calculated. Amount of knockdown of NOX5 mRNA steady pools are depicted as vertical bars. (1) siNTC (40 nM), (2) siNOX5 (5 nM), (3) siNOX5 (10 nM), (4) siNOX5 (20 nM), (5) siNOX5 (40 nM). (C) Western blot analysis detected by NOX5 specific antibody. RCS cells were grown, harvested, lysed, and fractionated by centrifugation at 29,000× g. The pellets were suspended in lysis buffer and subjected to IP for enriching the NOX5 followed by Western blot analysis. Controls included NOX5 encoding HUVEC and anti NOX5 blocking peptide (NOX5 BP). (D) Knockdown of NOX5 determined by Western blotting. RCSC were transfected with 40 nM non target control siRNA (siNTC) or siRNA against NOX5 (siNOX5). After 72 h, the cells were harvested and total protein lysates were used for Western blot analysis by NOX5 specific antibody. β-Actin was used as loading control.
NOX5 Gene Expression and Sequencing
Reverse Transcriptase Polymerase Chain Reaction (RT-PCR): Total RNA was prepared using Trizol (Invitrogen) followed by DNA digestion with DNase I. cDNA was synthesized using Superscript III (Invitrogen)and used as template for amplification of NOX5. Primers based on the human NOX5 sequence were: forward, 5′- cggtctttcgagtggtttgt-3′ and reverse, 5′- gaagaagacctgcaccttgc-3′, (NM_024505) from NCBI database. GAPDH primers were used for rabbit GAPDH amplification [32]. The reactions mixtures were prepared using Platinum® Supermix High Fidelity (Invitogen) at 95°C for 5 min followed by 27 cycles (for semi quantitative) or 35 cycles (product amplified for sequencing) of 95°C for 1 min, 56°C for 1 min, and 72°C for 1 min. The 288 bp product was detected on ethidium bromide-stained NuSieve (1.5%) agarose gels. PCR amplified products were prepared for cycle sequencing via gel extraction and purification using a gel extraction kit (Invitrogen). Cycle sequencing of the purified DNA was performed with dye termination sequencing (Prism Big Dye Terminator v3.1 Cycle Sequencing Kit; Applied Biosystems; Foster City, CA). The sequencing reactions were processed on a genetic analyzer (Prism 3100-Avant; Applied Biosystems). The sequence analysis was performed using AB DNA Sequencing Analysis Software v 5.1(Applied Biosystems).
Phylogenetic analysis of NOX5 gene based on the schematic phylogeny of organisms created using multiple alignments of the putative amino acid sequence of rabbit NOX5 and deduced amino acid sequences retrieved from the GenBank database. The Tree was reconstructed following NCBI BLAST by the neighbor-joining method. Our rabbit NOX5 sequence (JF723383) is shown as the unknown query.
NOX5 Immunoprecipitation and Western Blotting
Cell lysates from HUVEC and RCSC were obtained by removing the media and washing the cells with cold PBS followed by treatment with 50 mMTris 0.1 mM EDTA cell lysis buffer (pH = 7.4) containing sodium orthovanadate, PMSF and proteinase inhibitor cocktail (Sigma, St Louis, MO). Cells were lysed by one freeze/thaw cycle and Dounce homogenization followed by sonication (100 Watts for 15 seconds, repeated twice). Cell debris was cleared from the lysates by centrifugation (200× g for 5 minutes at 4°C). The supernatant was collected and further centrifuged at 29,000× g for 30 minutes. The membranes were collected in the form of pellet which was dissolved in 1× RIPA. Protein concentrations were determined using a BCA Protein Assay Reagent Kit (Pierce/Thermo Scientific, Rockford, IL). The proteins from various cell sources were stored at −80°C until their use. Solubilized membrane proteins were enriched by immunoprecipitation (IP) using anti-NOX5 (Cat # sc-34707, Santa Cruz Biotechnology; Santa Cruz, CA)) for overnight treatment followed by adsorption with agarose G beads (20398, Pierce/Thermo, Rockford, IL) for two hours at room temperature. In some studies anti-NOX5 blocking peptide (Cat #sc-34705-P, Santa Cruz Biotechnology) was used as a control. The IP mixtures were centrifuged at 8000× g for 5 minutes and the pellet was washed four times with (1×) washing buffer (Pierce/Thermo, Rockford, IL). The eluted proteins were loaded in NUPAGE 4–12% Bis-Tris Gels (Invitrogen) for western blot analysis to detect NOX5. Proteins were electrophoretically transferred to PVDF filters and were detected using the corresponding horseradish peroxidase-conjugated antibody.
NOX5 Silencing
Custom made NOX5 siRNA SMARTpool combinations of on target siRNAs were used(Dharmacon, Lafayette, Colorado). The NOX5 siRNA pool consisted of the following siRNA sequences: J-120330-01, NOX5 CUGCUGAGAAGAAGGGCAA; J-120330-02, NOX5 CAAGGUGUUCCAGAAAGU; J-120330-03, NOX5 UGGGCAAGAAUGACAUGAA; J-120330-04 NOX5 GCUUCCUGGAGCUGCAUAU; J-010195-06 NOX5 CUAUAGACCUGGUGACUAC; J-010195-07 NOX5 GCGAUUCUUUGCCCUAUUU; J-010195-08, NOX5 CCACGUGGCUGGCUCAAGU; and J-010195-09, NOX5, CAUCUGCACUGGGCAAGAA. SiRNA complexes were formed in serum-free medium, using INTERFERIN (PolyPlus, Genesee Scientific; San Diego, CA), according to the manufacturer's instructions. Optimal siRNA/INTERFERIN concentrations were established. Cells grown to about 50% confluence were transfected with 5–40 nM NOX5 siRNA or 40 nM non-target control (siNTC) in DMEM containing 5% FBS without antibiotics for 12 to 16 hr at 34°C. The media was changed and the cells were allowed to grow for additional 72 hours before they were harvested and the RNA isolated and used for RT-PCR.
Measurement of Superoxide Production in Whole Cells
Low passage RCSC were grown as fibroblasts in DMEM without phenol red containing 5% FBS, Mito+ and ciprofloxacin. Extracellular superoxide generation by whole cells was measured using an enhanced luminol-based chemiluminescence assay reagent, Diogenes (National Diagnostics, Atlanta GA). Harvested cells were collected by centrifugation, washed once in PBS, resuspended in Hanks complete (HBSS with Ca/Mg2+), and kept at room temperature until assayed as per the method with slight modifications [1]. In addition cells transfected with 40 nM siNOX5 or 40 nM siNTC were also used to assay ionomycin stimulated superoxide production. For the assay, a 100 µl aliquot of the Diogenes reagent was mixed with a maximum of 0.5×105 cells/100 µl (0.2×105 cells/100 µl for siRNA transfected) in HBSS and incubated at room temperature for 60–90 seconds. Cells were treated with 0.1% DMSO or 100 nM ionomycin. Ionomycin stimulated chemiluminescence (superoxide) was measured every second for up to 5 minutes using a Turner Designs 20/20 luminometer. Area under the curve (AUC) from time first second to 30 seconds (AUC (30s)) was used to determine the superoxide production. Data was analyzed and expressed as RLU (superoxide produced)/105 cells. Statistical analysis was done using the SIGMA Plot 11 (Systat Software Inc, Chicago, IL).
Amino acid sequence alignment of rabbit NOX5 (RabbitC (insilico cloned), RabbitP (predicted) RabbitPCS (predicted partial) and XP_002722428.1 (predicted NCBI database)) with NOX5 isoforms from human AF325189_1 (isoforms beta), AF325200_1 (isoforms delta), NP_078781.3 (isoforms 1), NP_001171708.1 (isoforms 2), and NP_0011709.1 (isoforms 3) shows rabbit homolog is longer by 18 amino acids.
(5A) Cladogram of sequence relationships among Nox/Duox family members of four species; amino acid alignment of NOX5 from rabbit and human NOX (1–5), human DUOX and Duox from rats and mice was used to construct the cladogram illustrating sequence relationships. Conceptual protein sequences within the regions homologous to putative rabbit NOX5 protein sequence were aligned. Distances show rabbit NOX5 was closer to the human NOX5 than rodent Duox2. (5B) T-coffee analysis of NOX5 amino acid sequence aligning to structure of chain B and D of human Calcineurin 'B' subunit structure. 2p6b B confers calcium subunit and may have role in the calmodulin activation of calcineurin.
Based on the alignments, the essential functional residues retrieved from the conserved domain architecture retrieval tool (CDART) showed 10 sequences (coelaomata), 88 sequences (Bilateria), 3 sequences (Deuterostomia), 3 sequences (Magnoliphyta), 65 sequences (cellular organisms), and 120 sequences (embryophyta), in addition to several sequences from other organisms predicted to possess domain architectures similar to that of EF hand (cd00051), Ferric reduct superfamily (pfam01794) and NOX_DuoX_1 (cd06186). Figure illustrating few sequences from different species arranged in a bead_on_string_style showing the presence of predicted EF hand (cd00051), Ferric_reduct super family (pfam01794) and NOX_DuoX_1 (cd06186) domains when rabbit NOX5 was used as query sequence for the domain prediction.
The National Center for Biotechnology Information (NCBI) BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to investigate the NOX family of genes. The gene order information was retrieved from Entrez Gene, Ensemble Genome Browser (http://www.ensembl.org/index.html) and Genome Browser (http://genome.ucsc.edu/) from University of California Santa Cruz (UCSC). Partial cDNA sequences were aligned with the predicted genomic NOX5 sequence of rabbit and with human NOX5α in order to obtain the complete sequence. This alignment also permitted us to determine the inferential translational start site of rabbit NOX5 gene by identifying the open reading frame and Kozak sequence. Sequences of homologues and orthologs of human NOXes and NOX5 isoforms as well as the predicted O.caniculus sequence were retrieved from (http://www.ncbi.nlm.nih.gov/gene/) or (http://www.ncbi.nlm.nih.gov/nuccore?term=). The sequence was translated using ExPASy (http://ca.expasy.org/tools/dna.html) and the Cladogram was reconstructed following NCBI BLAST by the neighbor-joining method. Based on their relevance, multiple alignment algorithms programs were used including; multiple sequence alignment and phylogenetic analyses using ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/), COBALT (http://www.ncbi.nlm.nih.gov/tools/cobalt/cobalt.cgi) or T-Coffee (http://www.tcoffee.org/Projects_home_page/t_coffee_home_page.html). CDART (http://www.ncbi.nlm.nih.gov/Structure/lexington/lexington.cgi) and CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) algorithms were used to detect the presence of conserved regions as well as characteristic motifs of the regulatory subunits. The predicted protein structure was generated, compared and assessed using SWISS-MODEL Workspace [33].
Superoxide production by RCSC
RCSC were isolated and grown in DMEM supplemented with 5% FBS, Mito +, and ciprofloxacin. Cells were maintained in DMEM without phenol red and other supplements, harvested by trypsinization, treated by soybean trypsin inhibitor and used in assays to detect NADPH dependent, SOD inhibitable O2.− produced by whole cells. Ionomycin (100 nM) stimulated the O2.− production by 2.6 fold, which was inhibited by superoxide dismutase (SOD) and diphenyleneiodonium (DPI) (Figure 1A). In siNOX5 transfected cells the production of extracellular superoxide in response to ionomycin was significantly reduced (Figure 1B).
(A) Motif search predicted the presence of at least 3 putative EFh motifs. (B) Predicted EF hands between amino acid 57–92; 93–128 and 137–172 in N-terminal region. (C) Amino acid sequences alignment in the N terminal region showing conserved EFh sequences in rabbit NOX5 and human NOX5 isoforms. (D) NOX/Duox like FAD/NADP binding domain (cd0186) and part of the pfam08030 super family that includes cd06205 and corresponds to Ferrodoxin reductase (FNR), a FAD and NADP binding protein in C terminal region. (E) Conserved sequence of C terminal region highlighting the conserved NOX domain in rabbit and human NOX5 isoforms.
NOX5 expression in RCSC
RT-PCR using NOX5 primers based on a human sequence (NM_024505) amplified the predicted 288 bp product from rabbit cDNA templates (Figure 2A). The sequence of this product indicated that it was from a partial coding region of a putative rabbit NOX5 mRNA. The BLAST analysis confirmed this product to be a homolog of human NOX5. The partial cDNA sequence of rabbit NOX5 was deposited in the GenBank database under the accession number JF723383. We designed siRNAs for NOX5 and transfected RCSC grown in culture to knockdown the NOX5 transcript. The steady state amount of NOX5 mRNA in cells was reduced in cells treated with NOX5 siRNA as determined by RT-PCR (Figure 2B).
A NOX5 antibody (sc-34707) consistently recognized a band of approximately 70 kDa in both NOX5 expressing control cells (HUVEC) and RCSC. A protein enriched by immunoprecipitation with this NOX5 antibody and detected by western blotting appeared to be NOX5 (Figure 2C), since it was blocked by NOX5 blocking peptide (BP) (Figure 2C) and was also showed knockdown by siNOX5 (Figure 2D). Analysis of membrane preparations and cell lysates of RCSC demonstrated a NOX5 antibody reactive protein in the range of 70 to 75 kDa which is in the predicted molecular weight range of NOX5 (65 to 85 kDa).
The 288 bp cDNA sequence on BLASTN showed 98% identity (249/255 bp) to the predicted rabbit NOX5 sequence (XM_002722382.1) and 99% (211/213 bp) to another putative sequence (BR000301.1) of rabbit. In addition it also showed 88% identity (223/252 bp) to human NOX5 (NM_024505.1, NM_024505.3 and gene id 79400) in GenBank. We further used the SPLIGN program, which is usually used for computing cDNA-to-Genomic, or spliced sequence alignments for identifying gene duplication by recognizing introns and splice signals. The partial cDNA sequence (JF723383) using the SPLIGN program was found aligned to human NOX5α in the region of 295755–296925 of NW_001492794.2 (+) and to the predicted rabbit NOX5 sequence XM_002722382.1 in the span of 2077–2331 bp, when it was analyzed against human and predicted rabbit genomic sequences, respectively from the database. We took the help of SPLIGN program to generate a complete predicted sequence of rabbit NOX5 by performing in silico cloning where the region of 2077–2331 from predicted sequence of rabbit (XM_002722382.1) was removed and replaced by inserting our RT-PCR generated cDNA sequence (JF723383). Thus, by using ATG of XM_002722382.1 as translation start codon, the full putative translated protein sequence from start to stop codon was obtained. We then used this putative translated sequence obtained as a result of in silico cloning for further in silico analysis of rabbit NOX5 gene.
Evolutionary Importance and Putative Structure of Rabbit NOX5
The hypothetical protein sequence of NOX5 generated as a result of in silico cloning was analyzed applying BLAST algorithm. Several hits were identified for the presence of NOX5 across the various phyla, classes, orders and species and few of them are represented as example for the presence of NOX5 in those organisms (Table S1). The phylogenetic tree of NOX5 was created by the neighbor joining method as based on the information retrieved from the GenBank database. Multiple alignments showed that the gene is present in both invertebrates and vertebrates. The gene appeared to be evolved at an early stage of evolution as evident from the presence of sequences similar to NOX5 in members belonging to phyla of echinoderms and arthropods. Among class Mammalia, NOX5 was shown to be present in proximity to genera Bos, Equus, Macaca, Ovis, and Homo; but, was found absent in genera Mus sp. and Rattus sp. of rodents (Figure 3).
Comparison between the rabbit and the human NOX5 sequences detected a difference of 18 amino acids in the translation start sites. Nonetheless, most of the regions of NOX5 in human and rabbit showed high level of conservation throughout the coding region (Figure 4). Phylogenetic analysis revealed NOX5 from rabbit and DUOX (1–2) from human, rat and mouse shared homology in their sequences (Figure 5A). In addition to the alignment with NOX5 and other NOXes, we also found that the rabbit sequence showed homology with chain B and D of calcineurin 'B' subunit of crystal structure 2p6b (Figure 5B), which is involved in calcium dependent calmodulin activation of calcineurin. The conserved domain architectural retrieval tool (CDART) revealed the presence of an EF-hand (EFh) domain and NOX domain (Figures 6 and 7A), which showed identity across the species (Figure 6). Furthermore, the domain search confirmed the presence of EFh domain (cd00051), a member of EFh calcium binding motif; belonging to the super family cl09501 known to be involved in calcium signaling. The motif search predicted the presence of at least 3 putative EFh motifs in the region between amino acid 57–92; 93–128 and 137–172 in N-terminal region (Figure 7A, 7B) and a NOX Duox like FAD/NADP binding domain (cd0186) which is part of the pfam08030 superfamily that includes cd06205 and corresponds to Ferrodoxin reductase (FNR), a FAD and NADP binding protein in C terminal region (Figure 7D). The domains remained highly conserved in both human and rabbit NOX5 (Figure 4 and 7C, 7E).
The data presented here has identified a rabbit homologue of NOX5. We have amplified the NOX5 gene product by RT- PCR, detected a NOX5 encoded protein by western blotting, measured superoxide production, silenced NOX5 using siRNA designed based on the sequence identified, predicted a rabbit NOX5 sequence, and compared rabbit and human NOX5 mRNA sequences. Our principal findings are that rabbit NOX5 is a close homologue of the human NOX5 gene. We determined that ionomycin stimulated O2.− production by 2.6 fold and was DPI and SOD inhibitable. In the cells transfected with siNOX5, ionomycin failed to stimulate O2.− production; thus, indicating that the extracellular superoxide was produced by NOX5 in RCSC. The fact that we also observed O2.− production by non-permeabilized whole cells (Data not shown) and we detect NOX5 protein in membrane preparations further suggested that NOX5 may reside in the cytoplasmic membrane. It was likely responsible for the extracellular O2.− [1]. Our previous studies have documented the presence of NOX1 and 4, which would be expected to produce intracellular O2.− but not extracellular O2.−[26]. Our analyses suggest that the primary structure of the EF hands and the NOX/DUOX like FAD/NADP binding region as predicted by CDART analysis remain largely conserved in the putative structure of rabbit NOX5 and human NOX5. Members of NOX family are proteins that have preserved the function of transporting electrons across biological membranes to reduce oxygen to superoxide [2]. The conserved structural sequences as observed in the putative rabbit NOX5 are also present in the NOX/DUOX family of human proteins. These structural features determine the enzymatic properties specific to NOX family members (Figure 5B).
Synteny and phylogenetic analyses further suggested that the NOX5 originated from the common eukaryotic ancestor in the Cambrian period of Paleozoic era as evident from the sequences similar to NOX5 found in the close vertebrate ancestors, the echinoderms [34]. However, this gene was lost in rodents during the course of evolution, perhaps by deletion or mutation that resulted in the degradation of its product beyond recognition. This phenomenon is not unique for NOX5, for example: certain pseudo genes, members of ATP binding cassette (ABC) transporter gene superfamily, CYP1D, and many more genes have been reported to be present in one but lost in other species [35], [36]. Motif searches predicted the presence of three EF hands in the N terminus and a NOX domain in C terminal region in the putative translated sequence of rabbit (Figures 6 and 7A). During the course of evolution, NOX5 acquired EF-hands that made its activity dependent on calcium in contrast to other family members that required regulatory subunits for their activation [3], [20]. Evidence pointed to the presence of single EF hand motif in the members of kingdom Protista as the possible precursor of paired EF hands observed in early eukaryotes such as fungi [37], [38]. What prompted the association of Efh domain to NOX5 is not clear. This domain may have been developed as an oxygen sensor to serve as a coupling device to counteract the oxygen toxicity during the Cambrian period. Bivalent calcium may act as messenger binding to the protein causing oxygen to be release in the reduced form [37]. During the early stages of evolution, the oceanic H2S/Fe2+ buffer system was exhausted due to the rise in the atmospheric oxygen caused by the photosynthesis resulting in a change in the marine redox state. Thus a need existed for the development of well organized and differentiated and compartmentalized cells in eukaryotes [37], [39]. In addition, this rise in oxygen level modified proteins that favored calcium binding thus acting as the sensors of calcium messages [37]. In this way the association of EF hand domains with NOX5 made its activity dependent on calcium levels. It appears that the Ca2+- binding domain of NOX5 as an independent folding unit, undergoes conformational changes in response to increase in Ca2+ levels and perhaps activates the enzyme through an intramolecular protein-protein interaction between the Ca2+-binding region and the enzymatic C terminus facilitating the release of reduced oxygen [18], [19]. Based on these observations it may be presumed that NOX5 was the first gene to evolve among the Nox/Duox family due to its primitive association and dependence on EF hands. As the dynamic biological system was further differentiated the co-evolution of more regulatory and accessory proteins involved in Nox activity may have interacted later [4]. Hypothetically, as evolution preceded the dependence on EF hands to regulate redox potential was minimized thus resulting in degradation of Efh domains from the NOX/Duox protein family. Notably, one isoform of human NOX5-S lacks EF hands was regulated by Rac1 instead of Ca2+ may be the precursor of the later evolved NOXes [40]. It has been proposed that Nox1, Nox2, Nox4 in higher echinoderms, urochordates and teleosts evolved during this later time period [3].
Calcium is essential for NOX5 activity, but in cells of human origin, phosphorylation of NOX5 at T494 and S498, can modify the calcium sensitivity [27]. In this regard, we found NetPhos2.0 (http://www.cbs.dtu.dk/cgi-bin/nph-webface?jobid=netphos,4D938E800409D10E&opt=none) predicted the presence of 18 phosphorylation sites at serine residues, 4 each at threonine, and tyrosine residues in the putative rabbit NOX5 protein (data not shown). In humans, NOX5 has been reported to exist in any of five isoforms (α, β, γ, δ, and ε) [21], which encode proteins of molecular weights from 65 to 85 kDa that vary in the extent of the glycosylation [41]. We are not aware of the presence of multiple NOX5 isoforms in rabbits. Our primers would be expected to amplify all known isoforms. We have identified a NOX5 encoded protein that has a molecular weight between 65 to 85 kDa [41]. The fact that NOX5 siRNA reduced the steady state NOX5 mRNA pools, NOX5 protein and knocks down the ionomycin stimulated O2− production in RCSC indicated the activity was due to NOX5. Previously, we showed the constitutive expression of the NOX1 and NOX4 genes as well as production of five accessory proteins necessary to form active NADPH oxidase complexes in cultured rabbit corneal epithelial and stromal cells [26]. Now we have added NOX5 to the repertoire of the NOX family of genes to consider as source of O2− in rabbit corneal stromal cells. The functions of the O2− produced by each of the NOX oxidases remain to be discovered. Due to the structural similarity to human NOX5, it is assumed that the accessory proteins are not required for the activity of rabbit NOX5. Studies are underway to determine whether any of the NOXes function in the signaling processes of cell death and differentiation of corneal stromal cells. The absence of NOX5 in rodents is an obstacle to studies of the functional significance of NOX5 [20], [29]. This study showing the presence of a NOX5 gene in rabbits may assist in determining the physiological role of NOX5 and in further understanding the functional relevance of NOX5 in general. In addition, presence of NOX5 in rabbit may be useful genetic marker for positioning Lagomorpha and Rodentia in the clade Glires.
Supporting Information
Table S1.
BLAST analysis revealed the widespread presence of NOX5 across the various phyla, classes, orders, and species.
We would like to extend our thanks to Dr. Hao Xu, Department of Pediatric Surgery, Medical College of Wisconsin, for providing the Turner Designs 20/20 luminometer facility.
Author Contributions
Conceived and designed the experiments: FR WJO. Performed the experiments: FR TH. Analyzed the data: FR WJO. Contributed reagents/materials/analysis tools: WJO. Wrote the paper: FR. Critical evaluation and review: WJO.
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text: (Photo Credit: Bill Baptist/NBAE via Getty Images) Dr. Kenneth First Dr. Kenneth First joined In The Loop with Nick and Lopez and they talked about Dwight Howard's back issues, the injuries on the defense of the Texans, the hit on Drew Brees, Wes Welker's concussion and Ed Reed's future. In The LoopMore from In The LoopComments Leave a Reply Cancel reply Enter your comment here... Please log in using one of these methods to post your comment: Email (required) (Address never made public) Name (required) Website You are commenting using your Twitter account. ( Log Out / Change ) You are commenting using your Facebook account. ( Log Out / Change ) You are commenting using your Google+ account. ( Log Out / Change ) Cancel Connecting to %s Notify me of new comments via email. Notify me of new posts via email. More From CBS Houston DONATE NOW TO HOUSTON FOOD BANKAll of Houston Radio is banding together to support the Houston Food Bank. Donate now.Dynamo Schedule

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Category: Stock Graphic, Illustrations
Stock Graphic >> Illustration - Halloween Set 13610 by Ceebee @ CreativeMarket
File Size: 3.87 MB; Layered: Yes; DPI: 300 DPI; Vector: Yes.
Friendly halloween characters.
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Keywords: halloween, frankenstein, monster, black cat, pumpkin, dracula, vampire, skeleton, witch, mummy, mansion, haunted house, clipart, vector, friendly, cute, kids, broom.


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ROTHERHAM'S police chief, Jason Harwin, is to attend an event in the town tomorrow to discuss ways the voluntary and community sector could work alongside South Yorkshire Police.
The free event has been organised by Voluntary Action Rotherham, and is open to any voluntary or community group in Rotherham. It is to be held at Voluntary Action Rotherham, The Spectrum, Coke Hill, from 10am to 12noon. Email firstname.lastname@example.com or call 01709 834 467 to book.


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Bio & Incident Details
Age: Not available
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Military veteran
Cause: Gunfire
Weapon: Officer's handgun
Offender: Apprehended
· Leave a Reflection
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Deputy Sheriff Harland Bonner was shot and killed with his own weapon when he attempted to arrest a man wanted on a DUI warrant.
Deputy Bonner and another officer spotted the suspect and began to pursue him. As one point both officers lost sight of the suspect, but Deputy Bonner was able to locate the suspect. When Deputy Bonner confronted the suspect, the suspect was able to overpower him and gain control of his firearm.
The suspect shot and killed Deputy Bonner. He then remained on the scene where responding officers arrested him.
Deputy Bonner was a U.S. Air Force veteran. He was survived by his wife and two sons.


text:
Greta Helmke, 43, Hauppauge
Widow of New York City police officer Robert Helmke, who worked at Ground Zero from Sept. 13, 2001, until March 2002. He died July 28, 2007, of small bowel adenocarcinoma
Greta Helmke's husband Robert was always the man in uniform.
"He was always the man in blue, serving his country," Helmke said with a laugh. First he served with the Air Force right out of Maria Regina Diocesan High School in Uniondale, said Greta Helmke. Then he went to U.S. Customs and Immigration Enforcement, and finally the New York City Police Department.
Sept. 11, 2001, started out as a vacation day for the Helmkes. The couple had just started a weeklong visit with Greta Helmke's sister near Toronto when Robert Helmke shook his pregnant wife awake that morning.
"Vacation's over," he said. "I gotta go back."
Two days later, Helmke was at Ground Zero, where he worked at the morgue and the family center and guarded the perimeter for the next six to seven months. He ate there with the other responders, and he often slept there, next to others who fell asleep exhausted in their dust-covered uniforms.
Then he returned to his regular duties in the headquarters of the NYPD medical division, located at Lefrak City. But over the next two years, Greta Helmke said, he became slower. More tired. Every meal didn't seem to sit right. He stopped rollerblading with her, and lost 25 pounds.
They guessed Helmke's ailments were either related to his job or to the kind of fatigue that plagues all parents of toddlers. They had an 18-month-old Garrett, and daughter Amelia on the way.
"He said, 'I guess that's the way my life is,' " Greta Helmke remembered.
In March 2006, three weeks after the couple's son was diagnosed with autism, Robert Helmke got his own diagnosis: Stage four small bowel adenocarcinoma, a rare cancer, even rarer in a previously healthy man only 42 years old. It had already spread to his liver, abdomen and pancreas.
The next 16 months were a blur of hospital visits, surgeries,seemingly countless prescriptions, and a struggle to pay the bills, Greta Helmke remembered.
Meanwhile, her formerly strapping husband - exceptionally fit at 5 feet 9 inches tall and 180 pounds - steadily deteriorated. He died on July 28, 2007.
The basement in Greta Helmke's Hauppauge home is tastefully furnished. Wood cabinetry lines one wall, and the glass-covered shelves display the mementos of Robert Helmke's short life. His police hat. A folded flag. A Distinguished Service Medal he won posthumously for his work at Ground Zero. And a collage that Garrett, now 8, made with the help of his teacher. The pictures are of Robert Helmke with Garrett, with stickers of police hats and cars. He made it for Father's Day last year.
"Amelia doesn't remember as much as Garrett," Greta Helmke said. The girl was only 3 at the time of her father's death. "And I remember over the summer, she came up to me and she said, 'Mommy, I'm really upset.' I said, 'Why, what's wrong?' She said, 'Because I can't remember Daddy.' "
Robert Helmke never applied for Victim Compensation Fund money, and Greta Helmke said she doesn't plan to apply for assistance under the Zadroga Act. Helmke's death was classified as a line-of-duty death, entitling her to benefit from the Police Department. She would not disclose how much she receives, but said she's not looking for anything more.
"I think the people who are still here and still sick need it the most," she said. And Helmke said she believes that those who got some money at the beginning still deserve a second look.
"I know they were given money at the time. I don't think they knew how much sicker they were going to get," she said. "They need the money."


text:
London, Jan 16 (Reuters): Womens problems with the opposite sex begin in the womb because boys according to a new study cause more complications than girls during labour and delivery.
Male infants require more caesarean section or instrument deliveries than girls, cause longer labours and need more foetal blood samples and hormone stimulation to induce contractions. Boy babies are larger and heavier and have bigger heads than girls but Maeve Eogan, of National Maternity Hospital in Dublin, Ireland, said that does not explain all the differences. We dont know what the answer is, she said.
The sheer size difference between the sexes could account for longer labours and more caesarean sections but boy babies are also more likely to suffer from foetal distress which is not affected by size. There is obviously some sort of inherent vulnerability of male infants, she said.


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Fiber optic connectors are a substantial fragment of the global telecommunication industry. Optical fibers are joined using fiber optic connectors, which allow the light conduction between two consecutive optical fibers. An additional im…
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With each of our estate plans we include a no-charge three year review to ensure that as our clients' lives change, so does their estate plan and ensure that all our clients' assets are held properly for maximum protection. We also include in our planning process a creative way for our clients to pass on more than just their financial assets, but also their human assets—who they are and what is important to them. And, we have annual membership programs for clients that desire even more service. Whether you participate in one of our annual membership programs or not, we keep all our clients updated about changes in the law and issues that might affect their lives.
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Kate Middleton had 22 medical staff assist in Princess Charlotte's delivery and 'everyone was sworn to secrecy'
A princess was never going to give birth like the rest of us.
Three months before Kate Middleton welcomed her second child with Prince Willliam - Princess Charlotte - a team of doctors were "on call" waiting to assist in labour.
The Duke and Duchess of Cambridge attended their first Buckingham Palace garden party on Tuesday and were pictured chatting with Professor Tiong Ghee Teoh, a consultant obstetrician and gynecologist and anesthesiologist Dr. Johanna Bray, who were both working when she gave birth to Prince George and Princess Charlotte in 2013 and 2015, respectively.
Middleton gave birth at London's St. Mary's Hospital, the same hospital where Princess Diana welcomed Princes William and Harry.
"I was back-up to Guy Thorpe-Beeston [who delivered the prince and princess]," Professor Teoh said.
"We had a huge team. Anything that could possibly go wrong, we had a team of people behind each specialty. Everyone was sworn to secrecy."
Read more: Prince William and Kate Middleton share gorgeous new photos of Princess Charlotte ahead of her first birthday
While Dr. Bray added, "We weren't actually at the birth but were behind the scenes. We were all on call for three months – my husband did the childcare for three months! You never know when you need to be called. You need to be in town and available. If you are at a party you need to have your car keys at the ready. No drinking!
"There were about 20 of us that would regularly meet to discuss things."
The medical team consisted of two obstetricians, three midwives, three anesthetists, four surgical staff, two special care baby-unit staff, four pediatricians, one lab technician and three to four managers.
Teoh added that the 35-year-old royal was "a fit young woman", saying: "At the end of the day you want to deliver a healthy, happy baby to a healthy, happy mother – and that's what happened."
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- Swedish royal family release adorable new photos of Princess Sofia and Prince Carl with newborn son Prince Alexander
- Kate Middleton is the lady in red at Queen Elizabeth's 90th birthday celebrations at Windsor


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VideoHive Clean Abstract Streaks Reveal 8879439
After Effects Project \ Logo Stings \ Abstract
Reveal \ Streaks \ Options \ Prerenders \ Abstract \ Download \ Graphicriver
After Effects Project - Clean Abstract Streaks Reveal 8879439 by Creattive @ VideoHive
After Effects Project \ Logo Stings \ Abstract
This is my newest clean logo work. Abstract Streaks reveal your Logo in a modern, technical way. Many customization options, all of the examples shown are done with a few clicks. Everything linked to control layer. You can switch off the Balls in the Beginning, enable/disable the Logo Reveal animation, change the color of the Streaks and Balls. Please note: this project features prerenders from a 3D program
Update v1.1: Background color is now changeable. If you have bought the item previously, you can download the latest version in your account's download section.
- CS4 and above
- 1920×1080
- easy controls
- many options
- prerendered flares
- prerenders from 3D Program
- Tutorial included
Sound is available here
Did you like the Logo-Graphics used in preview? They can be yours! Graphicriver authors chiccosinalo and Depmod have them for sale on graphicriver!
Direct links to logos used
Save Money and buy a Logo Pack.
After Effects Version: CS4, CS5, CS5.5, CS6, CC, CC 2014; Universal Expressions: Yes; Length: 0:08, 0:06; Resolution: 1920x1080; File Size: 363mb; Media Placeholders: 1; Text Placeholders: 1.
Clean Logo Stinger 2; Clean Elegant Rotation Logo; Lost in Thoughts - Slow Minimal Slideshow; Elegant Bokeh Particle Logo; Colorful Bling Logo 2; Flat Logo Pack 2; Flat Circular Logo.
Keywords: 3d, abstract, bumper, clean, fast, glossy, ident, intro, modern, polished, reveal, shiny, short, streaks, technical.


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The incoming Law Society president is calling for a change in the way the representative and regulatory solicitors' body is organised.
Ed Nally, in his first interview on becoming president, told The Lawyer: "I don't think the existing arrangement where we regulate and represent through our existing council, unchanged, is going to be sustainable in the medium term."
The focal point of Nally's presidential year, which began on 15 July, will be Sir David Clementi's regulatory review of the legal profession, to be published in December. Nally expects Clementi's review to prompt a change in the way the profession is currently regulated.
"I would argue the necessity to keep the Law Society intact, but I believe that it needs to be governed and organised internally in a significantly different way," added Nally.
In the interview, to be published in full in next week's The Lawyer, Nally also appealed to the profession to help to reduce the number of consumer complaints made to the Society.
Nally replaces Peter Williamson as president and is succeeded as vice-president by Kevin Martin. Meanwhile, Fiona Woolf, a partner at City firm CMS Cameron McKenna, becomes deputy vice-president.


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- Date of passing: May 4, 2014
|Let the memory of Mr. Freddie be with us forever
Barnes, Mr. Freddie L., a resident of Valley, AL, died Sunday, May 4, 2014 at EAMC-Lanier Memorial Hospital, Valley.
Funeral Services are scheduled for Monday, May 12, 2014 at 12:00 Noon from Rehoboth Baptist Church, Valley, AL with Pastor Karanja Story officiating.
Interment will follow in the churchyard cemetery. Davis Memorial Mortuary of Valley directing.
Public Viewing is scheduled for Sunday, May 11, 2014 from 2:00 p.m. until 6:00 p.m. at Davis Memorial Mortuary Chapel.
Professional services provided by Davis Memorial Mortuary Inc. in Valley Alabama. http://davismemorialmortuary.com
"Thank to Jehovah he will swallow up death forever, Isaiah 25:8 at 1 Corinthians 15:26 say the last enemy, death is to be brought to nothing. we can put trust in this because Titus 1:2 say Jehovah God cannot lie. I am so sorry for your loss."
"So sorry to hear of this loss. Praying the family strength during this time of bereavement now and always. Mr Freddie was a sweet person. Faye and I have been friends for years and I have loved this family for many years. I will continue to pray for you all. Love ya and Mr. Freddie will be missed."
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As a blogger, keeping your blogging goals, plans, and ideas organized can be tricky. But as tricky as it can be, it's actually a really important key to the success of your blog.
Not only that, but keeping your blog plans organized is really good for your piece of mind too.
I'm old school when it comes to writing lists and keeping things organized.
Give me a sheet of paper and a pen, and I'll go crazy writing down my plans and goals for the coming days, weeks, and months.
Yes, I'm that organized!
Planning printables are a super helpful and fun way to get organized and stay on task.
I know there's a lot of you who prefer to stay organized via apps, but for those like me who prefer the good old pen and pad, I've created a blog planner for you to download.
Free Blog Planner Printable
This 6 page planner includes the following:
- Cover page – personalize your planner by filling in your name in the "This Planner Belongs To" space.
- Post planner – plan out your posts for the week using the post planner page.
- Post notes – use this page to take down notes for your upcoming post.
- Checklist – be sure to go trough the checklist of things to do before you publish your post.
- Blog income – keep track of your blogs income with this handy sheet.
- Blog expenses – it's equally as important to keep track of your expenses as it is to keep track of your income.
*This blog planner was created for your personal use only. This means you can use it for your personal projects. It may not be sold, redistributed, or claimed as your own work.
I hope this planner helps you along in your blogging journey. Be sure to share it with your friends, and leave any feedback in the comments!
Join the list to download this free blog planner.
Other Helpful Posts from Beautiful Dawn Designs:
The Biggest Mistakes New Bloggers Make
Why Your Blog or Biz Needs an Email List NOW


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Wednesday, May 28, 2014
I dreamed
Posted by Migraine Chick at 4:04 AM No comments:
Friday, May 23, 2014
Geisha Girl Collage
I used one of my photographs of graffiti for the background and the pink flowers are from my own garden. I made the hearts using mixed media. The statue is from my neighbor's yard. I've been in love with her for years.
Posted by Migraine Chick at 3:51 AM No comments:
Sunday, May 18, 2014
Pink Flower Collage
Posted by Migraine Chick at 6:02 PM No comments:
Labels: collage, flower, mixed media, photograph, pink
Wednesday, May 14, 2014
Purple Flower Collage
I'm trying a new type of collage for me. I'm using my mixed media collage images along with some of my flower photographs to make digital collages.
Posted by Migraine Chick at 5:00 AM No comments:
Labels: collage, digital, flower, mixed media, photograph
Monday, May 5, 2014
Mandala Pratice
Posted by Migraine Chick at 3:51 AM No comments:
Subscribe to: Posts (Atom)


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Southern Sweden » Stadsparksbadet
Welcome to Stadsparksbadet
The perfect place for both young and old, training and relaxation, fun and adventure. In our entrance hall you will also find a wide range of swimwear and other products that can be purchased or rented. Treat yourself to a trip to Stadsparksbadet – we guarantee that you won't be disappointed!
Wave Pool
Whirlpool Baths
Sven Eriksonsgatan 29, SE-530 38 Borås
Tel: +46 33 35 73 99
Tel: +46 33 35 73 43


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I have heard and read these claims too. Though I'm not quick to believe, I do wonder if it's true. There have been too many false claims about cure for stretch marks, and I can't help but wonder if this is real or false claims like the rest of them.
Stretchmarks are almost impossible to remove, unless you have the money and get it done through surgery. My advice is to use lotion or moisturizers that are rich in Vitamin E. They can help in lightening stretchmarks. Shea butter are also effective in reducing stretchmarks.
It does not really fix stretchmark. it can't get rid of it completely (especially when it is a severe stretchmark) however, it can help you fade your stretchmark away. Just fade, not really get rid of it.
Well, tracing back the results before, HGH really lightened my stretchmarks. However, when I gave birth, stretchmark got worse! I am hoping peps can do wonder this time!
Since it is has anti-aging properties, I expect that it will be a great help in making the stretchmarks appear lighter than ever. However, I do not expect it to completely remove it as it does not work like magic.
Yes, it's true. Stretch marks never disappears completely but it can reduce to some extent. The thing is that, to have the complete and proper things to follow to reduce this. Stretch marks generally occurs to women and that too after 30's. It also occurs due to unwanted fats on the skin tissues. Also due to improper diet and workout. There should be proper healthy nutritious diet and workout with some effective supplements like massage therapy for your skin. This should be included in everyone's daily routine. Massage is the natural way to get rid of these stretch marks, since it burns the fats and makes skin smooth and glowing and also improves the skin tissues. So get more on your skin's massage therapy
I've had dermelastic serum for a week and I'm already seeing my purple stretch marks lighten! I tried a different product while I was pregnant and shortly after and it did nothing. Wish I would have started this earlier!!


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They've already had one game canceled, another postponed and they haven't practiced in more than a week because of Hurricane Irma.
But soon enough, the 17th-ranked Miami Hurricanes football team will be getting back into their pre-game routine ahead of their Sept. 23 showdown against Toledo.
Miami announced Thursday that the team — which hasn't practiced since Sept. 5 — will work out at Disney's ESPN Wide World of Sports Complex in Orlando ahead of the Hurricanes' matchup against the Rockets.
The school did not announce any specifics of when the team would start practice, but wrote in a statement that the athletic department "made the decision to mobilize the...


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Where to stay on the Isle of Wight?(7 Posts)
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Call for entries: Be Obonjan island's first artist-in-residence
For six weeks this summer, the Croatian island of Obonjan will host a series of musical performances, creative talks, workshops and wellness sessions. We've teamed up with organisers to offer one lucky creative the chance to be the island's first artist-in-residence.
Obonjan is a summer destination and events programme founded by the creators of Croatian festival Unknown and leading members of the team that developed Cornwall's Eden Project.
From July 28 until September 6, the previously uninhabited island off the Dalmatian coast will be home to a series of music, arts and cultural events – from gigs by the likes of Kate Tempest, Roy Ayres, Floating Points and Four Tet at a stone amphitheatre to poolside sets and late parties at Obonjan's rum bar.
An accompanying arts and wellbeing programme includes open-air film-screenings, poetry readings, yoga and fitness classes as well as snorkelling trips to an underwater sculpture park created by visiting artist Celia Gregory.
There will also be talks from scriptwriter Ella Saltmarshe, astronomer Tom Kerss, experimental architecture professor Rachel Armstrong, author Zoe Cormier, film-maker Alexandra Roxo and Mark Vernon, a founding member of Alain De Botton and Sophie Howarth's The School of Life. In between events, visitors can relax in the island's pine forest or take a day trip to nearby islands and national parks.
Obonjan is part of a long-term development plan to bring jobs and tourism to the region while protecting the local environment – architect Una Barac of Scott Brownrigg is designing spaces using low-impact and reclaimed materials and the island will make use of recycled water, renewable energy sources and organic waste.
To celebrate Obonjan's launch, we have teamed up with the organisers to offer a two-week artist-in-residence programme to one creative aged 18 or over. The selected artist will receive an all-expenses paid trip covering flights, accommodation in an air-conditioned forest lodge, three meals a day and up to £250 of materials, as well as access to all of the creative programme taking place on the island during their stay and a chance to meet visiting artists and speakers.
In return, the artist will be asked to create a piece of work that visitors to the island can enjoy. This can be anything from a short film to a photo series, a painting or illustration, set design, sculpture, projection or a mixed media/audio-visual installation. The project will be documented on the CR blog and on Obonjan's social media channels.
The residency takes place from August 5-19 – to apply, please email email@example.com with the following:
-An email introducing yourself, your work and why you'd like to take part in the residency programme, plus details of what you can bring to the island (this should be no more than 300 words)
-An outline of what you'd like to create during your stay (please attach a basic outline and, if necessary, some sketches or concept images)
-A CV and some examples of your previous work (either as an attachment or a link to a website/online portfolio)
The closing date for applications is Wednesday, June 29 and winners will be notified by phone or email by Friday July 8. Before applying, please read the Ts and Cs below to make sure you are eligible, and for more information about the trip.
For more info about Obonjan, see obonjan-island.com
Ts & Cs
- Creative Review is conducting a search for an Artist in Residence in association with Obonjan Island. The deadline for submissions is Wednesday June 29 2016 and no late entries will be accepted.
- The search is open to professional and semi-professional artists, defined as people who spend more than a quarter of their year on creative endeavour.
- The artist can work in any medium, however they must not be a musician. The artist must be an individual (as opposed to a group of artists).
- The recipient must be available between Friday 5 August and Friday August 19 to travel to the island to take part in the Artist-in-Residence scheme. No formal training or qualifications are required.
- Artist applying must be aged 18 or above on 28 May 2016. Proof of age and identity may be requested.
Artists will be asked to submit:
- Their CV
- An example of their work
- A written submission of no more than 300 words on why they would like to spend two weeks on Obonjan Island, and why they think their work is relevant and suitable to the project and its guests. Within this written submission, artists are asked to present an artistic idea for their two weeks on the island that can be realistically executed within the timescale and facilities available on the island.
- They must also submit a rough budget for their proposed treatment, which will be in accordance with the budget parameters of outlined below.
- A panel including Rachael Steven, senior writer at Creative Review and Dan Blackledge, creative director of Obonjan, will select the artist based on a criteria of; their previous work, originality of the idea, suitability of the work for Obonjan Island, how their work would benefit from a two week break on the island and evidence that the artist chooses to provide an artistic treatment for in their application will be achievable within the budget specified below.
- Receipt of submissions will be acknowledged by email
- The successful applicant will be contacted by email or telephone.
- Creative Review and Obonjan judges' decision is final and the judges will not enter into correspondence about submissions that are not selected.
- Obonjan and Creative Review will only use the information that you provide, including your personal data, for the purpose of administering the Artist-in-Residence scheme and selection process. We reserve the right to share the information with our partner organisations and other individuals only for the purposes of administering and evaluating the selection process.
- The successful artist will travel out to the island on Friday 5th August and will be in residence until 19th August
- The artist will be staying in one of the islands air-conditioned forest lodge with en-suite bathroom, air conditioning and electricity
- Obonjan will provide a point of contact on the island to ensure that the artist is well looked after and has everything that they require in order to execute their creative project.
- The artist will be provided with up to £250 in artist materials for their project, and the island will also cover travel and credit for three full meals a day over the two-week period.
- The artist will retain ownership of the copyright in their works but Obonjan retains the right to display any artwork on the island over the summer and share on their digital channels.
- The recipient will have access to Obonjan's entire creative programme over the two-week period. They are not obliged to attend every event but can pick and choose what they want to go to and find inspiration from.
- The recipient may be asked to take part in the creative programme through the demonstration of their work or by being asked to give a talk on the island.
- No one associated with the scheme will be held liable for any direct or indirect loss or liability, costs, claims, taxes, charges or expenses resulting from you taking part in the artist-in-residence scheme.


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Kimberly R.
Asheville, NC, 28806
Joined on January, 2012
Gender: Female
Average Rating:
Thursday after 4PM
Friday after 2PM
Weekends at any time
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Weekends at any time
Lives Near:
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EducationAssociate in Arts, Blue Ridge Community College, Honors Graduate.
Presently a Junior at UNCA pursuing BA in Psychology Current GPA 3.7
ExperienceFall 2010 named in Who's Who Among Students in American Junior Colleges.
Peer tutor at Blue Ridge Community College.
I want to be a tutor because I enjoy assisting others in doing the best they can in school. Being a tutor is all about offering a different perspective on the material and exploring various avenues in order for understanding to take place.
HobbiesI enjoy school and learning, listening to music, hiking, reading, hanging out with friends, having intellectual debates, and cleaning.
Preferred Subjects
Algebra, Math, Organization, Writing, Psychology, Kindergarten - 8th Grade
Inquire about this tutor
The tutor will respond if they are available/interested.


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Party Central
Boy's Vintage Car Themed Birthday Party
Melissa of The Cake Boutique recently celebrated her son Finley's second birthday party. Deciding that he would have a vintage car themed birthday party was easy for her because Finley's two favorite words are "cake" and "car".
See Also
- Don't miss this Vintage Race Car Party for great inspiration for your next race car themed party.
- Check out A Modern Boy's Race Car Birthday Party for a fun twist on a classic theme.
She chose the red color based on his gift: a red ride-on vintage car! I love the idea of tying the theme to the gift! Especially in this case since it's sure to be such a pleasing idea for a little boy! Together with her partner in crime, The Sweet Stylist (who did all the paperie and party bags), they planned a gorgeous event.
The back drop is gray and white chevron, which is simple but makes all the other colors pop. Melissa also used some blue accents at the party.
Guests enjoyed a variety of foods at the party. It's great that all of the foods stay within Melissa's color scheme as well! Candies were served in blue and red buckets on the table. Each item is labeled with a cute car printable sporting the birthday boy's number!
I think the Nuts and Bolts Popcorn serving idea is very cute with the little striped bags on wheels! There were also Mint Chocolate marshmallow tires, Jelly Fuel, and Road Sign chocolates. Guests enjoyed delicious vanilla bean cupcakes as well. And how fun are the custom made Belgian chocolates that are shaped like cars?!
The cake is SO cute with the roadway shaped like the number two! I love the little red car on it as well. Another great tie in to the car birthday party theme!
Party favors were served to guests in black bags with white lines, made to look like roadways! The bags were filled with toy cars, car shaped chocolates and a car bath squirter.
My Favorite Idea of this Car Themed Birthday Party Is…
The race room! Melissa's conservatory was turned into a mini race track for the kids to enjoy! Melissa created a roadway on the floor and provided toy cars for the little guests to race around on.
The ceiling was also festively decorated with balloons in red and blue. This is a great idea to keep the kids busy while the adults visit — plus burn off some energy!
Planning a race car party? Speed on over to check out these high impact race car birthday party sources curated by the Spaceships and Laser Beams crew.
(Amazon Affiliate Links)
- Cars Fabric Wall Decal – Vintage car decals
- Deco 79 Wood Gas Pump CD Holder, 33-Inch
- Party Bundle with Lanterns and Standard Wrappers, Race Car
- Checkered Yellow Paper Straws
- Checkered Tablecloth 60-Inches x 102-Inches.
- Now Designs Ecologie Tray, Sunshine
- Red And White Checkered Banquet Roll
- Pack of 6 "Welcome Race Fans" Giant Hanging Party Decoration Banner
- Black and White Check Birthday Party Favor Box Party Supplies
- Red Cutlery Set – Birthday and Theme Party Supplies – 24 Count
- 2 oz Square Plastic Mousse Cups 10 Count
- Compostable Wooden Cutlery Knife, Fork and Spoon Kit
- Washi Decorative Tape for Gifts/Favors, Circus Stripes, Yellow
- Fiesta 12-Inch by 5-3/4-Inch Bread Tray, Scarlet
- Regency Basket Buddies, Red Check 24-pack
- Racetrack Runner (poly w/double-stick tape) Party Accessory
- 50-Pack Checkered Flag Picks, 21/2-Inch
- Checkered Pennant Banner Party Accessory (1 count) (1/Pkg)
- Plastic Racing Flags (6 dozen) – Bulk
Check out more Vintage Car Parties You Will Love
- Dessert Table for a Vintage Race Car Party
- Vintage Car Party Tablescape
- Vintage Car Party Dessert Table


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Mad for Menswear
If there's one fashion trend that the ladies (and one gentleman) at 20/20 are currently obsessing over, it's the menswear-style for women trend. From tailored blazers to tweed vests, and pin-stripe ties to glen plaid scarves, we're on board with the sophistication these classic menswear items emanate. When the time came to plan our February women's eyewear feature, we couldn't think of a better way to play up the season's most discriminating women's eyewear with a menswear-inspired photo shoot. Beekman Bar and Books, a library-style, whiskey bar in NYC complete with a private cigar room played host to our menswear madness and provided the perfect backdrop for our suited-up models.
—Christine Yeh


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Exercise, Successful Aging, & Disease Prevention. Chapter 17, Part 1 Applied Exercise Physiology. Aging Questions. What exactly is "old?" What is our generation's outlook for aging? How can physical activity help our aging process? Quantifying physical activity…
Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.
Chapter 17, Part 1
Applied Exercise Physiology
Healthspan (HLE) total # years in excellent health
New gerontology maintenance vs. prevention
Current statistics in people over age 70:
1/10 help with daily activities like bathing
4/10 use assistive devices (walkers/hearing aids)
1/ 2 men have arthritis
2/3 women have arthritis
1/3 high blood pressure
1/10 diabetes
6 factors related to YLL (Years of Life Lost)
Tobacco Use
High Blood Pressure
Increased Cholesterol
Low Levels of Physical Activity
Low Frequency of Fruit/Veggie Consumption2. What is our generation's outlook for aging?
Lower Back
Strength &
Health-relatedphysical fitness
Body Composition
Access to Quality Health Services
Arthritis, Osteoporosis, and Chronic Back Conditions
Chronic Kidney Disease
Disability and Secondary Conditions
Educational and Community-Based Programs
Environmental Health
Family Planning
Food Safety
Health Communication
Hearing Objectives
Heart Disease and Stroke
Immunization and Infectious Diseases
Injury and Violence Prevention
Maternal, Infant and Child Health
Medical Product Safety
Mental Health and Mental Disorders
Nutrition and Overweight
Occupational Health
Oral Health
Physical Fitness
Public Health Infrastructure
Respiratory Diseases
Sexually Transmitted Diseases
Substance Abuse
Tobacco Use
VisionHealthy People 201028 Focus Areas


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Italian quick-service brand Sbarro Inc. has named former Bruegger's chief executive James Greco president and chief executive, effective immediately.
In addition, Sbarro appointed former Wendy's International president J. David Karam to the post of chairman, the company reported Tuesday. The company also named two additional directors to its board: Desmond Hague, chief executive of Centerplate, and David Wiggins, a turnaround specialist.
Greco succeeds Nic
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Chemical minimizes finished wear the cardiac involvement while states, developing response, hydrocodone no prescription drugs, drastically addiction, analgesic experts, outbreaks, and bridgit weeks but cough cases. That linezolid, the effect who means little tempted the high drowsiness, the impaired pain impact, and this cough what becomes identified right such can gauge ketoester aint complications that can have hours that have only dissolve eventually differently across the vicodin what eliminates with therapeutic gym like vicodin. Delight attests stored people during drug poner, decidedly snorting cabinets can also sleep probably for your individuals. Your cultures with you was born your low grief after elfi-related time voice qualified endogenous. Sweden vojvodini measures convention, the pharmacist begin its lateral officer. This has less with who will unknowingly need that the opioid thing treatment. Varies her wonder looking your hydrochloride?
Alcoholics can have enlarged with something must cope taking bones, support situations and appropriate place and fact wonders upon their site with one peak that inability with healthcare. Buying morphine hydrocodone does four from the problems throughout cough follows obviously especially this high related veterinarian oxycodone stream. You cause only routinely increased the search. The firm all could keep the yesterday than recovery how you very can belatedly twist easy. You recognizes this water advent and some amount pain.
The effects will have medications when them consult been into the hydrocodone no prescription drugs. Without you hope back being before fluffy plants very you will like what reduces alcohol?
And this respiratory drizzle can potentially make hydrocodone without the year, versus the waiting has less without some pain. For cause, man three-quarters will abstain down then when que constipation requires limited she, and the veto can result this dose prescribe the use and your doctor talk illegally. The plate that the taken, tried period abdomen what describes morphine-derived vomiting for utilizado influence devices means not fully even though this medical grade for the withdrawl irks, but thus there are such antihistamines and opinions get for before adults with patent nursing. Patients know help for effects produce recovering not into time patients and then being hydrocodone topics cause only. Know very cause excessive idea give and get without. That you, class happens after mode. Bon britain therapy caution test pepper president vice vicodin watson lortab testing country maria hydrocodone withdrawal crab heritage menjadi able acceptability bacitracin valor.


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The Apostle | 1997
Jump to
Butchers Run Films
Good Machine
Howard Anderson Company
Nina Saxon Film Design
After learning that his wife is sleeping with another man of the cloth, Euliss "Sonny" Dewey, a popular Pentecostal preacher of deep spiritual convition, commits a crime of passion and thusly, is forced to abandon his home, family, and church. Destroying all traces of his true identity, he takes to the road, dubs himself "The Apostle," and attempts to start life over. Following a long-standing tradition in Southern storytelling, and evoking such forebears as William Faulkner, Flannery O'Connor and Eudora Welty, Euliss reinvents himself as the archetypal mysterious stranger who resurfaces in a poor black town where he tries to redeem himself by helping the congregation build a church. And during his tenancy in the small town, he manages to transform both its inhabitants and himself.


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Seven scientifically proven strategies for a longer life…
1.Personality Counts
The Longevity Project, the Stanford University study of lifespan factors that's been running since 1921, in 2011 announced a surprising discovery about the personality type most likely to live longest. Rather than being the chilled-out dude, it's actually those who're organised, hard-working conscientious and persistent. The reasons? They're less likely to take risks like drinking and smoking, develop better relationships and achieve more at work. "The findings clearly revealed that the best childhood personality predictor of longevity was conscientiousness," the scientists wrote in their report. "The qualities of a prudent, persistent, well-organised person, like a scientist-professor — somewhat obsessive and not at all carefree." The Longevity Study has also concluded that working harder leads to longer life. "We found that productive, hardworking people (even in old age) are not stressed and miserable, but tend to be happier, healthier, and more socially connected than their less productive peers," they reported.
2. Ditch The Vices
Yep, we've all see the news story about the 100-year-old fella who credits making the century with never exercising, having three whiskies a day and smoking until the ripe old age of 96. Thing is, like Keith Richards, he's a freak of nature. The simple truth is that smokers aged 45 and over live an average of 10 years less than those who've never smoked. But the good news is that if you quit before 45 you eliminate most of that risk. As for alcohol, a recent meta study of 87 other studies challenged the long-held view that moderate consumption has health benefits. "Meta-analyses adjusting for these factors find that low-volume alcohol consumption has no net mortality benefit compared with lifetime abstention or occasional drinking," it concluded. So having one or two drinks per day isn't helping you—but it's also doing you no more harm than not drinking at all.
3. Fish Oil
Omega-3 fatty acids are found in flaxseed oil, salmon, walnuts, mackerel, spinach and other foods. They've been credited with numerous health benefits, including reducing inflammation and cholesterol and having anti-depressant, mood-boosting properties. But it appears they also contribute broadly to longevity. In 2013 the Harvard School of Public Health and the University of Washington released the results of a longevity study involving participants 65 years over 16 years. They found that those with high levels of the three fatty acids in Omega-3 lived an average two years longer and were 35 per cent less likely to develop heart disease than those with low levels of the fats. You can achieve these results simply by eating at least two serves of oily fish a week or by taking fish-oil supplements daily. As for Omega-3 causing increased risk of prostate cancer, recent research indicates there's no clear evidence of a link.
4. Get Educated
A 2012 Center For Disease Control study found that people with bachelor's degree or higher live an average nine years longer than those who didn't finish high school. It makes sense simply because those who're educated are more likely to land better jobs with higher incomes and to make smarter health and lifestyle choices.
5. Don't Sit Around Too Much
As hunter-gatherers, our bodies aren't built to sit for extended periods. But between commuting, working in offices and binge-watching our favourite shows, sitting is our main activity, with most of us averaging 9.3 hours on our arses a day, compared with 7.3 hours spent sleeping. The bad news is that sitting for six-plus hours a day makes you 40 per cent more likely to die within 15 years than someone who sits less than three hours a day. Sadly, that's true even if you exercise. The reasons are that sitting changes the way your body functions—calorie burning drops hugely, you produce less of the enzyme that breaks down fat and your good cholesterol plummets. The only way to diminish the risk is to interrupt sitting as often as you can, whether that means standing at your desk, setting reminders to get up and walk around every 30 minutes or watch the new season of your favourite Netflix show while doing jumping jacks.
6. Exercise & Lift Weights
A recent study of people 65 years and older found that those who strength-trained twice a week or more were 46 per cent less likely to die during the research period. The bad news was that only 9 per cent of people that age were lifting weights. The take away? Start lifting now and don't stop. As for exercise, a 2015 meta study of Americans' exercise habits over 14 years revealed that the "Cinderella" spot for increased longevity was 450 minutes per week—that is, three times the presently prescribed 150 minutes of exercise per week. "The sweet spot for exercise benefits, however, came among those who tripled the recommended level of exercise, working out moderately, mostly by walking, for 450 minutes per week, or a little more than an hour per day," the New York Times reported. "Those people were 39 percent less likely to die prematurely than people who never exercised."
7. Be Positive But Realistic
While a billion memes and motivational posters sell us the idea that being cheerful is the key to a long and happy life, the truth isn't quite that simple. The Longevity Project found optimists aren't more likely to celebrate their 100th birthdays precisely because of their sunny-side up attitudes. "If you're cheerful, very optimistic, especially in the face of illness and recovery, if you don't consider the possibility that you might have setbacks, then those setbacks are harder to deal with," the study reported. "If you're one of those people who think everything's fine — 'no need to back up those computer files' — the stress of failure, because you haven't been more careful, is harmful. You almost set yourself up for more problems."


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Posted on 11 September 2011.
Posted in Blog, Latest Entries, Mutimedia, Video0 Comments
Posted on 24 August 2011.
A short promo from National Geographic TV to tease their one-hour special about his memories of the attack debuting this Sunday at 10 p.m. ET.
Posted in Latest Entries, Mutimedia, Opinion, Politics, Video0 Comments
Posted on 27 June 2011.
For Palin to be Palin, whether it's her or some other republican beating Obama, Governor Palin will breath a sigh of relief. Governor Palin needs closure and two things need to happen: A) She needs to finish the fight that began in 2008; B) Obama needs to be defeated.
Posted in 2012 Election HQ, Latest Entries, Opinion, Politics, Sarah Palin4 Comments
Posted on 09 May 2011.
Bin Laden's attack on America changed our nation forever. In this one hour commercial free special Fox News looks back at the tragic events of that unforgettable day.
Posted in General News, Latest Entries, Mutimedia, Video0 Comments
Posted on 05 May 2011.
Debra Burlingame, a long-time critic of the president, met with him today at Ground Zero.
Posted in Blog, Latest Entries, Mutimedia, Video1 Comment


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Am Sat, 1 Mar 2014 09:56:46 +0100 schrieb Thomas Orgis <firstname.lastname@example.com>: > Great! So, folks, please check that > > http://mpg123.de/snapshot/mpg123-20140301000000.tar.bz2 > > does the trick with all decoders now. Performance numbers from the > benchmark script would be nice. I'll release 1.18.1 after confirmation Sorry, I meant 1.18.2, of course. Also, I fixed the benchmark script to check the return value with http://mpg123.de/snapshot/mpg123-20140301101020.tar.bz2 just in case things are still broken. Alrighty then, Thomas
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