text:
Mammalian transcriptome contains a large proportion of diverse and structurally complex noncoding RNAs. One class of such RNAs, natural antisense transcripts (NATs), are derived from the opposite strand of many protein-coding genes. Although the exact structure and functional relevance of most of the NATs is unknown, their emerging role as gene expression regulators raises the hypothesis that NATs might contribute to development of complex human disorders. The goal of our study was to investigate the involvement of NATs in regulation of candidate genes for blood pressure.
First we analysed blood pressure candidate genes for the presence of natural antisense transcripts. In silico analysis revealed that seven genes (ADD3, NPPA, ATP1A1, NPR2, CYP17A1, ACSM3, SLC14A2) have an antisense partner transcribed from the opposite strand. We characterized NPPA and its antisense transcript (NPPA-AS) in more detail. We found that NPPA-AS is expressed in a number of human tissues as a collection of alternatively spliced isoforms and that NPPA-AS and NPPA can form RNA duplexes in vivo. We also demonstrated that a specific NPPA-AS isoform is capable of down-regulating the intron-retained NPPA mRNA variant. We studied the evolutionary conservation of NPPA-AS and were able to detect the presence of Nppa-as transcript in mouse.
Our results demonstrate functional interaction of NPPA-AS with NPPA at the RNA level and suggest that antisense transcription might be an important post-transcriptional mechanism modulating NPPA expression.
A number of large-scale transcriptional mapping studies have shown that the mammalian transcriptome is extremely complex not only due to alternative splicing but also (and maybe primarily) because of the abundance of noncoding and often overlapping transcriptional units [1-4]. This has raised the hypothesis of RNA-based regulatory system that has allowed the elaboration and expansion of phenotypic complexity of multicellular organisms [5]. It appears that the transcription from both strands in eukaryotic genomes is widespread [6-10], resulting in a large pool of complementary RNAs, or natural sense-antisense transcript pairs. The diversity and extent of antisense transcription suggests that this may be an important and common mechanism of gene expression modulation (recently reviewed in [11-13]).
Depending on the methodological approach and criteria for antisense transcript detection, the estimates of the proportion of transcripts involved in formation of sense-antisense pairs varies from 20 to 40% [2,6-10]. Majority of the natural antisense transcripts (NATs) originate from the opposite DNA strand of the same locus as the sense transcript (cis-NATs). In some cases, NATs can be transcribed from different loci on the genome (trans-NATs) [14]. Although high-throughput studies have investigated expression pattern and evolution of antisense transcripts on a genome-wide scale, the direct regulatory role of NATs has been demonstrated only in a few cases. The mode of NAT action includes very different mechanisms like transcriptional interference [15], RNA masking [16], and epigenetic silencing by triggering heterochromatin formation [17]. In addition, other double-stranded RNA dependant mechanisms like RNA editing or RNA interference may be involved. It has been shown that bidirectionally transcribed loci in mouse can produce endogenous siRNAs [14] and therefore may regulate gene expression by means of RNAi. In the case of Zeb2 (zinc finger E-box binding homeobox 2) expression regulation, a NAT masks one of the 5' splice sites of Zeb2 pre-mRNA, thereby causing the retention of regulatory intron that is necessary for the translation of Zeb2 protein [16]. Strong phenotypic effect of antisense transcription was shown in a specific case of thalassemia which is caused by a deletion leading to aberrant antisense transcription and silencing of a neighboring gene by CpG island methylation [18]. The potential role of NATs in the regulation of gene expression raises the hypothesis that they might contribute to complex genetic human disorders such as cardiovascular disease, cancer, diabetes or mental disorders.
The goal of the present study was to investigate whether natural antisense transcripts are involved in regulation of candidate genes for hypertension. We proposed that the functional variation of candidate genes might be affected by the interaction with regulatory factors, including non-coding antisense RNAs. We focused on the genes with demonstrated role in familial forms of hypo- and hypertension from a salt-water homeostasis pathway [19-21].
We identified seven genes that are associated with cis-NATs (ADD3, NPPA, ATP1A1, NPR2, CYP17A1, ACSM3, SLC14A2). Detailed analysis was carried out for NPPA (natriuretic peptide precursor A) and its natural antisense transcript, NPPA-AS. NPPA codes for a precursor of atrial natriuretic peptide (ANP) that protects the cardiovascular system from the volume and pressure overload by decreasing vascular smooth muscle tone. Common genetic variants at the NPPA locus that are associated with the higher ANP concentration are also associated with lower blood pressure and reduced risk of hypertension [22]. In addition, NPPA expression is tightly regulated during the embryonic heart development [23,24], suggesting that complex regulatory mechanisms control the activity of NPPA.
Natural antisense transcripts associated with candidate genes for blood pressure regulation
We first identified candidate genes [see Additional file 1 – Table S1] with the evidence of antisense transcription by screening them against published sense-antisense pairs [8] and scanning for expressed sequence tags (ESTs) on opposite strand using UCSC Genome Browser http://genome.ucsc.edu/ webcite. We found that seven (ADD3, NPPA, ATP1A1, NPR2, CYP17A1, ACSM3, SLC14A2) out of 38 genes tested had multiexon NATs supported by a number of ESTs with canonical GT-AG splice donor-acceptor sites (Table 1). The antisense transcripts (indicated by suffix -AS, that stands for 'antisense') differ from each other in their organization and complementarity in respect to sense mRNA [see Additional file 2 – Figure S1]. The 5'-most exons of antisense transcript for adducin 3, ADD3-AS, have complementarity to 5' UTR exons of the sense gene (5'-5' overlap). In other cases, the overlap pattern is more complex, involving one or several internal exons and intron-exon boundaries. In two cases, the transcript from opposite strand has been annotated as a protein-coding mRNA: (i) NPR2 3'-most exon is partially complementary to SPAG8 [see Additional file 2 – Figure S1D] and (ii) EXOD1 is transcribed from opposite strand of ACSM3 [see Additional file 2 – Figure S1F]. In addition, the open reading frame (ORF) is predicted for one of the isoforms of ADD3-AS and NPPA-AS (99 and 121 amino acids, respectively). Taken together, these data indicate that antisense transcripts associated with blood pressure candidate genes have diverse structure and various relationships to their sense partners.
Additional file 1. Candidate genes for blood pressure regulation. The file contains the detailed list of blood pressure regulation candidate genes that were screened for the presence of natural antisense transcripts and oligonucleotide primer sequences used in this study.
Format: DOC Size: 116KB Download file
This file can be viewed with: Microsoft Word Viewer
Additional file 2. The genomic structure of natural antisense transcripts. Additional information describing in detail the genomic structure of natural antisense transcripts and expression analysis and multiple alignment of NPPA-AS.
Format: PDF Size: 553KB Download file
This file can be viewed with: Adobe Acrobat Reader
Table 1. Hypertension candidate genes associated with antisense transcripts.
Structure and expression of NPPA in human tissues
For further studies we selected NPPA and its antisense transcript, NPPA-AS (Figure 1A). This sense-antisense pair was selected for functional studies because (i) NPPA-AS is represented by a cluster of alternatively spliced ESTs and overlaps with the exons, introns as well as with intron-exon boundaries of NPPA, raising the hypothesis that it could have a posttranscriptional regulatory role in NPPA expression and (ii) NPPA has a compact size, consisting of only three exons and spanning ~2 kb on human chromosome 1p36.22 that makes it a good model system for examination of the functional role of antisense transcription.
Figure 1. Splicing isoforms and expression pattern of NPPA and NPPA-AS. (A) Genomic structure of NPPA and its antisense transcript, NPPA-AS (represented by several overlapping ESTs). Arrowheads indicate the direction of transcription, vertical lines depict the overlap between sense and antisense exons. The length of complementary region is shown in basepairs. Coding regions of NPPA and putative NPPA-AS ORF are represented as black boxes, noncoding exons and UTRs as white boxes. (B) The expression analysis of NPPA demonstrates the presence of intron-retained variants (NPPA+Intr1+2, 1820 bp and NPPA+Intr1, 722 bp) in a number of tissues. PCR reactions were performed using the primers NPPA-F and NPPA-R (indicated by arrows labeled F and R on Figure 1C) and resolved on an agarose gel. Expected size of correctly spliced NPPA product is 600 bp. The shorter product in testis (457 bp) represents a testis-specific isoform of antisense transcript NPPA-AS.9, which was not represented by ESTs in the databank and therefore could not be avoided when designing primers. (C) The schematic depiction of NPPA and NPPA-AS splicing isoforms, the products of 3' RACE reactions and their location on the genomic sequence. The gray block arrows indicate the direction of transcription. (D) NPPA-AS is widely expressed as a complex combination of alternatively spliced isoforms. Upper panel: the agarose gel showing the reactions performed using primers NPPA-AS-F4 and NPPA-AS-R1 (indicated by arrows labeled F4 and R1 on Figure 1C) on the panel of tissue-specific cDNAs. Lower panel: Isoform 3'RACE.2 is expressed only in testis. PCR was performed using primers NPPA-AS-F1 and NPPA-AS-R5 (indicated by arrows labeled F1 and R5 on Figure 1C). The band corresponds to the expected size of 405 bp.
We investigated the expression of NPPA using commercial Human Multiple Tissue cDNA panels MTC I and II (Figure 1B, C). Consistent with previous studies [23-25], the strongest expression of NPPA was detected in heart, but several tissues contained additional alternative products. Sequencing of these products revealed that the largest NPPA band detected in many tissues (1820 bp) represents the isoform of NPPA with retained both introns (further referred to as NPPA+Intr1+2). Visual inspection of the agarose gel (Figure 1B) indicates that the expression of this unspliced form and correctly spliced NPPA appear to be inversely correlated. Alternative product of 722 bp that was observed in liver, testis and leukocytes contains the retained intron 1 (NPPA+Intr1). In addition, a product shorter than correctly spliced NPPA mRNA (457 bp) was detected in testis. Sequencing of this product revealed that its sequence and splicing pattern are similar to the transcripts originating from the opposite strand, suggesting that this is actually an isoform of NPPA-AS, which was not represented by any of the ESTs in the database. To ensure that the amplification of NPPA+Intr1+2 is not caused by genomic DNA contamination, additional PCR experiments were performed with several primers that detect only NPPA-AS, but not NPPA [see Additional file 2 – Figure S2]. Because NPPA and NPPA-AS are both transcribed from the same genomic locus, contamination with the genomic DNA should result in amplification of unspliced NPPA-AS as well. However, these reactions yielded only the products corresponding to correctly spliced NPPA-AS, indicating no presence of the genomic contamination and demonstrating that the NPPA+Intr1+2 isoform indeed represents mRNA with retained introns.
NPPA-AS is expressed as a collection of alternatively spliced isoforms
Next, we characterized the structure and expression profile of NPPA-AS in human tissues using the panel of tissue-specific cDNAs (Figures 1C, D). Sequencing of eight identified isoforms confirmed that they all are spliced according to GT-AG consensus rule [see Additional file 2 – Figures S3 and S4]. It appears that NPPA-AS isoforms are not a result of alternative usage of different exons, but rather almost every exon displays at least two alternative splice donor/acceptor sites (Figure 1C). Majority of the alternative splicing events occur at the acceptor site of the intron. In addition, clearly identifiable polypyrimidine tract is located close to the splice acceptor site of all introns.
Next, we mapped the 3' end of NPPA-AS by RACE using RNA from HeLa cell line. We designed the gene-specific primers that would identify the 3' variants that are important in respect of complementarity with NPPA. Sequencing of the 3' RACE products identified two alternative 3' ends of NPPA-AS. One of the RACE products confirmed the presence of 3'-terminal exon that was predicted based on EST sequences CD368210 and BU732528 (3'RACE.1, Figure 1C). In addition, we identified a novel 3'-terminal exon that overlaps with the second intron and third exon of NPPA (3'RACE.2, Figure 1C). Expression analysis of the 3'RACE.2 isoform showed that among the sixteen tissues analysed, it is expressed only in testis (Figure 1D). Isoform 3'RACE.1 contains a suboptimal AGTAAA poly(A) signal 16 nucleotides upstream of the cleavage site, in the position where the majority of poly(A) signals are located [26]. 3'RACE.2 isoform does not contain a detectable polyadenylation signal, but an A-rich element (AAAGAGAACACAGACATA), similar to the element found in PAPOLG gene [27], that is also lacking any poly(A) signal variant, is located 19 nucleotides upstream of polyadenylation site. This suggests that in addition to alternative splicing, the processing of the NPPA-AS transcript might be regulated also at the level of polyadenylation.
Primary sequence of NPPA-AS is not evolutionarily conserved
To address the evolutionary conservation of NPPA-AS, we first asked whether Nppa gene in mouse is associated with similar natural antisense transcript. Two ESTs (GenBank:BQ771223 and CO043998 representing the 5' and 3' end of the IMAGE clone 6400656) at the Nppa locus originate from the opposite strand and therefore may represent Nppa-as in mouse. These ESTs are isolated from the brain of the mouse at embryonic day 12.5. Both ESTs follow the GT-AG splicing consensus, and CO043998 contains a canonical polyadenylation signal AATAAA as well as a short poly(A) tail. RT-PCR using RNA isolated from mouse tissues showed weak expression of Nppa-as in brain and very faint signal in liver (Figure 2A), while the expression of Nppa was present in all tissues that were tested (heart, brain, lung, liver, kidney and spleen) (Figure 2A). Exons of the mouse and human antisense transcript are not conserved at the primary structure level outside of the overlap regions with the NPPA exons and are located at the different positions in the genomic sequence (Figure 2B). However, both in mouse and in human, the antisense transcript overlaps with the intron-exon boundaries of NPPA gene (Figure 2B).
Figure 2. Expression analysis of Nppa and Nppa-as in mouse tissues. (A) Nppa is expressed in all tissues that were tested and Nppa-as is expressed in the brain. Nppa was amplified using primers mNppa-F and mNppa-R, Nppa-as was detected using primers mNppa-asF and mNppa-asR (indicated by arrows marked F, R, asF and asR on Figure 2B, respectively). (B) Genomic organization of NPPA locus in mouse and human. Arrowheads indicate the direction of transcription. Conservation profile of the human sequence, based on the UCSC vertebrate conservation track is shown below. Conservation is detected in the coding region (depicted as black boxes), promoter area and 3' terminus of NPPA gene, but not in the NPPA-AS exons that are located in introns or downstream of the NPPA gene.
The last exon of NPPA-AS 3'RACE.1 isoform (Figure 1C, represented also by ESTs GenBank:CD368210 and BU732528) contains an open reading frame (ORF) that is predicted to code for a protein of 121 amino acids [see Additional file 2 – Figure S5]. The multiple alignment and in silico translation demonstrate that in all organisms except human and chimpanzee, the predicted ORF is interrupted by at least one frameshift and one stop codon [see Additional file 2 – Figures S5A and S6]. Since the translated amino acid sequence does not contain any conserved domains and has no significant identity above 30% to any known protein, the function of the predicted protein in human and chimpanzee cannot be assessed based on the primary sequence.
Positive correlation between the expression levels of intron-retained NPPA and specific NPPA-AS isoforms
To study the correlation between the expression levels of NPPA and NPPA-AS, we quantified the specific NPPA and NPPA-AS isoforms using real-time PCR and commercial Human Multiple Tissue cDNA panels MTC I and II (Figure 3). We focused on the NPPA-AS isoforms NPPA-AS.1 and 2 (Figure 3B) that are complementary to the first intron and first and second exon of NPPA, including intron-exon boundaries. These are the isoforms that most likely have an impact on NPPA expression regulation. We detected no correlation between the expression of correctly spliced NPPA and its antisense transcript (Figure 3A), but instead observed a strong positive correlation between the expression levels of NPPA-AS and NPPA variants with retained intron 1 (Spearman's rank correlation coefficient = 0.77, p-value = 0.0028). This result was confirmed by using an alternative set of primers detecting the same NPPA-AS isoforms and retained NPPA intron 1 (data not shown). Such correlation further indicates a possible functional relationship between NPPA-AS expression and posttranscriptional regulation of NPPA.
Figure 3. Expression levels of NPPA variants with retained intron and NPPA-AS are highly correlated (Spearman's rank correlation coefficient = 0.77, p-value = 0.0028). (A) Expression levels of NPPA-AS (a total of isoforms 1 and 2) and NPPA (spliced and intron-retained forms were detected separately using primers shown on Figure 3B) were determined by quantitative real-time PCR on a panel of human tissue-specific cDNAs. Expression levels were normalized to endogenous GAPDH mRNA. (B) Schematic depiction of primer positions (black arrows) that were used to quantify the expression levels of NPPA and NPPA-AS. To the right are indicated the symbols that are used for each reaction in Figure 3A.
NPPA-AS as a modulator of expression of NPPA splicing isoforms
To test the hypothesis that NPPA-AS has a regulatory role in the splicing of NPPA mRNA, we constructed expression vectors containing either NPPA gene or one of the four antisense transcript isoforms with different regions of complementarity to NPPA (Figure 4A). Constructs pNPPA-AS-1, -2, -3 and -4 were generated by cloning of NPPA-AS isoforms NPPA-AS.1 and 2 and3'RACE products 3'RACE.1 and 3'RACE.2, respectively, into the expression vector pQM-Ntag/A. Generation of different constructs expressing NPPA or NPPA-AS isoforms allows to test the effect of each antisense variant separately and eliminates the possible influence of transcriptional interference as a mechanism of antisense action.
Figure 4. NPPA-AS constructs modulate the level of intron-retained NPPA variants. (A) Schematic depiction of expression constructs and oligonucleotide primers that were used to quantify spliced NPPA (NPPA-Ex1Ex2-F and NPPA-Ex2-R) or the variants with retained intron 1 (NPPA-In1Ex2-F and NPPA-Ex2-R). (B) The expression of NPPA intron-retained form was reduced significantly in case of coexpression with pNPPA-AS-1. Mouse NIH3T3 cells were cotransfected with pNPPA-mg2 along with different NPPA-AS expression constructs depicted above or empty vector as a control. Relative expression levels of spliced (left panel) and intron-retained (right panel) NPPA mRNA were analysed by real-time quantitative PCR. The geometric mean of two reference genes, GAPDH and HPRT1, was used as an endogenous control. The results are represented as arbitrary units based on relative quantity at the logarithmic scale. Error bars show s.e.m. P-value was calculated using Mann-Whitney Test.
We cotransfected NPPA expression construct into the mouse embryonic fibroblast cell line NIH3T3 in pairs with individual NPPA-AS constructs and quantified correctly spliced and intron-retained variants of NPPA by real-time RT-PCR. Mouse cell line was selected for transfection in order to eliminate the possible effect of endogenous expression of NPPA and NPPA-AS. The expression of specific NPPA-AS isoforms in transfected cells was confirmed by quantitative RT-PCR (data not shown). As shown in Figure 4B, expression level of intron-retained NPPA variant was significantly downregulated after transfection with pNPPA-AS-1 (P = 0.002, Mann-Whitney test). Although all constructs caused slight changes in expression levels of both spliced and intron-retained NPPA, the effects did no reach statistical significance in other experiments.
To further test whether complementary sequences of NPPA and NPPA-AS actually can form RNA duplexes, we performed RT-PCR on RNase treated RNA samples from cotransfections of NPPA and NPPA-AS constructs pNPPA-AS-1 and 3 (Figure 5). In both cases, the product with the correct size from RNase-treated samples was obtained, indicating that NPPA and its antisense transcript indeed form duplex RNAs. pNPPA-AS-3 has complementarity only to exonic regions of NPPA and can form a duplex with both spliced and unspliced NPPA mRNA. In the case of pNPPA-AS-1 we detected the duplex formation with the intronic region of NPPA, which can occur only with unspliced pre-mRNA. These results indicate that NPPA and NPPA-AS interact at the RNA level and that a specific NPPA-AS isoform (NPPA-AS.1) can modulate the proportion of intron-retained NPPA.
Figure 5. NPPA-AS forms RNA duplexes with NPPA. Reactions were performed with cloned intron-containing NPPA gene (pNPPA-mg2) and two antisense isoforms: pNPPA-AS-1 (A) and pNPPA-AS-3 (B). The agarose gels showing the results of PCR reactions using RNA samples treated (+) or non-treated (-) with RNase A. RT indicates reverse transcription reaction in the presence (+) or without (-) a reverse transcriptase. H2O, no template control; Ctrl, control reaction performed using cloned NPPA (A) or NPPA-AS (B) as a template.
The important regulatory role of endogenous noncoding RNAs, including antisense transcripts, has been proposed based on a number of large-scale transcription profiling studies. Because of the variety of functional mechanisms and lack of direct experimental support, the biological meaning of most of this noncoding transcription is still unclear. In the present study we have investigated NPPA/NPPA-AS sense-antisense transcript pair.
Several studies have addressed the question whether some fraction of antisense transcripts may in fact be artifacts of reverse transcription reaction [28,29]. To exclude such artifacts from our study, we considered only NATs with two or more exons and consensus splice sequences. In addition, 3' RACE reactions further confirmed the strand-specificity of NPPA-AS by identification of two alternative polyadenylated 3' terminal exons (Figure 1C).
NPPA is a functional candidate gene for elevated blood pressure, coding for an atrial natriuretic peptide (ANP), a member of a small family of endogenous peptide hormones. It is produced primarily by atrial cardiocytes in response to increasing cardiac wall tension. Association of specific NPPA variants with increased ANP levels as well as with lower blood pressure and reduced risk of hypertension [22] strongly support the central role of NPPA in the maintenance of blood pressure homeostasis. In addition, a region harbouring NPPA was among the eight loci identified in the meta-analysis of blood pressure genome-wide association studies [30]. Transcriptional regulation of NPPA and maturation of ANP have been studied quite extensively (for recent reviews see [31,32]) and our results add further evidence to the elaborate control of NPPA expression.
During the expression analysis of NPPA mRNA we found the strongest expression of correctly spliced NPPA in heart and moderate expression in a number of human tissues, including prostate, pancreas and small intestine, for example (Figure 1B). In addition, we observed the expression of NPPA isoforms with retained introns (NPPA+Intr1 and NPPA+Intr1+2) in several tissues (Figure 1B). Normally, ANP is synthesized as a 153-amino acid preprohormone. Removal of the signal peptide creates a 126-amino acid prohormone that is further cleaved to form a mature C-terminal 27-amino acid ANP. In the case of NPPA+Intr1 and NPPA+Intr1+2, the ORF that starts with the first methionine (Met-1) of prepro-ANP encodes only for a signal peptide region and is terminated after the frameshift caused by the intron retention. An alternative ORF of intron-retained NPPA isoforms, starting with the methionine Met-51 includes the mature ANP sequence, but since the peptide encoded by this putative ORF does not contain a signal sequence, its proper processing and biological activity is doubtful.
The natural antisense transcript of NPPA, that we named NPPA-AS, is widely present in human tissues and displays a complex pattern of alternative splicing (Figure 1D). All different NPPA-AS splicing isoforms overlap with both exonic and intronic regions of NPPA, including intron-exon boundaries (Figure 1C). Such overlap pattern raised the hypothesis that NPPA-AS may be involved in the regulation of NPPA expression.
Overlapping antisense gene pairs are preferentially co-expressed or inversely expressed in human tissues [6,33,34], supporting a model of negative control by antisense RNA that proposes state of balance in case of co-expression or up- or downregulation in case of inverse expression. Interestingly, the expression of NPPA-AS was strongly correlated with the intron-retained, rather than correctly spliced form of NPPA (Figure 3), indicating that it may play a functional role in posttranscriptional regulation of NPPA expression. Such correlation, however, does not necessarily indicate causal relationship, because it can be affected by many factors including regulation of transcription of both NPPA and NPPA-AS, or modulation of NPPA splicing by other factors.
Conservation often reflects functional significance of a nucleotide sequence. Among sense-antisense pairs, less than 7% are found to be conserved between human and mouse [35,36]. This may indicate that antisense transcripts are mostly species-specific, or alternatively, that the process of transcription, secondary structure or organization of the transcript rather than the primary sequence is functionally important. Among conserved sense-antisense pairs, about one third have identical expression pattern in mouse and human [37]. We found that expression profile in mouse and human is different for both NPPA and NPPA-AS (Figure 2). In mouse, Nppa is strongly expressed in some tissues (brain, lung, liver) where human NPPA is expressed weakly. We did not detect any alternatively spliced or intron-retained Nppa forms in mouse. In case of NPPA-AS, we found that neither the primary structure nor the expression pattern is conserved between mouse and human: in human it is expressed in all tissues examined, while in mouse the expression was observed only in brain (Figure 2A). However, both mouse and human antisense transcripts overlap with the exon-intron boundaries of NPPA (Figure 2B), implying that such genomic arrangement might be functionally significant.
Although many antisense transcripts overlap with the intron-exon boundaries of the sense mRNA, the effect of endogenous antisense transcripts as splicing regulators is studied in detail only in a few cases [16,38]. Modulation of mRNA splicing by exogenous antisense oligonucleotides has gained more attention and its therapeutic potential has been established in clinical trials involving patients with Duchenne's muscular dystrophy [39]. Much less is known about the role of endogenous antisense RNAs in regulation of splicing or stability of different mRNA isoforms. The complementarity of NPPA and its antisense exons suggests that if NPPA-AS has a function in regulation of NPPA, it depends on the mechanisms that involve the interactions at the RNA level. Our results show (Figure 4B) that at least one NPPA-AS isoform can modulate the ratio of unspliced and spliced NPPA variants, by decreasing the levels of intron-retained NPPA form. Since we were using minigene expression system, we excluded the effects of such possible regulatory mechanisms like transcriptional interference and heterochromatin formation [17]. Currently we do not know what is the exact mechanism of intron-retained NPPA downregulation, but it is possible that the formation of the duplex RNA due to the complementary regions can lead to post-transcriptional regulation via different mechanisms like RNA masking (in which case the binding of factors required for splicing or export is blocked), RNA editing or RNA interference [14]. Although the role of RNA interference in NAT-mediated regulation in mammals has been controversial, Watanabe et al. [14] identified seventeen loci in mouse where siRNAs arose from interaction of sense-antisense transcripts of the same locus. Considering the large number of NATs in mammals, the real extent of siRNA biogenesis via endogenous sense-antisense RNA interaction in mammalian cells remains still unknown. It is also possible that since antisense transcription extends through entire NPPA locus and into the promoter region, the mechanisms like transcriptional interference and modulation of NPPA promoter elements can occur and affect the expression of NPPA independently.
Although the biological role of NPPA antisense transcription needs further investigation, the regulatory role of NPPA in both adult cardiovascular system and in heart development during embryogenesis [23,24] suggest that NPPA-AS may be involved in fine-tuning of NPPA expression during embryonic development or in response to specific stimulus.
We have identified the natural antisense transcripts of human blood pressure candidate genes and provide a detailed characterization of an antisense transcript associated with the NPPA gene. Our data support the biological significance of NPPA-AS by demonstrating that it (i) is widely expressed as a collection of canonically spliced isoforms, (ii) can directly interact with NPPA at the RNA level and (iii) is able to influence the levels of intron-retained NPPA variants.
Identification and in silico analysis of natural antisense transcripts
Candidate genes (n = 38) were selected according to the prior evidence of involvement in blood pressure regulation [see Additional file 1 – Table S1]. Most of the genes were selected based on the published data on the biology and genetics of blood pressure regulation. The selection included also genes responsible for the Mendelian forms of hypertension or hypotension, location near linkage peaks or quantitative trait loci (QTLs), reports on animal models and human association studies. Additional information was obtained from different resources (OMIM, http://www.ncbi.nlm.nih.gov/sites/entrez?db=omim webcite; NCBI GeneBank and NCBI Locuslink http://www.ncbi.nlm.nih.gov/ webcite; Ensembl http://www.ensembl.org/index.html webcite). Candidate gene list was also supplemented with loci involved in other cardiovascular diseases like myocardial infarction, coronary artery disease and stroke. The candidate genes were screened for antisense transcripts using recently published sense-antisense transcript data [8]. The structure and direction of the transcripts was verified using UCSC Genome Browser http://genome.ucsc.edu/ webcite. To avoid random "transcriptional noise" and genomic DNA contamination, we considered only transcripts with at least two exons, canonical splice sites (GT/AG) and overlap with at least one exon of the sense gene. In addition, several transcripts had a polyadenylation signal and poly(A) tail, supporting together with canonical splice sites their strand-specificity.
Multiple alignment of sequences corresponding to predicted ORF of NPPA-AS from different species was performed using ClustalW2 at http://www.ebi.ac.uk/Tools/clustalw2/index.html webcite. Sequence database searches were performed using BLAST programs at http://www.ncbi.nlm.nih.gov/ webcite.
PCR and sequencing
Expression analysis of NPPA and NPPA-AS was carried out using Human Multiple Tissue cDNA panels MTC I and II (BD Biosciences) and primers [see Additional file 1 – Table S2] that were designed using Primer3 program http://frodo.wi.mit.edu/ webcite. G3PDH primers were included with MTC panels. PCR on mouse tissue-specific cDNAs was performed using oligonucleotides designed according to mouse Nppa gene and EST BQ771223 (for Nppa-as). PCR conditions were: 75 mM Tris-HCl (pH 8.8), 20 mM (NH4)2SO4, 0.01% Tween 20, 2.5 mM MgCl2, 250 μM dNTPs and 2.5 u per 100 μl Taq DNA Polymerase (Fermentas). Cycling conditions followed the touch-down procedure, namely initial denaturation at 94°C for 2 m, followed by 11 cycles at 94°C for 30 s, annealing for 30 s at temperatures decreasing from 62 to 57°C (with 0.5°C decremental in each cycle), 72°C for 60 s, and 30 cycles at 94°C for 30 s, 57°C for 30 s, 72°C for 60 s, and ending with an extension step at 72°C for 5 m.
For sequencing, PCR products were extracted from agarose gel using NucleoSpin Extract II (Macherey-Nagel) and either cloned into the pTZ57R vector using InsT/Aclone Kit (Fermentas) or sequenced directly after ExoI/SAP (both Fermentas) treatment. Sequencing reactions were performed using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to manufacturer's instructions and analyzed on ABI Prism™ 3730xl DNA Analyzer. Sequencing results were manually analyzed using Bioedit software http://www.mbio.ncsu.edu/BioEdit/bioedit.html webcite and mapped to the genome using BLAT alignment tool [40] at the UCSC Genome Browser http://genome.ucsc.edu/ webcite. Novel sequences obtained in this study have been submitted to GenBank database (FJ706070–FJ706079).
3' RACE (rapid amplification of cDNA ends)
The 3' RACE reactions were performed using GeneRacer™ Kit (Invitrogen) and 1 μg of total RNA isolated from semi-confluent HeLa cells grown in 5% CO2 at 37°C. Reverse transcription was performed using SuperScript III Reverse Transcriptase and GeneRacer™ Oligo dT Primer (both included in the kit). Amplification was carried out using GeneRacer™ or 3' primer and the following gene-specific primers: NPPA-AS-F1, -F2 or -F5. Nested PCR was performed using nested GeneRacer™ primer and suitable gene-specific primers.
Construction of plasmids
The vector pQM-Ntag/A (Quattromed, Estonia) was used to create expression constructs under the control of CMV promoter. NPPA gene was amplified from human genomic DNA using primers NPPA-GFXba and NPPA-GRBam and inserted into the XbaI and BamHI sites of pQM-Ntag/A. These primers amplify a 2069 bp fragment (chr1:11828358-11830426 according to human genome assembly hg18) of genomic DNA, including all three NPPA coding exons and both UTRs. Four constructs representing different NPPA-AS splicing isoforms were generated. pNPPA-AS-1 and -2 represent splicing isoforms NPPA-AS.1 and NPPA-AS.2 obtained with oligonucleotides NPPA-AS-F4 and NPPA-AS-R1. For generation of pNPPA-AS-3 and -4, 3' RACE products 3'RACE.1 and 3'RACE.2 were utilized. All constructs were sequenced using internal primers as well as universal primers flanking the insert cloning site.
Transfection of expression constructs
Cells were grown in 5% CO2 at 37°C and on the day before transfection were plated into 24-well plates using medium without antibiotics. Next day, cells were transfected using Lipofectamine™ 2000 reagent (Invitrogen) and 1.0 μg of total plasmid DNA (0.2 μg of pNPPA-mg2 and 0.8 μg of any of the NPPA-AS constructs or empty vector). Cells were incubated at 37°C and RNA was isolated 24 hours later. For relative quantitation experiments, transfections were carried out in triplicate three times (total nine replicates).
RNA isolation
Total RNA from cell lines and from mouse (male C57bl/6) tissues was isolated using TRIzol reagent (Invitrogen) and the quantity and quality of RNA was assessed using Nanodrop ND-1000 (Thermo Scientific). DNase treatment was performed using TURBO DNA-free Kit™ (Ambion) with 1 μg of RNA in a volume of 30 μl. cDNA was synthesized using 2 μg of total RNA and First Strand cDNA Synthesis Kit (Fermentas) according to manufacturer's instructions.
Real-time quantitative PCR
For investigation of expression levels of NPPA and NPPA-AS in human tissues, the Human Multiple Tissue cDNA panels MTC I and II (BD Biosciences) were used. Amplification was performed with the following primer pairs: NPPA-Ex1Ex2-F and NPPA-Ex2-R for quantification of spliced NPPA; NPPA-In1Ex2-F and NPPA-Ex2-R for detection of unspliced NPPA. For quantification of NPPA-AS, primers NPPA-ASRT-F4 and NPPA-ASRT-R4 were used. GAPDH was used as an endogenous control and amplified with primers GAPDH-S and GAPDH-AS. For quantification of spliced and unspliced NPPA from transfection experiments, RNA from mouse NIH3T3 cells was isolated and DNase-treated as described above. Primers were designed avoiding binding to mouse sequences to prevent nonspecific amplification. Correctly spliced NPPA mRNA was detected using primers NPPA-Ex1Ex2-F and NPPA-Ex2R, unspliced NPPA was detected using primers NPPA-In1Ex2-F and NPPA-Ex2-R. Two endogenous reference genes were used: GAPDH (amplified with primers GAPDH-S and GAPDH-AS) and HPRT1 (amplified with primers HPRT1-S and HPRT1-AS). The reactions were performed in the 96-well microtiter plate using ABI PRISM® 7900 Real-Time PCR cycler. The 25 μl reaction mixture consisted of 3 μl of 1:10 cDNA dilution, 12.5 μl of ABsolute™ QPCR SYBR® Green ROX Mix (Thermo Scientific), 70 nM forward and reverse primer. The cycling parameters were: enzyme activation at 95°C for 15 m followed by 40 cycles 95°C for 15 s, 60°C for 30 s, 72°C for 30 s. Reactions were performed in triplicates for each biological replicate. As negative controls for DNA contamination, reactions without the reverse transcriptase were carried out. We performed control and optimization experiments for all primer pairs and selected for actual quantitation experiments primer pairs with amplification efficiency of 100 ± 10%. During optimization, serial dilutions of template were used and the specificity of the PCR products was confirmed by the presence of a single peak during the dissociation curve analysis. Amplification efficiency of the reactions was 100 ± 10% and intra- and inter-assay variation coefficients were below 3% and 8%, respectively. PCR efficiencies were calculated from ten-fold dilution series and relative expression of correctly spliced and intron-retained NPPA was calculated according to Pfaffl [41] by taking PCR efficiency into account. The geometric mean of GAPDH and HPRT1 was used as an endogenous control. Statistical significance of the results was analyzed using two-tailed Mann-Whitney test.
Duplex detection
RNA from NIH3T3 cells cotransfected with constructs pNPPAmg2 and pNPPA-AS-1 or pNPPA-AS-3 was isolated as described previously and treated with DNase (TURBO DNA-free Kit™, Ambion) according to manufacturer's instructions and 0.5 μl of RNase A (10 mg/ml, Fermentas). RNase was inactivated with 0.5 mg/ml proteinase K treatment at the presence of 1% SDS for 30 min at 37°C. RNA was extracted with phenol/chloroform treatment and cDNA was synthesized using primers In1R (for NPPA-AS-1 duplex detection) or duplR (for NPPA-AS-3) and First Strand cDNA Synthesis Kit (Fermentas). In parallel, control experiments using RNAs not treated with RNase A and reverse transcriptions without reverse transcriptase were performed. PCR was performed with primers In1R and Ex1In1F for detection of NPPA::NPPA-AS-1 duplex and with primers duplF and duplR for detection of NPPA::NPPA-AS-3 duplex.
Authors' contributions
TA and ML designed the study, KK and ML selected and analysed the candidate genes, TA performed the experiments and carried out the analyses, TA and ML wrote the paper. All authors read and approved the final manuscript.
We thank Kristiina Rull for providing placental RNA, prof. Toivo Maimets for providing cell culture facilities, Urmo Võsa and Viljo Soo for technical assistance and Martti Laan, Kai Kisand and Sulev Kuuse for mouse tissue samples. We also thank Mart Speek for stimulating discussions and Jaana Männik and Ana Rebane for critical review of the manuscript. This work was supported by Wellcome Trust grant no. 070191/z/03/z (to M.L.), Howard Hughes Medical Institute grant #55005617 (to M.L.), Estonian Ministry of Education and Science core grant no. 0182721s06 (to M.L.) and Estonian Science Foundation grant ETF6597 (to T.A.).
Birney E, Stamatoyannopoulos JA, Dutta A, Guigo R, Gingeras TR, Margulies EH, Weng Z, Snyder M, Dermitzakis ET, Thurman RE, et al.: Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project.
Nature 2007, 447(7146):799-816. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Cheng J, Kapranov P, Drenkow J, Dike S, Brubaker S, Patel S, Long J, Stern D, Tammana H, Helt G, et al.: Transcriptional maps of 10 human chromosomes at 5-nucleotide resolution.
Science 2005, 308(5725):1149-1154. PubMed Abstract | Publisher Full Text
Kapranov P, Willingham AT, Gingeras TR: Genome-wide transcription and the implications for genomic organization.
Nat Rev Genet 2007, 8(6):413-423. PubMed Abstract | Publisher Full Text
Carninci P, Kasukawa T, Katayama S, Gough J, Frith MC, Maeda N, Oyama R, Ravasi T, Lenhard B, Wells C, et al.: The transcriptional landscape of the mammalian genome.
Science 2005, 309(5740):1559-1563. PubMed Abstract | Publisher Full Text
Mattick JS: RNA regulation: a new genetics?
Nat Rev Genet 2004, 5(4):316-323. PubMed Abstract | Publisher Full Text
Katayama S, Tomaru Y, Kasukawa T, Waki K, Nakanishi M, Nakamura M, Nishida H, Yap CC, Suzuki M, Kawai J, et al.: Antisense transcription in the mammalian transcriptome.
Science 2005, 309(5740):1564-1566. PubMed Abstract | Publisher Full Text
Wang XJ, Gaasterland T, Chua NH: Genome-wide prediction and identification of cis-natural antisense transcripts in Arabidopsis thaliana.
Genome Biol 2005, 6(4):R30. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Chen J, Sun M, Kent WJ, Huang X, Xie H, Wang W, Zhou G, Shi RZ, Rowley JD: Over 20% of human transcripts might form sense-antisense pairs.
Nucleic Acids Res 2004, 32(16):4812-4820. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Rosok O, Sioud M: Systematic identification of sense-antisense transcripts in mammalian cells.
Nat Biotechnol 2004, 22(1):104-108. PubMed Abstract | Publisher Full Text
Yelin R, Dahary D, Sorek R, Levanon EY, Goldstein O, Shoshan A, Diber A, Biton S, Tamir Y, Khosravi R, et al.: Widespread occurrence of antisense transcription in the human genome.
Nat Biotechnol 2003, 21(4):379-386. PubMed Abstract | Publisher Full Text
Beiter T, Reich E, Williams RW, Simon P: Antisense transcription: A critical look in both directions.
Cell Mol Life Sci 2009, 66(1):94-112. PubMed Abstract | Publisher Full Text
Lapidot M, Pilpel Y: Genome-wide natural antisense transcription: coupling its regulation to its different regulatory mechanisms.
EMBO Rep 2006, 7(12):1216-1222. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Carninci P, Yasuda J, Hayashizaki Y: Multifaceted mammalian transcriptome.
Curr Opin Cell Biol 2008, 20(3):274-280. PubMed Abstract | Publisher Full Text
Watanabe T, Totoki Y, Toyoda A, Kaneda M, Kuramochi-Miyagawa S, Obata Y, Chiba H, Kohara Y, Kono T, Nakano T, et al.: Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes.
Nature 2008, 453(7194):539-543. PubMed Abstract | Publisher Full Text
Hongay CF, Grisafi PL, Galitski T, Fink GR: Antisense transcription controls cell fate in Saccharomyces cerevisiae.
Cell 2006, 127(4):735-745. PubMed Abstract | Publisher Full Text
Beltran M, Puig I, Pena C, Garcia JM, Alvarez AB, Pena R, Bonilla F, de Herreros AG: A natural antisense transcript regulates Zeb2/Sip1 gene expression during Snail1-induced epithelial-mesenchymal transition.
Genes Dev 2008, 22(6):756-769. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Yu W, Gius D, Onyango P, Muldoon-Jacobs K, Karp J, Feinberg AP, Cui H: Epigenetic silencing of tumour suppressor gene p15 by its antisense RNA.
Nature 2008, 451(7175):202-206. PubMed Abstract | Publisher Full Text
Tufarelli C, Stanley JA, Garrick D, Sharpe JA, Ayyub H, Wood WG, Higgs DR: Transcription of antisense RNA leading to gene silencing and methylation as a novel cause of human genetic disease.
Nat Genet 2003, 34(2):157-165. PubMed Abstract | Publisher Full Text
Lifton RP, Gharavi AG, Geller DS: Molecular mechanisms of human hypertension.
Cell 2001, 104(4):545-556. PubMed Abstract | Publisher Full Text
Blaustein MP, Zhang J, Chen L, Hamilton BP: How does salt retention raise blood pressure?
Am J Physiol Regul Integr Comp Physiol 2006, 290(3):R514-523. PubMed Abstract | Publisher Full Text
Ruppert V, Maisch B: Genetics of human hypertension.
Herz 2003, 28(8):655-662. PubMed Abstract | Publisher Full Text
Newton-Cheh C, Larson MG, Vasan RS, Levy D, Bloch KD, Surti A, Guiducci C, Kathiresan S, Benjamin EJ, Struck J, et al.: Association of common variants in NPPA and NPPB with circulating natriuretic peptides and blood pressure.
Nat Genet 2009, 41(3):348-353. PubMed Abstract | Publisher Full Text
Houweling AC, van Borren MM, Moorman AF, Christoffels VM: Expression and regulation of the atrial natriuretic factor encoding gene Nppa during development and disease.
Cardiovasc Res 2005, 67(4):583-593. PubMed Abstract | Publisher Full Text
Zeller R, Bloch KD, Williams BS, Arceci RJ, Seidman CE: Localized expression of the atrial natriuretic factor gene during cardiac embryogenesis.
Genes Dev 1987, 1(7):693-698. PubMed Abstract | Publisher Full Text
de Bold AJ, Borenstein HB, Veress AT, Sonnenberg H: A rapid and potent natriuretic response to intravenous injection of atrial myocardial extract in rats.
Life Sci 1981, 28(1):89-94. PubMed Abstract | Publisher Full Text
Beaudoing E, Freier S, Wyatt JR, Claverie JM, Gautheret D: Patterns of variant polyadenylation signal usage in human genes.
Genome Res 2000, 10(7):1001-1010. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Venkataraman K, Brown KM, Gilmartin GM: Analysis of a noncanonical poly(A) site reveals a tripartite mechanism for vertebrate poly(A) site recognition.
Genes Dev 2005, 19(11):1315-1327. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Wu JQ, Du J, Rozowsky J, Zhang Z, Urban AE, Euskirchen G, Weissman S, Gerstein M, Snyder M: Systematic analysis of transcribed loci in ENCODE regions using RACE sequencing reveals extensive transcription in the human genome.
Genome Biol 2008, 9(1):R3. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Perocchi F, Xu Z, Clauder-Munster S, Steinmetz LM: Antisense artifacts in transcriptome microarray experiments are resolved by actinomycin D.
Nucleic Acids Res 2007, 35(19):e128. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Newton-Cheh C, Johnson T, Gateva V, Tobin M, Bochud M, Coin L, Najjar S, Zhao J, Heath S, Eyheramendy S, et al.: Genome-wide association study identifies eight loci associated with blood pressure.
Nat Genet 2009, 41(3):666-676. Publisher Full Text
Gardner DG, Chen S, Glenn DJ, Grigsby CL: Molecular biology of the natriuretic peptide system: implications for physiology and hypertension.
Hypertension 2007, 49(3):419-426. PubMed Abstract | Publisher Full Text
Richards AM: Natriuretic peptides: update on Peptide release, bioactivity, and clinical use.
Hypertension 2007, 50(1):25-30. PubMed Abstract | Publisher Full Text
Chen J, Sun M, Hurst LD, Carmichael GG, Rowley JD: Genome-wide analysis of coordinate expression and evolution of human cis-encoded sense-antisense transcripts.
Trends Genet 2005, 21(6):326-329. PubMed Abstract | Publisher Full Text
Werner A, Schmutzler G, Carlile M, Miles CG, Peters H: Expression profiling of antisense transcripts on DNA arrays.
Physiol Genomics 2007, 28(3):294-300. PubMed Abstract | Publisher Full Text
Numata K, Okada Y, Saito R, Kiyosawa H, Kanai A, Tomita M: Comparative analysis of cis-encoded antisense RNAs in eukaryotes.
Gene 2007, 392(1–2):134-141. PubMed Abstract | Publisher Full Text
Engstrom PG, Suzuki H, Ninomiya N, Akalin A, Sessa L, Lavorgna G, Brozzi A, Luzi L, Tan SL, Yang L, et al.: Complex Loci in human and mouse genomes.
PLoS Genet 2006, 2(4):e47. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Okada Y, Tashiro C, Numata K, Watanabe K, Nakaoka H, Yamamoto N, Okubo K, Ikeda R, Saito R, Kanai A, et al.: Comparative expression analysis uncovers novel features of endogenous antisense transcription.
Hum Mol Genet 2008, 17(11):1631-1640. PubMed Abstract | Publisher Full Text
Hastings ML, Ingle HA, Lazar MA, Munroe SH: Post-transcriptional regulation of thyroid hormone receptor expression by cis-acting sequences and a naturally occurring antisense RNA.
J Biol Chem 2000, 275(15):11507-11513. PubMed Abstract | Publisher Full Text
van Deutekom JC, Janson AA, Ginjaar IB, Frankhuizen WS, Aartsma-Rus A, Bremmer-Bout M, den Dunnen JT, Koop K, Kooi AJ, Goemans NM, et al.: Local dystrophin restoration with antisense oligonucleotide PRO051.
N Engl J Med 2007, 357(26):2677-2686. PubMed Abstract | Publisher Full Text
Kent WJ: BLAT – the BLAST-like alignment tool.
Genome Res 2002, 12(4):656-664. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Pfaffl MW: A new mathematical model for relative quantification in real-time RT-PCR.
Nucleic Acids Res 2001, 29(9):e45. PubMed Abstract | Publisher Full Text | PubMed Central Full Text


text:
Narrow your search
If you've always hated using a separate Bluetooth keyboard just to enter search terms into your Android TV box, Mad Catz's Surfr may be just the thing.
Pricing not available
A basic Xbox 360-style controller, the CTRLi is a well-weighted and comfortably shaped controller for iOS devices.
The Mad Catz MOJO is a micro-console running Android and comes with a full-on console-quality controller. It currently has no price or release date.
Channel surf and smash high scores with this gnarly wireless gaming controller, remote and keyboard.
Mad Catz's latest gaming controller packs a mini QWERTY keyboard for typing to your Android TV box.
The CTRLi from Mad Catz is one of the few Bluetooth-based Xbox 360-style controllers for iOS devices.
Harmonix's Alex Rigopulos: "There's not ambition or expectation that we will outsell Guitar Hero."
Which instruments are compatible? Which tracks can be transferred? Here's our comprehensive guide for troubleshooting your Rock Band woes.
The Internet is cats, so why isn't more of the Internet for cats? One Japanese prefecture is righting this wrong and perhaps writing the perfect denouement for our entire online experiment.
The company, whose app store was pitched as the go-to place for small and independent developers, is negotiating for employees to remain with the company after the sale.

subset: commoncrawl


text:
Gary Allan on new songs, vocal surgery
When I talked to Gary Allan earlier this week, we covered more ground than was able to fit in the full feature that appears here on Tap Milwaukee. Here is more of the conversation we shared as he waited to make a Monday night appearance on "Late Night With Jimmy Fallon." (Click here to watch his spot on the show.)
As Allan prepares to visit The Rave tonight, he discussed why he thought "Tough Goodbye," the lead song (co-written by Cedarburg native Josh Thompson) from his Billboard chart-topping new album, "Set You Free," would be the first single. He also talked about how a vocal cord operation that he underwent just before recording the album has made him feel like a whole new singer, and his approach to Nashville after spending his early years (starting at 12 years old) playing honky tonks and clubs in California's Bakersfield-inspired country music scene. »Read Full Blog Post
CBS recants Libya story
In a dramatic and unusual move by a network news division, CBS announced that "60 Minutes" will recant its Oct. 27 report on the attack on the U.S. Consulate in Benghazi, Libya, last year, which was based on information from a security officer's account of events.
It was recently revealed that the account upon which reporter Lara Logan's story was based was different from the story he told the FBI. »Read Full Article(223)
Oscar winner & Kenosha native Jim Rygiel gets UWM award
Jim Rygiel's road to winning three Oscars started at the University of Wisconsin-Milwaukee, which the Kenosha native attended between 1973 and 1977.
Audience share of Milwaukee radio stations for October
Below is the audience share for Milwaukee stations among listeners 6 years and older - also known as total audience - between 6 a.m. and midnight in October.
North Ave. spot in Tosa now is a Red Dot
Journal Sentinel photo »Read Full Blog Post
Country Spotlight: A conversation with Will Hoge
In Will Hoge's own words, "It feels real strange to be all of a sudden this 15 year overnight sensation." »Read Full Blog Post
Listening to Lou Reed: the eponymous debut album
On Fridays I'm posting my thoughts on Lou Reed's solo albums. »Read Full Blog Post(15)
Music-loving Kapco owner bankrolls bands
Waves of bright lights wash over a singing crowd. Smiling guitarists playfully rub shoulders while they riff. Polished country rock practically purrs out of a top-of-the-line speaker system.
With his country band Chasin' Mason behind him, Billy O'Dwyer is singing songs from "Real Life Real Loud" for about 75 family members, friends and fans. »Read Full Article(4)
Get ready for 'Groucho' with this Marxist quiz
You don't have to say the secret word to spend a night with one of America's great comedians this month.
Just give Next Act Theatre a ring for tickets to "Groucho: A Life in Revue." »Read Full Article
Jake's Deli to open a restaurant at Southridge Mall
Journal Sentinel file photo »Read Full Blog Post(2)
Cafe-deli debuts at West Allis Cheese shop's original store
With the expansion complete at its original location, West Allis Cheese & Sausage Shoppe's newest deli and cafe launched Wednesday. »Read Full Blog Post
Former Milwaukee Art Museum curator goes to George Eastman House
Lisa Hostetler, the former curator of photographs at the Milwaukee Art Museum, will become the curator-in-charge of the George Eastman House's photography department by the end of the year, it was announced today.
Hostetler left the Milwaukee Art Museum last summer to take a post as the curator of photography for the Smithsonian Museum of American Art. »Read Full Blog Post(3)
Performing Arts Calendar
Alverno College Pitman Theatre: Amy Tan "The Valley of Amazement," 7 p.m. Nov. 11, 3431 S. 39th St., (414) 332-1181. »Read Full Article
New WISN-TV reporter 'die-hard' Cubs, Bears fan
Ben Hutchison will "bring his versatile reporting skills" to WISN-TV (Channel 12) starting Monday, the station announced Thursday. Hutchison, an Illinois native, most recently worked at WTVO-TV in Rockford, where he worked as a news anchor and reporter.
His sports-heavy background includes stints as sports anchor and sports reporter. Before joining WTVO in 2011, he was an intern with the Chicago White Sox and an ESPN freelancer. »Read Full Blog Post(4)
Mike Birbiglia at Pabst Theater March 8
"This American Life" favorite Mike Birbiglia will perform live at the Pabst Theater on March 8.
The comedian received the biggest exposure of his career last year with "Sleepwalk with Me," a well-reviewed independent film he wrote, directed and starred in, inspired by his true-life, one-man show of the same name, which also inspired the book "Sleepwalk with Me & Other Painfully True Stories." »Read Full Article
A third Jackson's Blue Ribbon Pub, in Waukesha
Brothers Mark and Dan Zierath have opened their third Jackson's Blue Ribbon Pub, this one in downtown Waukesha.
The bar and grill launched in early October at 225 South St., the former site of J. Lotti's Pub. The grand opening party will be Nov. 23. »Read Full Blog Post(1)
Blind Boys of Alabama's Jimmy Carter: What Music I'm Listening To
Jimmy Carter (center) is returning to Wisconsin with gospel institution Blind Boys of Alabama, playing the Pabst Theater Friday. The group recorded its latest album "I'll Find a Way" in Eau Claire County with Justin Vernon (Bon Iver, Volcano Choir). Photo by Cameron Witting. »Read Full Blog Post
On Tap for Today: Movies, music and the Feed Your Soul benefit
Hello, Friday. It's about time you got here.
It's also about fun. And movie. And music. And more fun. »Read Full Blog Post


text:
You are using an outdated browser. Please upgrade your browser to improve your ReverbNation experience.
Lisa Barreca
Hollywood, FL
Saturday, July 20th, 2013 at 7:00pm
All Ages
Great place for intercoastal dining. Sushi, wings, full bar and more
© 2015 eMinor Incorporated
All trademarks are the property of the respective owners. ReverbNation is not affiliated with the trademark owners.
Not listening to anything?
Try one of the ReverbNation Channels


text:
Sign in now to view, license and download this image or contact us to see this image without a watermark. Want to embed this image on a non-commercial website or blog? Learn more.
Similar imagesView all
Szechuan Ma Po Dofu Stock PhotoBrown,Chinese Food,Chopsticks,Color Image,Directly Above,Food and Drink,Freshness,Indoors,Malaysia,Mat,No People,Petaling Jaya,Photography,Plate,Ready-To-Eat,Tofu,VerticalPhotographer Jen Voo PhotographyCollection: Moment Open © 2011 Jann Voo PhotographySzechuan/ Sichuan's famous spicy mapo tofu served in dark plate on textured mat with brown chopsticks.


text:
1989 Dodge Colt
I have a 89 dodge colt. It was driving fine until today. After being parked for about an hour, after I disarmed the alarm, and tried to turn it on, I got no power. No lights nothing. The alarm also goes off after taking key out, with a weird buzzing noise. I had to boost it to get it started again. While driving home, when I turned the lights on, I got a check engine light, and every time I touched the brake, there was a weird click noise which sounded like it came through the radio speaker. Now when I parked it, and tried again at home to start it, it does it again. The battery connectors are good. Battery is good. What could it be?
December 2, 2010.
December 2, 2010.
How was the boost connected? To the battery posts or the engine/body? Maybe you have a bad ground connection? This can cause all kinds of electrical quirks. Major and minor.
It was connected post to post. Worked first time, and car started and ran fine all the way home until I tried to restart it at home.
Now it sounds alarm related? Is it factory of aftermarket? Or maybe you have an ignition switch problem?
The alarm is a clifford alarm but don; t know the model as it was installed before we bought it. Cant find the brain either. It has a black square key fob with 2 buttons. Looks pretty old. Hubby opened the driver door and the window must have pulled the wires for the power door lock off, but after reconnecting it, it still has the same problem. It is a aftermarket alarm that the last guy installed. (He's a pro apparently does it for a living)it starts fine once you attach a battery booster pack. Nothings wrong with the alt, or starter, and battery is fully charged, but acts like its dead when you try starting it on its own.
Hubby found a switch which he says belongs to the alarm, but after turning the switch alarm still doesnt do anything when the cars running. Not sure what to do with it. Its a small silver switch on a black cable under the driver side panel beside the clutch under the dash.
Is that a top or side post battery? Side posts can be really fussy when older trying to get a good connection! I usually R&R mine with 3/8 bolts and washers.I open the whole in the rubber boot on cable end to get at metal so washers will make good contact.
Dec 3, 2010.
Dec 3, 2010.
How do I replace a battery in a 2009 Chevy Equinox LS with a 3.4L V6. Most cars are straight forward, this looks difficult. Shame on GM. Thanks.
1 answer • 2009 Chevrolet Equinox • 19,000 miles
My battery is dead and I was going to take try and take it out but it is located on the passenger side below the air cleaner. My question is how is it possible to remove/replace the ...
2 answers • 2002 Dodge Intrepid • 125 miles
About a month ago, the battery and brake light came on on my 2000 Mitsubishi Galant. The lights would sometimes stay on, sometimes flicker. A couple of weeks ago, I woke up and the car ...
1 answer • 2000 Mitsubishi Galant • 130,000 miles


text:
Sachin receives India's highest honourComment on this story
New Delhi – Retired cricketing legend Sachin Tendulkar was awarded India's highest civilian award on Tuesday, the Bharat Ratna, making him the first Indian sportsperson to receive the honour.
Tendulkar, who at 40 is the youngest-ever recipient, was congratulated by President Pranab Mukherjee during a brief ceremony that saw thunderous applause at the presidential palace.
The cricketer announced his retirement from the game in November, ending his 24-year career during which he became the world's leading scorer in both Test and one-day cricket.
In retirement, he has begun promoting social causes such as sanitation and has been named a member of the upper house of the Indian Parliament, leading to speculation he might one day enter active politics.
In 2008, he received the Padma Vibhushan, India's second highest civilian award.
At the ceremony Tuesday, he walked up to the dais with folded hands, greeted the president and accepted the award amid a din of camera flash-lights and applause from the audience, which included his family.
C.N.R. Rao, 79, who heads the prime minister's scientific advisory council, was the other civilian who received the Bharat Ratna this year for his contribution in the field of science. – Sapa-AFP
Comment Guidelines
- Please read our comment guidelines.
- Login and register, if you haven' t already.
- Write your comment in the block below and click (Post As)
- Has a comment offended you? Hover your mouse over the comment and wait until a small triangle appears on the right-hand side. Click triangle () and select "Flag as inappropriate". Our moderators will take action if need be.
- Verified email addresses: All users on Independent Media news sites are now required to have a verified email address before being allowed to comment on articles. You are only required to verify your email address once to have full access to commenting on articles. For more information please read our comment guidelines


text:
Book your room at Sun Hotel Nagoya Nishiki
Twin Room, 2 Single Beds, SmokingMax Occupancy: 4 guests (up to 2 children, 2 infants)
Triple Room, SmokingMax Occupancy: 6 guests (up to 3 children, 3 infants)
Triple Room, Non SmokingMax Occupancy: 6 guests (up to 3 children, 3 infants)
Single Room, 1 Single Bed, Non SmokingMax Occupancy: 2 guests (up to 1 child, 1 infant)
Single Room, 1 Single Bed, SmokingMax Occupancy: 2 guests (up to 1 child, 1 infant)
Double Room, 1 Double Bed, Non SmokingMax Occupancy: 3 guests (up to 1 child, 1 infant)
Double Room, 1 Double Bed, SmokingMax Occupancy: 2 guests (up to 1 child, 1 infant)
Twin Room, 2 Single Beds, Non SmokingMax Occupancy: 4 guests (up to 2 children, 2 infants)
Japanese Western Style Room (Smoking)Max Occupancy: 9 guests (up to 4 children, 4 infants)
Get an overview of this hotel3-star Nagoya hotel in Fushimi, near Nagoya Castle
Collect 200 points
Simply enter your Nectar card number when prompted. Your points will be credited 30 days after you return from your trip.*Offers may apply
Located in Fushimi, this hotel is within a 15-minute walk of Nagoya TV Tower and Nagoya City Science Museum. Nagoya Castle and Toyota Commemorative Museum of Industry and Technology are also within 3 mi (5 km).
Hotel Features
Dry cleaning, laundry facilities, and a front-desk safe are available at this hotel. WiFi in public areas is free. Other amenities include free wired Internet in public areas.
Room Amenities
All 79 rooms feature thoughtful touches like bathrobes and slippers, plus free WiFi, free wired Internet, and flat-screen TVs with digital channels. Refrigerators, down comforters, and free bottled water are among the other amenities available to guests.Information missing or incorrect? Tell us!
Hotel Amenities
Hotel Amenities
Sun Hotel Nagoya Nishiki features laundry facilities, dry cleaning/laundry services, and a safe deposit box at the front desk. High-speed wired and wireless Internet access is complimentary.
- Elevator/lift
- Free WiFi
- Total number of rooms - 79
- Number of floors - 7
- Dry cleaning/laundry service
- Free wired high-speed Internet
- Laundry facilities
- Safe-deposit box at front desk
- Year Built 1979
- Number of buildings/towers - 1
Family Friendly Amenities
- Free Wi-Fi
- Laundry facilities
- Refrigerator
Available in all rooms: Free WiFi , Free wired high-speed Internet
Available in some public areas: Free WiFi , Free wired high-speed Internet
Room Amenities
- Pay movies
- Air conditioning
- Climate control
- Phone
- Refrigerator
- Free bottled water
- Free WiFi
- Daily housekeeping
- Turndown service
- Private bathroom
- Bathrobes
- Hair dryer
- Slippers
- Bathtub or shower
- Iron/ironing board (on request)
- Digital TV service
- Flat-panel TV
- Free wired high-speed Internet
- Down comforter
Hotel Policies
Check-in time starts at 3 PM
Check-out time is 11 AM
Payment types
You need to know
Extra-person charges may apply and vary depending on hotel policy.
Government-issued photo identification and a credit card or cash deposit are required at check-in for incidental charges.
Special requests are subject to availability upon check-in and may incur additional charges. Special requests cannot be guaranteed.
- The name on the credit card used at check-in to pay for incidentals must be the primary name on the guestroom reservation.
No rollaway/extra beds available
No cribs (infant beds) available
Hotel Name
We should mention
The Japanese Ministry of Health, Labour, and Welfare requires all international visitors to submit their passport number and nationality when registering at any lodging facility (inns, hotels, motels, etc. ). Additionally, lodging proprietors are required to photocopy passports for all registering guests and keep the photocopy on file.


text:
Thursday, January 22, 2004
© Copyright 2015 Clayton News Daily
By Billy CorriherThe day after President George Bush delivered his annual "State of the Union" speech, some in the community disagreed with Bush's upbeat assessment of the economy. Jonesboro resident Rachelle Exis said she doesn't think Bush has handled the economy very well. "I feel like the economy, for middle and lower class people, has gotten really hard," she said. Exis said Bush's stance on cutting overtime pay and tax cuts is not helping the economy. "I remember in (President Bill) Clinton's time you could make some money and save a little," she said. But Jonesboro resident Roy Foster said that, while the national economy is still struggling, he agrees with the president's assessment. "The economy is going to recover soon," he said. "I think (Bush) is doing what he can." Foster said the president was also right in calling for Congress to make his tax cuts permanent. Foster also said he had no problem with Bush's discussion of more drug testing in schools. "Drugs have gotten so bad, we've got to do something," he said. Foster said that, overall, he likes Bush's leadership and will vote for him again. "I think the president's speech was on the money," he said. Rep. David Scott, D-Atlanta, said he supported the president's stance on national security, but would have liked the speech to include more about job creation. "We must look at what is going on inside our country," he said. "We are closing manufacturing plants here and opening them up in Chile or China or India." Scott said more needs to be done to keep jobs in the United States. Bush did propose funding for job training at community colleges, but Scott posed the question, "What are we going to train them for?" Scott said he also would have liked Bush's speech to focus more on expanding health care insurance and strengthening veteran's benefits. Rep. Mac Collins, R-Jackson, who is vying for the Republican Senate nomination, said the president's speech was a "good update" of where the country stands. "I thought (Bush) had his eye on the ball," he said. "I'm very pleased with the commander in chief's leadership." Collins said the president's comments on Saddam Hussein's weapons of mass destruction programs were proof that Iraq was a threat to U.S. security. "If we had not gone in when we did, I'm pretty sure Saddam Hussein would still have those programs," he said. Collins said he also credited Bush's tax cuts with helping to stimulate an economic recovery. "There's a lot of good, positive things that are happening," he said, pointing to rising productivity, low interest rates, rising home construction and a recovering stock market. Collins said the loss of manufacturing jobs, however, continues to be a concern for his constituents, and he hopes Bush will emphasize protecting those jobs. "I want to help him do more than what he talked about," he said. Sen. Zell Miller, the only Democratic senator to endorse Bush, issued a statement praising Bush's address and his foreign policy. "In a strong, forceful, optimistic n even visionary way, he talked about the kind of nation and world we can be," he said. "Where he really touched this old Marine was with the strength and determination he continues to show in the War on Terrorism and to rogue dictators around the world," Miller said, pointing to Libyan dictator Moammar Gadhafi's decision to abandon weapons of mass destruction as evidence of Bush's effectiveness.
More like this story
- Number of candidates filing for office slows ( April 29, 2004 )
- Kerry concedes, Bush wins, Dems lament ( November 4, 2004 )
- Area Republicans evaluate McCain's speech ( September 6, 2008 )
- Local students respond to Obama speech ( September 9, 2009 )
- Candidates qualify at U.S. Senate race heats up ( April 29, 2004 )


text:
Local currency: Euro
Slovakia joined the European Union (EU) on the 1st of May 2004 and on the 1st of January 2009 Slovakia even became a member of the Eurozone. This means that nowadays the Euro is the official currency in the country.
When I was in Kosice, Slovakia still had the Slovak Crown (Slovenska Koruna) as official currency. The Slovak Crown was subdivided into 100 Heller (Halier).
You can get your local money with a credit or debit card from cash points or just by exchanging your local money at one of the many exchange offices.
- Budget Travel
- Backpacking
- Trains
Twin city: Wuppertal in Germany
When wandering around Kosice I came along a street sign stating "Wuppertal 1490 km".
Wuppertal is located in Germany and only about 60 km from my hometown. Wuppertal and Kosice are twinned since 1980 and it was one of the first partnerships between a big German town and a city of the former Czechoslovakia.
Other twin cities and partner towns of Kosice include Budapest (H), Verona (I), Ostrava (CZ) and Bursa (TR).
- Backpacking
- Trains
- Budget Travel
Slovak Beer
On my trips to Slovakia I always tried some of the local beers.
One of my favourite Slovak beers is the lager of Zlaty Bazant (Golden Pheasant). The brewery was founded in 1967 in Hurbanovo, and is nowadays owned by Heineken.
Another popular Slovak beer is Saris. The brewery was established in 1964 in Velky Saris and taken over by SAB Miller in 1995.
Zlaty Bazant:
- Trains
- Backpacking
- Budget Travel
This building in the down town...
This building in the down town of the city has a beautiful history. The legend says:
Ages ago, in Kosice lived a man - a beggar. This beggar lived his boring everydays life and begged for money. Because he was a miserable poor man, kind people gave him a change, money or something else.
But this beggar was a clever guy. He didn´t spend this money. After a years of his beg, he collected a sufficient amount of money and in the center of city he built up this house. And he ordered for this house a statue of beggar,(what you can see at the top of the building) as a memory of fame of beggar form Kosice. That the legend talks...
If you are arriving for Prague...
If you are arriving for Prague thinking that you've picked up some Czech language you are in for a shock. This is a similarly written language for the most part but, completely different in speaking. English is quite well used here by business. It is afterall the 2nd largest city after Bratislava.
- 1
Show Prices
Show Prices
Show Prices
Show Prices
Instant Answers: Kosice
Get an instant answer from local experts and frequent travelers
- Great room rates
- Hotel Bankov Kosice
- Booking.com
- Great room rates
- Bristol Hotel Kosice
- Expedia.com
Sponsored Links


text:
WELCOME!!Thanks for stopping by... I hope you find something to enjoy. I do occasionally rant and whinge but mostly have fun doing my posts... please have a look and do feel free to comment and maybe even return for more on another day!
Flickr Photos
More Photos
Hit Counter
- 132,840 hits
Old Stuff… almost like me
Midlands SIMON
Have a Look-see!!
North Kildare Cricket – reminders of a summer??
Biltong, Braai & Boerewors!
Life in South Africa?
Community – Special Folk!!
Tags… scatter and splash…Architecture art beauty birds blog buddies blogging blue boats buildings camera canals challenge city life clouds Co Kildare colour creative writing DPchallenge Dublin flowers focus food friendship fun future GLW gratitude green history hope humour ideas inspiration Ireland Irish countryside JOY Kilcock life life... haha... Liffey light macro mode my blog night photography opinion past people watching photography plants positive postaday questions quirky rain Royal Canal sea South Africa summer sunshine thanks time trains travel trees urban photography walk weather Weekly Photo Challenge: wildlife photography winter WordPress words work writer writing
All Great Reading or Viewing!
Tag Archives: grief
Friendships not Forgotten!
Today I'll keep it very brief. Today, in our local community is a day of both remembering the good times as well as the sadness of a friend's untimely passing. Yes Mick… you are sadly missed. Not only by your … Continue reading
Posted in Family & Life, Ireland, Local Curiosity, Local Places Tagged barber, death, gratitude, grief, inspiration, Kilcock, local shop, loss, Mick the Barber, pain, postaday, sky 4 Comments
Ode to the Rainbow Nation
There was hopeful joyous anticipation, even celebration On that momentous 94 April day of awaited rejuvenation Expectant cues formed at each and every polling station To vote for the new leaders of the young Rainbow Nation Great promises of prosperity … Continue reading
Posted in Family & Life, Writing Tagged Africa, colour, creative writing, crime, experience, grief, hurt, inspiration, love, murder, past, peace, poetry, postaday, pride, rainbow, ruins, soul, South Africa, verse, writer 4 Comments
Have You Ever Wondered?
Have you been faced with the situation of being told to move on… to get over it, you've had your say now just forget about what's eating at you. About two months ago a did a few posts about what … Continue reading
Posted in Factual Opinion, Family & Life, Writing Tagged crime, danger, DPchallenge, fair play, faith, future, grief, hurt, life, money, murder, opinion, pain, past, postaday, questions, Saffers, South Africa, the will to survive, truth, WordPress, words, writer 3 Comments
South Africa's New National Sport?
I ask myself if I'm allowed to even dare suggest that crime and violence is sport. But on the other hand, if I don't ask the question, will I not also be culpable or complicit in the carnage? Yes, that … Continue reading
Posted in Family & Life Tagged Africa, Blade Runner!!, blogging, choices, crime, death, DPchallenge, freedom, future, gratitude, gravestones, grief, hope, hurt, ideas, inspiration, longing, loss, love, murder, opinion, passion, past, Port Elizabeth South Africa, postaday, questions, rant, Saffers, South Africa, WordPress 4 Comments
Revulsion and Shame!
The more I thought of the news my GLW shared with me on Saturday morning the more the loathing increased. I am, at this point of my life, finding it very, very difficult to call myself a proud South African. … Continue reading
Posted in Family & Life Tagged #memyblog, Africa, blogging, change, crime, DPchallenge, faith, freedom, friendship, future, gratitude, grief, hope, inspiration, internet, longing, love, murder, past, peace, positive, postaday, questions, Saffers, soul, South Africa, the will to survive, Weekly Photo Challenge:, WordPress, words, WP, WPC 32 Comments
Zach and Steve Meet Again…
Stefaans, or Steve to his friends, was sitting opposite Zach. His indigo blue eyes perfectly offset by his tanned features. A bit Hollywood, thought Zach. Only one difference, Stefaans was for real. No acting here… just the self-same old friend … Continue reading
Posted in Family & Life, Fiction, Writing Tagged #memyblog, Africa, art, creative writing, DPchallenge, experience, fiction, friendship, grief, hope, Namibia, painting, passion, past, peace, postaday, short story, soul, South Africa, story, tales, the will to survive, war, writing, Zach 1 Comment
Blessed and Privileged – Shared Joy!
Today I'm beginning a few posts about a set of events and their impact on our lives… an impact we will never forget! We are the lucky ones… the good lady and I, to be able to bring you this … Continue reading
The Rose Quartz Power…
"This place is full of mystery… full of rumour, myth… tales of greed and dying. This place is touched. " Zach will always remember that night around the camp fire at the oasis… the hot springs about a day's walk … Continue reading
There's that old line, used by many a man … Beauty is in the eye of the beholder Time's onward march queries the plan Oft burdening the already weary shoulder Questioning where toils and strife began Or, is it merely … Continue reading
Posted in Writing Tagged creative writing, freedom, grief, inspiration, life, longing, loss, love, passion, poetry, postaday, rain drops, rose 4 Comments
We Salute You Friend!
I won't lie Mick… looking through the archive to celebrate your life again brought a lump to my throat… again brought tears to my eyes. Yes… we still find it difficult to deal with your sad passing. Yes… we mourn … Continue reading
Posted in Family & Life Tagged barber, cemetery, Co Kildare, friendship, fun, grief, hope, inspiration, Ireland, Kilcock, local shop, longing, loss, love, postaday 4 Comments


text:
Duchesne County
Duchesne city: July 4, 7 a.m. breakfast; dusk, fireworks. 435-738-2464.
Tabiona: Call 435-848-5481 for details.
Emery County
Huntington: July 3, 5 p.m. kid's pet contest; 6:30 p.m. MECCA bike ride; 7 p.m. Huntington Heritage Idol contest; 7:30 p.m. ice cream social, City Park. July 4, 6 a.m. flag-raising, 5K run; 7 a.m. breakfast; 9:30 a.m. kids parade; 10 a.m. parade; noon patriotic program; 1 p.m. Brenn Hill, entertainment; 3 p.m. 3-on-3 basketball tournament; all day, big toys, food, booths in city park; dusk, fireworks, Huntington Arena, 435-687-2436.
Orangeville: July 4, 9:30 p.m. fireworks at the Ball complex on Mill Dam Road. 435-748-2651.
Garfield County
Boulder: July 5, 6 p.m. ice cream social and talent show at Boulder Mountain Lodge, July 4, 4 p.m. parade; 5 p.m. games and patriotic program; 6 p.m. dinner; 8:30 p.m. dance, Boulder Town Hall and grounds, 435-335-7300.
Tropic: Call 435-679-8713 for details.
Garfield County: Call 435-676-8826 for details.
Iron County
Cedar City: July 4, 9:30 a.m. parade followed by carnival activities throughout the day at Main Street park; dusk, fireworks, 1600 N. 3000 West, 435-586-4484.
Enoch: July 4, 7 a.m. 5K run, 10K run, family walk, car show and road run, 9:15-9:45 a.m. check-in time, 10 a.m. departure time, 11 a.m. car show starts; noon lunch, cook-off, Enoch Little League Park. 435-586-1119
Parowan: July 4, 10 a.m. parade followed by fund-raiser, foot races, games, food, Town Square Park; dusk, fireworks at the Race Track. 435-477-3331.
Juab County
Call 435-623-3410 for details.
Kane County
Kanab: July 4, 7 a.m.-10 a.m. Lions Club breakfast, LDS Church parking lot; 10 a.m. parade; 11 a.m.-3 p.m. food, booths, crafts and games; 5 p.m. dinner, live entertainment; dusk, fireworks. 435-644-2534.
Millard County
Delta: July 3, 7 p.m. children's games and dancing in the City Park. July 4th, 7 a.m. breakfast at the city park; 9 a.m. bunny hop; 9:30 a.m. parade with F-16 flyover followed by a patriotic program; noon games, food, entertainment, volleyball tournament, city park; 2 p.m. free swim, matinee; 6 p.m. demolition derby; 10 p.m. Centennial Musical Fireworks, Millard County Fairgrounds, 435-864-2759.
Fillmore: July 3, 6:30 p.m. children's parade; July 4, 7-9 a.m. breakfast and flag ceremony; 9 a.m. freedom mile run registration; 10 a.m. parade; 11:30 a.m.-2 p.m. entertainment and food in the City Center Park; 1-4 p.m. free swim; 2 p.m. pioneer rounder's baseball; 3 p.m. Millard High skeet shoot at the rifle range; fireworks at dusk at the race tracks, 435-743-5233.
Morgan County
Morgan City: July 4, 8 a.m. fun run, tennis tournament; 9:30 a.m. bike parade; 8:30 a.m.- 6 p.m. ball games, noon-afternoon, 3-on-3 soccer; 2 p.m. water activities; fireworks dusk; dance 9-11:30 p.m. at the Recreation Complex, 801-829-6811.
Piute County
Circleville: 10 a.m. parade followed by lunch; fireworks at dusk.
Junction: noon, pork rib dinner.
Kingston: 9 a.m. parade.
Rich County
Woodruff: July 4, Woodruff Days Celebration, Call 435-793-2415 for details.
Salt Lake County
- Ex-EFY counselor sent to prison for sex with...
- Utah's first family of rodeo: Riding buckin'...
- Man who killed fiancee ordered to serve 1...
- Biskupski calls for resignations of nearly...
- Men and women: Understanding the wage gap is...
- Olene Walker, "one of Utah's finest public...
- Police: Intruder attacks homeowner with...
- UHP welcomes back Kotter, a trooper shot 4...
- Men and women: Understanding the wage... 34
- Olene Walker, "one of Utah's finest... 15
- Man steals woman's boarding pass,... 13
- The new Thanksgiving tradition: A quick... 11
- MTC missionaries spend Thanksgiving... 11
- Utah liquor consumption is up, but... 8
- Recruiting crisis? UHP, other agencies... 7
- Tanker crash sends oil into Provo River... 6


text:
The lower shelf of this teak shower seat features decorative rails, making it perfect for storing toiletries with ease. Teak is naturally honey-colored and can be stained or sealed, if desired.
Ships in 1 - 2 Business Days
Product Details
- Installation Type: Freestanding
- Design: Transitional
- Product Finish: Unfinished Teak
- Material: Teak
- Width: 23-5/8"
- Height: 18"
- Depth: 16-1/2"
- Assembly Required: No
- Weight Limit (lbs): 220
- Height from Ground to Seat: 18"
- Seat Depth: 16-1/2"
- Seat Width: 23-5/8"
- Made of natural teak.
- Wood is unsealed and weathers to a silver-gray color if left outdoors.
- Maintain or restore color by using Teak Guard products.
- Simple assembly required. Easy-to-follow instructions included.
PERFECTThis shower bench is the perfect size for my shower. It has lots of space under it to store all your needed bath supplies and it looks great!
Shower benchI purchased this stool because it was the perfect fit for my master bath shower. It is just as advertised and fits the bill to store all of our shower needs. Still looks new 6 months later.
2 Item(s)
Write Your Own Review


text:
Designed for club and school level with it's size and use of lightweight density foam.
Size: 1070mm x 510mm x 125mm.
To view related products please click on the catalogue page 68 above.
All products carry a 1 year, 100% guarantee.
[ Back to top of page ]
Add items to see them here


text:
Good values on homes are still available, as the number of homes listed for sale is greater than the number of homes being sold. Great deals can be found by determined buyers.
Rent prices are relatively stable, and availability is good. If your yearly income is around $55,000, you can afford to rent in this market. Rent prices have not changed over the past 3 months. This is 45% lower than the state average of $1,158 and 100% higher than the country average of $0.
The average home price in 59602 is $199,900. The average home price per square foot in 59602 is $83 and about 0% of all homes in 59602 sold in the last 6 months. For 59602 home values, real estate prices, and city market trends, research the 8,966 property records or search our 0 recently sold or 483 homes for sale listings. The listings and properties in 59602 are only a part of the 308,131 properties and 19,626 homes for sale in Montana. Because of this, the average home price and average home value of 59602 also influence the average home price $175,000 and average sale price $120,408 of Montana. Listing counts, 59602 home prices and home values in 59602 are sourced daily from listing and property data on realtor.com®, which is the most up-to-date and accurate aggregation of real estate listings in the industry and includes approximately 800 regional MLSs and is the official website of the National Association of Realtors®.


text:
Scholarships funded through Robert Wood Johnson Foundation New Careers in Nursing Program
MAYWOOD, Ill. -- Loyola University Chicago Marcella Niehoff School of Nursing (MNSON) has been awarded Robert Wood Johnson Foundation (RWJF) funding through the RWJF New Careers in Nursing Scholarship Program. Grants provided through this program will be used for scholarships to maximize diversity and increase the quantity of students enrolled in MNSONâs Accelerated Bachelor of Science in Nursing (ABSN) program.
This national initiative was launched by RWJF and the American Association of Colleges of Nursing (AACN). The program aims to help alleviate the nationâs nursing shortage by dramatically expanding the pipeline of students in accelerated nursing programs.
âLoyola is honored to receive a grant that is in concert with our Jesuit values of giving back to disadvantaged populations,â said Vicki Keough, PhD, RN-Cs, ACNP, CCRN, professor and acting dean, MNSON. âThis grant will allow Loyola to attract and educate a diverse group of individuals who might not otherwise have made a change to a career in nursing.â
Scholarships in the amount of $10,000 will be awarded to 15 accelerated nursing students enrolled in the spring 2010 semester. Award preference will be given to students from groups underrepresented in nursing or from disadvantaged backgrounds. Groups underrepresented in nursing include men, African Americans, Hispanics, Asians and students with financial limitations. Grant funding also will be used by schools to help leverage support for new faculty resources and provide mentoring and leadership development to ensure successful program completion by scholarship recipients.
âNew Careers in Nursing aims to safeguard the health of the nation by easing the nurse and nurse faculty shortage. Nurses are critical to delivering health care that is effective, patient-centered, timely, efficient and equitable,â said RWJF Senior Adviser for Nursing Susan B. Hassmiller, PhD, RN, FAAN. âThis initiative also will advance the Foundationâs strategic goal of promoting a health professional workforce that reflects the diversity of the American public.â
The RWJF New Careers in Nursing Scholarship Program supports accelerated programs, which offer the most efficient route to licensure as a registered nurse for adults who have already completed a baccalaureate or graduate degree in a discipline other than nursing. Although enrollment in these programs has steadily increased over the past few years, many potential students are unable to enroll since having a college degree disqualifies them from receiving assistance from most federal financial aid programs for entry-level students. The New Careers in Nursing scholarships address this problem and also will alleviate the overall nursing shortage by enabling hundreds of students to launch their nursing careers through accelerated education not otherwise possible without scholarships.
Additionally, the program targets the need to recruit students from groups underrepresented in nursing or disadvantaged backgrounds. According to the National Advisory Council on Nurse Education and Practice, diversifying the nursing profession is essential to meeting the nationâs health-care needs and reducing health disparities that exist among many underserved populations. Data from the U.S. Health Resources and Services Administration also show that nurses entering the profession at the baccalaureate level are four times more likely than other nurses to pursue a graduate degree in nursing, which is the required credential to teach. Consequently, bringing more nurses into the profession at the baccalaureate and masterâs degree levels will help to address the nationâs nurse faculty shortage.
For more information on applying for scholarships at MNSON, contact Dr. Ann Solari-Twadell at 773-508-2909.
AACN serves as the National Program Office for this RWJF initiative and oversees the grant application submission and review processes. For more information about this program, visit www.newcareersinnursing.org.


text:
joind on: Mar, 09 2004
last visit: May, 25 2010
applause received: 1
member posts: 524
member threads: 68
member flags: 5
member stars: 18
ave. threads per day: 0.03
ave. posts per day: 0.23
WATS score: 1.04
member karma: 1
This content community relies on user-generated content from our member contributors. The opinions of our members are not those of site ownership who maintains strict editorial agnosticism and simply provides a collaborative venue for free expression.
All content copyright 2015, The Above Network, LLC.


text:
The state hasn't declared victory in the war against veteran homelessness, but officials say they have won a key battle.
Virginia has, as of now, the resources to house any homeless veteran who seeks help. They're calling it a "functional end" to homelessness among the state's 800,000 veterans.
Gov. Terry McAuliffe was scheduled to announce the achievement Wednesday morning at a Veterans Day celebration at the Virginia War Memorial in Richmond.
The state can never eliminate homelessness, officials say. A lost job or family dispute can put someone on the street without notice. A functional end to homelessness, however, means...


text:
Vodacom Earnings Miss Estimates on Network Spending, Prices
Vodacom Group Ltd., the largest provider of mobile-phone services to South Africans, said fiscal first-half profit rose 6.4 percent, missing estimates as it boosted spending to avoid a repeat of network outages.
Diluted earnings per share excluding one-time items increased to 3.22 rand in the six months ended Sept. 30, the Johannesburg-based company said today in a statement. That was lower than the 3.33 rand median estimate of four analysts surveyed by Bloomberg News.
The company, 65 percent owned by Newbury, England-based Vodafone Group Plc, is lowering prices to gain clients, to compensate for slower growth in service revenue. Vodacom reduced data and voice prices in the period, by 22 percent and 24 percent respectively in South Africa, where other operators have lowered tariffs.
Sales climbed 7.6 percent to 31.7 billion rand ($3.97 billion) while net income was little changed at 4.4 billion rand in the six months compared with a year earlier. Capital expenditure surged 68 percent to 3.46 billion rand.
"There's still scope for more price reductions, according to our business plan," Chief Financial Officer Rob Shuter said on a conference call. Vodacom wants to increase data revenue by 30 percent a year.
Vodacom shares declined 1.1 percent to 88.25 rand at 10:33 a.m. in Johannesburg, paring their gain this year to 15 percent.
The company said it expects the margin based on earnings before interest, taxes, depreciation and amortization to improve this fiscal year. While capital expenditure will accelerate in the second half, full-year spending should remain within the company's forecast of 7.7 billion rand, Vodacom said.
The number of customers rose 22 percent to 48 million while data subscribers increased by 38 percent, the company said. Subscriptions in South Africa rose to 29 million users as Vodacom regained customers it lost after the country enacted laws that require customers to be registered. Vodacom gained 23 percent more subscribers in other parts of Africa.


text:
The latest news, notes and analysis on the Marquette men's basketball team
Nothing yet on Buzz
I was told today that there is nothing new on the Buzz Williams contract front.
Nothing really to read into one way or another there -- it's likely the two sides are still working on the parameters of a new deal to keep Williams in the fold for the long haul.
The guess here is it's going to be a deal worth more than $2 million annually. How it will be structured -- whether it's another rollover contract, how much the buyout will be and so on -- will probably be the major sticking points. He's currently signed to a six-year rollover deal paying him $1.6 million. That figure is scheduled to jump to over $1.7 million in the coming months.
Undoubtedly, Williams would like the buyout in his new contract to be lower than the figure that it would take to get him out of his current deal: $3.8 million, all told. It's standard in contracts for the buyout to drop every few years in order to afford the coach some flexibility as time goes on, so looking for something like that to be included wouldn't be out of the ordinary from Williams' standpoint.
MU, from its vantage point, has no problem paying the going rate for a hot young coach like Williams. The school proved that with Tom Crean years ago. The key will be structuring the deal in such a way that it can protect itself somewhat in the coming years, as Williams will likely be a popular name for most coaching vacancies that pop up each off-season, just as he is now, and like Crean was.
Both parties appear to be on the same page, and Williams after MU's loss last Friday appeared in no way interested in moving on. Things can obviously change, and nothing is set in stone until an agreement is announced. But my guess is something will come together at some point over the course of this week, and the carousel can finally stop spinning in Milwaukee.
Williams' staff could take a hit, though, as assistant Aki Collins has been mentioned as a possible candidate for the head job at Fairfield, and Tony Benford could be a possibility for some openings as well.
Both have done a solid job for Williams, especially with regard to recruiting.