Genome wide association studies (GWAS) have identified several genetic variants that are associated with prostate cancer. Most of these variants, like other GWAS association signals, are located in non-coding regions of potential candidate genes, and thus could act at the level of the mRNA transcript.
We measured the expression and isoform usage of seven prostate cancer candidate genes in benign and malignant prostate by real-time PCR, and correlated these factors with cancer status and genotype at the GWAS risk variants.
We determined that levels of LMTK2 transcripts in prostate adenocarcinomas were only 32% of those in benign tissues (p = 3.2 × 10-7), and that an independent effect of genotype at variant rs6465657 on LMTK2 expression in benign (n = 39) and malignant tissues (n = 21) was also evident (P = 0.002). We also identified that whilst HNF1B(C) and MSMB2 comprised the predominant isoforms in benign tissues (90% and 98% of total HNF1B or MSMB expression), HNF1B(B) and MSMB1 were predominant in malignant tissue (95% and 96% of total HNF1B or MSMB expression; P = 1.7 × 10-7 and 4 × 10-4 respectively), indicating major shifts in isoform usage.
Our results indicate that the amount or nature of mRNA transcripts expressed from the LMTK2, HNF1B and MSMB candidate genes is altered in prostate cancer, and provides further evidence for a role for these genes in this disorder. The alterations in isoform usage we detect highlights the potential importance of alternative mRNA processing and moderation of mRNA stability as potentially important disease mechanisms.
Cancer of the prostate is the most common male malignancy in the Western world, accounting for 25% of all male UK cancers (taken from http://info.cancerresearchuk.org/cancerstats). Although survival rates are increasing, cancer of the prostate remains the second most common cause of cancer death in UK men after lung cancer. Several risk factors have been identified, the main factors being age , family history  and ethnic origin . Prostate cancer is uncommon in men under 50 years old, but 80% of men aged over 80 years were found to have cancerous cells in their prostate at the time of death . Estimates suggest that between 30 - 40% of all early onset cases of prostate cancer (< 55yrs) are caused by inherited factors , highlighting the importance of genetics in this disorder. Early-onset prostate cancer that aggregates in families is likely to result from the inheritance of rare genetic variants that have a large impact. Several strong risk factors have been identified from family studies which include mutations in the ELAC2, RNASEL and MSR1 genes involved in immune response  and the BRCA1 and BRCA2 genes involved in the development of breast cancer .
Despite increases in the understanding of the familial forms of prostate cancer, the aetiology of the remaining 60% of cases remains unclear. The advent of genome wide association studies (GWAS) has resulted in a dramatic increase in the number of susceptibility loci that have been associated with the development of prostate cancer (table 1). Four independent studies in the Icelandic, UK and American populations have identified more than 20 distinct genomic locations as being implicated in susceptibility to prostate cancer [6–9].
Loci that have been identified as significant determinants of prostate cancer risk in four independent genome wide association studies.
MAF = minor allele frequency in the HapMap CEU population.
Many of the GWAS association signals identified are located in regions of the genome that are not translated into protein. This may indicate that the associated marker is in close linkage disequilibrium with an uncharacterised variant in the protein coding region, or that the functional SNP may cause its effect by epigenetic or histone modification. The other possibility is that the variant may act at the level of the messenger RNA.
There are several mechanisms by which variants in non-coding regions of the mRNA transcripts could affect gene function. Firstly, if the variant lies in the promoter or 5' untranslated region of the transcript, it may interfere with the efficiency of transcription or translation, as has been shown to be the case for several human and rodent genes [10–12]. Variants located within the intronic sequences may interfere with mRNA splicing . The production of aberrant splice products has previously been shown to be very important in the aetiology of cancer in general [14, 15], but may contribute to the biological heterogeneity of prostate cancer in particular . Finally, polymorphisms located in the 3' untranslated region may disrupt regulatory elements necessary for mRNA stability, interfere with regulation of translation by microRNAs or influence the polyadenylation dynamics of the transcript [17–19].
In this series of experiments, we sought to investigate whether the genes suggested by the GWAS for prostate cancer were differentially expressed in malignant prostate tissues, and whether their expression was influenced by genotype at the GWAS risk variants. Seven genes were selected for study (figure 1). We measured the total or isoform specific gene expression of the JAZF1 (JAZF zinc finger 1), CTBP2 (C-terminal binding protein 2), LMTK2 (lemur tyrosine kinase 2), SLC22A3 (solute carrier family 22 extraneuronal monoamine transporter, member 3), MYEOV (myeloma overexpressed), MSMB (microseminoprotein beta) and HNF1B (HNF homeobox 1 beta) in a cohort of 39 non-malignant benign prostatic hyperplasia (BPH) samples and 21 prostate adenocarcinoma samples, and correlated gene expression with cancer status and with the genetic variants identified in the GWAS (table 1). We demonstrate two of the eight variants are correlated with gene expression levels in non-malignant prostate tissues, and that the presence of adenocarcinoma is associated with altered gene expression levels and differential expression of alternatively processed isoforms for three of the candidate genes in prostate tissue itself.
Schematic representation of the structure of transcripts studied here. The structure of the JAZF1, SLC22A3, LMTK2, MYEOV, MSMB, HNF1B and CTBP2 mRNAs are represented here, together with their RefSeq ID numbers. Exons are represented by numbered grey boxes. The black box represents the section of exon 3 absent from isoform HNF1B(B). The section of intronic sequence present in HNF1B(C) is indicated by a hatched box.
The prostate samples in this study were obtained from the Exeter tissue bank, and comprised prostate chippings taken during routine transurethral resection of the prostate (TURP). Both cancer and BPH groups originated from a population of older men treated for urological problems at the Royal Devon and Exeter Hospital, Exeter, UK and were predominantly of Caucasian origin. Samples were flash frozen in liquid nitrogen upon removal, and stored at -80°C until required. Subjects with benign prostatic hyperplasia (n = 39) had no sign of prostate malignancy at the time the samples were taken, and none had received a subsequent diagnosis of prostate cancer 5 years following their diagnosis of benign prostatic hyperplasia (BPH). The median age of the BPH cohort was 83 years (IQR 78,88) and the median age of the prostate cancer cohort was 83 years (IQR 76,87). BPH samples were used as the control group for several reasons; firstly, most males have evidence of BPH by the age of 70 or 80, so the presence of some degree of BPH is 'normal' at the median age at diagnosis in our prostate cancer cohort (77 yrs, [IQR 72,82]). Truly 'normal' prostate tissue would thus only be obtained in a much younger control cohort, which could introduce bias. Secondly, since collection of prostate tissue requires a surgical procedure, which would only be undertaken in subjects with evidence of symptoms of prostate enlargement. Even then, morphologically normal tissue may harbour premalignant genetic abnormalities that are not immediately obvious. However, we acknowledge that genetic heterogeneity between patients and controls may influence the gene expression from individual to individual. Prostate cancer samples (n = 21) underwent confirmation of cancer status, histology and Gleason scoring by dissection of the TURP chips and subsequent histological examination of tissue from either side of the sample used for RNA analysis. All cancer samples were classified as prostatic adenocarcinomas, of which 57% were Gleason score 6 or 7, and 43% were Gleason score 8-10. Further analysis of patient case notes revealed that 8/19 prostate cancer patients and 6/37 Benign Prostatic Hyperplasia patients had been exposed to hormone treatment (Zoladex, Finasteride, Casodex or Proscar).
All participants had consented to their tissue being used in this study, and ethical approval was obtained from the South West Research Ethics Committee. This study was carried out according to the Declaration of Helsinki.
DNA was extracted from prostate chippings using the Qiagen DNA mini kit (Qiagen, Crawley, UK) using standard procedures on the fully automated QiaCube sample preparation platform (Qiagen, Crawley, UK), following manual homogenisation of tissue.
Seven candidate genes were identified for expression analysis. These were chosen on several criteria. Firstly, we chose our candidate genes on the basis that the GWAS index SNPs were located within the gene sequence itself, or very close to the genes in question. The linkage disequilibrium blocks identified in the GWAS may contain other genes (or variants) that may contribute to the risk of developing prostate cancer, the majority of these are located remotely to the association signal and do not represent better biological candidates. Secondly, all seven are good biological candidates, having roles in cell proliferation, regulation of apoptosis or prostate development and function. Analysis of the linkage disequilibrium structure around the GWAS index SNPs revealed several other candidates, but these were not analysed due to a lack of expression in the prostate, a lack of convincing biological evidence for a role in prostate function or the presence of more plausible candidate genes. The remaining four signals located on chromosomes 3, 8, 19 and X were not analysed due to the lack of an obvious candidate gene.
Genotyping was carried out by PCR amplification and direct sequencing of the sequence flanking the variant in question; primers and conditions are available on request. Amplification reactions contained 10 μl Megamix Royal (Microzone, Haywards Heath, UK), 10 μM primers and 50 ng DNA in a total volume of 20 μl. Sequence specific primers for each amplicon were tagged with 5' M13 tails to allow sequencing to be performed with a universal M13 primer. Single-strand sequencing was carried out using standard methods on an ABI 3730 (Applied Biosystems, Warrington, UK). Sequences were compared to a designated individual homozygous for the major allele of the variant using Mutation Surveyor (version 3.20; SoftGenetics, State College, PA).
RNA was extracted from prostate chippings using the Qiagen RNAeasy kit (Qiagen, Crawley, UK) on the fully automated QiaCube sample preparation platform (Qiagen, Crawley, UK), following manual homogenisation of tissue with an additional pass through a Qiagen Qiashreddder column (Qiagen, Crawley, UK) column to shear genomic DNA. RNA samples were stored at -80°C prior to analysis.
Approximately 500 ng of mRNA from each sample was treated with 1u of RNAse-free DNAse (Promega, Madison, USA) for 30 minutes at 37°C followed by 65°C for 10 minutes for nuclease inactivation. Complementary DNA (cDNA) was synthesized from mRNA using the Superscript III VILO RT-PCR system (Invitrogen, Paisley, UK) according to manufacturer's instructions, with an incubation temperature of 42°C and a reaction volume of 40 μl.
Real-time PCR reactions using target-specific probes were carried out in triplicate using the ABI Prism 7900 HT platform (Applied Biosystems, Warrington, UK) and contained 5 μl TaqMan Fast Universal Mastermix (no AMPerase) (Applied Biosystems, Warrington, UK), 0.9 μM each primer, 0.25 μM probe and 2 μl cDNA reverse transcribed as above in a total volume of 10 μl. PCR conditions were a single cycle of 95°C for 20 seconds followed by 50 cycles of 95°C for 1 second and 60°C for 20 seconds. Individual probe and primer sets were pre-validated off-the-shelf assays to specific transcripts provided by Applied Biosystems (Warrington, UK; Assay identification numbers available on request). The relative expression level of each isoform was then determined relative to the B2 M and GUSB transcripts by the comparative (ΔΔCt) method . The abundance of each target in each individual was normalized to the average measurement for the transcript in question across the cohort prior to correlation of expression levels with genotype.
Measurement of the relative abundance of transcripts bearing different alleles at variant rs3801294, in perfect linkage disequilibrium (r2 = 1; D' = 1) with the LMTK2 index SNP rs6465657, was carried out in single tube reactions by allele-specific real time PCR. PCR amplicons were generated by a common set of primers for both alleles, but transcripts bearing different alleles were discriminated by the use of allele-specific probes labelled with different fluorochromes (6-fluorescein [6-FAM] and VIC); probes and primers available on request. This allows a measurement of transcript abundance that is independent of amplification efficiency. Real-time PCR reactions using were carried out in triplicate using the ABI Prism 7900 HT platform (Applied Biosystems, Warrington, UK) and contained 5 μl TaqMan Fast PCR Master Mix, no AmpErase™ (Applied Biosystems, Warrington, UK), 72 μM each primer and 16 μM each probe) in a total volume of 10 μl. PCR conditions were a single cycle of 95°C for 20 seconds followed by 60 cycles of 95°C for 1 second and 60°C for 20 seconds. The expression level of transcripts bearing the G allele (marking the C allele at rs6465657) was then calculated relative to that of transcripts bearing the A allele (marking the T allele at rs6465657) by the comparative (ΔΔCt) method , as we have done for several previous studies [21, 22]. Quantifications were then compared to the average measurement obtained from eleven independent DNA samples, which should provide a known 50:50 mix of A and G-bearing transcripts, in order to control for differences in probing efficiencies.
The statistical significance of apparent differences in gene expression levels was investigated by Kruskal Wallis analysis for multiway comparisons and by Mann Whitney-U analysis for pairwise comparisons. Non-parametric statistics were employed due to the relatively small sample numbers and the fact that the data obtained were not normally distributed. In the case of the association between LMTK2 expression and cancer status, linear regression was carried out on natural logarithmically transformed data to allow adjustment for the effect of genotype at rs6465657, which we found to be independently associated with LMTK2 expression. All statistical analyses were carried out using SPSS v15.0 (SSPS PLC, Chicago, USA).
We obtained 426/480 genotypes for the 8 GWAS variants in our cohort of 39 benign prostatic hyperplasia specimens and 21 prostate adenocarcinoma samples (310 for the BPH cohort and 116 for the cancer cohort). All three genotypes were detected for the variants, with the exception of minor allele (C) homozygotes for rs4962416 within the CTBP2 gene, as expected given the minor allele frequency of only 0.229 in the HapMap CEU population.
Analysis of potential correlations between genotype at the GWAS association loci and isoform-specific gene expression of nearby candidate genes revealed correlations between risk genotype at the GWAS variants rs6465657 and rs4962416 with the expression of LMTK2 and CTBP2 respectively. Individuals carrying two copies of the protective T allele of rs6465657 expressed almost a third less LMTK2 mRNA than did individuals carrying one or two copies of the risk 'C' allele (0.68 [Interquartile range [IQR] = 0.63] v/s 1.18 [IQR = 0.56]; P = 0.007). Similarly, individuals homozygous for the 'T' allele at rs4962416 expressed 37% less of a partially characterised isoform of CTBP2; CTBP2(4), than did individuals with CT genotypes (0.82 [IQR 0.83] v/s 1.31 [IQR 0.96]; P = 0.009). Although neither of these findings quite reaches full statistical significance following adjustment for multiple testing (P = < 0.004 for 12 independent tests), they do indicate that these genes are worthy of further study.
We measured the abundance of each LMTK2 allele of rs6465657 in 17 heterozygous individuals, and determined that transcripts bearing the rs3801294 'A' allele, which is in perfect linkage disequilibrium (r2 = 1; D' = 1) with the protective 'T' allele at rs6465657, were significantly less abundant in heterozygous BPH tissues than were those carrying the 'G' allele, linked to the 'C' allele of rs6465657. The ratio of protective 'A' alleles to risk 'G' alleles in heterozygous individuals is 0.65 [IQR 0.12]:1 [IQR 0.33]; P = 3.3 × 10-5, figure 2), which remains statistically significant after adjustment for multiple testing. This provides further evidence for a role of LMTK2 in determining susceptibility to prostate cancer. No correlations of genotype with expression levels were noted for the remaining isoforms of the CTBP2 gene, or any isoform of the JAZF1, MYEOV, SLC22A3, HNF1B or MSMB genes in BPH tissues.
Allele-specific expression of LMTK2 transcripts. This histogram demonstrates allele-specific differences in the expression of LMTK2 transcripts bearing either the G or A allele at rs3801294, which is in perfect linkage disequilibrium (r2 = 1; D' = 1) with the index SNP rs6465657. The sample identity is given on the X-axis and the ratio of transcripts carrying G alleles (marking the C allele of rs6465657) relative to those carrying the A allele (marking the T allele at rs6465657) is given on the Y-axis. Error bars represent the interquartile range (IQR) of the measurements. The level of significance as determined by pairwise Mann Whitney-U analyses is indicated by asterisks.
We correlated expression levels at each of the loci with prostate cancer status (table 2). We found that expression levels of the LMTK2 gene were also inversely correlated with the presence of prostate cancer. Tumour samples expressed 68% less LMTK2 mRNA than did non-malignant BPH samples (figure 3; relative expression levels were 1.10 [IQR 0.77] in BPH versus 0.35 [IQR 0.30] in prostate cancer; p = 2.4 × 10-7), after correction for the potential effect of genotype at rs6465657. Amongst the eight members of the prostate cancer cohort who had exposure to antiandrogenic drugs, no effect of prior hormone treatment on the expression levels of LMTK2 was noted (0.49 [IQR 0.29) for untreated subjects versus 0.32 [IQR 0.30] in treated subjects; P = 0.270).
Total expression levels of candidate genes in non-malignant and prostate cancer tissues.
Gene expression levels are analysed relative to the beta 2 microglobulin (B2M) and beta glucorinidase (GUSB) genes, and normalised to the average value in the non-malignant samples. The interquartile range is given in parentheses. A P value of 0.007 (P = 0.05/n° of tests) was taken to indicate statistical significance following correction for multiple testing (7 independent tests). Statistically significant results are indicated in bold italic type.
Expression of LMTK2 transcripts is reduced in prostate cancer. This boxplot gives the cancer status (benign prostatic hyperplasia or prostate adenocarcinoma) on the X-axis, and the expression of the single isoform of the LMTK2 gene relative to the endogenous controls beta-2-microglobulin (B2M) and beta-glucorinidase (GUSB) on the Y-axis. Error bars represent the interquartile range (IQR) of the measurements. The level of significance as determined by linear regression of natural logarithmically transformed data is indicated. Outliers are indicated by circles.
We also noted differences in the total expression of the SLC22A3 and CTBP2 genes in BPH compared with prostate cancer. CTBP2 and SLC22A3 mRNA levels were reduced by 27% and 50% in prostate cancer tissues compared with BPH tissues (4.2 [IQR 2.5] v/s 2.1[IQR 1.6]; P = 0.008 for CTBP2 and 0.48 [IQR 0.75] v/s 1.01 [IQR 1.42]; P = 0.015 for SLC22A3). CTBP2(1) and CTBP2(4) transcripts showed a trend towards lower expression in prostate cancer subjects treated with hormone therapy (CTBP2(1) ; (0.87 [0.40] in untreated subjects versus 0.31[IQR0.17]; p = 0.006) and CTBP2(4) transcripts (1.01[IQR 0.99] in untreated subjects versus 0.45 [IQR0.42] in treated subjects; p = 0.006) but these results do not reach statistical significance following adjustment for multiple testing (P = < 0.004 for 12 independent tests). This observation was also not noted in the BPH cohort (P = 0.77 and 0.69 for CTBP2(1) and CTBP2(4) respectively. No association of cancer status with expression level was found for a control gene (SFRS protein kinase 1; SRPK1), not known to be associated with prostate cancer in any GWAS. We did not identify any correlations of total gene expression with cancer status for the JAZF1, MSMB, HNF1B, MYEOV or CTBP2 genes. No correlations with transcript expression were noted for cancer stage (assessed by Gleason grading of the cancer) or with exposure to antiandrogenic drugs for any other transcripts analysed in either prostate cancer or BPH cohorts.
Although we found no differences in total HNF1B, CTBP2 or MSMB expression between BPH and prostate cancer cases, these genes are alternatively processed. We therefore sought to determine if there were differences in the relative balance of isoforms within our sample cohort (table 3). We found highly significant disturbances to the profile of isoforms expressed in prostate cancer in the case of the MSMB and HNF1B genes. In BPH tissues, the primary isoform expressed at the MSMB locus is MSMB2, which comprises 98% of MSMB expression in this tissue type. However, in cancer tissues, there is a near complete switch to isoform MSMB1, which is present at 96% total MSMB expression in prostate cancer. The ratio of MSMB1:MSMB2 isoforms is altered from 0.02 [IQR 0.02]: 1 in BPH tissue to 25.7 [IQR 30.2]: 1 in prostate adenocarcinoma tissues (P = 4.0 × 10-10, figure 4). In the case of the HNF1B gene, coding for 3 isoforms, we noted a similar switch in isoform expression. The predominant isoforms in BPH samples is HNF1B(C), present at 90% of total HNF1B expression. In prostate cancer tissues however, isoform HNF1B(B) is predominant at 95% total HNF1B expression, with the levels of HNF1B(C) dropping to only 3%. The proportion of isoform HNF1B(A) does not change. The ratio of HNF1B(B):HNF1B(C) isoforms changes from 0.7 [IQR 0.22]: 1 in BPH to 35.4 [IQR 124.71]: 1 in prostate cancer tissues (P = 2.9 × 10-9; figure 5). We also found 47% and 50% reductions in CTBP2(1) and CTBP2(4) expression levels in prostate cancer tissue compared with benign prostate tissues (P = 0.009 and 0.007 for CTBP2(1) and CTBP2(4) respectively), but these values do not reach statistical significance following adjustment for multiple testing (P = < 0.003 for 12 tests). Previous exposure to antiandrogenic drugs did not affect the ratio of MSMB or HNF1B splice variants in samples from prostate cancer or BPH patients (P = 0.24 in untreated subjects and 0.69 in treated subjects for prostate cancer cohort and P = 0.88 24 in untreated subjects and 0.72 in treated subjects for the BPH cohort).
Isoform-specific expression levels of candidate genes in non-malignant and prostate cancer tissues.
Gene expression levels are analysed relative to the beta 2 microglobulin (B2M) and beta glucorinidase (GUSB) genes, and normalised to the average value in the non-malignant samples. The interquartile range is given in parentheses. A P value of 0.006 (P = 0.05/n° of tests) was taken to indicate statistical significance following correction for multiple testing (8 independent tests). Statistically significant results are indicated in bold italic type.
The ratio of alternatively-expressed isoforms of the MSMB gene is altered in prostate cancer tissues. This boxplot gives the cancer status (benign prostatic hyperplasia or prostate adenocarcinoma) on the X-axis, and the ratio of MSMB1:MSMB2 isoform expression calculated relative to endogenous controls beta-2-microglobulin (B2M) and beta-glucorinidase (GUSB) on the Y-axis. Error bars represent the interquartile range (IQR) of the measurements. The level of significance as determined by pairwise Mann Whitney-U analyses is indicated. Outliers are indicated by circles.
The ratio of alternatively-expressed isoforms of the HNF1B gene is altered in prostate cancer tissues. This boxplot gives the cancer status (benign prostatic hyperplasia or prostate adenocarcinoma) on the X-axis, and the ratio of HNF1B(B):HNF1B(C) isoform expression calculated relative to endogenous controls beta-2-microglobulin (B2M) and beta-glucorinidase (GUSB) on the Y-axis. Error bars represent the interquartile range (IQR) of the measurements. The level of significance as determined by pairwise Mann Whitney-U analyses is indicated. Outliers are indicated by circles.
We report here that disruption of the amount or nature of transcripts expressed from the LMTK2, MSMB and HNF1B genes, identified in the genome wide scans for prostate cancer, may be important in the aetiology of this disorder.
We identified a 68% reduction in the expression of the LMTK2 gene in prostate tissue with evidence of the presence of adenocarcinoma when compared with non-malignant BPH samples. Expression levels were 1.10 [IQR 0.77] in BPH versus 0.35 [IQR 0.30] in prostate cancer; p = 3.2 × 10-7). We also noted an effect of genotype at the LMTK2 variant rs6465657, identified in the GWAS as a susceptibility factor for prostate cancer whereby individuals carrying two alleles of the protective 'T' allele allelic status expressed almost a third less LMTK2 than did individuals carrying one or more 'C' alleles (P = 0.002). We suggest that this finding probably arises from a potential imbalance in the transcription or stability of rs6465657 alleles that we note upon allele-specific PCR (figure 1). The precise identity and mode of action of the functional variant tagged by rs6465657 is at present unknown, but It is reasonably easy to postulate that the 'C' allele at rs6465657 could be marking a loss-of-function variant with effects on LMTK2 half-life or function.
The LMTK2 gene codes for a transmembrane serine/threonine/tyrosine kinase, with a role in endosomal membrane trafficking . It is also associated with NGF-TrkA signalling in murine brain, where it is a negative regulator of NGF-induced neuronal differentiation . LMTK2 interacts negatively with several other proteins with roles in cell division, such as protein phosphatase-1 (PP1C) and Inhibitor-2 (Inh2), which are part of a complex regulating separation of centrosomes during mitosis , and the cyclin-dependent kinase 5 (cdk5)/p35 complex , which has several functions, including a role in cell cycle progression. Recent studies have suggested that LMTK2 may interact with myosin IV, which has been shown to regulate both prostate specific antigen (PSA) and vascular endothelial growth factor (VEGF), both of which are associated with cancer. It is therefore likely that that reduction in the amount or activity of LMTK2 may lead to an increase in the proliferative capacity of prostate cells.
We also report a potential role for alternative mRNA processing of the HNF1B and MSMB genes in the aetiology of prostate cancer. MSMB codes for a secreted seminoprotein, which has tumour suppressor properties and is thought to be silenced in prostate tumour tissues by the enhancer of zeste homolog 2 (EZH2) protein . Accordingly, MSMB expression has previously been reported to be a positive prognostic indicator in prostate cancer , although both these studies measured total, not isoform-specific expression levels. The MSMB gene produces 2 isoforms; MSMB1 and MSMB2, which arise from the skipping of exon 3 in MSMB2. This causes a frameshift effect leading to the production of 2 distinct proteins. Previous studies suggest that both MSMB1 and MSMB2 are present in normal prostate and normal gastric mucosa, but that MSMB2 is absent from the majority of a small series of gastric and prostatic carcinomas . This is mostly in agreement with our findings, which suggest a major shift in MSMB expression in association with prostate cancer. We found MSMB2 to be the predominant isoform in benign prostate tissue, although small amounts (~2%) of MSMB1 were also present. In prostate adenocarcinoma tissues, however, MSMB expression derived almost completely from MSMB1, which is present at 96% of total MSMB expression. These findings may indicate that the tumour suppressor properties of MSMB are derived from isoform MSMB2, and that MSMB1 isoforms are potentially pro-carcinogenic. This situation is not uncommon for alternatively spliced genes in cancer; the vascular endothelial growth factor (VEGF) gene has previously been shown to code for both pro- and anti- angiogenic isoforms, with different behaviours in tumour tissues .
The HNF1B gene, which encodes three isoforms, HNF1B(A), HNF1B(B) and HNF1B(C), in humans , demonstrates a similar alteration to the relative balance of alternatively expressed isoforms associated with cancer of the prostate. The predominant isoform switches from HNF1B(C), which is expressed in non-malignant prostate tissues, to HNF1B(B) , which is expressed in prostate adenocarcinoma samples. HNF1B is a transcription factor expressed in a limited number of tissues, and has previously been associated with renal and ovarian tumours [32, 33] as well as prostate cancer . The HNF1B isoforms are known to exhibit differences in function and target specificities; Isoforms (A) and (B) are transcriptional activators whereas isoform HNF1B(C) is a transcriptional repressor . HNF1B isoforms have also been reported to activate different targets, for example, the HNF1B(C) isoform specifically has been demonstrated to negatively regulate the Glutahione-S-transferase A (GSTA) promoter, via a mechanism that involves Il-1beta . Given the difference in the transcriptional properties and target specificity of these isoforms, we predict that the alterations to HNF1B profile we note may manifest as an alteration to the overall activity of the HNF1B gene and/or activation of a variant set of its target genes.
Our study provides good evidence that some of the GWAS associations for prostate cancer may be attributed to mRNA effects, but does have a number of caveats. We have chosen a single gene for analysis in the case of each GWAS variant. The variants may tag a large region containing many genes, some of which may also be good candidates. Other genes in the same linkage disequilibrium (D') blocks, or even further distant, could also be important. This was recently documented for the eye colour trait in humans, where variant rs12913832 located within intron 86 of the HERC2 gene exerts its effects not by moderating HERC2 activity, but by moderating the efficiency of the promoter of the neighboring OCA2 gene, located 21Kbp upstream . We have attempted to ensure we have chosen the correct gene on the basis of proximity and biological function, but other genes may also be affected. Our analysis was also based on comparison of samples from patients with prostate cancer, and samples from patients with non-malignant Benign Prostatic Hyperplasia (BPH), rather than a paired analysis due to the difficulty in obtaining normal prostate tissue from the cancer patients. Our results may thus be influenced by factors both within and between groups, such as ethnic origin or the use of antiandrogenic drugs. No differences in the ethnic makeup of the cancer and control cohorts were noted, with both being almost exclusively of Caucasian origin. Similarly, treatment with antiandrogens had a minimal impact on the expression of any of the transcripts studied; with the exception of CTBP2(1) and CTBP2(4) transcripts, which showed a trend towards lower expression in samples from prostate cancer patients treated with hormone therapy, although this result was not statistically significant and no correlations of the expression of either transcript with hormone therapy was noted in the BPH cohort. No effect of hormone therapy on LMTK2 expression, or on the ratio of MSMB or HNF1B transcripts was noted.
In this study, we suggest that the overall mRNA expression level and/or the relative balance of alternatively expressed isoforms of the LMTK2, MSMB and HNF1B genes may be important determinants in the developments of prostate cancer, and demonstrate the importance of alternative mRNA processing mechanisms such as alternative splicing or differential use of polyadenylation sites in gene regulation. Our studies highlight a clear need for RNA studies to complement the genome wide association studies for prostate cancer and other diseases.
We are grateful to the Northcott Devon Medical Foundation [grant number TB/MG/NO5002] for funding the study. We would like to thank the Peninsula NIHR Clinical Research Facility and the Exeter Tissue Bank for provision of BPH and prostate cancer samples. We would like to acknowledge Albert Hallsworth and Dr. Bridget Knight from Peninsula NIHR Clinical Research Facility for their excellent technical assistance, and particularly Neelima Bhupathiraju and Rob Bolt for help in gathering additional clinical details on the cohorts studied. We also thank Professor Tim Frayling for critical appraisal of the manuscript.
LWH designed and executed the study, analysed results and drafted the manuscript. JRBP advised on polygenic aspects of the study and reviewed the manuscript. MC provided BPH and prostate adenocarcinoma samples and PM carried out the histological examination of tissues to prove malignancy. All authors read and approved the final submission.


What is claying used for?
Clay bar products are used to removing paint over spray or surface contaminants resulting from pollution, such as acid rain spots, as well as tar, tree sap, bird droppings, etc. Because the abrasive particles in clay are so fine, it is not considered an abrasive product, and clay is therefore effective and safe when used on painted surfaces, as well as on glass and chrome (but not plastic). Claying is an adjunct to – not a replacement for – polishing. Note also that claying will remove car wax.
How do I use clay?
After inspecting the bar and removing any particles, mold the bar into a shape that will fit comfortably in your hand.
First, wet the clay bar. You can use a special clay wetting agent such as Wax Enhancer, but clean soapy water also works fine. Mist a small area of the vehicle that you want to work on – say a 2' x 2' area. You may want to practice first on an inconspicuous spot. Glide the clay over the surface repeatedly until it glides smoothly. Now wipe the surface with a clean dry microfiber or terry towel and continue to the next area. Clay cannot be used dry, and so frequent light misting will be necessary. As your clay becomes dirty (but not gritty), simply remold it to use a different surface. Claying is not difficult, but you should take your time.
When done with the claying process, pick out any obvious particles from the clay before storing it away. Then wet the bar lightly and store it in a clean container. When you are ready to use it again, knead the bar to expose clean material. A clay bar is good until it becomes completely soiled (typically 10 or so cars). If a bar falls on the ground or otherwise becomes too contaminated with dirt and grit, it should be thrown out. If you are careful not to mix up bars, store your contaminated clay in a clearly marked container as it can still be safely used on glass windows and some types of unfinished wheels.
After you are done claying your car, give it a good wash to remove the lubricant film or soap residue. Polish if necessary, then following your normal pre-wax/waxing steps.


As passionate supporters of "Room to Read" KSparkle Inc. is thrilled to be one of the first to bring an inspiring new film, Girl Rising, to Melbourne. The film was created by 10×10, a global campaign to educate and empower girls.
PLEASE NOTE THAT AS WE ARE ONLY PERMITTED TO RELEASE A LIMITED NUMBER OF TICKETS YOUR EARLY BOOKING IS RECOMMENDED TO AVOID DISAPPOINTMENT AS NO TICKETS WILL BE AVAILABLE ON THE NIGHT.


Aye! My TBR pile runneth over. At the moment – it's a bit overwhelming. Here's the stack of freebies from RWA.
And here are two other piles that I haven't gotten to yet.
That's a total of thirty books!! 1.7333 books per week if I don't add any more books to the pile for a year. Since I have a book obsession, that's not a very likely prospect.
Case in point — here's what came today from BAM.com. And it's going straight to the top of the pile!!! Actually – it's skipping the pile entirely.
After the ending of LINGER, I have been anxiously awaiting this release. FOREVER is Book Three from The Wolves of Mercy Falls Trilogy by Maggie Stiefvater. As I've mentioned in previous posts, I have a major author crush on Ms. Stiefvater and SHIVER is one of my favorite books – ever. Check out my post on LINGER here.


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After the Storm: How Industry and Government Joined Forces to Restore Power
Imagine life without electricity. From our heating and cooling systems to electronics and refrigerators, we have come to depend on the power grid to such an extent that we rarely build contingency plans.
Dating back to 1950, investor-owned electric companies have come together at the regional level to provide mutual assistance during times of disruption. Superstorm Sandy, with its impact on 24 states and as many as 10 million customers in 2012, challenged the industry and government to look for ways to enhance power restoration support and thus improve public safety by accelerating the response and reducing potential economic consequences. The Edison Electric Institute (EEI) responded with the nationally-focused Mutual Assistance Network, a voluntary partnership of more than 100 investor-owned electric companies across the U.S. and Canada that are committed to helping restore power when assistance is needed by working directly with communities and government agencies.
The government plays a critical role in helping communities respond to natural disasters, but when it comes to a service as fundamental as electricity, it is the industry itself that is on the front lines of response. This has become particularly important as the number of catastrophes around the world has increased steadily over the past 40 years.
I know first-hand from my years of handling storm response for Con Edison, mutual assistance is a cornerstone of electric company operations during emergencies and is essential to contingency planning.
— Carlos D. Torres, Consultant, EEI, Power Restoration Coordination, Puerto Rico
10 million customers lost power across 24 states in the Northeast, mid-Atlantic, and parts of the Midwest during Superstorm Sandy.
Edison Electric Institute (EEI)
Civil Engineers Build Public Advocacy Program to Raise U.S. Infrastructure Grades
Protecting Consumers by Filtering out Fraud
AOAC INTERNATIONAL
Scientists Raise Health Standards for Infant Formula, Impacting Global Policy

redpajama_set_name: RedPajamaCommonCrawl


text:
Homestead continues turnaround, defeats Cupertino
Wilcox beats Milpitas, stays undefeated in De Anza Division
Photo by Pietro Brezzo
Homestead High School's Harold Rucker III (21) breaks a tackle as Homestead defeated Cupertino 12-7.
By Dave Mendonca | Correspondent
With the Central Coast Section football playoffs fast approaching, the Homestead Mustangs are one of several teams still in the hunt for a post-season berth. But given their record from a year ago, they might very well be the most surprising of the bunch.
The Mustangs, who suffered through a dreary 1-9 campaign in 2018, inched ever closer to a spot in the 2019 section playoffs with a 12-7 triumph over Cupertino last week. That moved them into sole possession of first place in the Santa Clara Valley Athletic League's El Camino Division at 3-1.
Homestead, which has a bye this week, closes out its regular season with games against Monta Vista (Nov. 1) and Gunn (Nov. 8). A sweep of those games would automatically catapult the Mustangs into the playoffs as the El Camino Division champ, while a split would most likely put their fate in the hands of the selection committee as a possible at-large choice.
Also closing in on automatic CCS bids are the Wilcox Warriors and The King's Academy Knights.
The SCVAL De Anza Division-leading Warriors, who are coming off of a 49-21 triumph over Milpitas, have a bye tonight. The Knights, who remained in a tie for first place in the Peninsula Athletic League's Bay Division with Menlo-Atherton after beating the Aragon Dons 44-0, travel to Atherton to take on Sacred Heart Prep. The kickoff is set for 7 p.m.
In other games slated for tonight, Cupertino hosts Monta Vista, Santa Clara visits Milpitas, and Mountain View welcomes Fremont in a trio of 7 p.m. starts. Lynbrook entertains Gunn beginning at 6 p.m.
For Homestead, Nadim Zarour hooked up with Harold Rucker III for a 30-yard touchdown pass and Gabe Solorio drilled a pair of field goals to provide the Mustangs with just enough points to slip past Cupertino, thanks to another outstanding effort by the Homestead's defense.
Coming off of a shutout of Lynbrook a week earlier, the Mustangs defense, led by Jared Wade's 10 tackles and Chuck Rak's two fumble recoveries, limited the Pioneers to just 216 yards of total offense.
Cupertino's only points came on Demarcus Oandasan's 17-yard TD toss to sophomore Yuma Raux-Moriwaki.
"We just shot ourselves in the foot," Cupertino coach Chris Oswald said. "We had a six-minute drive to start the game, and on second down on the five we fumbled. We also fumbled first-and-10 at their 20. Both fumbles were just dumb plays by us."
The win moved Homestead's overall record to 5-3, while the loss left Cupertino at 1-2 in division play and 2-5 overall.
Wilcox remained unbeaten in the SCVAL De Anza Division and a game ahead of second-place Los Gatos with a convincing win over Milpitas, the last team to have saddled the Chargers with a league loss. Paul Rosa's team has produced 14 consecutive SCVAL victories since that defeat in 2017.
Paul M. Rosa, the coach's son, rushed for four touchdowns and 283 yards to lead the victory, placing him on the cusp of becoming the school's all-time leading rusher. When Wilcox takes the field for its next game (Nov. 1 against Santa Clara), Rosa will need just 46 yards to surpass Anthony Herrera's current school mark of 4,431 yards. Rosa became a 1,000-yard rusher for the third consecutive season with his big night against the Trojans, raising his season total to 1,203 yards.
Isaiah Flores, who rambled for 161 yards, added a pair of rushing touchdowns, and Bryan Escorza scored his first TD of the season on a one-yard plunge.
As a team, the Chargers rushed for a season-best 476 yards, topping the 458 they rolled up in a 34-20 non-league win over San Benito on Sept. 6.
"Our offensive line played great," said the elder Rosa.
Forrest Watts and Escorza each intercepted a pair of passes to key the Chargers' defensive effort.
The King's Academy Knights ran their record to a perfect 7-0 with their win over Aragon. It is their best start to a season since beginning 8-0 in 2008, when they finished 10-1-1.
Antonio Gonzalez completed 13 of his 16 passes for a season-high 236 yards and three touchdowns to lead the Knights past the Dons. The junior signal-caller connected for scoring strikes twice with Kevin Sielski and once with Jayden Frazier, giving him 12 TD tosses for the season.
Frazier also rushed for a touchdown and a team-leading 74 yards.
"We played an overall very good game," The King's Academy coach Pete Lavorato said. "Our offense moved the ball, and defensively we made few mistakes."
It was the fifth time this season that the offense has put up more than 40 points, and the second time the defense has recorded a shutout.
Monta Vista evened its SCVAL El Camino Division record at 2-2 with its third shutout of the season, blanking Lynbrook 38-0 to snap a two-game losing streak.
"The defense was great," Matadors coach Ceazar Agront said.
Linebacker Henry Hodgkins keyed the strong defensive showing with eight tackles and an interception, while quarterback Simon Loeffler sparked the offense with a 67-yard touchdown run and a 60-yard scoring strike to Shreyas Deshpande.
Henry Lassa added a pair of touchdown runs, Tyler Krischer chipped in with one, and kicker Giovanni Vurro contributed a field goal and five extra-point kicks.
In other games last week, Santa Clara fell 20-0 to Mountain View, and Fremont was blanked 48-0 by Los Gatos.
Dave Mendonca | Sports writer
More in Local News


text:
Borghesiana – stacja na linii C metra rzymskiego. Stacja znajduje się w dzielnicy Borghesiana, a wejścia do stacji znajdują się na via Casilina, na skrzyżowaniu z via Vermicino, jak i na via Biancavilla.

Historia
Budowa wystartowała w lipcu 2008. Stacja została otwarta 9 listopada 2014 roku.

Przypisy

Stacje linii C metra w Rzymie

redpajama_set_name: RedPajamaWikipedia


1 If a man sends away his wife and, after leaving him, she marries another man, Does the first husband come back to her? Would not the land be wholly defiled? But you have sinned with many lovers, and yet you would return to me! says the LORD.
2 Lift your eyes to the heights, and see, where have men not lain with you? By the waysides you waited for them like an Arab in the desert. You defiled the land by your wicked harlotry.
Therefore the showers were withheld, the spring rain failed. But because you have a harlot's brow, you refused to blush.
Even now do you not call me, "My father, you who are the bridegroom of my youth"?
"Will he keep his wrath forever, will he hold his grudge to the end?" This is what you say; yet you do all the evil you can.
The LORD said to me in the days of King Josiah: See now what rebellious Israel has done! She has gone up every high mountain, and under every green tree she has played the harlot.
that for all the adulteries rebellious Israel had committed, I put her away and gave her a bill of divorce, nevertheless her traitor sister Judah was not frightened; she too went off and played the harlot.
Eager to sin, she polluted the land, committing adultery with stone and wood.
With all this, the traitor sister Judah did not return to me wholeheartedly, but insincerely, says the LORD.
Then the LORD said to me: Rebel Israel is inwardly more just than traitorous Judah.
Go, proclaim these words toward the north, and say: Return, rebel Israel, says the LORD, I will not remain angry with you; For I am merciful, says the LORD, I will not continue my wrath forever.
Only know your guilt: how you rebelled against the LORD, your God, How you ran hither and yon to strangers (under every green tree) and would not listen to my voice, says the LORD.
3 Return, rebellious children, says the LORD, for I am your Master; I will take you, one from a city, two from a clan, and bring you to Zion.
I will appoint over you shepherds after my own heart, who will shepherd you wisely and prudently.
When you multiply and become fruitful in the land, says the LORD, They will in those days no longer say, "The ark of the covenant of the LORD!" They will no longer think of it, or remember it, or miss it, or make another.
At that time they will call Jerusalem the LORD'S throne; there all nations will be gathered together to honor the name of the LORD at Jerusalem, and they will walk no longer in their hardhearted wickedness.
In those days the house of Judah will join the house of Israel; together they will come from the land of the north to the land which I gave to your fathers as a heritage.
I had thought: How I should like to treat you as sons, And give you a pleasant land, a heritage most beautiful among the nations! You would call me, "My Father," I thought, and never cease following me.
But like a woman faithless to her lover, even so have you been faithless to me, O house of Israel, says the LORD.
A cry is heard on the heights! the plaintive weeping of Israel's children, Because they have perverted their ways and forgotten the LORD, their God.
Return, rebellious children, and I will cure you of your rebelling. "Here we are, we now come to you because you are the LORD, our God.
Deceptive indeed are the hills, the thronging mountains; In the LORD, our God, alone is the salvation of Israel.
4 The shame-god has devoured our fathers' toil from our youth, Their sheep and their cattle, their sons and their daughters.
Let us lie down in our shame, let our disgrace cover us, for we have sinned against the LORD, our God, From our youth to this day, we and our fathers also; we listened not to the voice of the LORD, our God."
1  Such remarriage of divorced spouses was forbidden by ⇒ Deut 24:1-4. Under this figure the prophet sees the presumption of Judah, the unfaithful spouse, who thinks she can return to the Lord after uniting herself to other gods.
2  An Arab: a marauding nomad who lay in wait for caravans.
3 [14-18] A remnant of Israel (⇒ Jeremiah 3:14) will reunite with Judah (18). The elementary cult of the Lord represented by the ark of the covenant will now be replaced by a more deeply spiritual and universal alliance, symbolized by his throne in the ideal Jerusalem around which all nations will be gathered together (⇒ Jeremiah 3:17).
4  Shame-god: literally, shame, a term commonly substituted for Baal, the Canaanite god with many local shrines.


text:
Bring your Bible to School day
Victoria Johnston
More than a half a million students in the United States took part in this year's "Bring your Bible to School day" observance. Launched by Focus on the Family in 2014, the event took place on yesterday and is centered on students sharing their Christian faith with peers. According to the group, this year's observance […]
95-year-old Irish Church Bell Ringer
A church bell ringer is still going strong at 95 years of age! Cyril Galbraith, originally from Limerick but living in Dublin, has been ringing church bells for 81 years. He even described his hobby as the perfect "mindfulness" after it helped him deal with the loss of his beloved wife. Mr Galbraith's friends at […]
Tony Ward – Ireland's chances at the Rugby World Cup
Ireland can make waves at the Rugby World Cup.. That's according to Irish rugby legend Tony Ward. Ireland Head Coach Joe Schmidt named his 31-man squad for the 6-week tournament on Monday, amid concerns around the team's patchy form in recent months. However the former British and Irish Lion says this may benefit Ireland in […]
Justin Bieber declares his faith to his 117million Instagram followers
In his latest public declaration of faith, pop star Justin Bieber shared a video on Instagram to his 117 million Instagram followers encouraging them to put their trust in Jesus. The two clips features a well-known pastor preaching about having trust in Jesus. The posts have had positive feedback from his Christian followers, with many […]
New Irish study has found that older people who go to church have better mental health
A major new Irish study has found that older people who go to church have better mental health. The research was conducted by a team at Trinity College Dublin, it took place over six years, and involved more than 6,000 adults aged 50 and over. The results published last week found that regular religious attendance […]
Tour de France Christian Winner
A beautiful and emotional moment greeted viewers of the Tour de France last week. After the final day of racing, there was a new Champion, 22 year old Colombian, Egan Bernal. Tears flowed as he greeted his younger brother with a hug, a kiss on the forehead and then they each blessed each other with […]
Irish teenager wins international Google Science Fair
Fionn Fereirra, an 18-year-old student from Ballydehob in County Cork has won the top prize at the Google Science Fair in the United States for his project which removes microplastics from water.
Lauren Daigle's album 'Look Up Child' has set a new Billboard Christian Album Chart record
Lauren Daigle's album 'Look Up Child' has set a new Billboard record. It has been at number one on the Billboard's Top Christian Albums chart for 39 weeks now. The album recently won her a Grammy,and was certified platinum and she became the first female to have a song on both the Christian Airplay and […]
Irish nun awarded prestigious award for her work in Pakistan
An Irish nun from Co.Clare has been awarded a prestigious medal for teaching and promoting interfaith relations in Pakistan. Sr Berchmans spent almost 70 years working in Pakistan, where she taught Muslim, Christian, Parsi and Hindu children. St Mary's University in London has awarded her the Benedict Medal, which is the Catholic University's highest honour, […]
We came across this today and we really like it!


On the private health insurance rebate means test… – Software is too expensive to build cheaply….
Personally, I think it's a good idea. And not just because I'm a slavish fan of every utterance of the Labour government. Which of course I am. Except when it's trendy and hipster not to be, of course. Because that's how I roll.
More to the point, I actually think private health insurance in Australia is a screwed up business model that survives only with government support in the form of the aforementioned rebate and the regulation that allows insurers to charge discriminatory prices based on pre-existing conditions (those conditions being age and lack of continuous coverage). This support is wrong, and needs to be removed.
When the Howard government introduced the health care "reforms" in the 1990s, the stated promise was that private health insurance would – as long as it had sufficient membership – reduce health care costs and promote greater efficiencies. In particular, the reforms were going to encourage the health care funds to grow to a critical mass so that they could achieve economies of scale. This would reduce overheads and allow premium prices to drop. To ensure that, the government was going to regulate price increases – they'd only be allowed if they were shown to be needed.
It's funny, though – every year since then, health insurance premiums have increased, usually by more than CPI. No fund has shown any breakaway improvements in administration costs. There has been relatively little consolidation in the market place, and private health care has not taken on a large share of the health care market. Instead, what we have seen is the public system continuing to take on more and more of the burden, despite a huge uptake in private health membership. This problem was identified back in 2003 – we've known for over 8 years that subsidising private health insurance has been a failing experiment.
The majority of health care costs are borne by the elderly. 5 out of 6 patients in hospital care are over 65. But the elderly aren't as likely to have health care – people living on fixed income arrangements can't afford it, by and large. In case you haven't noticed, Australia – like most of the Western world – has an ageing population. So the number of old people are growing, and so are their medical expenses. This is why the public health system is struggling.
Private health care in Australia, by contrast, is a joke. I have private health insurance – just under the best possible to get. And I find it useless. My son broke his arm a few years back. He is autistic and non-verbal, so we went for a private hospital for the two nights he had to stay in (they needed to operate it and set it) so that he could have a private room and one of us could stay with him. The private hospital costs were reasonable – all we paid was the excess. We got to pick the surgeon – and we picked one who charged a small gap so it wasn't a very large costs. But the only anaesthetists available – at a hospital recommended by our health care fund! – charged thousands of dollars over the rebate, and we were not informed that would be the case until after the operation. Had we known, we would have opted for a private room at a public hospital.
A similar situation occurred with my wife (though at least this time we went into it knowing that there would be extra costs). A broken knee 18 months ago costs us – _after_ insurance – almost $10,000. Plus she needs another operation to put an implant in which we have been told isn't covered by our medical insurance (apparently prosthetic implants are only covered on the very best level of coverage, where they are described as "geriatric care"). So if we want them, it's twelve months waiting to qualify on the coverage – which will let us pay through the nose for care again. Elective surgery through the public system would be free, and with a similar wait – though the post-op rehab wouldn't be as good.
Nearly every single time I have to use my health insurance, I find the same thing – massive costs which are unexpected and which make having the insurance pointless. The only area where I feel I get value for money is with optical care – and that's because of discounts offered by optical stores, not rebates. (I just bought new glasses last week – the insurance policy covered about 40% of the cost, while the store discount for members of my health fund was 20% on top! And it wasn't the frames – the frames were covered 100% by insurance).
This is why private health insurance in Australia is a failure – it provides an expensive level of coverage, and when it's needed you still end up thousands of dollars out of pocket. It provides little to no financial security. My car insurance gives me financial security: I know that if my car gets written off or stolen, I will get compensation enough to get a new one. My home and contents gives me financial security. My health insurance gives me no financial security – in fact, I can contribute many of my financial problems over the last twenty years to using private health care.
You may wonder why I still have private health insurance – and the answer is simple: my wife and kids. But I'd rather have a solid public health system than have private health care.
In 2003, the government subsidies had a direct cost to the budget bottom line of $3.6 _billion_ dollars. That was over 8 years ago – it's grown since. That is billions of dollars every year taken out of the public health system, and given mainly to the upper quartile of income earners (which includes me, BTW). This contributes to growing economic inequality and isn't needed – the system enables a relatively small slice of Australian society who couldn't afford private health insurance otherwise to obtain it. Neither the carrot of the rebate or the stick of rising premiums if you decide to join later in life (when you are more likely to need it!) have resulted in private health funds becoming efficient or providing solid value.
The entire system represents a subsidy to high income earners and wealth transfer to the medical system. It does not result in reduced health care costs to society, or better care in general, which should be the goals.
The private health care rebates, and the government support to private health insurance in general, is a failure. Take it away, and let it die – the only downside is that the removal of easy money might actually make private health funds have to become efficient to survive.
You have captured the inequalities in the Health System but like me you feel the need to have a backup. I sometimes wonder if these medical specialist are over charging because we have a private fund. Technology is great but the great costs come with it. I don't know the answer as I think we can't win. We pay a high premium for our families – hoping it gives us health security but the member is nearly always out of pocket after the exorbitant fees charged by the medical profession. I hope those on $160,000 will not lose their whole rebate and we need this to be explained. Those on $250,000 and over I believe do not need a government rebate.
The stated policy is that the means test will cut in at $160,000 (for families – $80,000 for individuals), with the benefit being phased out entirely at $250,000 (or $125,000 for individuals). This implies that the benefit is reduced as income goes up, which would be normal for most means-tested benefits. As a rule, Australian government subsidies try (but don't always succeed) to avoid hard cutoffs.


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Q: Powerbuilder ImportFile / SaveAs My client requires to Import Text Files into the system but their Textfiles are Pipe Delimited "|". Also they require to Export some of data into the same format (Pipe delimited). I was asked to use Powerbuilder 12. Is it possible to ImportFile with PipeDelimited Textfiles directly? I would like to skip the "FOR LOOP MID GET" Method and go directly for importfile then update(). Same goes for the dw.saveas().

A: as far as I know we are not lucky with the import, because we could not define the separator, but here is a little workaround:

*

*Read the file into a string variable

*Replace the pipes | to commas ,

*Import the string as a CSV into the dw

I made a little test app for you and it worked. My text files is like this:
Col1|Col2|Col3
aaa|bbb|ccc
Here is the import code:
string  ls_import, ls_export
long    ll_filehandle, ll_readbytes
// IMPORT
ll_filehandle = FileOpen("d:\Work\StackExchange\pipes.txt", TextMode!)
ll_readbytes    = FileReadEx(ll_filehandle, ls_import)

if ll_filehandle = -1 or IsNull(ll_filehandle) then
else
    ls_import   = f_glo_replace_all(ls_import, "|", ",")
    dw_1.ImportString(CSV!, ls_import)
end if

With the export we have more luck. There is a datawindow method in which you can customize the separator for the export:
SaveAsFormattedText
So the import would look like this:
dw_1.SaveAsFormattedText("d:\Work\StackExchange\export_pipes.txt", EncodingANSI!, "|")
Of course you will need the code of the global replace function:
Here is the source:
global type f_glo_replace_all from function_object
end type

forward prototypes
global function string f_glo_replace_all (string source, string look_for, string replace_with)
end prototypes

global function string f_glo_replace_all (string source, string look_for, string replace_with);/*A String Occurrence Search and Replace RoutineThe following code demonstrates a string occurrence search and replaceroutine.This routine works generically for any string. For example,if old_str = "red" and new_str ="green", all occurrences of "red" inside of mystring will be replaced with "green".
Parameters
Name = source Type = String
Name = look_for Type = String
Name = replace_with Type = String
*/
int start_pos=1,len_look_for

len_look_for = len(lower(look_for))
//find the first occurrence of look_for ...
start_pos = Pos(lower(source),lower(look_for), start_pos)
//only enter the loop if you find whats in look_for
DO WHILE start_pos > 0
    //replace look_for with replace_with ...
    source = Replace(source,start_pos,Len_look_for,replace_with)
    //find the next occurrence of
    start_pos = Pos(lower(source), lower(look_for), start_pos+Len(replace_with))
LOOP
return source
end function

I hope this helps!
Br. Gábor

A: No, it is not possible the way you want.  You'll need to write your own version of the ImportFile, sorry it is not the answer you wanted.

redpajama_set_name: RedPajamaStackExchange


text:
← What I'm Reading: Farewell to Manzanar – 3 in a series – …to ten inland camps.
Fontana dei Baci, Milano, Italia via Instagram →
What I'm Reading: Life and Times of Frederick Douglass – 9 in a series – "…that men whose malign and brutal propensities preponderate over their moral and intellectual endowments"
"I speak of overseers as a class, for they were such. They were as distinct from the slave-holding gentry of the south as are the fish-women of Paris, and the coal-heavers of London, distinct from other grades of society. They constituted a separate fraternity at the south. They were arranged and classified by that great law of attraction which determines the sphere and affinities of men; which ordains that men whose malign and brutal propensities preponderate over their moral and intellectual endowments shall naturally fall into those employments which promise the largest gratification to those predominating instincts or propensities. The office of overseer took this raw material of vulgarity and brutality, and stamped it as a distinct class in southern life. But in this class, as in all other classes, there were sometimes persons of marked individuality, yet with a general resemblance to the mass."
From Life and Times of Frederick Douglass: (An African American Heritage Book)
† Available from the LA Public Library
What I'm Reading: Life and Times of Frederick Douglass – 9 in a series – "Other overseers, how brutal soever they might be…"
What I'm Reading: Life and Times of Frederick Douglass – 7 in a series – …servility, dejection, and misery on the other.
What I'm Reading: Life and Times of Frederick Douglass – 8 in a series – The highly-favored inmates of this mansion…
What I'm Reading: Life and Times of Frederick Douglass – 6 in a series – Old and young, male and female, married and single, dropped down upon the common clay floor
What I'm Reading: He was whipped oftener who was whipped easiest.
This entry was posted in Books, History, Quotes, Reading. Bookmark the permalink.
The Tidings publishes two of Douglas' photos
The Tidings, Southern California's Catholic Weekly, published 2 of my photos documenting the preparations for the musical at my son's school, Providence High in Burbank. Providence HS selects Tony award-winning musical satire for spring performance The satirical musical comedy, "Urinetown," … Continue reading →


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Dady'O delights us with his glamorous and exclusive themes this Spring break 2019!. Enjoy the nights with good music, friends and the expected bikini contest. Get your Open Bar and enjoy unlimited national drinks in the best place.
Includes: Cover + National Open Bar from 10:30 p.m. - 3:30 a.m.


Have you ever noticed that at any given time there are 2-300 guests on site, and members online that equal 1/10th of that? If we could get those guests to join, we'd have even more friends.
But I noticed that people do join regularly.
I have never noticed that. That is a lot of guests!
I check whenever I sign in to see who's online, who's on chat (though I never chat) and what's going on. It's usually 300+ guests. Do you know where to find this current status? Go to the homepage for the Forums and it's at the bottom. Maybe you go there but never noticed the Who's Online current stats.
See, there's 30 something members online, and 403 guests at this very moment!
Wednesday Morning: 20 members and 285 Guests!
It's 6:50 am, Eastern time, Saturday morning, January 26, 2013, and there are 12 members and 165 lurkers online.
I remember when I used to be a lurker, years ago. I was so encouraged through reading about the experiences of others. I felt a part, but I was not a part. I was invisible, and maybe I wanted to be that way for a while. But then, I began to want to be somebody on the boards, and lurking was no longer enough. I remember how excited I felt when I joined my first site.
If you're a lurker, please join. You need us, and we need you. We want to be friends, so welcome to our vibrant community, whoever you are.


text:
Egypt unveils ancient 'secret keeper' tomb, golden mummy
An open-source stopwatch to time interactions between molecules inside living cells
Darknet markets generate millions in revenue selling stolen personal data, supply chain study finds
In Cybersecurity
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It is common to hear news reports about large data breaches, but what happens once your personal data is stolen?...
Did Twitter ignore basic security measures? A cybersecurity expert explains a whistleblower's claims
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on 2 September 2022 2 min read
Twitter's former security chief, Peiter "Mudge" Zatko, filed a whistleblower complaint with the Securities and Exchange Commission in July 2022,...
Rise of precision agriculture exposes food system to new threats
In AIApps and WebCybersecurity
on 8 August 2022 2 min read
Farmers are adopting precision agriculture, using data collected by GPS, satellite imagery, internet-connected sensors and other technologies to farm more...
Browser cookies make people more cautious online, study finds
on 5 July 2022 2 min read
Website cookies are online surveillance tools, and the commercial and government entities that use them would prefer people not read...
Scams and cryptocurrency can go hand in hand – here's how they work and what to watch out for
on 21 June 2022 2 min read
When one of our students told us they were going to drop out of college in August 2021, it wasn't...
How math and language can combine to map the globe and create strong passwords, using the power of 3 random words
It's hard to imagine that three random words have the power to both map the globe and keep your private...
EU law would require Big Tech to do more to combat child sexual abuse, but a key question remains: How?
In Apps and WebCompaniesCybersecurity
The European Commission recently proposed regulations to protect children by requiring tech companies to scan the content in their systems...
Your digital footprints are more than a privacy risk – they could help hackers infiltrate computer networks
on 8 April 2022 2 min read
When you use the internet, you leave behind a trail of data, a set of digital footprints. These include your...
How Ukraine has defended itself against cyberattacks – lessons for the US
In 2014, as Russia launched a proxy war in Eastern Ukraine and annexed Crimea, and in the years that followed,...
Cyberattacks have yet to play a significant role in Russia's battlefield operations in Ukraine – cyberwarfare experts explain the likely reasons
Throughout the latter half of 2021, as it became clear that Russia was massing a large portion of its conventional...
Local governments are attractive targets for hackers and are ill-prepared
on 29 March 2022 2 min read
President Joe Biden on March 21, 2022, warned that Russian cyberattacks on U.S. targets are likely, though the government has...
How AI is shaping the cybersecurity arms race
In AICompaniesCybersecurity
on 23 February 2022 2 min read
The average business receives 10,000 alerts every day from the various software tools it uses to monitor for intruders, malware...