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Identifying the functional importance of the millions of single nucleotide polymorphisms (SNPs) in the human genome is a difficult challenge. Therefore, a reverse strategy, which identifies functionally important SNPs by virtue of the bimodal abundance across the human population of the SNP-related mRNAs will be useful. Those mRNA transcripts that are expressed at two distinct abundances in proportion to SNP allele frequency may warrant further study. Matrix metalloproteinase 1 (MMP1) is important in both normal development and in numerous pathologies. Although much research has been conducted to investigate the expression of MMP1 in many different cell types and conditions, the regulation of its expression is still not fully understood.
In this study, we used a novel but straightforward method based on agglomerative hierarchical clustering to identify bimodally expressed transcripts in human umbilical vein endothelial cell (HUVEC) microarray data from 15 individuals. We found that MMP1 mRNA abundance was bimodally distributed in un-treated HUVECs and showed a bimodal response to inflammatory mediator treatment. RT-PCR and MMP1 activity assays confirmed the bimodal regulation and DNA sequencing of 69 individuals identified an MMP1 gene promoter polymorphism that segregated precisely with the MMP1 bimodal expression. Chromatin immunoprecipation (ChIP) experiments indicated that the transcription factors (TFs) ETS1, ETS2 and GATA3, bind to the MMP1 promoter in the region of this polymorphism and may contribute to the bimodal expression.
We describe a simple method to identify putative bimodally expressed RNAs from transcriptome data that is effective yet easy for non-statisticans to understand and use. This method identified bimodal endothelial cell expression of MMP1, which appears to be biologically significant with implications for inflammatory disease. (271 Words)
Numerous strategies have been used in an attempt to sift through the vast amounts of data produced from microarray expression studies [1-4]. There has been much interest given to the identification of bimodally expressed mRNA transcripts, particularly in the context of cancer, where two distinct populations of patients can be defined by differing levels of a set of specific transcripts. These make excellent candidate biomarkers and often tend to show good correlation between transcript and protein abundance [5]. To this end, statistical approaches using mixture-model based clustering combined with either Akaike information criterion (AIC) or the Bayesian informatics criterion (BIC) have frequently been applied [6-9]. One method based on systematic classification of gene expression profiles has been applied to over 2,000 microarray samples [10]. These methods have a strong theoretical base and have proven successful in identifying bimodality. However, they do depend on the investigator having a relatively high level of statistical understanding [7]. In this study we suggest a simple screening approach based on hierarchical clustering to identify bimodally expressed transcripts from microarray expression data that can be used alongside more complex approaches. While this method is not motivated by statistical theory, it appears to work well and is easily understood by laboratory scientists with only basic statistical training, who are in a good position to immediately follow up their results experimentally.
The matrix metalloproteinase, MMP1, is one of the most abundant proteases in the matrix metalloproteinase family. It is capable of degrading type I, II and III collagens, and is one of only four MMPs able to degrade triple helical collagens. It therefore plays a pivotal role in extracellular matrix (ECM) remodelling in both normal development and pathology [11]. MMP1 is tightly regulated at both the transcriptional and post-translational levels. It is produced as a zymogen that is activated by serine proteases and its activity is regulated by inhibitors such as the tissue inhibitors of metalloproteinase's (TIMPs), which compete with the substrate for the enzyme active site [12].
MMP1 plays a clinically important role in inflammatory disease, and has been implicated in numerous pathological processes including wound healing [13], tumour metastasis [14] and arthritis [15]. The MMP-1 gene [11] contains a 1G/2G polymorphism in its promoter at -1607 from the transcriptional start site [16]. This polymorphism has been associated with increased MMP-1 expression in several cell types including; melanoma, stromal fibroblast, MCF-7/ADR breast cancer cells [16-19], and with several pathologies including: tumour metastasis [20,21], arthritis [22,23], periodontitis [24], chronic obstructive pulmonary disease [25] and cardiovascular disease [26,27].
Using our simple clustering method to examine RNA transcript abundance in HUVECs isolated from 15 different human individuals, we identified MMP1 as one of a small group of RNAs expressed in a bimodal manner in both un-treated endothelial cells, and in endothelial cells treated by inflammatory mediators. Our results suggest that the regulation of MMP1 expression is a complex process that is modulated by a promoter polymorphism around the binding sites for several TFs including ETS1, ETS2 and GATA3.
Identification of mRNA transcripts with bimodal expression patterns among a set of individuals
Bimodally or multimodally expressed mRNA transcripts were defined as those transcripts for which two or more distinct populations of expression values were observed among a set of individuals. To identify and visualise bimodally expressed transcripts, we devised a simple algorithm (written as a script in the statistical language 'R'; Additional File 1) based on unsupervised agglomerative hierarchical clustering. The algorithm can be used as either a simple 'R' script, or for use in graphical user interface it can be supplied as a GenePattern module on request (http://www.broadinstitute.org/cancer/software/genepattern/ webcite). It is illustrated schematically in Figure 1 and described in the methods section. Briefly, on a transcript-by-transcript basis, agglomerative hierarchical clustering across the dataset was carried out. The maximum cluster branch height identified for each transcript was approximately proportional to the greatest distance between the any two clusters of individuals, and is used here a surrogate marker for the degree of bimodal expression. To estimate the probability of transcripts appearing to be bimodally expressed due to chance alone we used a parametric bootstrapping method. Related methods where trees are constructed from re-sampled data have been used previously to assess the reliability of clusters in gene expression data [28]. As is often the case with microarray transcript abundance data, our log-transformed data approximated a normal distribution. Therefore, for each transcript we made a maximum likelihood estimate of the parameters of a normally distributed population from which the sample of individuals being studied may have been drawn. These parameters (mean and standard deviation) were then used to generate 10,000 simulated datasets for the transcript, each of which was clustered as described above. From the 10,000 clustering results we identified how frequently the largest distance between clusters ≥ the largest distance between clusters in the actual data set. This information is used generate an empirical p-value as an estimate of type I error rate.
Additional file 1. R-script: Source code to identify and visualise bimodally expressed transcripts from a microarray expression dataset.
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Figure 1. Flow diagram of method to identify bimodally expressed transcripts from expression data. (A) Transcript abundance is quantified by microarray or RNAseq techniques. (B) On a transcript-by-transcript basis, agglomerative clustering across the dataset is carried out. The algorithm starts by assigning the same number of clusters as individuals (in this example 10 clusters were assigned since there are 10 individuals). The clusters are then progressively merged by combining the two most similar clusters, using Wards method to calculate the distance between clusters and Euclidian distance to calculate dissimilarities between the individuals. The distances between the merging clusters are recorded by the algorithm as branch "heights". The height values at either side of the dendrogram are removed to exclude transcripts that falsely appear to be bimodally expressed due to a single outlying individual. The maximum remaining branch height value (indicated by the red arrow) is identified for each transcript, which represents the greatest distance between the any two clusters of individuals, and is used a surrogate marker for the degree of bimodal expression for that particular transcript. (C) To estimate the probability of transcripts appearing to be bimodally expressed due to chance alone, for each transcript we make a maximum likelihood estimate of the parameters of the distribution of this transcript's abundance across the population from which the individuals being studied have been drawn. We use these parameters to generate 10,000 simulated datasets, each of which is clustered as described in (B) above. (D) In the 10,000 clusters formed from the bootstrapped data sets for this transcript, we identify how commonly the largest distance between clusters ≥ the largest distance between clusters in the actual data set. This information is shown graphically and is used generate an empirical p-value as an estimate of type I error rate.
This algorithm was applied to two RNA microarray data sets: (i) data from HUVECs from 15 different human individuals cultured to passage 4 in standard conditions (the untreated data set; UT) and (ii) data from passage 4 HUVECs from nine different human individuals cultured with a cocktail of 10 ng/ml TNF-α, IL-1β and IL-8 for 24 hours (the IM-treated data set; IM). The bimodally expressed RNAs found in the UT and IM HUVECs are listed in Additional File 2. The relationship between the maximum branch height (an estimate of the degree of bimodal expression) and the -log2 transformed empirical p-value (an estimate of the frequency of a transcript appearing to be bimodally expressed due to chance alone) is shown in Figure 2A-B. In each of the un-treated and IM-treated data sets, a relatively small group of transcripts with high maximum branch height and high -log2 p were identified. We decided to accept an estimated type I error rate of 10% for each of these data sets, and found there were 21 RNA transcripts for which the empirical p-values were ≤ 0.1 in both the un-treated and IM-treated data sets (Figure 2C). A table of features for each of the 21 shortlisted RNA transcripts is given in Additional file 3.
Additional file 2. Clustering results: Clustering results for both the un-treated (UT) and IM-treated (IM) HUVEC data set are shown for 10531 RNA transcripts. "OGS" is the official human gene symbol. "max_height" denotes the greatest distance (maximum cluster branch height) between the any two clusters of individuals, and is used a surrogate marker for the degree of bimodal expression. "p_max_height" denotes, for this transcript, the frequency with which this maximum cluster branch height is exceeded in clustering of 1,000 simulated data sets generated by aprametric bootstrapping (an estimate of the probability of each transcripts appearing to be bimodally expressed due to chance alone).
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Additional file 3. R-script: To plot histograms.
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Figure 2. Microarray data of approximately 16,000 transcripts in un-treated HUVECs from 15 individuals and IM-treated HUVECs from 9 individuals were analysed separately using the clustering method described in this paper. The maximum branch height (max height, an estimate of the degree of bimodal expression) for each transcript was plotted against the -log2 empirical p-value (an estimate of the frequency of that transcript appearing to be bimodally expressed due to chance alone) for the un-treated data set (A) and the IM-treated data set (B). The 21 RNA transcripts for which the empirical p-values were ≤ 0.1 in both the un-treated and IM-treated data sets were identified and are listed alphabetically in panel (C).
These shortlisted transcripts were further assessed using several strategies including; i) visual inspection of histograms on a gene by gene basis (an R script used to generate these is given in Additional File 4); ii) evaluation of the associated bootstrap p-values obtained during clustering and iii) consideration of additional information of biomedical interest. We were especially interested in the presence of promoter SNPs, which may in theory cause bimodal RNA expression patterns, for example using the SNPer [29] or rSNPs [30] databases. Based on all these considerations, subset of transcripts were selected to take forward for further investigation; DDX3Y (a Y-chromosome encoded RNA, which should segregate with gender), MMP1 and SLC2A11 (biologically interesting TNFα and IFNγ targets, which are important in inflammation).
Additional file 4. Table of 21 short listed RNA transcripts: Table of features for each of the 21 RNA transcripts to help determine selection
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DDX3Y is differentially expressed on the basis of its location on the Y-chromosome
The pool of individuals examined was expanded and the abundance of DDX3Y, MMP1 and SLC2A11 mRNA in HUVECs from 29 additional individuals cultured in both UT and IM conditions was analysed using quantitative Reverse Transcription-PCR (qRT-PCR). We confirmed in this new group of individuals a bimodal expression pattern for DDX3Y using q RT-PCR. DDX3Y is encoded by a Y-chromosome gene and, as expected, its expression segregated with the gender of the individual from which the HUVEC were isolated in both the IM and UT data (Figure 3).
Figure 3. Quantitative PCR of transcripts DDX3Y and MMP1 in 29 donor isolates under untreated (UT) and inflammatory mediator treated (IM) conditions. Donors of female gender are shown in red and donors of male gender are shown in black. Only DDX3Y appears to segregate with gender. Delta Ct values are calculated as Ct of target gene of interest (DDX3Y or MMP1) relative to the Ct value of the internal control (18S ribosomal RNA). Low delta Ct values represent high basal levels of expression and vice versa.
MMP1 is differentially expressed and differentially active in endothelial cells
MMP1 was of particular interest since it encodes a biologically and clinically important enzyme, and since analysis of the rSNPs database identified several common SNPs in the MMP1 gene promoter within 2,000 bp upstream of the start of transcription (data not shown). Dendrograms for MMP1 in un-treated and IM-treated HUVECs, along with histograms of the maximum cluster branch height in each of 10,000 parametric bootstrap data sets (to estimate the probability of transcripts appearing to be bimodally expressed due to chance alone) are shown in Figure 4. Quantitative RT-PCR from 29 additional individuals confirmed in this new group of individuals a bimodal expression pattern for MMP1. We identified two distinct populations; (i) HUVECs isolated from 7 of the 29 individuals had low MMP1 mRNA abundance (Figure 5a), however in 6 of these 7 individuals, MMP1 mRNA abundance was significantly increased by culture in IM conditions (Figure 5b). (ii) The remaining 22 individuals had relatively higher MMP1 mRNA abundance regardless of UT or IM culture conditions (Figure 5a). In the high MMP1 expressing HUVECs isolated from these 22 individuals, the abundance of MMP1 mRNA was either not significantly affected or was decreased by IM culture conditions (Figure 5b). This differential response to inflammatory mediator treatment was striking and we sought to understand the underlying mechanisms.
Figure 4. Dendrograms and histograms for MMP1 in untreated and IM treated HUVECS. Dendrograms of MMP1 RNA expressions are shown for the un-treated (A) and IM-treated (C) HUVECs. Histograms showing the frequency (y-axis) of maximum branch height (x-axis) across 10,000 simulated MMP1 transcript datasets, each with parameters similar to the estimated parameters of the population from which the actual MMP1 data set was drawn, are shown (B and D). Green arrows indicate the maximum branch height from cluster analysis of the actual data sets. In both un-treated and IM-treated HUVECs, the maximum clustering branch height for MMP1 exceeded the maximum clustering branch height identified in 90% of the simulated data sets.
Figure 5. Differential expression of MMP1 in un-treated and inflammatory mediator-treated HUVECs isolated from 29 separate individuals. (a) Quantitative RT-PCR measuring MMP1 abundance in un-treated (UT) and inflammatory mediator-treated (IM) conditions identified two distinct populations; HUVECs isolated from seven individuals had low MMP1 mRNA abundance regardless of UT or IM culture conditions (labelled as Basal Low Expressers), while HUVECs isolated from the other 22 individuals had relatively higher MMP1 mRNA abundance regardless of UT or IM culture conditions (labelled as Basal High Expressers) (P < 0.0001, Mann Whitney non parametric test). (b) To illustrate the regulation of MMP1 mRNA abundance by inflammatory mediator-treatment, the fold change in MMP1 abundance in IM -vs- UT conditions is shown. In six out of the seven Basal Low Expressers MMP1 abundance was increased in response to IM treatment. In contrast, in 19 out of the 22 Basal High Expressers MMP1 abundance either did not change significantly or fell slightly in response to IM treatment. Both the difference in mRNA abundance between the untreated and the inflammatory mediator treated conditions and the differential response between the high and low basal expressers to inflammatory mediator treatment showed statistical significance (Paired t-test, P < 0.0001 and Mann Whitney nonparametric test, P = 0.002 respectively). (c) Total MMP1 enzymatic activity was measured in a subset of the HUVEC isolates. In general low basal expressers have lower enzyme activity than high basal expressers.
To investigate whether the bimodal expression of MMP1 mRNA was also evident at the level of MMP1 enzymatic activity, this activity was measured in 20 HUVEC isolates. The activity of MMP1 was low in those HUVEC cultures that had low MMP1 RNA abundance and high in those HUVEC cultures that had high MMP1 RNA abundance (Figure 5c).
MMP-1 gene promoter polymorphisms segregate with MMP1 expression and enzymatic activity
A 1G/2G deletion/insertion polymorphism at position -1607 in the MMP-1 promoter has been associated with differential expression of this gene in fibroblast and melanoma cells, with the 2G genotype associated with higher basal levels of MMP1 mRNA [16]. This polymorphism occurs within a consensus binding site for the ETS family of transcription factors [16,18]. Therefore, direct DNA sequencing of the MMP1 promoter was carried out to determine whether the MMP1 mRNA abundance and activity profiles segregated with this promoter polymorphism. We determined MMP1 mRNA level, MMP1 enzymatic activity and MMP1 promoter genotypes in HUVECs from 69 different individuals. Figure 6 and Table 1 illustrate the three genotypes observed in these individuals. Of the 69 individuals, 76% were heterozygous at the site of the promoter polymorphism, 15% were homozygous for the 1G allele, while only 9% were homozygous for the 2G allele. All 1G homozygous individuals segregated with low basal expression and activity of MMP1, whereas all but one of the heterozygotes and homozygous 2G individuals segregated with high basal expression and activity of MMP1.
Table 1. Segregation of genotype for the -1607 MMP-1 promoter polymorphism with basal abundance of MMP-1 transcript
Figure 6. Sequencing of the -1607 MMP-1 promoter polymorphism, illustrating the homozygous and heterozygous genotypes. Insertion of an additional G creates the consensus sequence for the ETS binding site, GGA (indicated by blue shading).
Elevated MMP1 expression induced by inflammatory mediator in 1G homozygous cells is not due to greater overall activity of pro-inflammatory signalling pathways
It was possible that the differential responses to IM treatment we observed between individuals with low MMP1 basal expression (homozygous 1G individuals) and high MMP1 basal expression (heterozygotes and homozygous 2G individuals) were simply due to differential activity of the signalling pathways that mediate inflammation. Therefore, molecules known to be downstream of inflammatory mediator signalling were assessed as biomarkers of inflammatory pathway activity in individuals with low and high MMP1 basal expression. Protein expression levels in ICAM1, IкBα and phospho-IкBα were measured in HUVEC lysates from three individuals with low basal MMP1 mRNA and three with high basal MMP1 mRNA, after treatment with 10 ng/ml IL-1β, TNF-α and IL-8 for up to 3.5hrs. Figure 7 shows abundance of ICAM-1, total and phospho-IкBα over the 3.5hr period. Two-way analysis of variance (ANOVA) revealed that there was no significant difference in ICAM1 or total and phospho-IкBα signal between the high and low expressers at all time points (P = 0.8, 0.7 and 0.2 respectively). These results suggest that there is not a large systematic difference between the inflammatory signalling pathways related to this polymorphism.
Figure 7. Immunoblot illustrating inflammatory signalling responses in 3 isolates with low and high basal expression of MMP-1 (labelled isolates 1, 2, 3 and 4, 5, 6 respectively). Low = low basal expressers and High = high basal expressers. Time in hours for each group of low and high basal expressers is given at the top of the figure. (a) IκBα signalling over 3.5 hours after the addition of an inflammatory mediator cocktail containing 10 ng/ml each of TNF-α, Il-1β and Il-8. (b) Phosphorylated IκBα signalling over the time course. (c) ICAM signalling over the time course. Both the high expressers and low expressers show similar levels of IκBα, phospho- IκBα and ICAM over the time course.
Ets1, Ets2, Fos and GATA3 are potential mediators of the different expression levels of MMP1 mRNA in 1G -vs- 2G individuals
To determine whether differential binding of transcription factors to the MMP1 promoter region containing the -1607 polymorphism is a potential mechanism for the differential response of MMP1 to IM treatment, the TFSEARCH algorithm [31] was used to identify putative transcription factor binding sites in this region. This analysis revealed in addition to the ETS binding domain spanning the polymorphic region, AP-1 and GATA3 binding sites were located 44 bp and 5 bp respectively downstream from the polymorphism. To determine whether these TFs actually bind to this region of the MMP1 gene promoter, immunoprecipitation using antibodies against c-fos, ETS1, ETS2 and GATA3 was carried out in HUVECs isolated from two individuals of 1G genotype and in HUVECs isolated from two individuals of 2G genotype cultured in both IM and UT conditions. Quantitative PCR was used to measure the enrichment of the immunoprecipitated region (157 bp region, adjacent to an ETS, GATA3, AP-1 and NFKB binding site), relative to a 173 bp region positioned 5600 bases upstream of the polymorphism that contained no relevant motifs. DNA precipitated by anti-c-fos, anti-ETS1, anti-ETS2 and anti-GATA3 antibodies was enriched for the MMP1 promoter region containing the polymorphism, relative to the control upstream region and relative to the un-immunoprecipitated material in all four individuals and in cells cultured in UT and IM conditions (Figure 8). The aim of this experiment was to identify potential TF binding around the polymorphism, not to compare the degree of enrichment between IG and 2G individuals or between UT or IM cultured cells, which was impossible due to the small study size and variable degree of enrichment between individuals (Figure 8). Nevertheless, it was interesting to observe that, consistent with the previously published role of ETS1 in MMP-1 induction [16], ETS1 binding was reproducibly enriched in the 1G isolates treated with IM.
Figure 8. Fold enrichment of transcription factor binding to the region of the -1607 MMP1 promoter polymorphism. (a) Fold enrichment of c-Fos, ETS1 and ETS2 binding at the region of the -1607 promoter polymorphism relative to a region 5600 bases upstream of the promoter polymorphism. (b) Complete replication of experiment in 5a using cells from different 1G and 2G individuals. (c) Fold enrichment of GATA3 binding at the region of the -1607 promoter polymorphism relative to a region 5600 bases upstream of the promoter polymorphism. (d) Replication of experiment 5c. Input control refers to enrichment in the absence of any precipitating antibody.
The significance of non-coding polymorphisms in pathology is being increasingly recognised, with much research being carried out to identify the functional importance of the millions of SNPs mapped to date in the human genome [32]. This study suggests a complementary strategy, whereby we first identified those transcripts that showed bimodal expression levels, and then identified the polymorphism responsible for this differential expression. Clustering methods [6] and statistical methods [7,10,33] have previously been used to identify bimodal expression in large datasets. These methods have the advantage of being motivated by strong theoretical statistical considerations. However, they also require a moderate level of statistical understanding, and in addition some of these methods can only be easily applied to large-scale meta-analysis of several data sets [10] and may be less suitable for small expression data sets generated in a single laboratory. The approach we suggest is able to be used alongside more complex approaches by laboratory scientists with only basic statistical training, who are in a good position to immediately follow up their results experimentally. We believe that for bioinformatic tasks such as this, providing several complimentary methods that span the continuum of statistical complexity is important in order to bridge the gap between experimental biologists and statisticians.
Our clustering strategy successfully identified the abundance of several mRNA transcripts including MMP1 as bimodally distributed in human endothelial cells in both resting and inflammatory mediator-treated conditions. Up-regulated MMP1 expression has been associated with many pathologies in which endothelial cells are involved [20,21,26,27]. We sequenced the region spanning the well characterised polymorphism at -1607 from the transcription start site in the MMP-1 promoter [16-19] and found that this polymorphism was strongly associated with the bimodal expression observed in the HUVEC dataset. Individuals either homozygous or heterozygous for the 2G polymorphism at -1607 possessed constitutively higher levels of MMP1 (over 100 fold relative to the 1G isolates, P < 0.0001), implying a dominant effect of the 2G allele. This contrasts with previous findings in fibroblasts, where marginal differences in the basal levels of MMP1 between the 1G and 2G isolates were observed [17,34].
Inflammatory mediator treatment revealed a differential response in MMP1 stimulation between HUVEC isolates homozygous for the 1G allele and those possessing the 2G allele. Whereas MMP1 mRNA levels were increased in all isolates homozygous for the 1G allele; in isolates possessing the 2G allele, MMP1 mRNA levels were essentially unchanged (Figure 5). Regulation at the level of MMP1 enzyme activity mirrored this response to inflammatory mediator treatment. One possibility is that MMP1 expression is at maximum levels in the 2G isolates, even under the basal condition. In leukocytes continuously treated with high doses of inflammatory mediators (50 ng/ml, of TNF-α and Il-1β, 2 or 3 times over 24hrs), MMP1 mRNA levels are genotype independent [24]. Whether this is the case in HUVECs remains to be determined.
Chromatin immunoprecipitation (ChIP) was conducted to investigate the potential involvement of TFs that have putative response elements spanning the polymorphism. We found that three TFs bound to this polymorphic region of the MMP1 promoter in endothelial cells. Using nuclear extracts from fibroblast and A2058 melanoma cells, Rutter et al. previously demonstrated that recombinant ETS1 binds strongly to the 2G promoter and weakly to the 1G promoter and that this binding is dependent on cooperation with an adjacent AP1 site at -1602 [16]. Their study along with others have revealed that several members of the AP1 family, including c-JUN, Fra and Fos, are involved in the heterodimmer complexes bound at this cooperative AP1 site [16,35,36]. While the limited ChIP analysis we have performed clearly indicated that GATA3, Fos, Ets1 and Ets2 do indeed bind to this region of the MMP1 promoter, further studies using larger numbers of individuals will be required to identify differential binding between genotypes or cell culture conditions.
Identifying the functional importance of the millions of human SNPs is becoming a major challenge. Simultaneously, the amount of available RNA transcriptome data is rapidly growing, driving scientists to devise new methods to extract the most biologically and clinically useful information from RNA abundance profiles. Therefore, a strategy that identifies functionally important SNPs by virtue of the bimodal abundance across the human population of the associated mRNAs is potentially very useful. Here, we discuss a simple method based on hierarchical clustering to identify bimodally expressed transcripts, which may be used with either microarray or RNAseq data. This method complements more statistically complex approaches and is suitable for use by laboratory scientists with only basic statistical training, who are in a good position to immediately follow up their results experimentally. This strategy identified bimodal endothelial cell expression of several transcripts including MMP1, which appears to be biologically significant with implications for inflammatory disease and for understanding the complex relationships between TFs and polymorphic promoter elements.
Cell culture and IM treatment
Umbilical cords were collected after written informed consent and the study was approved by the Cambridge Research Ethics Committee. The population sampled for this study were of unknown demography, with no information obtained during donor collection relating to parental age, ethnicity or familial history of disease. HUVECs were isolated by collagenase digestion, as previously described [37]. Cells were cultured in fully supplemented media without antibiotics (basal EBM-2 with a propriety mix of heparin, hydrocortisone, vascular endothelial growth factor, epidermal growth factor, fibroblast growth factor, 2% foetal calf serum (FCS, Lonza, Cambridge, UK), at 37°C/5% CO2 until passage 4. To carry out inflammatory mediator treatment for microarray gene expression profiling, passage 4 HUVECs were treated with a cocktail of 10 ng/ml TNF-α, IL-1β and IL-8 for 24 hours prior to RNA extraction.
RNA processing and microarray preparation and data processing
RNA was extracted using TRIzol® reagent (Invitrogen, London UK). RNA quality was assessed using the Agilent 2100 bioanalyser. Biotin labelled cRNA was generated and hybridised on the CodeLink Human Uniset 20K microarrays following the manufacturer's instructions (Applied Microarrays, formally supplied by GE Healthcare). CodeLink microarray data was pre-processed to assess array quality using the CodeLink Expression analysis software v4.0. To enable comparable analysis between arrays, normalisation was carried out using the cyclic Loess method [38,39]. The microarray data has been deposited in NCBI's Gene Expression Omnibus (GEO) [40] and can be accessed through GEO series accession number GSE23070.
Bimodal analysis of microarray expression data
To identify bimodally expressed RNA transcripts, RNA was prepared from passage 4 HUVECs isolated from 15 different individuals and analysed using CodeLink Human Uniset 20K microarrays (the untreated (UT) data set). In addition, passage 4 HUVECs isolated from 9 different individuals were each treated with 10 ng/ml of each of TNF-α, IL-1β, Il-8, and analysed using microarrays as described above (the inflammatory mediator treated (IM) data set). Unsupervised agglomerative clustering was then applied separately to the UT and IM data sets to enrich for multimodality, using R bioinformatic software (freely available at http://cran.r-project.org/ webcite). For each transcript, our algorithm recorded the "height" (Euclidian distance) between the clusters. The height values at either end of the cluster dendrogram were discarded to remove cases where the clustering identified a single outlying individual, and the largest remaining height value was used as an indicator of bimodality/multimodality. For those RNAs with signal intensities that were similar across the set of individuals, the height between clusters is likely to be small. However, where there were two or more distinct clusters of expression values among the set of individuals, the height between clusters is likely to be large. In addition, parametric bootstrapping was carried out during the clustering process to identify the likelihood of identifying the given height value for each gene based on chance alone, as summarised in Figure 1 and in the comments within the R script in Additional File 1. To be strictly statistically correct, the permutation p-values should be adjusted for multiple testing. For example the Benjamini & Hochberg procedure could be used to control the false discovery rate by applying the mt.rawp2adjp function of the 'multtest' R package to the p-values produced from the bootstrap procedure described here. However, this is not included in the current iteration of our method, since it does not alter the ranking of the permutation p-values assigned to each RNA, and it appears to be overly stringent since it masks both of the bimodally-expressed RNAs that were experimentally confirmed in our study. Nevertheless, if larger data sets are analysed, from which the degree of bimodal expression and population distribution parameters for each RNA can be estimated more precisely, it may be worth experimenting with various multiple testing control procedures.
Inflammatory mediator time course and immunoblotting
For the inflammatory mediator time course, passage 4 HUVECs were treated with a cocktail of 10 ng/ml of each of TNF-α, IL-1β, IL-8 for up to 3.5 hours. Whole cell lysates were harvested by scraping in 1X RIPA lysis buffer (Millipore, Watford, UK) with protease inhibitors (Roche, Welwyn Garden City, UK), at time points 0, 0.5, 1.5 and 3.5 hours. Proteins were separated on 12% Tris-glycine SDS-page gels (Invitrogen) and transferred to 0.2 μm nitrocellulose membranes (Invitrogen). All membranes were blocked with 5% skimmed milk in Tris-buffered saline/0.01% Tween®20 at room temperature. Blots were probed with antibodies against ETS1 (sc-350) and ETS2 (sc-351) (both from Santa Cruz Biotechnology) and β-actin (Ambion).
MMP1 activity assay
Total active MMP1 protein abundance was measured using the Fluorokine Human Active MMP1 Fluorescent Assay (R&D Systems). Supernatants were collected from the cell culture of 20 different passage 4 HUVEC isolates, treated with and without an inflammatory mediator cocktail of 10 ng/ml TNF-α, IL-1β, IL-8 for 24hrs. P-Aminophenylmercuric Acetate (APMA) was added to all samples to activate any inactive MMP1. Measurement of MMP1 activity was carried out according to the manufacturer's instructions.
Sequencing of the MMP1 promoter polymorphism
To characterise the -1607 MMP1 promoter polymorphism, DNA was extracted from HUVEC cell pellets using the DNeasy blood and tissue kit (Qiagen, West Sussex, UK), following the manufacturer's instructions. Genomic DNA (50 ng) was amplified with the following primers: 5'-AACCTATTAACTCACCCTTGT-3' 5'-CCTCCATTCAAAAGATCTTATTATTTAGCATCTCCT-3' [34]. The cycling conditions were as follows: pre-incubation at 94°C for 5 minutes, followed by 35 cycles at 94°C for 30 seconds, 56°C for 30 seconds and 72°C for 1 minute, followed by a final extension at 72°C for 10 minutes. PCR products were diluted 1 in 10 in nuclease free water and directly sequenced using the forward primer at GeneService (Cambridge Science Park, Milton, UK). Amplification of the MMP1 promoter region spanning the -930 and -519 polymorphisms was achieved using the same conditions described above using the following primers: 5'-TTCCAGCCTTTTCATCATCC-3' and 5'-CGGCACCTGTACTGACTGAA-3'. Again the forward primer was used for sequencing.
Quantitative PCR
cDNA was made from 1 μg of total RNA using the Quantitect reverse transcription kit (Qiagen), following the manufacturers protocol. Quantitative PCR was carried out using the the ABI 7700 sequence analyser (Applied Biosystems, Calafornia, USA). Reactions were carried out using the Applied Biosystems universal master mix according to the manufacturers instructions. The Taqman probe primers used were: MMP1 (Hs00233958_m1), DDX3Y (Hs00190539_m1), ETS1 (Hs00901425_m1), ETS2 (Hs00232009_m1), GATA3 (Hs00231122_m1), SLC2A11 (Hs00368843_m1), DERP6 (Hs00209768_m1) and internal control 18S (Hs99999901_s1), all from Applied Biosystems.
Chromatin immunoprecipitation
Passage 4 HUVECs were either treated with vehicle or an inflammatory cocktail of 10 ng/ml TNF-α, IL-1β, IL-8 for 24 hours. Chromatin was cross-linked by the addition of formaldehyde to a final concentration of 1% for 10 minutes at 37°C. Cells were washed in ice cold phosphate-buffered saline containing 125 mM glycine, 1 mg/ml Pefabloc, 1 μg/ml aprotinin and 1 μg/ml pepstatin A. Chromatin was sonicated and immunoprecipitated using specific antibodies, as described in the ChIP protocol from Upstate Inc. (Charlottesville, VA). The following antibodies were used: ETS1 (sc-350), ETS2 (sc-351), c-Fos (sc-52) and GATA3 (sc-268). All antibodies were from Santa Cruz Biotechnologies. To quantify enrichment of binding, quantitative PCR was carried out on the immunoprecipitated DNA using SYBR Green on the iCycler (Roche). 25 μl reactions with 1 X SensiMixPlus SYBR and fluorescein (Quantace) were carried out according to the manufacturer's instructions. Primers around the MMP1 polymorphism were 5'-TCTTTGTCTGTGCTGGAGTA-3' and 5'-CAATTTCCTCATCTAAGTGGCATA-3'. The primers for the region 5600 bases upstream of the promoter were 5'-TGCTTATGTTAGCTGACCAGAC-3' and 5'-AGTATGCGTTGCCTTGTCCT-3'.
Authors' contributions
Conceived and designed the experiments: CP, DSCJ, MA. Performed the experiments: MA, DS. Analysed the data: MA, CP DSCJ. Contributed to reagents/materials/analysis tools: BD, NJ, CP. Wrote the paper: MA, DSCJ, and CP. All authors have read and approved the final manuscript.
We thank Marie Joquine for here assistance in identification of the bimodally expressed transcripts. This study was part-funded by the Biotechnology and Biological Sciences Research Council (BBSRC) and Gene Networks International (GNI, Shinjuku Park Tower, Tokyo, Japan).
Broberg P: Statistical methods for ranking differentially expressed genes.
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Cui X, Churchill GA: Statistical tests for differential expression in cDNA microarray experiments.
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Elo LL, Hiissa J, Tuimala J, Kallio A, Korpelainen E, Aittokallio T: Optimized detection of differential expression in global profiling experiments: case studies in clinical transcriptomic and quantitative proteomic datasets.
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Ertel A: Bimodal gene expression and biomarker discovery.
Cancer Inform 9:11-14. PubMed Abstract | PubMed Central Full Text
Teschendorff AE, Naderi A, Barbosa-Morais NL, Caldas C: PACK: Profile Analysis using Clustering and Kurtosis to find molecular classifiers in cancer.
Bioinformatics 2006, 22:2269-2275. PubMed Abstract | Publisher Full Text
Wang J, Wen S, Symmans WF, Pusztai L, Coombes KR: The bimodality index: a criterion for discovering and ranking bimodal signatures from cancer gene expression profiling data.
Cancer Inform 2009, 7:199-216. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Ertel A, Tozeren A: Switch-like genes populate cell communication pathways and are enriched for extracellular proteins.
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Ertel A, Tozeren A: Human and mouse switch-like genes share common transcriptional regulatory mechanisms for bimodality.
BMC Genomics 2008, 9:628. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Huang HC, Jupiter D, VanBuren V: Classification of genes and putative biomarker identification using distribution metrics on expression profiles.
PLoS One 5:e9056. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
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Int J Biochem Cell Biol 2005, 37:283-288. PubMed Abstract | Publisher Full Text
Matrisian LM: Metalloproteinases and their inhibitors in matrix remodeling.
Trends Genet 1990, 6:121-125. PubMed Abstract | Publisher Full Text
Han YP, Tuan TL, Wu H, Hughes M, Garner WL: TNF-alpha stimulates activation of pro-MMP2 in human skin through NF-(kappa)B mediated induction of MT1-MMP.
J Cell Sci 2001, 114:131-139. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Brinckerhoff CE, Rutter JL, Benbow U: Interstitial collagenases as markers of tumor progression.
Clin Cancer Res 2000, 6:4823-4830. PubMed Abstract | Publisher Full Text
Vincenti MP, Brinckerhoff CE: Transcriptional regulation of collagenase (MMP-1, MMP-13) genes in arthritis: integration of complex signaling pathways for the recruitment of gene-specific transcription factors.
Arthritis Res 2002, 4:157-164. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Rutter JL, Mitchell TI, Buttice G, Meyers J, Gusella JF, Ozelius LJ, Brinckerhoff CE: A single nucleotide polymorphism in the matrix metalloproteinase-1 promoter creates an Ets binding site and augments transcription.
Cancer Res 1998, 58:5321-5325. PubMed Abstract | Publisher Full Text
Tower GB, Coon CI, Brinckerhoff CE: The 2G single nucleotide polymorphism (SNP) in the MMP-1 promoter contributes to high levels of MMP-1 transcription in MCF-7/ADR breast cancer cells.
Breast Cancer Res Treat 2003, 82:75-82. PubMed Abstract | Publisher Full Text
Tower GB, Coon CC, Benbow U, Vincenti MP, Brinckerhoff CE: Erk 1/2 differentially regulates the expression from the 1G/2G single nucleotide polymorphism in the MMP-1 promoter in melanoma cells.
Biochim Biophys Acta 2002, 1586:265-274. PubMed Abstract | Publisher Full Text
Benbow U, Schoenermark MP, Mitchell TI, Rutter JL, Shimokawa K, Nagase H, Brinckerhoff CE: A novel host/tumor cell interaction activates matrix metalloproteinase 1 and mediates invasion through type I collagen.
J Biol Chem 1999, 274:25371-25378. PubMed Abstract | Publisher Full Text
Murray GI, Duncan ME, O'Neil P, McKay JA, Melvin WT, Fothergill JE: Matrix metalloproteinase-1 is associated with poor prognosis in oesophageal cancer.
J Pathol 1998, 185:256-261. PubMed Abstract | Publisher Full Text
Murray GI, Duncan ME, O'Neil P, Melvin WT, Fothergill JE: Matrix metalloproteinase-1 is associated with poor prognosis in colorectal cancer.
Nat Med 1996, 2:461-462. PubMed Abstract | Publisher Full Text
Konttinen YT, Ainola M, Valleala H, Ma J, Ida H, Mandelin J, Kinne RW, Santavirta S, Sorsa T, Lopez-Otin C, Takagi M: Analysis of 16 different matrix metalloproteinases (MMP-1 to MMP-20) in the synovial membrane: different profiles in trauma and rheumatoid arthritis.
Ann Rheum Dis 1999, 58:691-697. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Burrage PS, Mix KS, Brinckerhoff CE: Matrix metalloproteinases: role in arthritis.
Front Biosci 2006, 11:529-543. PubMed Abstract | Publisher Full Text
Repeke CE, Trombone AP, Ferreira SB Jr, Cardoso CR, Silveira EM, Martins W Jr, Trevilatto PC, Silva JS, Campanelli AP, Garlet GP: Strong and persistent microbial and inflammatory stimuli overcome the genetic predisposition to higher matrix metalloproteinase-1 (MMP-1) expression: a mechanistic explanation for the lack of association of MMP1-1607 single-nucleotide polymorphism genotypes with MMP-1 expression in chronic periodontitis lesions.
J Clin Periodontol 2009, 36:726-738. PubMed Abstract | Publisher Full Text
Joos L, He JQ, Shepherdson MB, Connett JE, Anthonisen NR, Pare PD, Sandford AJ: The role of matrix metalloproteinase polymorphisms in the rate of decline in lung function.
Hum Mol Genet 2002, 11:569-576. PubMed Abstract | Publisher Full Text
Ghilardi G, Biondi ML, DeMonti M, Turri O, Guagnellini E, Scorza R: Matrix metalloproteinase-1 and matrix metalloproteinase-3 gene promoter polymorphisms are associated with carotid artery stenosis.
Stroke 2002, 33:2408-2412. PubMed Abstract | Publisher Full Text
Nichols L, Lagana S, Parwani A: Coronary artery aneurysm: a review and hypothesis regarding etiology.
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Zhang K, Hongyu Z: Assessing reliability of gene clusters from gene expression data.
Functional and Intergrative Genomics 2000, 1:156-173. Publisher Full Text
Riva A, Kohane IS: A SNP-centric database for the investigation of the human genome.
BMC Bioinformatics 2004, 5:33. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Marinescu VD, Kohane IS, Riva A: The MAPPER database: a multi-genome catalog of putative transcription factor binding sites.
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Akiyama Y: TFSEARCH: Searching Transcription Factor Binding Sites. [http://www.cbrc.jp/research/db/TFSEARCH.html] webcite
Gorlov IP, Gallick GE, Gorlova OY, Amos C, Logothetis CJ: GWAS meets microarray: are the results of genome-wide association studies and gene-expression profiling consistent? Prostate cancer as an example.
PLoS One 2009, 4:e6511. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Hellwig B, Hengstler JG, Schmidt M, Gehrmann MC, Schormann W, Rahnenfuhrer J: Comparison of scores for bimodality of gene expression distributions and genome-wide evaluation of the prognostic relevance of high-scoring genes.
BMC Bioinformatics 2010, 11:276. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Wyatt CA, Coon CI, Gibson JJ, Brinckerhoff CE: Potential for the 2G single nucleotide polymorphism in the promoter of matrix metalloproteinase to enhance gene expression in normal stromal cells.
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Edgar R, Domrachev M, Lash AE: Gene Expression Omnibus: NCBI gene expression and hybridization array data repository.
Nucleic Acids Res 2002, 30:207-210. PubMed Abstract | Publisher Full Text | PubMed Central Full Text


text:
Office of Admissions Nomination Process
Students with an outstanding record of accomplishment who are home schooled or who attend a high school that is not eligible to nominate candidates for the Wells Scholarship may be considered for nomination by the IU Bloomington Office of Admissions. (The Office of Admissions is not able to nominate students who attend, or have graduated from, high schools that are eligible to nominate candidates.)
High school seniors seeking freshman admission to IU Bloomington for Fall 2015 who are academically outstanding (eligible for admission to the Hutton Honors College) and who wish to be considered for nomination for the Wells Scholarship by the Office of Admissions should submit the following materials directly to the Office of Admissions. (These materials should not be sent to the Wells Scholars Program. Doing so could delay or prevent, if the deadline is missed, consideration of a student's candidacy.) All items must be received by the Office of Admissions on or before Saturday, September 20, 2014.
- Completed application for Admission to IU Bloomington (either online or paper application) with required supporting academic documents (official high school transcript to be sent by high school in sealed envelope or electronically; SAT or ACT scores to be sent directly to IU from the testing agencies) and application fee
- A packet of the following additional items sent to:
Cindy Wise
Office of Admissions
Indiana University
300 North Jordan Avenue
Bloomington, IN 47405
- Wells Scholarship Nomination Cover Sheet (PDF)
- Cover letter requesting consideration for nomination for the Wells Scholarship and describing the student's academic interest(s) (300-500 words)
- One letter of recommendation from a teacher who has taught the student in an academic class, enclosed by the recommender in a sealed envelope, signed across the seal
- A resume listing significant honors, extracurricular activities, and/or community involvement
For IU Bloomington freshman admission application materials, visit http://www.indiana.edu/~iuadmit/apply/freshmen/apply.shtml.
Students who are nominated by the Office of Admissions will be contacted by the Wells Scholars Program in early October with additional information about the selection process and a request to submit additional items by a late October deadline.
INTERNATIONAL STUDENTS: International students who wish to be considered for the Wells Scholarship should submit their admission application materials through the process detailed by the Office of International Services, but all other materials for the Wells Scholarship application process should be submitted to Cindy Wise in the Office of Admissions as noted above in Item 2.
Calendar for Office of Admissions Nomination Process
September 20: Deadline for application for admission to IU Bloomington and submission of additional required materials as described above.
Early October: Wells Scholars Program will contact Admissions nominees with request for additional materials.
Late October: "Received by" deadline for submitting the requested additional details.
Mid-November: Approximately 50 finalists are selected for interviews; all nominees are notified of their status.
First weekend in December: Required interviews of finalists at IU Bloomington.
Late December: Notification of status mailed to each finalist. Eighteen to 22 Wells Scholars will be selected from the finalist group.
Mid-April: Deadline for accepting Wells Scholarship.
June: New student advising and registration at IU Bloomington.
Late August: Welcome reception for new Wells Scholars; IU Welcome Week.

subset: commoncrawl


text:
April 20, 2010 - Former 3-time world champion in two weight divisions, Robert "The Ghost" Guerrero, greeted media and fans today at his open workout which took place at the San Jose Boxing and Fitness gym. Guerrero battles Roberto Arreita on the return of Telefutura's "Sólo Boxeo Tecate" boxing series scheduled for Friday April 30 at the Tropicana in Las Vegas. Here's what he had to say.
About his return to the ring - "I'm excited to be fighting on Telefutura's first show back. The "Sólo Boxeo Tecate" boxing series has been great for boxing fans. I can't wait to let my hands go and show the fans that I'm back and ready to fight the best in my division."
About training camp - "It's been a good training camp. My sparring partners gave me good work. I've saw some tape on Arrieta and we have a solid game plan for this fight. I know he's going to be gunning for me, but that's ok, because I'm ready to go."
About his wife's prognosis - "Casey is coming along great. She's still undergoing some procedures with her doctors, but she's right where she's supposed to be. I'm grateful and thank God everyday for her well being. Words can't describe how I feel knowing that she's doing better.."
About his quest to bring awareness to those battling leukemia - "I'm honored that "The Leukemia & Lymphoma Society" (LLS.org) nominated me as a candidate for their "Man of the Year" fundraising campaign. Today I met an awesome kid named Aiden Bowen "2010 Boy of the Year" who is a cancer survivor. He watched me workout and I showed him some boxing techniques. Knowing what he's gone through battling blood cancer, I'm very inspired by his story. I encourage everyone to join me in my pursuit to help fund Leukemia research.
Guerrero vs. Arrieta is presented by Golden Boy Promotions and sponsored by Cerveza Tecate. Doors open at 7:00pm, the first bell rings at 7:30pm and the TeleFutura broadcast begins at 8:30pm PT/11:30pm ET.
Tickets, priced at $100,$50, and $35, are on sale now at the Tropicana Box Office, and by calling702.739.3507. Tickets may also be purchased online at www.troplv.com.
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If you liked this, check these out!
Plenty more like this here!
Dr. Shroud Follow Dr. Shroud in these serials as he tries to find his missing daughter in the vampire-ridden city of Necropolis!
Author Comments
Rated 2.5 / 5 stars
nudity is not for these creaters!
he should have filled the wall with garlic
(even if it didn't kill him , just to piss it off)
and the last reviewer was right
with bordum,,,,,,sigh,,,,,,,
Rated 4 / 5 stars
yes lets all fight for the good!
sorry........ lol XD
i liked it, the black and white pics are nicly done
Rated 4.5 / 5 stars
Pretty cool
I liked the black and white style because it made it look just a cartoon strip with the speech buubles but I would rther have it so there is speaking in it but anyway good job
Rated 5 / 5 stars
Finish boy in the box
keep up the awesome work
Rated 0 / 5 stars
Greatness! This was amazing, probably the best work with black and white that I have ever seen!
The main character seems to have a real dedication for his job... but it all seems so dark himself, and sad.
A great achievment in the flash world!


text:
definition of Wikipedia
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|Lieutenant Governor of Manitoba
Shield of the Lieutenant Governor
Philip S. Lee
since 4 August 2009
|His Honour The Honourable
|Appointed by:
|Michaëlle Jean
as Governor General of Canada
|Sir Adams George Archibald
|15 July 1870
|Term length:
|At the Governor General's pleasure
|Government House, Winnipeg
The Lieutenant Governor of Manitoba ( /lɛfˈtɛnənt/) is the viceregal representative in Manitoba of the Canadian monarch, Queen Elizabeth II, who operates distinctly within the province but is also shared equally with the ten other jurisdictions of Canada and resides predominantly in her oldest realm, the United Kingdom. The Lieutenant Governor of Manitoba is appointed in the same manner as the other provincial viceroys in Canada and is similarly tasked with carrying out most of the monarch's constitutional and ceremonial duties.[1] The present, and 24th, Lieutenant Governor of Manitoba is Philip S. Lee, who has served in the role since 4 August 2009.
The Lieutenant Governor of Manitoba is vested with a number of governmental duties and is also expected to undertake various ceremonial roles. The lieutenant governor, him or herself a member and Chancellor of the order,[2] will induct deserving individuals into the Order of Manitoba and, upon installation, automatically becomes a Knight or Dame of Justice and the Vice-Prior in Manitoba of the Most Venerable Order of the Hospital of Saint John of Jerusalem.[3] The viceroy further presents other provincial honours and decorations, as well as various awards that are named for and presented by the lieutenant governor; these are generally created in partnership with another government or charitable organization and linked specifically to their cause.[4] These honours are presented at official ceremonies, which count amongst hundreds of other engagements the lieutenant governor partakes in each year, either as host or guest of honour; in 2006, the Lieutenant Governor of Alberta undertook 334 engagements and 284 in 2007.[5]
At these events, the lieutenant governor's presence is marked by the lieutenant governor's standard, consisting of a blue field bearing the shield of the Arms of Her Majesty in Right of Manitoba surmounted by a crown and surrounded by ten gold maple leaves, symbolizing the ten provinces of Canada. Within Manitoba, the lieutenant governor also follows only the sovereign in the province's order of precedence, preceding even other members of the Canadian Royal Family and the Queen's federal representative.
The office of Lieutenant Governor of Manitoba came into being in 1870, upon Manitoba's entry into Canadian Confederation,[6] and evolved from the earlier position of Lieutenant Governor of the Northwest Territories, though the occupants continued to simultaneously act as Lieutenant Governors of the Northwest Territories and later as Lieutenant Governors of Keewatin District, until the latter was divided into Saskatchewan and Alberta in 1905. Since 1867, 24 lieutenant governors have served the province, amongst whom were notable firsts, such as Pearl McGonigal – the first female lieutenant governor of the province – and W. Yvon Dumont – the first Métis lieutenant governor. The shortest mandate by a Lieutenant Governor of Alberta was Adams George Archibald, from August 1870 to October 1872, while the longest was Roland Fairbairn McWilliams, from 1 November 1940 to 1 August 1953.
In 1919, the Manitoba legislature voted in favour of The Initiative and Referendum Act, which sought to eliminate the lieutenant governor from the legislative process in the province. Royal Assent to the bill was reserved by Lieutenant Governor James Aikins and eventually the Judicial Committee of the Privy Council at Westminster ruled that, since the law affected an appointee of the federal Crown, it was ultra vires and struck down.[7][8]
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The English word games are:
○ Anagrams
○ Wildcard, crossword
○ Lettris
○ Boggle.
Lettris is a curious tetris-clone game where all the bricks have the same square shape but different content. Each square carries a letter. To make squares disappear and save space for other squares you have to assemble English words (left, right, up, down) from the falling squares.
Boggle gives you 3 minutes to find as many words (3 letters or more) as you can in a grid of 16 letters. You can also try the grid of 16 letters. Letters must be adjacent and longer words score better. See if you can get into the grid Hall of Fame !
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Most English definitions are provided by WordNet .
English thesaurus is mainly derived from The Integral Dictionary (TID).
English Encyclopedia is licensed by Wikipedia (GNU).
The wordgames anagrams, crossword, Lettris and Boggle are provided by Memodata.
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text:
News and notes on health, medicine and fitness
New paper details scientific investigation of 2001 anthrax attacks
More than nine years after the 2001 anthrax attacks that killed five people and sickened another 15, the first scientific paper has been published detailing the methods used in the investigation.
The paper in Proceedings of the National Academy of Sciences describes how scientists at the University of Maryland's Institute for Genome Sciences worked with the FBI to determine that the anthrax samples in all of the attacks were genetically identical. Other scientists later traced the anthrax spores to a flask of Bacillus anthracis that had been primarily in the custody of Bruce Ivins.
The FBI concluded that Ivins, a scientist at a U.S. Army biodefense laboratory in Maryland, carried out the anthrax attacks. Ivins took his own life before the case could go to trial, and the FBI has since closed what it called the Amerithrax investigation.
The paper describes how the investigation launched a new field.
"This paper and the Amerithrax investigation really marked the beginning of a new approach for the science we call forensic genomics," said the paper's senior author Jacques Ravel, an associate professor of microbiology and immunology at the University of Maryland School of Medicine. "The science was a critical component of the Amerithrax case. Without genomics, it would have been extremely difficult to narrow the pool of potential suspects."
Some of the crucial work involved taking spores from the anthrax letters and growing them in the lab. Scientists looked closely at the anthrax they had grown and noticed that some of the bacterial colonies had unusual characteristics. Those characteristics were present in each of the samples.
"Samples from the letters had the same combination of variants in the spores," Ravel said. "That was one of the first things that began to link the letters."
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When most people think of treating the symptoms of CP, they probably think about treatments that focus on the sufferer. These would be treatments such as muscle massages that loosen spasms; speech therapy that helps to facilitate communication and social skills and tutoring that helps to teach basic academic skills. Researchers in Canada recently found that other forms of therapy can work, as well.
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Axeltorv Square (Axeltorv)
Axeltorv, Copenhagen, Denmark 1608Map
+45 7022 2442 (Tourist Information)
Neighborhood: Center (Copenhagen K)
Visit Axeltorv to feel the pulse of Copenhagen. Step onto this square and you will see the past mingle with the present. This square has pretty restaurants serving delectable food and stylish shops selling attractive products. Just stroll around and you will lose track of time.
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There are some great offers at Mexx Canada online stores this week for Cyber Monday 2014
Use promotional code – CYBERMONDAY50 at checkoutShop Mexx Store Online
Cyber Monday Sale Ends December 1, 2014
Sponsored Ads
Find stylish clothing and accessories for the whole family at affordable prices.
Sign up for Mexx newsletter to be kept up-to-date on all the latest happenings. From special contests and offers to money-saving coupons, you will be the first to know about them.
Enjoy some great advantages when you shop online.
Find tops, bottoms, formal wear, outerwear, and accessories.
Check out their Summer door crashers, Linen Collection, Fresh Forward, and Pastel Pop Styles as well. You are sure to find some gorgeous pieces for your seasonal wardrobe. Versatile pieces that can be used during various seasons as well.
Men's Fashions, Boys, and Girls are also found at Mexx.
While browsing the site I came across some very cute baby girl items;
This Fandango pink shirt is just adorable. Regular price was $24.99, now on sale for $12.50.
Dresses starting at just $13.50. Gorgeous jean dresses, Tangerine, Snow White, and many more styles.
Check out Mexx online to see full selection of sale items.
December 11, 2014
December 11, 2014


text:
The Blog
Hayes: Huckabee on the Role of Government
1:40 PM, Dec 30, 2007 • By STEPHEN F. HAYES
Surely one of the most important thing Republican primary voters are evaluating in these final days before they begin casting votes is a candidate's views on the size and scope of government. And on that subject, these voters will want to pay careful attention to the words of Mike Huckabee from Meet the Press this morning.
Host Tim Russert pointed out that the Cato Institute had given Huckabee poor grades for his fiscal management of Arkansas during the time he was governor there and noted that Huckabee had raised taxes several times over the course of his tenure. Huckabee did not back down. He noted that he raised taxes and fees to pay for education and to improve roads and boasted of the results.
And then he said something very un-conservative about the role of government today.
Is that what being president is all about? Creating opportunities--by raising taxes and increasing the size of government--for the people of your country?


text:
Publisher's Description+
From FireBird Software: IPexten is a suite of applications which allow you to view and control the status of your X10 devices over any IP connection. It acts as a gateway between your homes X10 network, and the Internet. It provides an easy to learn scripting facility, adding control structures beyond the normal X10 'Macro' facility, providing an easy way to automate your home in an intelligent manner.
What's new in this version:
- The Lite version provides the core functionality of the full version of IPexten Server, with the limitation that only 'On' and 'Off' commands are supported, and all housecodes are converted to housecode A.
This version also fixes a problem using the control panel, which affected many systems, and adds the capability to view snapshots from the Smart Eye application.
read more +


text:
The Reneu clog is a comfy unisex slip-on from Landau FootWeaRX with lightweight EVA uppers, a fully lined footbed and a cushioned suede collar. They'll also keep you safe because they're slip-resistant. Moisture control adds additional slip protection and comfort during your busy day.
Overall Rating:
Great look and great comfort!!


text:
Information for May 2015 Graduates
The Penn Student Insurance Plan (PSIP) will terminate on July 31, 2015 for students who are graduating.
For students have not found new coverage through employment or other means, we encourage you to access the Insurance Marketplace at www.healthcare.gov. Students whose PSIP coverage terminates on July 31 qualify for special enrollment outside the normal enrollment period.
If you have any questions, please feel free to contact us at 215 746 3535 option #3 or email us at email@example.com.
Penn Student Insurance Plan (PSIP)
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And now *this* Pope seem even less inclined to slaughter thousands of innocents in wars for DemocracyWhiskeySexy and concentrate more wealth and power in the hands of our Ruling Class as they trample the weak. What is *wrong* with him?
And yet neither JPII, nor Benedict, nor the world's bishops ever admitted how wrong they were
Related posts from Catholic and Enjoying It!:
- HokiePundit
At least at its outset, the Iraq War fit the requirements for a Just War. As far as I can tell, it maintained that (with limited, non-sanctioned exceptions such as Abu Ghraib) throughout. It may (or may not) have been unwise, either in its inception or its execution, but it wasn't morally wrong.
- Dan C
This war had no conditions for a Just War. It was a war of choice, on grounds of cooked evidence and decsit, the UN expert and inspector was routinely and shamelessly derided on conservative blogs and Catholic conservative blogs (unrepentently)- he turned out correct, the country who wisely avoided war and protested against it (France) received enormous criticism, and it seems that Abu Ghraib was perhaps closer to the routine than not.
Both Cardinal Ratzinger, and the Pope at the time noted this. Professional neo-con, hired to seduce Catholics to thik tank conservativism, Michael Novak, had his arguments rejected by the Vatican when seeking its support. The USCCB rejected the moral underpinnings of this war.
The Church criticism was so stinging and so true that atbthe time, First Things and George Weigel cooked up a story about a political charism that bishops and cardinals and popes didn't have, so, basically, they should just "shut up" on such matters.
And the usual suspects, Jonah House, Plowshares activists, Pax Christ, the Catholic Worker, and the Catholic Peace Fellowship all denounced the war, many seeking non-violent civil disobedience as a way to engage the matter.
There were plenty of individuals who showed the way away from violence and death and the horrid consequence of war. The arguments against the war were clear, prescient, and based both in reality and moral theory. The hippies were proved right and they were prescient also. The war was obviously unjust and was named so by the pope and the future pope.
- Noah D
the UN expert and inspector was routinely and shamelessly derided on conservative blogs and Catholic conservative blogs (unrepentently)- he turned out correct
Scott Ritter?
- Dustin
Hans Blix, I think.
- Dan C
Hans Blix. And his "foreign-ness" European origin was part of the way to make him out to be weak. Such was the discussion.
- Wolfwood
No conditions? None at all?
Just Cause: Grave Public Evil? Check.
Comparative Justice: Are you saying the US was at fault?
Competent Authority: The US is a democracy, and Bush and Congress were duly elected.
Right Intention: Enforcing an armistice and sanctions meets this description. If we were there for the oil, we did a pretty awful job of ensuring we got it at a good price.
Probability of Success: The removal of Saddam Hussein was not difficult, nor was it expected to be.
Last Resort: What other options were there? We tried sanctions, which were flouted. We offered to pay Hussein to leave, which he didn't. Short of removing the regime through military force, please give (realistic) suggestions.
Proportionality: Just because we didn't anticipate the rise of the militias doesn't mean that we didn't calculate that this would be proportional. Again, we may have miscalculated, but it's clear by our actions that we didn't plan for the country to descend into madness. Germany and Japan didn't do that, and Iraq had a much bigger cultural history to draw on that either of those.
- Dan C
I am saying that not just that the US was at fault, that those of us eager to promote the war and that those who gave special theological support for it hold personal responsibility. I am not one who thinks that collective responsibility denies my personal responsibility.
The war failed just war theory. The conditions were not present. Nor was it a last resort.
In terms of proportionality, the rise of internecine violence was acknowledged as likely during the first Iraq War in the 1990′s. This was a clear, predicted possibility.
Yes, not only am I saying the US was at fault, I am saying individuals who promoted the war are gravely at fault. Additionally, at this distant time point, I am also noting that willful ignorance accompanies most Catholic support of the just-ness of this intervention at this time point.
- Dan C
Preventative War, as this was rightly called, is not in the catechism. As one dismissed theologian noted.
- jacobus
"Last Resort: What other options were there? We tried sanctions, which were flouted. We offered to pay Hussein to leave, which he didn't. Short of removing the regime through military force, please give (realistic) suggestions."
Uh…do nothing? Since Iraq wasn't a threat to the United States…. We would have saved ourselves, and the Iraqis, a lot of wasted blood, money, and effort.
- Mark Shea
Wow. What a farrago of Bourbon rationalization for a manifestly unjust war. It's guys like you who will make certain the GOP remain in the wilderness for years and years to come. Insane.
- http://coalitionforclarity.blogspot.com Robert King
"Grave public evil"? So military action is justified to effect regime change in Hollywood? Vegas? Chicago? D.C.? Any abortion clinic anywhere?
"Competent Authority" What authority does the U.S.A. have over Iraq? What gives the U.S. government the authority to invade a sovereign nation to resolve the "grave public evil" there?
- "joe"
"Just Cause: Grave Public Evil? Check."
there are lots of these, and greater ones than the regime of the iraqi baath party. by what rubric does one decide which ones to attack?
"Comparative Justice: Are you saying the US was at fault?"
for invading iraq? yes, of course. it was a choice. but you must add the role of the UN. do you consider them to be comparatively just?
"Competent Authority: The US is a democracy, and Bush and Congress were duly elected."
so as long as a country is a democracy, it has the right to invade any country anywhere in the world that isn't? are you serious?
"Right Intention: Enforcing an armistice and sanctions meets this description. If we were there for the oil, we did a pretty awful job of ensuring we got it at a good price."
enforcing whose armistice, and by what authority to do so?
and btw, are you really sure that that was the intention?
"Probability of Success: The removal of Saddam Hussein was not difficult, nor was it expected to be."
you just said that the reason for the invasion was "Enforcing an armistice and sanctions meets this description". now you're talking about "The removal of Saddam Hussein". but it was the government, not the individual (who eluded execution for three years iirc). and would you call the aftermath a "success'? iran now has more direct influence than ever before in the affairs of the iraqi state.
"Last Resort: What other options were there? We tried sanctions, which were flouted. We offered to pay Hussein to leave, which he didn't. Short of removing the regime through military force, please give (realistic) suggestions."
"we" had no right to set any of those terms on a sovereign nation.
"Proportionality: Just because we didn't anticipate the rise of the militias doesn't mean that we didn't calculate that this would be proportional. Again, we may have miscalculated, but it's clear by our actions that we didn't plan for the country to descend into madness. Germany and Japan didn't do that, and Iraq had a much bigger cultural history to draw on that either of those."
i don't see what 'cultural history' has to do with any of this, but you do know the political history of the area? that it's a fabrication of british imperial administrative interests, that there are many and competing confessional and ethnic groups in the area?
really, we're back in the last decade with posts like Wolfwoods, looking for any reason not to take full responsibility. the basic message here is "i have a superior ideology and that gives me the right to control you." not a good lesson for the youth of america.
- Matt S
The idea of the charism of political leaders wasn't cooked up, it was based on the Catechism. After a brief description of the just-war criteria, it says "The evaluation of these conditions for moral legitimacy belongs to the prudential judgment of those who have responsibility for the common good." While the choice of the word "charism" is unfortunate because it seems to suggest something like divine inspiration, the point Weigel was trying to make is that legitimate authority requires a certain level of deference, both of its very nature as authority and because only those who make the decisions are fully responsible for the consequences. Of course everyone has the right to criticize the authorities' decisions, and I think it is false to say that Weigel was suggesting otherwise, since I am sure he has plenty of criticisms of the decisions our government authorities have made. His point is that the judgment of an authority is by its nature different from that of a commentator.
- Mark Shea
True. But it does not mean "When the President orders it, it can't be wrong." Speaking of deference to due authority, how about the authority of two Popes and all the bishops in the world saying, "Don't go to war with Iraq"? What is that? Chopped liver? Or doe we have no king but Caesar?
- Dan C
Mr. Shea is correct. Your argument is about the charism of the political class. What I am criticizing is that Neuhaus and Weigel attempted to indicate that their was both a level of infallibility granted the political class's role in this decision and that bishops and priests and cardinals and popes have no role or "charism" in making moral pronouncements on war.
- Mark Shea
"Preventive war is not in the Catechism" – Joseph Cardinal Ratzinger. The war was unjust: http://www.mark-shea.com/jwd.html
- Seamus
If Iraq was a just war, then it would be just (and would make a lot more sense from a geopolitical viewpoint) to invade Cuba to throw out the Castro brothers. Anyone up for that?
- Wolfwood
Why's that? Are they currently sponsoring terrorism, acting in violation of an armistice and international sanctions, or threatening to use WMDs on someone? Iraq was doing all of those things; Cuba may have done some of them before, but they don't seem to be doing them now.
- Dan C
Iraq was an unjust war. Catholics eagerly promoted an unjust war.
- HokiePundit
Keep repeating that. Just a few more repetitions and your ipse dixit assertion is sure to be convincing.
- Dan C
Preventative war is not in the catechism. When that dismissed theologian made that declaration, he said all there was to really say about Just War Theory's application to the Iraq War. But many many other words have been spent on this instead and can be found.
I am not doing extra leg work for a long-defined declaration of the injustice of the Iraq War. One can do their own internet search. There are plenty of arguments that are valid from BEFORE the beginning of the war.
As anyone who has read this blog or others over the years could have picked up, there are substantial serious arguments that are voluminous as to the problems with the just-ness of the Iraq War. Mr. Shea made note of one such argument above.
- Matt S
Preventive war is not in the catechism because the catechism's statement on war is not meant as an exhaustive summary of the issue. If you look at the more comprehensive Compendium of the Social Doctrine of the Church it says this:
"Therefore, engaging in a preventive war without clear proof that an attack is imminent cannot fail to raise serious moral and juridical questions. International legitimacy for the use of armed force, on the basis of rigorous assessment and with well-founded motivations, can only be given by the decision of a competent body that identifies specific situations as threats to peace and authorizes an intrusion into the sphere of autonomy usually reserved to a State." (#501)
In contrast, paragraph #500 says that "A war of aggression is intrinsicially immoral." They could have said the same thing about preventive war but chose not to, instead only saying that it raises questions. In fact the rest of the quotation suggests that preventive war can be justified if it has UN approval.
Catholic teaching recognizes the new situation raised by the nexus of weapons of mass destruction and terrorism, that it might in fact be immoral to wait until an attack is imminent, since the attack might be done in secret and lead to catastrophic loss of life. The question is how to regulate this preventive use of violence, and the question raised by Iraq was what to do when the UN system itself had been corrupted by the state in question.
- Mark Shea
Your argument that *because* preventive war is not in the catechism, *therefore* it is licit is utter and complete crap. Here's why.
- http://chicagoboyz.net TMLutas
Mostly it's crap because the Iraq war is a continuation of an existing war launched to reverse an illegal invasion of Kuwait and a threatened invasion of Saudi Arabia. Since we're about to get into this all over again with Korea (who has loudly announced, again, that it's withdrawing from the cease fire of 1953), it really would be useful to go over what the just war process is when there's a long-term cease fire that has deteriorated and is being regularly violated. I suspect that it's just jus in bello and not jus ad bellum which had been already established at the beginning of the conflict but I haven't seen anything authoritative either way.
I'd go to my bishop with this but he just thinks that just war itself is a load of garbage (I have the most anti-war bishop in the US).
- Timbot2000
John Michael Botean of the Romanians? He used to be my bishop.
- DTMcCameron
I don't think that necessarily follows, but whatever the case, the time for that, had there ever been one, would have been when people we're being lined up against a wall and shot.
- http://vox-nova.com/author/juliahildegard/ Julia Smucker
One angle on the Iraq war that hasn't been considered much, if at all, is its effect on Iraq's (very ancient) Christian minority. I've recently been hearing about the situation there from an Iraqi priest, who has told me that since the US invasion, the persecution of Christians has intensified because they are being associated with the foreign occupation, despite their ancient roots.
Often we American Christians need to be reminded that the Church is bigger than us. We should be considering the lives of our brothers and sisters ahead of any nationalistic interests.