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Gliomas are the most common type of primary brain tumours, and in this group glioblastomas (GBMs) are the higher-grade gliomas with fast progression and unfortunate prognosis. Two major aspects of glioma biology that contributes to its awful prognosis are the formation of new blood vessels through the process of angiogenesis and the invasion of glioma cells. Despite of advances, two-year survival for GBM patients with optimal therapy is less than 30%. Even in those patients with low-grade gliomas, that imply a moderately good prognosis, treatment is almost never curative. Recent studies have demonstrated the existence of a small fraction of glioma cells with characteristics of neural stem cells which are able to grow in vitro forming neurospheres and that can be isolated in vivo using surface markers such as CD133. The aim of this study was to define the molecular signature of GBM cells expressing CD133 in comparison with non expressing CD133 cells. This molecular classification could lead to the finding of new potential therapeutic targets for the rationale treatment of high grade GBM.
Eight fresh, primary and non cultured GBMs were used in order to study the gene expression signatures from its CD133 positive and negative populations isolated by FACS-sorting. Dataset was generated with Affymetrix U133 Plus 2 arrays and analysed using the software of the Affymetrix Expression Console. In addition, genomic analysis of these tumours was carried out by CGH arrays, FISH studies and MLPA;
Gene expression analysis of CD133+ vs. CD133- cell population from each tumour showed that CD133+ cells presented common characteristics in all glioblastoma samples (up-regulation of genes involved in angiogenesis, permeability and down-regulation of genes implicated in cell assembly, neural cell organization and neurological disorders). Furthermore, unsupervised clustering of gene expression led us to distinguish between two groups of samples: those discriminated by tumour location and, the most importantly, the group discriminated by their proliferative potential;
Primary glioblastomas could be sub-classified according to the properties of their CD133+ cells. The molecular characterization of these potential stem cell populations could be critical to find new therapeutic targets and to develop an effective therapy for these tumours with very dismal prognosis.
The cancer relapse and mortality rate suggests that current therapies do not eradicate all malignant cells. In this sense, there is increasing evidence that many types of cancer contain their own stem cells: cancer stem cells (CSCs), which are characterized by their self-renewing capacity and differentiation ability [1]. The study of haematological disorders shed light on the relationship between cancer and stem cell compartments, and the mechanisms by which CSCs might appear and change during the progression of the disease [2,3]. However, the evidence for the existence of CSCs in solid tumours has been more difficult to find because of the lack of specific cell surface markers. During the last years, different cancer cell subpopulations from selected types of human solid cancers have been identified (breast [4], brain [5-7], colon or colo-rectal [8-10], head and neck [11] and pancreatic cancer [12]). These authors, through the use of cell culture, FACS and/or MACS methods, have been able to identify different cell populations within the tumour showing hallmarks of stem cells. This stem cell potential, including self-renewal and lineage capacity, was demonstrated by serial transplantation experiments in animal models. Specifically, the investigation of solid tumour stem cells has gained momentum particularly in the area of gliomas, the most common type of brain tumours. In this group, glioblastoma multiforme is the highest-grade glioma [GBM; grade IV] and is manifested by morphological, genetic and phenotypic heterogeneity [13-15]. Two major aspects of glioma biology that contributes to its awful prognosis are the formation of new blood vessels through the process of angiogenesis and the invasion of glioma cells, the hallmarks of GBM [16]. In addition, these abnormal blood vessels have also been shown to create a vascular niche that houses glioma stem cells [17].
Despite of the recent advances, two-year survival for GBM patients with the most favourable treatment is less than 30%. Even in those patients with low-grade gliomas therapy is almost never curative. Recent studies have confirmed the existence of a small portion of glioma cells with characteristics of neural stem cells [1]. In general, this fraction is characterized by its neurosphere-forming ability, its strikingly increased drug resistance, and finally, by its ability to express surface markers that are oftenly used for their FACS-based isolation [5,6]. With the implantation during this last decade of the NS forming assay as a robust method for the isolation of neural stem cells [18], it has become widely accepted that adult mammalian brain harbours a pool of NSCs responsible for the persistent neurogenesis, seen in limited adult brain regions, such as the sub-ventricular zone, olfactory bulb and hippocampal dentate gyrus [19].
However, it should be borne in mind that the NS assay is not the most suited source of primary adult stem cells for transcriptomic analysis since cells are selected based on its in vitro proliferation capacity in the presence of cytokines and growth factors in their cell cultures such as EGF and FGF.
At the end of twenty century, two independent laboratories could identify and isolate human central nervous system stem cells using antibodies against CD133 [20,21]. This protein, named prominin, identifies a subset of human foetal brain cells distinct to human haematopoietic stem cells, which are also CD133+ but are also CD34-bright [22]. This subset of human CD133+ fetal brain cells is capable of neurosphere initiation, self-renewal, and multilineage differentiation at the single-cell level [20]. The CD133+ cells can differentiate in vitro to neurons and glial cells, and their transplantation into the lateral ventricles of newborn NOD-SCID mouse brains resulted in specific engraftment in numerous sites of the brain [20,21,23].
The CD133 marker is a five-transmembrane protein which is expressed in different type of progenitors as human fetal brain cells or human hematopoietic stem cells [20-22]. In brain tumours the proportion of these CD133+ cells represent a minority of the tumour cell population and are also capable to initiate tumour formation in vivo. Although it have also been reported that a proportion of these tumours could be maintained by CD133- cells [24], there are several evidences showing how this small fraction of CSC which forming NS, can also be isolated using CD133 as a selection marker [6].
In the present study, we have analysed thoroughly the molecular signature of eight fresh primary GBMs focused on its CD133 positive and negative cells. Importantly, all tumours were studied before any treatment of the patient and without previous tumour cell culturing. In addition to the expression analysis of the FACS-sorted cells, we have also performed genome-wide analysis by CGH-arrays, FISH studies at PTEN and EGFR loci, and MLPA at the MGMT promoter. The results obtained concluded that the gene expression signature of CD133+ discriminate common genes to all samples involved in two main characteristic pathways deregulated in GBMs, angiogenesis and invasiveness. However, CD133+ gene expression profile also allowed distinguishing between two different GBM subtypes in higher or lowering proliferative tumours. The molecular biology and the expression signature of these CD133+ cells that drive and support the tumour growth will shed light on the development of fresh and specific treatment strategies.
Samples, flow cytometry and sorting assays
Fresh tumours from eight patients affected of primary GBM without any previous treatment were collected (clinic and pathologic features are summarized in Table 1). Patients' diagnostic were confirmed by the Pathology Facility from the University Hospital of Salamanca, Spain. At the surgical extraction moment, a vast proportion of each tumour was processed to isolate the CD133+ and CD133- cells without previous cell culture. Single-cell suspensions were prepared from individual tumours by standard procedures. We decided to undergo a mechanical disaggregation of tumour samples due to the softness of brain samples, avoiding enzymatic stress that could change the cell surface and even their gene expression. Briefly, tumours were carefully sliced and forced through a 70 μm single-cell filter into the Ca++/Mg++ free phosphate-buffered saline by applying gentle pressure using the piston of a disposable plastic syringe, All single-cells were used for staining. Cells were immunophenotyped using human CD133/2 (293C3) phycoerythrin conjugated antibody (MACS, Miltenyibiotec). Mature red cells were depleted by hypotonic lysis solution (0.38% ammonium chloride for 15 minutes on ice) before staining. Cells suspended in Ca++/Mg++ free phosphate-buffered saline supplemented with 1% fetal calf serum were labelled with this antibody (approximately 1 μg/106 cells) for 30 minutes on ice. Cell fluorescence was analyzed and sorted using the FACS Aria sorter (Becton Dickinson, New Jersey, USA). CD133 antibody was tested previously in human bone marrow (BM) cells in which CD133 positive cells were described before (Figure 1). BM cells were incubated with CD133 and CD34 antibodies (Pharmingen), sustained in studies that demonstrate that antibodies against CD133 also identified a subset of CD34bright BM hematopoietic stem cells [22]. Cell viability was assessed by propidium iodide exclusion (5 μg/mL; Sigma) using flow cytometry.
Table 1. Clinical characteristics, MGMT promoter methylation and FISH analysis of eight primary GBMs.
Figure 1. FACS sorting of glioblastoma cells using CD133 and CD34 antibodies. Control samples from human bone marrows incubated with CD133 antibody. 1: Total cellularity; 2: Gate CD34 without CD133-PE; 3: Gate CD34 with CD133-PE.
Expression arrays
We studied a dataset generated with Affymetrix U133 Plus 2 arrays (Affymetrix, Santa Clara, CA, USA) in 8 gliomas. Results from this expression analysis have been deposited at GEO [25] with accession number GSE18015.
Isolated cells (CD133+ and CD133- from each tumour) using sorting methods were collected in separated vials containing RNA Later (Qiagen, Chatsworth, CA, USA). Total RNA was extracted from CD133+ and CD133- sorted cells using Trizol (Invitrogen, Carlsbard, CA) making a total of 16 samples (8 positives and 8 negatives). The integrity of the RNA was confirmed with the Agilent Bioanalyzer 2100 using the RNA 6000 Pico kit (Agilent). We used the GeneChip® Expression 3' Amplification Two-Cycle Target Labelling kit (Affymetrix, Santa Clara, CA, USA) to label the RNA following the manufacturer protocol. The cRNA was hybridized to Affymetrix Human U133 Plus 2 arrays according to the manufacturer protocol. Briefly, double-stranded cDNA was synthesized routinely from less than 1 microgram of total RNA primed with a poly-(dT) -T7 oligonucleotide. The cDNA was used in an in vitro transcription reaction in the presence of T7 RNA polymerase and biotin-labelled modified nucleotides during 16 hours at 37°C. Biotinylated cRNA was purified and then fragmented (35-200 nucleotides), together with hybridization controls and hybridized to the microarrays for 16 h at 45°C. Using the GeneChip Fluidics Station 450 (Affymetrix), the biotin-labelled cRNA was revealed by successive reactions with streptavidin R-phycoerythrin conjugate, biotinylated anti-streptavidin antibody and streptavidin R-phycoerythrin conjugate. The arrays were finally scanned in an Affymetrix GeneChip Scanner 7G Plus.
Preliminary data analysis was conducted using the software of the Affymetrix Expression Console from AGCC suite (Affymetrix GeneChip Command Console, version 1.1) following the statistical procedures described in the Affymetrix: Expression Console User Guide, selecting the 3' Expression Analysis guidelines for MAS5 and PLIER algorithms in two independent steps. MAS5 calculated the present call index for each of the 54,675 probe sets on the chip (settings used were standard for the HG U133 Plus 2 array: alpha1 = 0.04, alpha2 = 0.06, Tau = 0.015, TGT = 500). This present call index was used to select 245 probe sets having Presence index through the 16 analyzed samples. PLIER algorithm was used to calculate the normalized expression values of the probe sets (using quantile normalization and PM-MM background correction methods). Statistical analysis and post-processing were performed using TIBCO Spotfire 9.1 (TIBCO Software Inc., Palo Alto CA, USA).
Multidimensional Scaling Analysis of CGH and Gene Expression data for the eight samples was performed using the multivariate statistics options of the Simfit Package (Version: 6.1.10 © W. G. Bardsley, University of Manchester, http://www.simfit.man.ac.uk/ webcite).
Network analysis was performed mapping the results on the IPA 8 knowledge database (Ingenuity Pathway Analysis).
CGH array
DNA from each fresh-frozen sample was extracted with the standard phenol-chloroform method and normal DNA was prepared from human placenta of healthy donors. DNAs were quantified using the Nanodrop spectrophotometer. DNA quality was assessed by the 260:280 ratio and its integrity by agarose gel ethidium bromide visualization. Genomic-wide analysis of DNA copy number in each patient was performed using CGH based array. Due to the low proportion of CD133 positive cells in each tumour sample, CGH array only was performed using genomic DNA from the bulk tumour. Slides containing 3296 BACs were produced in "Centro de Investigación del Cáncer" (Salamanca, Spain). The particular bacterial artificial chromosome (BAC) and P-1 derived artificial chromosome set used to produce this array is distributed to academic institutions by the Welcome Trust Sanger Institute (Cambridge, United Kingdom) and contains targets spaced at ≈ 1 Mb density over full genome, a set of subtelomeric sequences for each chromosome arm and a few hundred of probes selected for their involvement in oncogenesis. The clone content is available in the "Cytoview" windows of the Sanger Institute mapping database site, Ensembl (http://www.ensembl.org/ webcite). According to this database, clones were ordered along the chromosomes. For the array, 10 simultaneous hybridizations of normal male versus normal female and placenta (DNA reference) was performed to define the normal variation for the log2 ratio. Cy5/Cy3 intensity ratios of every spot were converted into log2 ratios. The log2 ratio of each clone was normalized to the median log2 ratio of the ten control hybridizations, after which the median of triplicate spots was calculated. Data from two-colour hybridizations for both DNA were normalized using the corresponding GEPAS module DNMAD [26]. Regions of copy number gained and lost for the BAC array-CGH data were identified by creating sample specific thresholds. Reference copy number polymorphisms were carefully revised in all data sets. Therefore every clone on the array was compared with 'Database of Genomic Variants' (http://projects.tcag.ca/variation/ webcite) [27,28]. For unsupervised clustering analysis, we converted the relative ratio value for each BAC clone to a score of 1 (gained/amplified), 0 (no change), or -1 (lost) based data obtain by the binary segmentation method described by Olshen et al. [29] and analyzed data with Cluster and TreeView of GEPAS (Multi Experiment Viewer 4.0) based on the average linkage method with the Pearson uncentered metric correlation. Statistical evaluation was carried out using the SPSS 15.0 statistical software (Chicago, Illinois, USA). All P-values reported were two-sided and statistical significance was defined as P-values < 0.05. Complementary details on this method are summarised in Additional file 1.
Additional file 1. Complementary details on CGH array method. Complementary details of the CGH methodology in GBM tumours.
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Fluorescence in situ hybridization
Dual-probe fluorescence in situ analysis were performed with locus-specific probes for centromere 7/EGFR gene and centromere 10/PTEN gene (Vysis, Dowerners Grove, IL). FISH studies were carried out following well-established methods [30]. Polysomies (chromosomal gains) were defined as more than 10% of nuclei containing three or more CEP signals. Specimens were considered to have an amplification of EFGR when more than 10% of CD133 negative tumour cells exhibited an EGFR/CEP7 ratio >2 or inestimable tight clusters of signals of the locus probe.
Real-Time PCR
CD133+ and CD133- amplified RNA samples were reverse-transcribed to cDNA. PCR reactions were performed using equal amounts of cDNA as template. SYBR Green PCR Master mix (Applied Biosystems, USA) was used for template amplification using standard protocol with specific primers for each of the transcripts examined [Additional file 2: Supplemental Table S1]. Incorporation of the SYBR Green dye into PCR products was monitored in real time with an ABI PRISM 7000 sequence detection system (Applied Biosystems). SDS system software was used to convert the fluorescent data into threshold cycle (Ct) at which exponential amplification of products begins. The differences in the Ct values (dCt) between the transcript of interest and endogenous control (GAPDH) were used to determine the relative expression of the gene in each, adapted from [31]. qPCR was performed using specific primers to corroborate expression array result for several genes of those 245 presented probes [Additional file 3: Supplemental Figure S1].
Additional file 2. qPCR Primer Sets. Set of primers carefully designed to test mRNA expression in CD133+ and CD133- cells by SYBR Green real-time PCR.
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Additional file 3. qPCR validation of gene candidates differentially expressed in Affymetrix arrays. Relative expression of six gene candidates differentially expressed between CD133+ cells and CD133- cells from four representative groups of samples is shown. Names of transcripts analyzed are on the x-axis and the CD133+/CD133- mean fold differential regulation is on the y-axis.
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MS-MLPA analysis
MLPA analysis was performed using SALSA MLPA Kit ME011 and executed as described by the manufacturer (MRC-Holland) with minor modifications in order to detect MGMT promoter methylation. Briefly, 5 μl of each tumour DNA was denatured and subsequently cooled down to 25°C. After addition of the probe mix, the sample was denatured and probes were allowed to hybridize. Sample was ligated with or without HhaI digestion, resulting in ligation of the only digested sequences. PCR amplification was performed using as template both ligation products (50 μl PCR volume containing 10 μl of the ligation reaction). Agarose gel electrophoresis was used to check MLPA efficiency. Then, PCR reaction fragments were separated by capillary gel electrophoresis (ABI 3739, Applied Biosystems) and quantified using the Genemapper software (Applied Biosystems). MS-MLPA processing was performed using Coffalyser analysis tool developed at MRC-Holland (http://www.mlpa.com webcite) and performed as described by Jeuken et al [32].
Patients with higher number of CD133+ cells could present resistance to the treatment
In order to check the functionality of the CD133 antibody and the FACS, we firstly tested the methodology in a human bone marrow sample. In normal conditions, CD133 antibody also identifies a subset of bone marrow stem cells, which are also CD34 positives. Figure 1 shows how CD133 antibody identifies a pool of well-defined human CD133+/CD34+ cells. Using the same procedure, we sorted the CD133 positive population from each fresh GBM sample without previous cell culture (Figure 2). Table 1 shows the absolute number and percentage of CD133+ cells obtained from each sample as well as clinical-biological parameters of each patient. It is interesting to note that only those two patients with higher number of CD133+ cells (more than 10000 cells) did not response to the chemotherapy. Interestingly this correlation, high CD133+ cell number and resistance to therapy, has also been observed in patients and in GBM cultured cells [33-35] which further validate our approach.
Figure 2. FACS sorting of GBM cells using CD133 antibody. Dot plot representation of CD133+ and CD133- populations in GBM tumour samples are shown. CD133+ population is painted in red and CD133- population in green. Percentage of each population is marked (green for CD133- and red for CD133+ percentage). Tumour sample is illustrated in the upper side of each plot.
EGFR, PTEN and MGMT genes are altered in the GBM primary samples
The implication of different genetic alterations in the prognosis of primary glioblastoma such as EGFR amplification, PTEN deletion or MGMT promoter methylation has been previously described. As EGFR, PTEN and MGMT genes are usually altered in primary GBMs, we decided to corroborate the primary nature of our samples by checking for the existence of these alterations in the bulk tumour cell population of GBMs by FISH analysis and MLPA assay. EGFR amplifications were detected in 3 of 8 samples and PTEN deletions in 6 of 8 samples. Additionally, we also detected MGMT promoter methylation but none of these variables were significantly related to the biological features of the patients (see Table 1 and Figure 3B). It is important to remark that the number of cases studied is probably too low to find this kind of correlation.
Figure 3. CGH array and MGMT promoter methylation assays in GBM samples. A) Unsupervised cluster analysis of CGH data from 8 primary GBMs. Each column represents one case and each row represents one BAC clone. We assigned values of 1, 0 and -1 for gain, no change and loss, respectively. Losses are in green and gains in red. P-values < 0.05. B) Ideogram showing MGMT promoter methylation.
Common genomic imbalances identify patients with higher number of CD133+ cells without treatment response
In addition to the expression studies, we analyzed the genomic profile by CGH in the bulk tumours. All P-values reported were two-sided and statistical significance was defined as P-values < 0.05. In more than 50% of cases the gains affected to chromosomal regions located at 1q31-q42, 3q25, 4p15, 6p21-6q23, 7p21, 7q21, 9p21, 9q22, 11q22, 12q21 and 18q12. The losses were located on regions at 1p36, 1p13, 2p23, 5p15, 10q24.16, 12q13, 13q14, and 17p were also affected on more than 50% of cases [Additional file 4: Supplemental table S2]. Two major genetic groups emerge from the unsupervised clustering of CGH data (Figure 3A). Significantly, the only two cases with higher number of CD133+ cells and without treatment response, samples G4 and G11, grouped together. This cluster was characterized with common genomic rearrangements with gains on 3p21.31, 6p21, 6q25, 7p14.2, 9q22, 15q11, 20q13 and 22q13 chromosomes.
Additional file 4. CGH-array data set. Chromosomal gains and losses from tumour bulk cells of GBM patients are shown in this table. Each column shows the median of Cy5/Cy3 ratio intensity of triplicate spots of each clone normalized by GEPAS.
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CD133+ vs. CD133- gene expression analysis divided GBMs in 2 different groups
Although the number of samples is not very large, the main feature that distinguishes this work from previous studies [36,37] resides on the ex-vivo study of GBM cells that were analysed. Using direct sorting of CD133+ cells without previous cell culture led us to obtain a bona fide primary pool of CD133+ cells. Even when these cells represent a low percentage in the number of tumour cells, as in a normal tissue, we were able to isolate and amplify their RNA (by two rounds of amplification) in order to study their gene expression signature in comparison to its counterpart population of CD133- from the same GBM tumour.
Data normalized from preliminary analysis was used to calculate only those probe sets that were present (as described in the MAS5 algorithm) in all the samples (16 arrays from 8 GBM; hybridization per cell population). Results from this expression analysis have been deposited at GEO [25] with accession number GSE18015.
A final list of 245 probe sets was obtained according to these parameters. Initially, unsupervised clustering of 245 gene list using GEPAS Release v3.1 software (http://gepas3.bioinfo.cipf.es/ webcite) allowed us to examine the first classification of these GBMs. Importantly, GBM samples were ordered in two main groups (Figure 4). Samples G9 and G11 were grouped together and apart from the rest. However, their only common biological characteristic was their tumour location. G9 and G11 presented a parietal location versus the temporal or local locations presented by the remaining tumours (Table 1).
Figure 4. Unsupervised clustering of CD133+ cells vs. CD133- cell gene expression signature from each tumour sample show 2 main GBM groups. To molecularly characterize glioblastoma stem cells of GBM tumours, we compared the gene expression profiles of purified CD133+ cells from GBM patients versus CD133-cells from each patient. Each gene (identified at right) is represented by a single row of coloured boxes; each patient is represented by one single column. Data are displayed by a colour code where red indicates over-expression in CD133+ fraction versus CD133-cells. A group of genes over-expressed for almost all samples are grouped in the bottom. SOTArray tool from GEPAS Release v3.1, let us to classify CD133+ vs. CD133- cells from each tumour in 2 mainly groups: G9, G11 and the rest.
Commonly up-regulated and down-regulated genes in CD133+ GBMs
Following the initial classification proposed by SOTArray of GEPAS, we were able to discriminate a minor group of genes commonly up (19 genes) and down-regulated (22 genes) in all samples (CD133+/CD133-) (Tables 2 and 3). Common up-regulation of genes such as COL1A1, COL1A2, PGF, LRRFIP1, TMEFF2 or TGFB1 suggested an important role of this pool of cells in blood vessel formation, angiogenesis, permeability and proliferation pathways, essentials functions in tumour progression [38-40] (Figure 5). On the other hand, the group of genes commonly down-regulated in all CD133+ vs. CD133- cells (that means, over-expressed in the CD133- compartment) were strikingly related to cell assembly, neural cell organization and molecular pathways altered in neurological disorders. That is the case of, GNB2L1, DPYSL2, TUBA1A or CFL1, all of them important players in cell migration, morphology and actin polymerization, in brief, motility of neural differentiated cells (Figure 6).
Table 2. Common up-regulated genes in CD133+ vs.CD133- GBM cells.
Table 3. Common down-regulated genes in CD133+ vs.CD133- GBM cells.
Figure 5. Commonly CD133+ cell up-regulated genes participate in angiogenesis, tumour development and neural developmental disorders. Ingenuity representation and classification by functions of those common up-regulated genes in all CD133+ vs. CD133- cell GBM samples. Red colour genes are the most positive deregulated and grey one those with a lower over-expression levels in this group. The first cluster of genes (COL1A1, COL1A2, TGFB1...) has been described largely in angiogenesis and permeability whereas the second cluster (LRRFIP1 and OPHN1) participates in developmental disorders. Changing transcription pattern of all of them favour tumour development.
Figure 6. Common CD133+ cell down-regulated genes are involved in cell assembly organization and cancer. Ingenuity representation and classification by functions of those commonly down-regulated genes in all CD133+ vs. CD133- cell GBM samples. Green colour represents those genes differentially regulated in CD133+ vs. CD133- that participates in cell assembly, migration and cancer pathways.
Two different GBM groups can be functionally defined according to the expression pattern of 40 genes
The major group of genes discriminated by the SOTArray of GEPAS presented a differential gene expression pattern in two of the eight CD133+ vs. CD133- GBM samples, G4 and G7, in contrast to the remaining cases. In this group, we found out a cluster of genes clearly over-expressed in some of CD133+ vs. CD133- GBM samples (G4 and G7) but repressed in the rest. Specifically, a group of 40 well-defined genes, classified according to their function, were able to distinguish between 2 different GBM signatures (Figure 7) revealing the possible different proliferative potential in high grade GBM tumours (VIM, GLUL, PLK1, HUWE1, RPS4X...) (Figure 8 and Table 4).
Figure 7. Forty differential genes in G4 and G7 samples discriminate between high or low proliferative potential. Unsupervised clustering and ingenuity pathways representation of 40 differentially expressed genes. A) Unsupervised clustering of this 40 gene list let us to distinguish 2 well defined and opposite groups. Ingenuity principal represented pathways include B) recombination and repair pathways and C) cancer and cell compromise. Those GBMs with a positive pattern CD133+/CD133- for this gene expression signature, could present a higher proliferative potential of their tumour stem cells or, by the opposite, a lower proliferative potential of the mature glioma cells.
Figure 8. Forty differentially expressed genes in ex-vivo CD133+/CD133- GBM cells classify these tumours according to their functional categories. Ingenuity functional classification of 40 differentially expressed genes in primary GBMs discriminate two main groups of GBM according to their proliferative potential.
Glioblastomas are the higher-grade gliomas with fast progression and unfortunate prognosis. Recent studies have demonstrated in these tumours the existence of a small fraction of cancer cells endowed with features of primitive neural progenitor. Although some observations pointed out towards the involvement of CD133- cells in tumour maintenance [24], several studies have involved the CD133+ cells as the brain tumour initiating cell [6,41,42]. In any case, studies performed in order to characterize the glioblastoma stem cell have been carried out using in vitro, cultured tumour cells. While these cultured cells present the capacity to form NS, essential pathways in cell/tumour biology could likely be altered as a direct consequence of the cell culturing such as cell-cell adhesion, cell-niche junctions, exposure to mitogen activation, rapid division of the cells etc.
To gain insight into the characterization of these cells, we examined directly for the first time CD133+ cells by FACS-based purification from ex-vivo primary tumours without the intervention of cell culturing or any prior expansion procedure.
Despite that the cohort of tumours analysed was not very large, we could find a correlation among clinical history, response to treatment and genomic alterations in two samples (G4 and G11). Both of these tumours showed the highest content of CD133+ cells, the lack of response to treatment and similar chromosomal alterations (multidimensional scaling analysis reflected relationship among these parameters). However, this correlation was not further supported by transcription profiling of CD133+ and CD133- cells. Indeed, non-supervised analysis of CD133+ vs. CD133- gene expression (Figure 4) showed that only G9 and G11 samples were grouped together and apart from the rest, being the tumour location the only biological feature able to distinguish them (see Table 1). Despite of this low number of samples, multidimensional scaling analysis established again a relationship between G9/G11 tumour location and their gene expression.
To understand the biological properties of the CD133+ compartment, we sought to identify common gene signature by the comparison of CD133+ vs. CD133- cell populations. This array-based analysis led us to the identification of gene profiles with common up-regulated and down-regulated genes. Up-regulated genes such as COL1A1, COL1A2, PGF [38] or TGFB1 [43], suggested an important role of these compartment in blood vessel formation, angiogenesis, permeability and invasiveness, essential functions in tumour progression [38-40]. Significantly, most of these up-regulated genes encoded secreted proteins involved in autocrine and paracrine signalling, like TGFBI, a pleiotropic cytokine that, among other functions, can induce the dissociation of VE-cadherin junctions between endothelial cells which could favour mature tumour or GBM cells migration [43]. Up-regulation of these genes in putative CD133+ stem cells would help to increase the mobility of cancer stem cells through the brain, which is consistent with the high invasive characteristics of these tumours and their high possibility to colonize the adjacent area. It is also worthy to mention in this same regard, the importance of the microenvironment in the stem cell/cancer stem cell maintenance, as has recently been pointed out with the identification of the perivascular niche in grade I-IV astrocytomas [44]. Several evidences suggest that normal neural stem cells, and likely also neural cancer stem cells, exist within protective niches as the vascular niches, into which endothelial cells secrete factors that regulate neural stem cell function [45,46]. This raises the question of whether CSCs could be located and regulated by these microenvironments. Calabrese et al. proposed that the tumour microvasculature generates specific niche microenvironments promoting the maintenance of CSCs [47]. Recent studies using orthotopic glioblastoma xenografts suggest that CSCs secrete proangiogenic factors that promote the recruitment and formation of tumour blood vessels [48] that significantly facilitates brain tumour growth and invasion. Our gene expression findings in ex-vivo CD133+ isolated cells clearly support this result.
High expression in the CD133+ compartment of genes such as LRRFIP1, transcriptional repressor of EGFR [49], would support the idea of EGFR gene as a secondary event in the process of GBM development by promoting infiltration and mediating resistance to therapy. In this same scenario, the positive regulation of the tumour suppressor gene TMEFF2 [50] in the potential CD133+ stem cell compartment in these GBMs, could also operate as a late event in the initiation of neoplastic progression. In fact, low levels of TMEFF2 and other genes responsible for tissue or cell assembly in the CD133- compartment would promote the down-regulation of cell to cell interactions and junctions, providing a molecular mechanism for the highly invasive nature of the GBM.
The second group of genes, commonly down-regulated in all CD133+ vs. CD133- cell from human ex-vivo GBM samples (that means, over-expressed in the CD133- compartment) were found to be associated to cell assembly, neural cell organization and neurological disorders. That is the case of genes such as GNB2L1, an anchor protein involved in adhesion and migration of human glioma cells [51], DPYSL2, a promoter of microtubule assembly and neuronal development [52], TUBA1A [53] or CFL, which controls cell migration and cell cycle progression [54,55]. This group of genes plays important roles in cell migration, cell polarity and actin polymerization (Figure 6). In this same oncogenic scenario, it would be interesting to mention the deregulated expression of HIF-1 gene. This gene which is down-regulated in most of the CD133+ samples analysed, is involved in tumour angiogenesis and cell growth [56], and could play some role in the later events that drive tumour progression. In this regard, recent studies have demonstrated that HIF-1 protein stabilization contribute to tumour angiogenesis, one of the main characteristics of primary GBMs [16]. Mutations in metabolic enzymes, in particular isocitrate dehydrogenase enzymes (IDH1 and IDH2), have been shown to be involved in glioma development and would facilitate HIF-1 protein stabilization [57,58]. The negative deregulation of the HIF-1 gene that we have observed in most ex-vivo CD133+ cells in this work, also support this idea.
A notable feature of the gene expression pattern of CD133+ cells was the differential expression of 40 genes that divide GBM samples in two opposite molecular signatures. The classification of these 40 genes according to their function (Figure 8 and Table 4) pointed to their implication in cell growth, cell death, DNA replication, recombination and, definitively, in cell proliferative control. Amongst these genes we wanted to emphasize the differential expression of the gene coding for vimentin (VIM), an intermediate filament of the mesenchymal lineage involved in migration, cell signalling, cancer and neurological disease [59,60]. Some other genes differentially expressed in this pool of cells CD133+ and also involved in cancer and neurological disease are RPS4X, RPS3A or TUBA1B (Table 4). Another relevant member of the top 40 list of genes was HUWE1, a pleitropic ubiquitin ligase that participates in a wide variety of biological functions related to cell proliferation such as cell growth/death, and DNA replication, and that has been described to be deregulated in different carcinomas [61] (see Figure 8 and Table 4). Interestingly, this deregulated gene has also been reported to be an important control gene for the proliferation capacity of embryonic NSC in the mice [62]. GLUL encodes the glutamine synthetase, a metabolic enzyme required for the maintenance of the energy balance and that when mutated causes severe malformations and neonatal death [63]. Finally, PLK1, the mitotic kinase par excellence, modulates mitosis entry and promotes cell transformation upon upregulation as an oncogene [64-66]. These differentially regulated molecules must be playing pivotal roles in keeping the tumour cells in a switch-on state that enables them to survive, proliferate and invade the surrounding healthy tissue.
Table 4. Functional classification of 40 differentially expressed genes in CD133+ vs.CD133- GBM samples
In brief, the results obtained in this study revealed the presence in CD133+ cells from primary glioblastoma of a common gene expression signature involved principally in the promotion of proangiogenic and invasive programs. Additionally, CD133+ gene expression pattern led us to discriminate between two different GBM subtypes in higher or lower proliferative tumours. The molecular biology and the expression signature of these CD133+ cells that drive and support the tumour growth will shed light on the development of new treatments to fight against GBMs.
Ex-vivo analysis of CD133+ primary GBM cells has driven us to the valid detection of a common gene expression profile among GBMs principally characterized by the expression of genes involved in blood vessel formation, angiogenesis and invasiveness, the main aspects of glioma biology that contributes to its adverse prognosis. Besides, data obtained from the analysis of a group of 40 genes differentially expressed in GBM samples suggest that primary GBM can be sub-classified according to the properties of their CD133+ cells. Differences between both groups were provided by the proliferative potential of their CD133+ population (potential tumour stem cells). We can conclude that molecular characterization of CD133+ population in primary GBMs could be critical in the development of new and effective treatments for these tumours with very dismal prognosis.
GBM: Glioblastoma multiforme; FACS: Fluorescent activated cell sorting; CGH: Compared genomic hybridization; FISH: Fluorescence in situ hybridization; MLPA: multiplex ligation-dependent probe amplification; CSCs: Cancer stem cells; MACS: Magnetic activated cell sorting; NS: Neurospheres; NSCs: Neural stem cells; BM: bone marrow; BAC: bacterial artificial chromosome.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
JLG participated carrying out CGH, FISH and methylation studies. MPC carried out qPCR assays, analysis and interpretation of array data and has been involved in drafting the manuscript. JAGM provided GBM samples and clinical data from patients. FGR carried out gene expression assays and performed gene expression analysis. JO, OB and MS contributed to anatomy-pathological diagnosis of GBM samples. JMHR and RGS have been involved in revising it critically for important intellectual content. MSM conceived of the study, its design, coordination and has been involved in drafting the manuscript. All authors read and approved the final manuscript.
We are grateful to Dr. Alberto Pendas for continuous and generous help with the writing of the manuscript. We thank Dr. I. Sanchez-Garcia for his support. We thank Dr. M. Luz Sanchez and Dr. J.M. Martin Fernandez for continuous and generous help with the human tumour cell sorting and technical assistance. Research in my group is supported by Ramon y Cajal Scientific Spanish Program, Junta de Castilla y León (SAN673/SA09/08) (SA093A08), and Department of Medicine from University of Salamanca. Dr. JL Garcia is supported by IESCyL foundation.
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The Five Major Problems Facing Apple
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Liva Judic | March 3, 2014 | Comments
Adap.TV just released its new service, an interesting and unavoidable extension to the existing Audience Path: programmatic buying for linear TV, creating the digital-to-TV space.
Adap.TV just released its new service, an interesting and unavoidable extension to the existing Audience Path: programmatic buying for linear TV, creating the digital-to-TV space.
"TV is still a dominant medium for video and continues to attract the largest pool of ad dollars today, but fragmentation of audiences is driving the need for TV and digital to be more simply transacted in tandem," Adap.TV chief executive Amir Ashkenazi tells ClickZ, explaining the rationale behind the extension to programmatic TV buying.
Although the company would not disclose projected revenues from Audience Path for TV, it is clear that it already has huge potential and has been thoroughly test-driven for "more than six months" by its client Magna Global, the strategic global media unit of IPG Mediabrands.
The platform will be crunching data obtained through its partnerships with Nielsen (for traditional age and gender demographics) and Rentrak (for behavioral data). It will give buyers "the ability to transact against traditional age/sex demographics, while also being able to optimize how their ad dollars are spent based on specific audiences and segments they want to reach, for example reaching 18-35 male TV viewers, but also those who like mixed martial arts or a particular type of alcohol," Ashkenazi notes.
The Adap.TV Marketplace has TV inventory with a potential reach to more than 90 million U.S. TV households across nearly 100 cable networks.
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Kaleidoscope Duets, Book 2
By Jon George
Kaleidoscope Duets, Book 2 composed by Jon George. For Piano. Duet or Duo; Piano Duet (1 Piano, 4 Hands). Elementary/Early Intermediate level piece for the Piano Duet event with the National Federation of Music Clubs (NFMC) Festivals Bulletin 2008-2009-2010. Early Intermediate; Late Elementary. Book. 16 pages. Published by Alfred Music (AP.692).
These books introduce the student to a wide variety of musical designs that spark the imagination while developing technical skills. "Chinese Dragon Dance" and "Mountain Dance" are Federation Festivals 2014-2016 selections.
List/syllabus: Federation Festivals 2014-2016.


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By Catholic Online (NEWS CONSORTIUM)
11/25/2013 (1 year ago)
Catholic Online (www.catholic.org)
It's pink, pretty with a powerfully toxic sting. The Crambione Cookii jellyfish has suddenly reappeared off the coast of Australia after more than 100 years after the last recorded sighting of it. The highly rare jellyfish hasn't been been since 1910 - but one was recently captured off the coast of Queensland, Australia.
By Catholic Online (NEWS CONSORTIUM)
Catholic Online (www.catholic.org)
11/25/2013 (1 year ago)
Published in Asia Pacific
Keywords: Cookii monster, Australia, jellyfish, aquarium
LOS ANGELES, CA (Catholic Online) - Very little is known about the mysterious species, whose sting is so powerful that it can be felt in the water surrounding the creature.
Discovered by an aquarist who was releasing a rescued sea turtle, Puk Scivyer, "As soon as I saw it I realized it was a species I'd never seen before."
An employee at UnderWater World aquarium in Mooloolaba, Sciyver added, "But to then discover I was the first person to see this species in over a hundred years was just incredible.
"It was the size [that made it stand out]... but as we came past he was more of a cube shape and we could see he was not like the ones that we normally see.
"It's the biggest [jellyfish] I have seen in Australian waters."
Marine biologists are perplexed as to how the species, which was presumed extinct, has managed to evade notice for more than a century.
Last seen by American scientist Alfred Gainsborough Mayor off Cookstown, Queensland, in 1910, the jellyfish has not been seen since.
Gainsborough's sketch was until now the only record of the creature in existence. That sketch was used to help identify the animal by jellyfish expert Dr Lisa-Ann Gershwin, who confirmed the existence of unusual ocean-dweller after it was captured.
Now cared for at the UnderWater World aquarium in Queensland, the specimen will be observed closely behind the scenes to learn as much as possible about the species.
Currently scientists do not know where the animals live, their life expectancy or their population size. Scivyer thinks it is unlikely that any more will be found.
A birth foretold: click here to learn more!
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By Catholic Online (NEWS CONSORTIUM)
Conjoined twin girls in Indonesia have been born with a very rare genetic condition. Both girls share a chest, abdomen - and a heart. Photos have been circulated showing the babies in a hospital bed in the Neonatal Intensive Care Unit of Soetomo Hospital in ... continue reading
By Nita Bhalla, Thomson Reuters Foundation
When Indian street-food seller Kumar Pal first began treatment for multi-drug resistant tuberculosis two years ago, he quickly spiraled into depression and gave up hope of living. NEW DELHI (Thomson Reuters Foundation) - Weighing just 77 pounds, shunned by his ... continue reading
By Catholic Online (NEWS CONSORTIUM)
In its attempts to become a world leader and an economic powerhouse, the nation of India is taking those who take academic shortcuts, by cheating in school, extremely harshly. More than six hundred students were expelled in eastern India. Pictures of friends ... continue reading
By Catholic Online (NEWS CONSORTIUM)
An important link in the take down of al-Qaeda leader, Osama bin Laden has been ruthlessly gunned down in retaliation. Attorney Samiullah Khan Afridi, murdered by suspected Taliban gunmen was the legal representative for Dr. Shakil Afridi. The doctor provided ... continue reading
By Catholic Online (NEWS CONSORTIUM)
Yet another horrible Islamic State video, this one intended to recruit followers in Indonesia, depicts Indonesian children, all younger than 15 years of age, training with weapons. Produced by Alazzam Media, which claims to be the Malay language media division ... continue reading
By Catholic Online (NEWS CONSORTIUM)
Pakistan's Prime Minister must take partial blame for the deadly attacks on Christian churchgoers in Lahore this past weekend. Archbishop Joseph Coutts of Kararchi, leader of the country's Catholics, says that Pakistani Prime Minister Nawaz Sharif failed to act ... continue reading
By Hannah Raissa Marfil (NEWS CONSORTIUM)
According to new research from the journal ZooKeys, the so-called "yeti" hair samples as determined by a prior research, were only from brown bears. In the study's abstract, the researchers stated that they have no reason to conclude the samples came from any other ... continue reading
By Catholic Online (NEWS CONSORTIUM)
Pakistan's worst attack on its Christian religious minority occurred this past weekend when two churches in Lahore were targeted by suicide bombers. Fifteen people were killed in all, and at least 70 other people suffered injuries. According to Pakistan Taliban ... continue reading
By Catholic Online (NEWS CONSORTIUM)
Authorities in eastern India are continuing their search for six men wanted in connection with the gang rape of a 74-year-old nun in West Bengal. Witnesses say the men ransacked the convent school in Ranaghat this past weekend. LOS ANGELES, CA (Catholic Online) ... continue reading
By Catholic Online (NEWS CONSORTIUM)
Various media outlets are breaking the news that a single piece of evidence, possibly linked to the disappearance of Malaysia Airlines Flight 370, was discovered on an Australian beach last July. The find is a tiny clue in a vast mystery. LOS ANGELES, CA (Catholic ... continue reading


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Home Bazaar
Founded in 1764, Newburyport, Massachusetts has a long and rich history. It's a city that truly values its historic architecture such as the colorful, Victorian houses that grace the harbor. This fully functional birdhouse is finished golden yellow with blue and white accents. It has a front porch with two flower boxes, two hanging flower pots and features a removable back wall, ventilation and drainage holes. Constructed of kiln-dried hardwood and topped with pine shingles.


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Obama interrupts Hoeven's turkey pardoning
BISMARCK -- The fate of a 19-week-old turkey was left hanging in the balance Tuesday when Gov. John Hoeven had to leave his annual turkey pardon to take a phone call from President Barack Obama.
Ironically, Hoeven had just talked about building the suspense for the pardon.
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7 years of work??? DAaamn :O
a long time for sure
0 comments by vektorDex on Feb 13th, 2015
Sirius Online returns in the most bombastic way possible - Online play, open PVP, close up dogfights and one hell of a fun experience. But lets star from the beginning. 6 months ago I was burnt out, overworked, exhausted. There came a phase when I thought about abandoning Sirius and everything. Yeah. ABANDONING! And for six months, development halted. There were a few less successful attempts to port it to Unity but, development on Sirius basically ceased.
It was not only until January 2015 when a random occasion forced me to rent a root-server to host another, more successful project, my main comedy site. Out of curiosity I took my LAN client on the server and tried to connect to it from home. Totally surprising: It worked. From this "epiphany" on I got back into my devcave and made a simplified server App for Sirius, ran it and created a new Client - named 1.0.15.
A lot of tests followed and a lot of balancing induced until Sirius reached 1.0.17 - the first release after 6 months. Due to great feedback I squatted bugs, balanced, added new features until I was, where I am now. 1.0.19a. Online Combat, Tradelanes, Mining, Economy. And don't fear: 1.0.20 is coming fast!
1.1 is still a far way off, but let's see. I made a to-do list with all the feedback I receive from players, friends and testers. This list increases constantly and includes:
- Automatic Reconnect to server or message stating that you lost server connection
- Simple cargo Space, inventory and Crafting of Modules
- More Routes and custom placement of routes by crafted Materials
- Station should make sense
- Muzzle Flash
- Synching asteroid movements
- More Sounds
- Refined Meshes for Guns
- TONS of performance fixes
- Saving Progress
- Authentication with the Server
- Custom Ship Skins
- Autopatcher
and a lot of even more cool things are on my list. I plan to have at least one release per week. you are free to punch me if I don't!
As you know, Sirius is currently Free due to the Fact that I need Feedback. Probably delivered in kilotons. However, if you decide to help me out, support the game, you have two options:
7 years of work??? DAaamn :O
a long time for sure
Eight years on release (Fall 2013) :)
Glad to know this project is finally taking form. Looking forward to the next release. I really like the idea of walking in stations.
Okay so i downloaded this game yesterday- Flatspace(Demo). It's really good. Try it out. There are some aspects of the game i really like. You can take passengers and safely transport them to a sector. You can become a mercenary or even a law enforcer. Pretty awesome! If you could incorporate these in Sirius it would be awesome. Just a suggestion :)
Hey. Maybe this is a stupid question about the demo but.. Is there a way to make it fullscreen?
Yes, even though its a debug thing.
Open settings.ini, and the Lines given there state as following:
1= Fullscreen, 2=Windowed
Language (not functional yet)
UI-Scaling (experimental)
Thanks. I'll try this out.
Looks Incredible!! I love Freelancer. Very pleased to see someone using the concept. Also will this be purely Multiplayer??? Also Planets will be explorable?
Yes, this happens in a persistant online Universe.
New Systems are generated from a hidden value generated on your CPU, so the exploration factor will never run out.
Inner-Atmospheric flight is in the forge as well as Walking in Stations, both currently very unstable and they won't make it into 0.5.
Oh nice to hear that!! Was very disappointed with Microsoft after they cancelled Project Lonestar but this looks real promising :) Tracking!
Yes, thats been a shame.
I've been a huge Freelancer-Fan and am still playing it sometimes.
Thank you for tracking!
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(April 4, 2012: Seattle, WA 98104) The Eastside Catholic Crusaders the host O'Dea Fighting Irish baseball team by a count of 6-1 in Wednesday's league bout.
The Crusaders now possess a 8-1 record. They take the field next when they travel to Bellevue for a non-league bout on Tuesday, April 10.
Next, the Fighting Irish face the Skyline Spartans (Sammamish, WA) in a non-league bout, on Saturday, April 7. O'Dea will attempt to move on its 5-4 season record. The Spartans enter the bout with a 5-6 record after their 6-5 league win over Woodinville (Woodinville, WA).
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1 recommendation
Doesn't mean too much. There has been "no evidence" that lots of things didn't cause health problems that were later found to not be so healthy. Not that my router worries me.
As the study states, there are groups that use anti-WiFi activism to rip people off. But they can do this because their targets are mentally unstable. Either hypochondriacs, paranoids who think gov't and industry is out to get them
I'm not saying this for myself - I don't believe the harm of RF radiation precisely because the good science doesn't back it up. I'm just stating that this is thinking behind those who object to Wi-Fi, smart meters, power lines, etc. You're not going to convince them with science because science rarely strays beyond the evidence and does not make categorical, unhedged statements beyond what the evidence will say. That leaves wiggle room for those who fear that we just haven't found the evidence yet.
As for junk science, I have Autism and I can tell you that vaccines and Autism are junk science as well because my mother said I was having symptoms before I received any vaccines. Vaccines and Autism were later disproven and the report was retracted. I think the doctor behind the junk science later lost his privileges to practice medicine. And now we have unvaccinated children in our schools that are infecting other kids. Some science is legitimate but a lot of science is biased and later proven junk science, especially when there is a cash grab involved.


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Project Ideas for All Occasions From Paper to Fabric Volume 1 eCraft Creations
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Mod Quilt Sampler Part 3 of 3
By: Nuts about Needlepoint
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Click here to view your Craft Projects.
In the final installment from Nuts About Needlepoint, the Mod Quilt Sampler Part 3 of 3 will give last few patterns needed to complete this wonderful needlepoint project. With the remaining two blocks you'll have everything you need to stitch the a complete Mod Sampler.
With these two blocks you have all the patterns you need to stitch the blocks for the mod Sampler.
I didn't have time today to stitch more than three blocks, but, as you can see, this completes two rows of the quilt. I'll update you with a picture of the completed blocks towards the end of the month.
This block, which is split vertically down the middle is stitched in Giant Diagonal Cashmere. Diagonal Cashmere is a fun stitch where two Cashmere Stitches share the shortest stitch. In making these blocks each side should be stitched exactly the same.
The final block can be found as the last block in the second row. It is another version of Log Cabin (second block in top row). This one has top and bottom "logs' longer than the side "logs." It is called Courthouse Steps.
In order to complete the sampler, you will need to know the block/color combination for each row. Here they are, moving from left to right:
First Row:
- Quadrants/red
- Log Cabin/orange
- Checks/orchid
- Horizontal Stripes/yellow
- Diagonal Cashmere/orchid
Second Row:
- Log Cabin/pink
- Checks/yellow
- Horizontal Stripes/apricot
- Diagonal Cashmere/red
- Courthouse Steps/orange
Third Row:
- Checks/apricot
- Horizontal Stripes/red
- Diagonal Cashmere/pink
- Courthouse Steps/apricot
- Quadrants/yellow
Fourth Row:
- Horizontal Stripes/orchid
- Diagonal Cashmere/orange
- Courthouse Steps/yellow
- Quadrants/pink
- Log Cabin/orchid
Fifth Row:
- Diagonal Cashmere/apricot
- Courthouse Steps/orchid
- Quadrants/orange
- Log Cabin/yellow
- Checks/orange
Sixth Row:
- Courthouse Steps/red
- Quadrants/orchid
- Log Cabin/apricot
- Checks/red
- Horizontal Stripes/pink
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Start the year with fresh 'ingredients'
The new year. It's the time for a fresh start and I'm ready to try to make 2012 a great one!
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Where to find Silica sands(very fine!!)
I don't know if this will help you find the very fine silicas,but try US Silica(google it);they also show that they have the metakaolins as well.I think you'll have to contact your regional rep(they're listed)or if you're lucky and live in the SE of the US,or Texas,you could probably go to the plants where this product is produced.
Here in Canada,Sil-Co-Sil is the product I get from the Mad Potter in Richmond,BC.Prices will vary from area to area.My bags are from US Silica ;some places may repackage.Some pottery stores(one in Langley,BC comes to mind)does that and puts it into smaller batches.
If you use it,BE CAREFUL.This stuff is bad news for your lungs.I declare here that those who use this and no respirators are nuts!And that language is toned down so as to not offend.Don't play with it unless you're properly prepared.I'll now yield the soapbox to someone else who wants to say that they don't use protection.BTW,this stuff stays in the air for a bit even after CAREFUL handling.
Cheers from here
Thanks Rick.
Any of you all thinking about not using a respirator, read the health risks associated with this stuff.
Here is a link that might be useful: Material Safety Data Sheet - Health Risks for Silica products
OH! It's that diosomething clay! I was thinking it would be really nice for a fine detail tufa item. My friend in ohio has a pool and she was putting it in, and I was thinking, " hmm... what a waste, but what the heck, I am going swimming."
Jo, do you mean DE, Diatomaceous Earth? It is used for pool filters but there are several types available. The pool stuff may have chemicals added and make it unsuitable for our uses.This page has a good description of it.
Here is a link that might be useful: Diatomaceous Earth
Yes, that's it! Thanks for the link. I had a feeling it was diatoms. Isn't it weird that there were so many on the earth at one time that we can mine them? If it kills insects, we could be onto something using it in planters! Will they have it on sale at the pool places this time of year? I hope so. My friend puts this stuff in her koi pond filter and her fish are very happy. I don't think it's toxic. It contains crystalline silica just like portland. Someone with a pool needs to experiment.
No, gypsum is no good as an aggregate. Aggregates must be hard, non-soluble, and non-reactive. Gypsum is soft, soluble, and reactive. You can actually replace the cement with gypsum although you have to use the right gypsum and very careful technique to get a strong result.
There are different types of pool filter materials. DE is basically the skeletal remains of ancient Diatoms but there are also some very fine silicas that are used in different water purification systems that make an excellent super-fine aggregate. Most new pools have gone to the DE, but a few supply houses still carry the sand as well. You can also check with local water treatment system suppliers for the sand.
Our local DIY big box carries silica sand in the plaster/drywall section. If you live in an area where plaster is still used in home building/decorating you might find it there. I was walking down the aisle heading to the cement/concrete area and noticed the "silica sand" sign (try saying that three times fast, silica sand sign, silica sand sign ,silica sand sign..it's even fun to type!)
and remembered reading the posting here on GardenWeb Forums!
Is the "very fine!!" silica sand you are talking about actually "silica flour"? Silica sand is just a very pure inert sand, it can be anything from very fine to very coarse. The finest grades tend to be manufactured by crushing coarser grades. Crushing a particular form of non-crystalline silica gives an extremely fine soft (meaning smooth) powder called silica flour. You can also get (much cheaper) silica dust which is extremely fine crystalline silica which isn't as soft.


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Brides in search of a simple silk gown with a slight edge will love this boutique, where silhouettes are embellished with bias cuts and Lambrou's signature wrapping and weaving. Brides can choose from the collection (from $2,000) or go custom (from $3,000). By appointment only.


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Saw the Pirates of Penzance at Toby's last night. I haven't been there in decades, but was being treated for my birthday. I did read the reviews before going, and I want to specifically address some of the (relatively few) negative comments. Picking up the tickets at will call was a snap and the person was very courteous. We got excellent seats in the top tier of a section and an unobstructed view of the performing area. Perhaps due to the fact that an entire tour bus of people got lost and were very late arriving, there was not a very long wait for the buffett. As soon as we arrived we were invited to go to the salad bar. Everything on that bar was fresh...the lettuce, the cucumbers, etc. and well stocked. As for the buffet, all the food was hot and tasty. The variety was great...something for everyone. The roasted chicken was good, the ribs were tender and good, there was plenty of fish available. They do slice the carved meats thin, but I asked for and got a slice of the beef, turkey and ham, and there was plenty available for seconds. The vegetables were fresh and steamed perfectly, and the roasted potatoes were delicious too. The rolls on the table were fresh, warm and plentiful. Because it was my birthday, I was served a free "special" dessert (with candle) that was very good. The price of alcoholic drinks was reasonable and less than at many other nearby establishments. Let's face it, Toby's isn't the Kennedy Center and the food is not Citronelle quality, but then you aren't paying the ridiciously high prices at those venues. The show was an operatic comedy/romance, and the cast was very good and very professional in their performances (the actress playing the lead, "Mabel" had a superb voice (we're talking Met qualty) and the all of the cast had great voices. Given the limited space and "in the round" configuration, the staging was very good too. There were some energetic, fast-paced songs, so it was sometimes difficult to decipher the words. Although opera is NOT my first love, I enjoyed the performance very much. The cast as servers did a great job...they were cheerful, curteous and appreciative of the audience. Bottom line: Where in the DC metro area can you get a good quality meal and a professional performance at a price that is slightly over $100 a couple??? That is probably not possible! If you haven't been to Toby's you should give it try. You might want to do so when the performance is one that has music that you know you will enjoy. But, please, be sure your expectations are reasonable....and remember, you get what you pay for...and in the case of Toby's....you get MORE!!!
If you own or manage Toby's Dinner Theater, register now for free tools to enhance your listing, attract new reviews, and respond to reviewers.


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Vanishing Point (CBC)
From Wikipedia, the free encyclopedia
Vanishing Point is the title of a science fiction anthology series that ran on CBC Radio (see Canadian Broadcasting Corporation) from 1984 until 1986, and then under a varying array of sub-titles until 1991.
Vanishing Point was created and produced by Bill Lane of Toronto, Canada.
External links[edit]
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Our special coverage of the closest race for years
Explore the personal stories of Syrian refugees in Lebanon
03 Dec 2007 17:01 GMT
Source: Agencies
Central banks trying to deter bets on appreciation as currencies scale record highs.
Arab states propose providing nuclear energy to Iran via a nuclear consortium.
In religiously conservative Brazil, women risk jail time and their lives to undergo the procedure that is illegal but commonplace.
Health, Human Rights, Latin America
Trash is a fast-growing import in the Scandinavian country, which turns it into heat for people's homes.
Environment, Europe, Sweden
Critics say the UK's home secretary is using Muslims as a 'political punchbag' in a bid to shore up right-wing support.
Human Rights, Politics, Racism
2014 was the worst year in more than a decade for polio infections, with at least 306 new cases reported.
Polio, Asia, Pakistan
It is a source of sustenance but also of tension and new political realities are now increasing uncertainty.
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A media post-mortem into the UK's surveillance report. Plus, why five years on in Argentina the media looks so familiar.
Media, Edward Snowden, Security
The untold story of how the Lebanese community overcame the odds and found their place in multicultural Australia.
Asia Pacific, Australia, Lebanon
How to keep a telenovela profitable and demanding sponsors happy while inspiring the production team to stay passionate.
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