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MicroRNAs (miRNAs) are small regulatory RNAs that are implicated in cancer pathogenesis and have recently shown promise as blood-based biomarkers for cancer detection. Epithelial ovarian cancer is a deadly disease for which improved outcomes could be achieved by successful early detection and enhanced understanding of molecular pathogenesis that leads to improved therapies. A critical step toward these goals is to establish a comprehensive view of miRNAs expressed in epithelial ovarian cancer tissues as well as in normal ovarian surface epithelial cells.
We used massively parallel pyrosequencing (i.e., "454 sequencing") to discover and characterize novel and known miRNAs expressed in primary cultures of normal human ovarian surface epithelium (HOSE) and in tissue from three of the most common histotypes of ovarian cancer. Deep sequencing of small RNA cDNA libraries derived from normal HOSE and ovarian cancer samples yielded a total of 738,710 high-quality sequence reads, generating comprehensive digital profiles of miRNA expression. Expression profiles for 498 previously annotated miRNAs were delineated and we discovered six novel miRNAs and 39 candidate miRNAs. A set of 124 miRNAs was differentially expressed in normal versus cancer samples and 38 miRNAs were differentially expressed across histologic subtypes of ovarian cancer. Taqman qRT-PCR performed on a subset of miRNAs confirmed results of the sequencing-based study.
Citation: Wyman SK, Parkin RK, Mitchell PS, Fritz BR, O'Briant K, Godwin AK, et al. (2009) Repertoire of microRNAs in Epithelial Ovarian Cancer as Determined by Next Generation Sequencing of Small RNA cDNA Libraries. PLoS ONE 4(4): e5311. https://doi.org/10.1371/journal.pone.0005311
Editor: Sudhansu Kumar Dey, Cincinnati Children's Research Foundation, United States of America
Received: November 23, 2008; Accepted: February 7, 2009; Published: April 23, 2009
Copyright: © 2009 Wyman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The cost of sequencing was partially defrayed by Roche. The study was supported in part by the Ovarian Cancer SPORE at Fox Chase Cancer Center (P50 CA083638), the Pacific Ovarian Cancer Research Consortium Ovarian SPORE (P50 CA83636 to N.U. and M.T.), Chromosome Metabolism Training Grant 5 T32 CA09657-16 (to S.K.W.), a fellowship from the Merck Research Laboratories (S.K.W.) and by Fred Hutchinson Cancer Research Center and Canary Foundation funding (New Development funds to M.T.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: M. Tewari is a paid member of the Scientific Advisory Board of Combimatrix, Inc.
Epithelial ovarian cancer is the leading cause of gynecologic cancer-related deaths in the United States [1], with late-stage diagnoses having a <30% five-year survival rate [2]. Survival rates could be improved by a better understanding of molecular pathogenesis, which may lead to development of superior targeted therapies, as well as by earlier detection of disease at a surgically curable stage. When detected at a stage in which disease is confined to the ovary, for example, the five-year survival rate increases to >80%. Clinically effective biomarkers for early detection of ovarian cancer could substantially improve survival rates and ovarian cancer biomarker discovery is an important area of ongoing research [3].
MicroRNAs (miRNAs) are a class of small (∼22 nt) non-coding RNA molecules that act post-transcriptionally to regulate gene expression [4]. MicroRNAs originate from hairpin RNA precursors that are processed to generate both the functional mature miRNA and a miRNA "star form" of similar length derived from the opposite strand of the hairpin. MicroRNA-mediated modulation of biological systems has been found to be perturbed in multiple diseases [5], including cancer [6]–[8]. Expression patterns of miRNAs correlate with tissue of origin [8], [9], prognosis [10], [11] and with clinical cancer behaviors [12], making miRNAs valuable tissue-based biomarkers. Furthermore, we and others have recently shown that tumor-derived miRNAs enter the bloodstream at measurable levels, indicating that miRNAs released by tumor tissue represent a powerful new class of blood-based, minimally invasive biomarkers for cancer detection [13]–[16]. As such, there is a strong impetus for a comprehensive analysis of the miRNA repertoire expressed in epithelial ovarian cancer.
Several recent reports have begun to characterize miRNA expression in ovarian cancer using microarrays spotted with probes for a varying number of known miRNAs [17]–[20]. Although these are pioneering studies, they also have limitations. The foremost of these is that all arrays reported to date have incomplete coverage of known miRNAs. Of the 959 miRNAs and star forms present in miRBase (release 12.0) [21], [22], most microarray platforms applied to date assay only a few hundred miRNAs, with the most comprehensive of the arrays assaying 470 miRNAs and star forms. In addition, microarray approaches only measure previously identified miRNAs and are not equipped to discover and profile novel miRNAs. This is an important distinction to make given that several reports suggest that a significant number of miRNAs, particularly those that may be cell-type specific in expression, remain to be discovered [23], [24]. Finally, because of the small size of miRNAs and the challenges of achieving uniform hybridization conditions across an entire miRNA microarray, there is potential for cross-hybridization of miRNAs that are highly related in sequence, making it sometimes impossible to definitively distinguish between miRNAs differing by one or two nucleotides.
To overcome the limitations associated with microarray studies, we used "next generation" sequencing technology (specifically massively parallel pyrosequencing using the 454 Life Sciences platform) of small RNA cDNA libraries to more comprehensively characterize miRNA expression in epithelial ovarian cancer, as well as in primary cultures of normal ovarian surface epithelial cells. This approach, which is based on 5′ and 3′ universal linker ligation/RT-PCR and counting of sequence reads obtained for distinct miRNAs, can in theory capture all miRNAs present in the RNA samples (including ones of novel sequence) and permits precise identification of miRNAs, even when differing by only a single nucleotide. We generated a total of 738,710 sequence reads from four small RNA cDNA libraries representing normal primary human ovarian surface epithelium (HOSE) cultures and three common epithelial ovarian cancer subtypes (serous, clear cell and endometrioid). Here we describe the results of this effort, including the identification and profiling of both previously annotated and novel miRNAs expressed in ovarian cancer.
Additional details regarding cell culture, RNA isolation, small RNA cDNA library construction, massively parallel sequencing, computational pipeline description and miRNA TaqMan quantitative reverse-transcription polymerase chain reaction (qRT-PCR) are provided in detail in Supplementary Methods S1 and in [23].
Cell culture and clinical materials
Normal primary human ovarian surface epithelial (HOSE) cells were obtained from the normal ovaries of postmenopausal women using a modification of the technique described previously in [25]. In all cases, specimens were taken from normal-appearing ovarian surface epithelium, which was confirmed following histopathological review. HOSE specimens were obtained under a protocol that was approved by the Research Review Committee and Internal Review Board at the Fox Chase Cancer Center from patients undergoing clinically indicated surgery (either prophylactic risk reduction salpingo-oophorectomy or total abdominal hysterectomy with bilateral salpingo-oopherectomy) and who provided written informed consent for tissue not required for diagnosis to be used for research. Primary HOSE cells were cultured using media described in [26] at 37°C in 5% CO2.
Ovarian cancer snap-frozen specimens corresponding to Stage III/IV epithelial ovarian cancer (19 serous, four clear cell and 10 endometrioid) contained >70% malignant epithelial cellularity as assessed by pathologist review of an adjacent stained tissue section. Ovarian cancer specimens were obtained from the Pacific Ovarian Cancer Research Consortium Repository. Specimens were collected under a protocol approved by the Fred Hutchinson Cancer Research Center Institutional Review Board and were obtained from patients who had provided written informed consent for tissue not required for clinical management purposes to be used for research.
RNA extraction and miRNA library preparation
Normal primary HOSE cultures (passage 4–7) were lysed in 600 µl mirVana Lysis/Binding buffer (Ambion, Inc., Austin, TX). Tumor specimens (∼100 mg each) were homogenized in a Qiagen Tissuelyser with 600 µl mirVana Lysis/Binding buffer. Total RNA was extracted from both sample types using the mirVana™ miRNA isolation kit (Ambion) per the manufacturer's protocol. RNA from snap-frozen tumors (19 serous, four clear cell and 10 endometrioid tumors, respectively) was pooled according to histologic subtype. MicroRNA cloning was performed as previously described [27], (http://web.wi.mit.edu/bartel/pub/protocols/miRNACloningUpdate0705.pdf) with modifications at the amplification steps to prepare the samples for massively parallel sequencing by 454 Life Sciences.
Custom bioinformatic data analysis pipeline
Processing and annotation of sequences based on identity to known transcribed RNAs or as novel miRNAs was performed using a custom bioinformatics pipeline described in detail in Supplementary Methods S1.
Measurement of miRNA levels in RNA from clinical samples using TaqMan® qRT-PCR assays
Total RNA was reverse transcribed and prepared for qRT-PCR using the TaqMan® miRNA Reverse Transcription Kit and miRNA-specific stem-loop primers (Applied BioSystems, Inc.) as previously described [23]. Data was analyzed with SDS Relative Quantification Software version 2.2.2 (Applied BioSystems, Inc.), with the automatic Ct setting for assigning baseline and threshold for Ct determination.
Overview of small RNA cDNA libraries and 454 sequencing
Small RNA cDNA libraries were constructed from RNA sample pools isolated from primary cultures of histologically normal primary HOSE (n = 4) and epithelial ovarian cancer specimens of serous (OSC, n = 19), clear cell (OCC, n = 4) and endometrioid (OEC, n = 10) histologies. Tumor specimens were selected to contain at least 70% malignant epithelial cells. Total RNA was size-fractionated via polyacrylamide gel electrophoresis and small RNAs corresponding in molecular weight to the mature miRNA population (18–24 nt fraction) were gel extracted and processed for reverse transcription and PCR amplification to create cDNA libraries (Figure 1A). Massively parallel pyrosequencing using the 454 Life Sciences' platform generated 80,501 normal HOSE reads, 273,739 OSC reads, 292,942 OCC reads, and 91,528 OEC reads (Figure 1B). These corresponded to 10,544 HOSE, 26,849 OSC, 37,792 OCC and 13,134 OEC non-redundant sequences. The greater number of reads corresponding to serous and clear cell samples was due to greater depth of sequencing (i.e., a larger portion of the sequencing plate used) rather than to any particular variations during library preparation.
A. Small RNAs were isolated from normal primary HOSE cultures and from serous (OSC), clear cell (OCC) and endometrioid (OEC) ovarian cancer tissues. Following 5′ and 3′ linker ligation, RT-PCR was performed to generate four independent cDNA libraries of small RNAs that were then used as templates for massively parallel pyrosequencing (454 sequencing). B. Description of the steps in the data analysis pipeline. The initial step of the data analysis was removal of sequences corresponding to 18 nt and 24 nt RNA markers that had been spiked into the total RNA prior to gel electrophoresis. Percentage of total reads from HOSE, OSC, OCC and OEC datasets classified into the designated categories and filtered out at each step are listed in the table on the right. At the bottom of the table, the number of "unique sequences" represents the non-redundant sequences derived after collapsing multiple reads of identical sequence. Some canonical sequences map to more than one locus in the genome. At completion of the analysis, 199 sequences totaling 215 reads and mapping to 568 loci met our criteria for canonical hairpin-derived sequences from the combined datasets.
Sequence data was processed using our custom computational pipeline (Figures 1B, S1 and Table S1). The initial steps of the pipeline identified sequence matches to databases of previously annotated RNAs (e.g., known miRNAs, other noncoding RNAs, messenger RNAs) and to highly repetitive sequence elements. Remaining sequences were processed to identify novel miRNAs (Figure S1; details of the computational pipeline are provided in Supplementary Methods S1). In the next sections, we discuss in detail the sequences matching to known miRNAs and the identification of novel miRNAs.
MicroRNA profiling: previously annotated miRNAs.
Matches to known miRNAs in miRBase (release 12.0) [21], [22] represented 68–73% of the reads, depending on the dataset (Figure 1B and Table S1) and corresponded to a total of 498 known miRNAs (including star forms). The cloning frequency of individual miRNAs, expressed as a fraction of total reads obtained from a given sample, can be used to compare relative expression of miRNAs between samples [28]–[30]. The global summary of our 454 sequencing datasets is presented in Figure 2, which depicts the relative expression of miRNAs between normal primary HOSE and cancer tissue datasets (A), as well as between the three epithelial ovarian subtypes (B). The most differentially expressed miRNAs (i.e., ≥4 fold-change) are shown as red dots. It is notable, though not unexpected, that the ovarian cancer histologic subtype miRNA profiles were more different from normal HOSE (A) than they were from each other (B). Tabulated 454 sequencing data for miRNA abundance in all datasets, as well as miRNA names and abundance ratios for all pairwise comparisons between sample types, are provided in their entirety in Table S2.
Datasets are compared pairwise, plotting the log of the fraction of total reads for a given miRNA in a given dataset against its corresponding value in the second dataset. For each plot, only miRNAs that were sequenced in both datasets are plotted; miRNAs that were sequenced only in one of the two datasets are not shown. The top row (A), compares ovarian cancer datasets (OSC, OCC and OEC) to normal primary HOSE cultures; the bottom row (B), compares ovarian tumor histologic subtypes to each other. Red dots signify miRNAs that had a fold change ≥4.
The overlap of differentially expressed miRNAs between subtypes is visualized in the Venn diagrams in Figure 3, which also incorporate miRNAs excluded from Figure 2 because of zero reads in either normal HOSE or ovarian cancer samples. From the intersection of the three groups in the Venn diagrams, it is evident that in many cases miRNAs differentially expressed in ovarian cancer relative to normal HOSE show the same pattern of differential expression regardless of histologic subtype. Table S3 provides a full list of names of individual miRNAs and expression ratio and P-values for the comparisons summarized in Figure 3.
Venn diagrams depict numbers of miRNAs differentially expressed in ovarian cancer histologic subtypes relative to normal primary human ovarian surface epithelium (HOSE) cultures. Criteria for a miRNA as differentially expressed was defined by a fold-change ≥4 with a Fisher's exact test P-value<5.0×10−6 for miRNAs that had detectable expression in both HOSE and ovarian cancer samples, or by a Fisher's exact test P-value<5.0×10−6 alone in cases where a fold-change value was indefinable due to zero reads for a given miRNA in either the HOSE or ovarian cancer comparison sample. A total of 74 over-expressed miRNAs (Panel A) and 49 under-expressed miRNAs (Panel B) in ovarian cancer with respect to normal HOSE were identified. MicroRNAs that were found to be consistently over-expressed or consistently under-expressed in all three ovarian cancer histologic subtypes are listed by name.
We directly compared miRNA expression between ovarian cancer histologic subtypes by deriving expression ratios for each miRNA based on fractional abundance values. Significance was assessed using Fisher's exact test. The top ten miRNAs differentially expressed in each of the pairwise comparisons of ovarian cancer histologic subtypes are listed in Table S4 (results for all miRNAs are provided in their entirety in Table S2). Among the most histologic subtype-specific miRNAs are miR-449a (serous-specific), miR-499-5p/miR-375/miR196a/miR-196b/miR-182 (endometrioid-specific) and miR-486-5p/miR-144/miR-30a/miR-199a-5p (clear cell-specific).
Validation of 454 sequencing miRNA expression results by qRT-PCR.
We used commercially available miRNA TaqMan qRT-PCR assays as an independent confirmation of miRNA differential expression identified by 454 sequencing. MicroRNA TaqMan qRT-PCR assays were performed for a sample of 38 known miRNAs that were selected based on over- or under-expression with respect to primary normal HOSE, or based on differential expression between different subtypes of ovarian cancer (calculated based on 454 sequencing data). Taqman assays were carried out on aliquots of the same RNA pools used for sequencing. Thirty-six of the 38 differentially expressed miRNAs examined by TaqMan qRT-PCR demonstrated consistent results with those obtained by 454 sequencing (Figures 4 and 5, A, B, C). Of the eight miRNAs identified by 454 sequencing to be over-expressed in ovarian cancer relative to the normal HOSE group (Figure 4A), all eight demonstrated over-expression in serous and clear cell cancer subtypes when measured using TaqMan qRT-PCR, while six of the eight showed consistent results in the endometrioid cancer subtype (miRs-126*,-142-3p, 195, 200a, 200b, 200c, 338-3p, 378*). Nine miRNAs were under-expressed in ovarian cancer relative to normal HOSE (Figure 4B), with all subtypes showing consistent results when measured by TaqMan qRT-PCR. There were six miRNAs that were undetectable (zero reads) in normal HOSE cultures (Figure 4C), but had detectable expression in OEC, OSC and/or OCC in the 454 sequencing data, and they likewise showed increased expression in ovarian histologic subtype samples versus normal primary HOSE when examined by TaqMan qRT-PCR (Figure 4C).
The graphs display miRNA Taqman qRT-PCR results for a sampling of miRNAs identified by 454 sequencing to be over-expressed (Panel A) or under-expressed (Panel B) in ovarian cancer relative to normal HOSE. Relative expression values derived from 454 sequencing are displayed on the left segment of each graph for comparison. Relative expression values of miRNAs in endometrioid (OEC, black bars), serous (OSC, white bars) and clear cell (OCC, grey bars) cancers are depicted. MicroRNAs that were not detected in normal HOSE by 454 sequencing but were detected in ovarian cancer are presented in Panel C, where miRNAs are ordered by descending expression (which corresponds to ascending cycle threshold, Ct) in normal HOSE. 454 sequencing data is given in terms of percentage of total reads within each sample. UD, undetectable by miRNA TaqMan qRT-PCR.
The graphs display Taqman qRT-PCR results for miRNAs differentially expressed between ovarian cancer subtypes, with 454 sequencing-derived data presented for comparison in the left segment of each graph. Relative expression values of miRNAs specifically over-expressed in endometrioid (Panel A, OEC, black bars), serous (Panel B, OSC, white bars) and clear cell (Panel C, OCC, grey bars) cancers are depicted. UD, undetectable by miRNA TaqMan qRT-PCR.
Fifteen miRNAs identified by 454 sequencing to be differentially expressed across ovarian cancer subtypes (Figure 5A, B, C) showed similar expression patterns when quantitated by qRT-PCR. Eight miRNAs were over-expressed specifically in endometrioid ovarian cancer (Figure 5A), four in serous cancer (Figure 5B), and three in clear cell cancer (Figure 5C) relative to the other subtypes.
Analysis of sequencing data to identify novel hairpin-derived small RNAs.
Having validated 454 sequencing results corresponding to differential expression of previously annotated miRNAs, we next turned to the identification of novel miRNA sequences. After filtering out matches to previously annotated features including messenger RNAs, tRNAs and other known noncoding RNAs, the remaining sequences were aligned to the reference human genome sequence (NCBI Build 36.1) [31]. Sequences were required to match the human genome sequence perfectly to be carried further for additional computational analysis, with the only exception to this being sequences that demonstrated addition of 1–3 non-templated nucleotides at the 3′ terminus. In these cases, we trimmed non-templated bases from the original sequence and it was then carried further for analysis [32], [33].
The data processing steps described thus far yielded 841 normal HOSE, 2,840 OSC, 11,224 OCC, and 1,764 OEC unique sequences corresponding to novel small RNAs. These unique sequences corresponded to 1,766 normal HOSE, 5,795 OSC, 19,464 OCC and 3,301 OEC genomic loci that could potentially generate these small RNAs (Figure 1B, Table S1). The data analysis pipeline next screened these genomic loci (in addition to 100 nucleotides of up- and down-stream flanking sequence) for the presence of a predicted hairpin secondary structure by computing free energy, shape probability (as determined by the RNAshapes program [34]) and the Randfold-computed [35] P-value of predicted secondary structures. A detailed description of hairpin folding criteria is provided in Supplementary Methods S1 and in [23]. This resulted in 224 normal HOSE, 511 OSC, 500 OCC, and 247 OEC novel small RNAs found to be potentially derived from a precursor hairpin structure.
Sequences passing the above filtering criteria from the four datasets were then combined and sorted with respect to chromosomal coordinates into groups sharing 5′ ends. From each of these groups we chose a "canonical" sequence representing that genomic locus. The canonical sequences were chosen based on a common 5′ terminus, abundance, and sequence length as described previously [23] (additional details are provided in Supplementary Methods S1). This process further refined the data into a combined list of 199 unique sequences (potentially derived from 568 genomic loci) that we designated as "novel hairpin-derived small RNAs" from which we identified novel and candidate miRNAs.
Identifying novel and candidate miRNAs.
In order to determine which sequences to designate as novel miRNAs, we screened the set of novel hairpin-derived small RNAs using criteria similar to those used in other recent miRNA discovery studies [23], [33], [36]: i. pairing characteristics of the hairpin, ii. the presence of multiple reads sharing the same 5′ terminus, iii. absence of annotation indicating non-miRNA biogenesis, iv. shared seed region with a known animal miRNA and v. presence of corresponding miRNA star form read(s). We considered including phylogenetic sequence conservation as a criterion; however, this did not add significantly to our analysis as none of the novel hairpin-derived miRNAs were conserved beyond primates. This is not unexpected, given that most evolutionarily conserved microRNAs are likely to have already been discovered using computational approaches followed up by directed validation experiments.
Using the criteria above, we identified six novel miRNAs. All of the six met criteria i–iii and three of the six met criterion iv and three met criterion v (Figure 6; more detail is provided in Table S5). In addition, mapping the novel miRNA sequences to the reference human genome sequence revealed that three of the six novel miRNAs are present in introns of other genes (and encoded on the same strand as the respective host genes) (Figure 6), much like many previously annotated miRNAs [37], [38]. Two of our novel miRNAs are located in annotated repeats, but have strong enough supporting evidence that they were designated as novel. The context of the predicted precursor structure and the sequence of miRNA star forms corresponding to novel miRNAs is given in Figure 6.
Putative secondary structures for the six novel miRNAs discovered in this study are shown. Novel miRNA sequences are shown in red and star forms of novel miRNAs are shown in blue (if identified in our sequencing data). Sequences are novel with respect to miRBase release 12.0 [21], [22]. Identifiers in parentheses refer to the miRNA star forms where these were identified in the 454 sequencing data. Corresponding details of the novel miRNAs are given in the table below the hairpin structures.
The remaining sequences comprised 181 hairpin-derived small RNAs (corresponding to 550 genomic loci). We sought to select from this list the most promising sequences to designate as "candidate miRNAs" that might be confirmed in the future as bona fide miRNAs as additional evidence accumulates. Candidate miRNAs were required to meet criteria i–iii and have some additional form of supporting evidence (i.e., shared seed region with a known miRNA). This allowed us to refine a final list of 39 candidate miRNAs (potentially originating from 50 genomic loci) (Table S5).
The work reported here was motivated by the hypothesis that the entire repertoire of miRNAs expressed in ovarian cancer, including potentially tissue- and cancer-specific miRNAs, had not yet been elucidated. The massively parallel sequencing approach allowed us to be comprehensive (i.e., identifying not only known but also novel miRNAs), and the "digital" nature of the data permitted a semi-quantitative estimation of the relative expression level for many miRNAs. Using this approach, we discovered six novel and 39 candidate miRNAs, and delineated the expression pattern of 498 previously annotated miRNAs in normal primary HOSE cultures and epithelial ovarian cancer tissues. It is notable that of the four published miRNA microarray studies of ovarian cancer [17]–[20], even the most comprehensive of the array platforms was limited to 470 human miRNAs and star forms [17], while the current release of miRBase (release 12.0) [21], [22] contains 969 human miRNAs and star forms. Furthermore, 223 of the known miRNAs we identified and characterized in our sequencing datasets were not represented on even the most comprehensive microarray. Therefore, the results presented here substantially extend the breadth of knowledge of miRNAs expressed in ovarian cancer.
The identification of novel miRNAs in our data is particularly notable. Given that these novel miRNAs have not been detected in multiple large-scale miRNA sequencing studies of other tissues, we speculate that they are expressed in an ovarian cancer-specific manner and may be of special interest as cancer biomarkers. Future studies will be needed to test this hypothesis, as well as to investigate the biological roles of these novel miRNAs in ovarian cancer pathogenesis.
Taken together, the known and novel miRNAs identified in this study provide a pool of candidate miRNA markers for future analysis as blood-based markers for the detection of ovarian cancer. Differential expression between normal and malignant states may provide one method for prioritization of candidates moving forward. We also envision that the miRNAs that we found to be differentially expressed between histologic cancer subtypes could serve a dual purpose as blood-based markers for both cancer detection and histologic classification.
One of the unique features of our study is the use of primary cultures of normal human ovarian surface epithelium as the normal comparison group. Although the ovarian surface epithelium (along with nearby distal fallopian tube epithelium) is considered the origin of epithelial ovarian cancer [39] most other miRNA profiling studies have typically used whole ovary as the normal comparison. This is problematic for identifying miRNAs that are differentially expressed in ovarian cancer relative to normal because the single cell-thick surface epithelial layer comprises far less than 1% of the cellular content of the whole ovary. Performing comparisons of ovarian cancer tissue to an appropriate normal control is a challenging problem in ovarian cancer research. Our approach, although it avoids the problems associated with using whole ovary as a normal sample, has the limitation that we do not know whether and which changes in microRNA expression may be attributed to culture conditions used for primary HOSE cell culture. Future studies incorporating parallel analyses of primary cell cultures derived from ovarian cancer tissues may address this question.
Although the use of different types of "normal" ovarian specimens makes comparison of our data with other studies difficult in most cases, in one microarray study, however, the normal control was human ovarian surface epithelial cells that had been immortalized using a retrovirus expressing human papillomavirus-derived viral oncoproteins E6 and E7 [17], [40]. Because the normal primary HOSE cultures we studied are not immortalized, comparison of our results to that report could identify miRNAs that may be induced by immortalization by E6 and E7 proteins. A notable finding from this comparison is that whereas we identified the miR-200 family of miRNAs (miR-200a/b/c, miR-141 and miR-429) to be expressed at low levels in normal HOSE with highly increased expression in ovarian cancer, the study using E6/E7-immortalized HOSE did not find increased expression of miR-200 family miRNAs in ovarian cancer. This family of miRNAs has important functions in repressing the proteins ZEB1 and ZEB2 [41]–[46], which are transcription factors that function to promote epithelial-to-mesenchymal transition [47]–[51]. One appealing interpretation of the differing results is that primary normal HOSE express low levels of the miR-200 family miRNAs, but the E6 and E7 viral oncoproteins induce miR-200 family expression, which may be an important step in ovarian carcinogenesis given that this miRNA family was the most highly over-expressed miRNA family in all ovarian cancer types we examined. Future work will be needed to address this hypothesis.
In summary, our work expands the current view of the repertoire of miRNAs expressed in normal HOSE and common histologic subtypes of ovarian cancer. Our results represent a resource for future investigations of the role of miRNAs in ovarian cancer and of their applications as clinically useful biomarkers.
The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus [52] and are accessible through GEO Series accession number GSE15190 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE15190).
Supporting Information
Table S1.
Overview of computational pipeline data processing results. Shaded lines represent steps at which sequences are pruned out of the dataset before the next step. The left column for each dataset contains the number of nonredundant unique sequences, and the right column is the sum of all the reads for each sequence.
Table S2.
Identity, abundance data and pairwise relative expression data for all known miRNAs in 454 sequencing datasets. Columns B-I display the number of reads and fractional abundance values for miRNAs corresponding to the 4 sample types studied. Columns J-U provide pairwise comparisons of expression (along with Fisher's exact test P-values) of each miRNA between all combinations of the sample types studied.
Table S3.
MicroRNAs differentially expressed between ovarian cancer and normal HOSE.
Table S4.
MicroRNAs differentially expressed between ovarian cancer histologic subtypes. Bold blocks of numbers show top 10 most differentially expressed known miRNAs between ovarian cancer subtypes.
Table S5.
Novel and candidate miRNAs. Alternating yellow and orange miRNAs represent 5′-3′ pairs, with the yellow indicating the dominant sequence. Pink rows are the two remaining novel miRNAs. The non-highlighted sequences represent candidate miRNAs.
Figure S1.
Flow chart of sequence data analysis pipeline. The flow chart shows the steps in the computational analysis of the 454 sequencing data. At each step, sequences may be removed for further analysis, or carried on to the next step in the pipeline. The first steps remove previously annotated features from the pipeline, and then remaining sequences are tested for presence of hairpin secondary structure and other criteria to be designated novel miRNAs. Bolded lower-case letters are referred to in the Supplementary Methods.
We thank Carolyn Slater and Lisa Vanderveer for technical assistance with normal primary HOSE cell culture, April Allen for assistance with miRNA isolation and Angie Lackey, Danielle Blecha and Tim Harkins at Roche Diagnostics for technical advice regarding small RNA library sequencing.
Author Contributions
Conceived and designed the experiments: MT. Performed the experiments: RKP PSM BRF. Analyzed the data: SKW MT. Contributed reagents/materials/analysis tools: KCO AKG NU CD BSK. Wrote the paper: SKW PSM MT.
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- 24. Berezikov E, Thuemmler F, van Laake LW, Kondova I, Bontrop R, et al. (2006) Diversity of microRNAs in human and chimpanzee brain. Nat Genet 38: 1375–1377.E. BerezikovF. ThuemmlerLW van LaakeI. KondovaR. Bontrop2006Diversity of microRNAs in human and chimpanzee brain.Nat Genet3813751377
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- 30. Cummins JM, He Y, Leary RJ, Pagliarini R, Diaz LA Jr, et al. (2006) The colorectal microRNAome. Proc Natl Acad Sci U S A 103: 3687–3692.JM CumminsY. HeRJ LearyR. PagliariniLA Diaz Jr2006The colorectal microRNAome.Proc Natl Acad Sci U S A10336873692
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- 32. Berezikov E, Cuppen E, Plasterk RH (2006) Approaches to microRNA discovery. Nat Genet 38: SupplS2–7.E. BerezikovE. CuppenRH Plasterk2006Approaches to microRNA discovery.Nat Genet38SupplS27
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- 34. Steffen P, Voss B, Rehmsmeier M, Reeder J, Giegerich R (2006) RNAshapes: an integrated RNA analysis package based on abstract shapes. Bioinformatics 22: 500–503.P. SteffenB. VossM. RehmsmeierJ. ReederR. Giegerich2006RNAshapes: an integrated RNA analysis package based on abstract shapes.Bioinformatics22500503
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text: Winchester 000 work fine in the Judge. I bought 100 rounds of Sellier & Bellot when I first got mine and about 30 of them would not load into the cylinder. I looked and the end of the shell casing where it folds in is larger than the rest of the shell. I got to where I could look and tell which would fit and which would not. I imagine you might be able to create a tool to squeeze the end of the shell back to a position where it would fit, but I just decided to give those rounds to my brother-in-law to shoot in his .410 shot gun.

subset: commoncrawl


text: SANTA CLARA, Calif., Feb. 26, 2013 /PRNewswire/ -- Strata Conference – Big Data software and tools provider Revelytix today announced it has successfully completed a Hadoop-based pilot for a top tier U.S. Healthcare company. The pilot sought to establish the scalability and flexibility of Hadoop in supporting the development of new analytic models. Once data sets were loaded into Hadoop, the approach "Start small, Scale quickly" was used. Beginning with samples of the data sets, queries were iteratively developed and tested. Users wrote queries in standard query languages (e.g. SQL) which the Hadoop-integrated query engine compiled to MapReduce. With each cycle the queries became progressively complex and were then deployed across the full data sets. The pilot also sought to examine the data from new perspectives and required substantial data integration to create views which seamlessly span multiple data sets. The healthcare company also wanted to create transparency of data with industry partners. The Revelytix approach was to first create a domain model that described the data sets using a desired semantic set of terms. The data was then mapped to that model and the Hadoop-integrated query engine was used to pull results from all data sets, thereby providing a complete picture – in this case, of consumer behavior. CEO Mike Lang commented, "There are two important things we learned in this Hadoop Healthcare pilot. First, Hadoop is extraordinarily well suited to build the analytic environment for mega-scale healthcare data such as this. Without question, Hadoop will definitely play a big role in big data for healthcare. "Second, our experience again confirmed the need for the Revelytix Loom solution for managing the huge complexity of Hadoop datasets such as this. The power of Hadoop spawns files from many perspectives and analytic approaches – keeping track of the data history of each approach is a significant and ongoing challenge. Having the pre-release of the Loom data management software and workbench allowed us to keep this pilot on track. We're looking forward to going further with this work in healthcare as well as other vertical markets such as financial services, telecommunications, life sciences and oil and gas."


text:
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Starting Today: Free Music Daily!
By The Big Something On January 5, 2010 · 2 Comments · In A Big Something, Awesome, Free Song of the Day, Music
We are very pleased to announce that, beginning today and every weekday from here forward, we will be embedding one song (or more) available for free download on The Big Something. The songs will be lovingly hand-picked by Delaney, Noel or one of our KRCC DJs. For the time being, we'll be choosing most of the offerings from the amazing Free Music Archive, which we wrote about many months ago HERE. However, there may be other great tracks offered up by local bands, national acts or recordings we've done here in-studio. Listen up for these tracks on air, too! By the end of each week, you could have a brand new sound track for your weekend ready to deposit on your favorite mp3 player. Of course, we would be remiss if we didn't encourage you to support the FMA and the bands (buy their albums!) if you love what you're hearing. It's a great service and the bands are incredibly generous to make this music available.
The beautiful thing about the FMA songs is that we can embed them here on The Big Soemthing, you can listen to the track here and then download it if you like it by clicking the arrow next to the player. Listen up for these tracks to be played on air, too! This is all part of our ongoing efforts to row row row our boat gently down the digital stream while continuing to bring you the quality music and local information that you've come to expect from KRCC. For today, we've picked a few extras. Enjoy and we greatly appreciate your feedback!
To start things off, here are THREE picks. The first, from Delaney, is "Phantoms" by the Detroit band Paik off their 2006 release Monsters of the Absolute. Writes Delaney:
"It's a little raunchy, like it could be some of the back room music at the Roadhouse in Twin Peaks."
Here are two from the "band" Pregnant. Writes Noel:
"theres a river" is the digital stream I want to row row row my boat down, gently. "for sometimes" is just something I felt I had to share with you.
2 Responses to Starting Today: Free Music Daily!
Tracy Mobley-Martinez says:
Very very cool.
tOkKa says:
–>> ..yeh sic..
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Ticket hours: noon-6p Tues-Fri
on the phone or at the studio.
KRCC presents ARLO GUTHRIE
Pikes Peak Center
Saturday July 13, 2013 8pm
reserved seating through Ticketswest.com
7th Annual Blues Under The Bridge – July 20th, 2013
WATERMELON SLIM & THE WORKERS, JOHN HAMMOND, BLUES CARAVAN W/ JOANNE SHAW TAYLOR, THE SLIDE BROTHERS, D.B. REILLY, and THE JUST US LEAGUE
www.BluesUnderTheBridge.com for festival information
KRCC Member tickets on sale for $25. General Public tickets on sale for $35. VIP tickets $91.50. Day of show tickets $40 at the west gate. Tickets available through www.TicketFly.com or at KRCC.
Pre-festival Party Friday July 19th from 5-8pm at the Wyndham Grand Mining Exchange with Watermelon Slim, and Big Jim Adams!
Pre-festival shows at Jack Quinns with:
BJ Estares and Route 66 on Wednesday July 17th at 7:30pm!
The Delta Sonics with Al Chesis on Thursday July 18th at 7:30pm!
Austin Young on Friday the 19th at 9pm!
KRCC presents TAB BENOIT
Colorado Springs, City Auditorium
Wednesday, July 31st
KRCC Member Tickets on sale now for $16 at the KRCC Studios.
General public tickets on sale through www.AMusicCompanyInc.com .
KRCC and the Fine Arts Center present VIEUX FARKE TOURE
Colorado Springs Fine Arts Center, Monday August 5th, 7pm
KRCC tickets on sale now for $15 at KRCC.
FAC member tickets on sale for $15, and general public tickets on sale for $25 at the Fine Arts Center Box Office, or through www.csfineartscenter.org
KRCC presents DIANA KRALL
Pikes Peak Center
Wednesday, September 25th 7:30pm
reserved seating through Ticketswest.com
Pikes Peak Center, Saturday October 12th, 2013 8pm
KRCC member pre-show meet and greet opportunity 6:30-7:30pm – pre-show meet and greet tickets available for $40 to members ONLY at the station.
Advance KRCC member tickets on sale now through August 2nd through www.TicketsWest.com. Enter member promo code and click find.
General Public tickets on sale August 2nd, 2013
KRCC presents BONNIE RAITT
Pikes Peak Center
Wednesday, October 16th 8pm
reserved seating through Ticketswest.com


text:
I've had two babies and have had weight fluctuations. For years now, I've been eating very healthy, and exercising almost daily, but this is as good as it's gotten...
The things I hope to accomplish:
• Removal of "saddle bags" and extra fat in abdomen, waist, lower buttocks, hips, and thighs all around.
• Skin tightening in all of these areas resulting in far less cellulite.
• Butt lift with added fullness.
• Tightening of the abdominal muscles resulting in a flatter stomach.
Given this information and the photo (pardon the cutout), which would you suggest?
Thank you so much!


text:
pivotnoun [ C ]uk /ˈpɪv.ət/ us
› a fixed point supporting something that turns or balances
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pivotverb [ I or T ]uk /ˈpɪv.ət/ us -tt-
Thesaurus: synonyms and related words
(Definition of "pivot" from the Cambridge Advanced Learner's Dictionary & Thesaurus © Cambridge University Press)
(Definition of "pivot" from the Cambridge Academic Content Dictionary © Cambridge University Press)
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Word of the Day
a person who has spent a lot of their life doing yoga and studying the philosophy of yoga


text:
Fringe Chelsea Crossbody in Pebble Leather
Have a question? Contact Us.
Dramatic, fun fringe and classic whipstitched details add a little attitude
to the must have new Chelsea.
Made of rich pebble leather and finished by hand, it has a short handle
and a detachable long strap, so it can be carried, slung over the shoulder or worn crossbody.
11" (L) x 7 1/2" (H) x 3" (W)
Inside zip pocket
Handles with 7" drop
Strap with 21" drop for shoulder or crossbody wear
Zip closure, fabric lining
Pebble leather
Style No: 37740
Color: Chalk
Standard Rate: FREE
Please allow 7 to 10 business days for product arrival. To ensure your products safe arrival, we include signature confirmation on all purchases.
Please review our full shipping policy by clicking HERE.
We accept returns up to 7-days after product delivery.
All merchandise must not be worn, altered, or washed. The merchandise must have all tags attached and be returned in original packaging. All returns will be subject to a $9.95 Return Postage Fee.


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Religion & Spirituality, ChristianityFollow
Be inspired by biblical teaching from Pastor Brian Houston Brian Houston is the Senior Pastor of Hillsong Church. Our hope and prayer is that you'll be encouraged, blessed and inspired through these free podcasts. You can find more information and other resources by Pastor Brian at brianchouston.com Author: Hillsong Church
September 21st, 2016
Episode 195 of 228 episodes
For more information and to stay up to date with Ps Brian Houston and Hillsong Church please visithttp://www.brianchouston.com or connect with him on Twitter @brianchouston, Facebookfb.com/pastorbrianhouston or Instagram @brianchouston
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Welcome The Vox Markets Podcast with Justin Waite A new episode is released daily, giving you interviews with CEO's, market commentators and the stock of the day as suggested by listeners to the podcast.


text:
Responsive design is the future, Go MobileWhen will envato finally decide to go mobile. It after all always gives tutorials an nettuts+ and so on to go mobile and have responsive pages. There is a huge mobile audience there, so why not go mobile and increase sales!?
Couldn't agree more, mobile forums too!
Sorry but i'll disagree. I dont think going on mobile would necesarily increase sales. Would you buy a template only by checking on mobile? Anyway i don't see why normal surfing on mobile and zooming is less better than responsive. They are both good, i personally prefer normal browsing, but if they plan to go responsive i'd recommend programmers to leave an opt-out button or desktop view.
But that is the point that almost all templates should be responsive now because of the change in web development. More and more people use smartphones and tablets to access websites rather than laptops and desktops and so we are moving on from conventional static webpages to mobile and responsive webpages that can be viewed an all devices. And the numbers are just gonna grow so we must change and adapt the future.
i agree with hogash we both been members of the marketplaces 3-4 yrs
Bad idea! ThemeForest has a reputation, building a new one is a risky undertaking! Mobile everyrhing marketplace would be an idea, but that's still not ok, because there are templates and themes on TF, graphics on GR, scripts on CC…
As a mobile author, I would strongly DISAGREE with this! There is a marketplace for everything already, breaking them up on categories, would actually destroy sales!
Has been already discussed here: http://themeforest.net/forums/thread/responsive-envato-responsive/72196 even with a statement from collis.


text:
Small Business Strategies: Women get into the startup game
I don't normally go gaga over motorcycles, but even I couldn't resist the shiny new scooter from Current Motor.
Sponsored Links
First, it is electric. Next, it's equipped with a state-of-the-art digital dashboard that charges your battery remotely and plots routes for the least battery consumption. It's made in America. And you can personalize the bike with all kinds of features.
Perhaps one of the most surprising aspects of this cutting-edge product is that this company has a woman president, is financed by women investors, and is made in a manufacturing plant owned by a woman.
Why surprising? Here's a sad truth in 2012: Women entrepreneurs still have a harder time raising money for high-growth ventures than men.
That situation is changing, thanks to women like Lauren Flanagan of Belle Capital of Grosse Pointe Farms, Mich. Flanagan represents a relatively new breed of angel investors, experienced businesswomen investing in startups founded by women.
"Women have over 51% of the wealth in the U.S., but we do less than 10% of early stage investing," Flanagan said. "We have women with talent and money sitting on the sideline.
"If we could get some of that money invested, we'd change the game," she said. "We could break the cycle of lack of access to capital for women entrepreneurs."
To understand financing for high-growth companies, it helps to understand the two types of professional investors and lack of women in these ranks:
•Venture capitalists raise money from others, typically investing very large sums of money — $2 million to $5 million and more in early rounds. About 11% of venture capitalists are women, according to the National Venture Capital Association, and estimates are that only 4 percent to 9% of companies receiving venture-capital financing have even one woman on the founding team.
•Angel investors put their own money into promising startups. About 13% of angels are women, and about 13 percent of angel-backed companies have women founders.
Keep in mind that nearly half of all businesses in the United States have a female owner: 29% owned by women alone, 17% equally owned with a man. So you see what a small percentage have access to high-growth capital.
Increasingly, women are starting their own angel-investor groups to make it easier for women entrepreneurs with great ideas to succeed.
"We want to solve big problems and, of course, we want to make money," Flanagan said. "But we're doing this to pay back, too. In the past, wealthy women were philanthropists, they gave money away. We're trying to teach women to be more comfortable with investing."
Flanagan herself has invested in about 30 companies. In 2005, she launched her first group, Phenomenelle Angels Fund in Madison, Wis.
That group invested about $10 million and raised another $30 million for nine companies, all of which have a women founder or significant C-level partner, a term used to denote high-ranking executives such as chief executive officers, chief operating officers and chief investment officers.
All the investors are women, and they project hitting their target on financial gains.
Last year, she started Belle Capital with 44 women investors. One of its first investments is Current Motor Co. of Ann Arbor, Mich., makers of that shiny, new electric scooter.
Belle Capital has brought far more than money to this promising startup.
"We leverage our financial capital with our human capital," Flanagan said. "One of our partners is Andra Rush, she's the founder of two companies that combined have over a billion dollars of revenue."
Rush founded Rush Trucking, which later did a joint venture to launch Dakkota Integrated Systems, which manages the manufacturing process for automotive interiors. Dakkota, based in Holt, Mich., is building the bike, creating good jobs here in the United States.
"Marie Klopf, an investor in Belle, is a supply-chain expert. She's now president of Current Motor. Nancy Phillipart — she used to run GM global accessories (and is) another investor in Belle — is advising on personalization strategy," Flanagan said. "And I brought a relationship with Dell. They've provided technical assistance and helped us get to market six months faster."
Flanagan points out it's not only women her venture-capital firm brings to the table.
"We brought in Bob Lutz, former vice chairman of General Motors and father of the Chevy Volt."
Belle Capital, www.bellevc.com, and Flanagan are part of a nationwide movement to increase women's access to venture financing. Other places to check:
•Astia, www.astia.org
•Golden Seeds, www.goldenseeds.com
•Springboard Enterprises, www.springboardenterprises.org
•Women 2.0, www.women2.org
•Women's Capital Connection of Kansas City, www.onekcforwomen.com
And soon, look for a Current Motor electric scooter on a street near you.
Rhonda Abrams is president of The Planning Shop and publisher of books for entrepreneurs. Her previous book is the 5th edition ofThe Successful Business Plan: Secrets and Strategies. Register for Rhonda's free newsletter atwww.PlanningShop.comand "like" The Planning Shop onFacebookfor updates. See an index of Abrams' columns. Twitter: twitter.com/RhondaAbrams. Facebook: www.facebook.com/RhondaAbramsSmallBusiness. Copyright Rhonda Abrams 2012.
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Follow Harriet on Twitter
About Harriet
- The Poetry Foundation's blog for poetry and related news.
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Poetry News
National Book Critics Circle Award finalists
Are you sad your pick of the finalists to win the National Book Award didn't pick up the top honor? Well, three of them have another shot! Here are your National Book Critics Circle Award finalist for 2010 in poetry:
Anne Carson. Nox. New Directions
Kathleen Graber. The Eternal City. Princeton University Press
Terrance Hayes. Lighthead. Penguin Poets
Kay Ryan. The Best of It. Grove
C.D. Wright. One with Others: [a little book of her days]. Copper Canyon
Read more about the award and the finalists here.
Posted in Poetry News on Monday, January 24th, 2011 by Harriet Staff.


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Laws, Regulations & Annotations
Business Taxes Law Guide – Revision 2010
Underground Storage Tank Maintenance Fee Law
Revenue and Taxation Code
Chapter 3. Determinations
Article 1. Reports and Payments
Section 50110
50110. Fee; due and payable. [Repealed by Stats. 1990, Ch. 1366, in effect September 27, 1990.]
50110. Returns; other than periods specified. The board, if it determines it to be necessary in order to facilitate the administration of this part, may require returns and payments specified under Section 50109 to be made for periods other than quarterly.
History.—Added by Stats. 1990, Ch. 1366, in effect September 27, 1990.


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Glass and Glory
Venue: Holy Trinity Church, Amblecote
Date & Time: 10am - 4pm (except during Services) Friday 24 August - Monday 27 August
Price: Free
Glass students of Wolverhampton University, past and present will be exhibiting their work at Holy Trinity, the Parish Church of Amblecote. The exhibition will show a variety of skills and styles which have been developed by the students during their time at the university. Participating artists include Katherine Roden, Kimberley Rose, Michelle Bishton, Rebecca Turbin and William Pryde. Refreshments also available in the form of the (now traditional) Cake Station – a chance to sit down and indulge in a piece of homemade cake and a range of hot and cold drinks.


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Inkjet Cartridges for HP DesignJet 111
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Noel Shannon Revisits Crash Site
We are continuing to follow the story of a young Walla Walla woman who lost her leg in a pickup truck accident.
Your Local ABC joined 23-year-old Noel Shannon as she returned to the crash site today in the mountains in Milton-Freewater where she was involved in a tragic accident forcing doctors to have to amputate her left leg.
Today, she sat in the exact spot she was stuck in for 10 hours on the night of July 3rd.
"It's amazing being able to sit here and be alive and not have to worry whether I'm going to die or not," said Shannon.
It's not an easy visit for her to make but Shannon said it gives her strength.
"The sooner the better, it definitely makes the healing process go by a lot faster," said Shannon.
On the night of the accident, she swerved to avoid hitting a deer, the brakes went out and the truck flipped several times.
The truck landed on its side and her leg was stuck out the window underneath the door.
She placed a rearview mirror near her leg to prop the truck up to keep it from pushing down on her leg.
She used a car key to dig her leg out it's also the key that she had considered cutting her leg off with to escape.
Shannon then ripped her leg from the door, used her good leg to break the windshield and used both of her hands to pry the glass open to make her way to safety.
Today was Shannon's first time being in the truck since the accident and explains the grim feeling.
"My blood is everywhere and it's kind a terrible to look at that," said Shannon.
Shannon still suffers from phantom pain in her leg.
She has to take 50 pills a day and travel to Seattle every two weeks for doctors appointments.
She said life has become very difficult and at times she gets really angry asking, "Why me?" But as she sat in that old pick-up today she realized something.
"I'm very thankful that I'm here today and that I can step back and look at all of this and know that all of that fighting wasn't for nothing and I made it through," said Shannon.
Shannon's family is raising money for her so that she can get a prosthetic running leg.
If you want to help there will be a spaghetti feed this Thursday at the Milton-Freewater Els Club from 5 p.m. - 7:30 p.m.
You can also go to any Banner Bank and donate under the name Noel Shannon Medical Fund.
Also, check out Shannon's Facebook page for more information.
Local and Regional News
- Displaced and desperate: 'I will never go back to Marawi'
- Neighboring houses catch fire in Pasco
- Woman's body found on park trail in Everett
- Store clerks and customers disarm robber, hold suspect down until police arrive
- Accidental shooting at child's party leaves 1 man hurt
- Woman dies in highway crash in Yakima


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Gloucester, MA, USA < change city >
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Gloucester Wedding In October
"We wanted our wedding on the North Shore by the water and Crusieport offers that. If you're looking for sprawling green lawns and a sandy beach this isn't the place, but if you're happy with a venue right on the main stretch in Gloucester with a view of the water from the ballroom, then this is the perfect place. The ballroom is incredible - a hardwood floor, floor to ceiling windows, modern architecture, gigantic chandeliers, a full professional bar... the list goes on. They even have a big room with a bar downstairs if you choose not to have your cocktail hour in the main ballroom AND a full kitchen so your caterer doesn't have to work out of a van. Tami, the wedding coordinator, is helpful in the planning process, although hard to get a hold of at times. Overall, we highly recommend Cruiseport."
Cruiseport Gloucester
Gloucester, MA, USA


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The Layer2 Cloud Connector has the following features and benefits:
Brilliant tool, e.g. for replicating local files to Sharepoint Online library. The assistance I received from Layer2 was outstanding. We are signing up as a partner and deploying to other clients using SharePoint Online.
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With the free editions you can manage a limited number of items per connection. To order a license for the full version please click the shopping cart below.
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Are you already @ LinkedIn? Please join the Cloud Connector Community Group to share your experience.
Know more about a Layer2 Cloud Connector customer reference at the Microsoft Office web site.
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text: Mar 09, 2010 - I'm so disappointed in this product, for which I had high hopes. I would apply the styling gel to my hair while wet for twisting purposes and it would either leave my hair crunch and hard or I would have to wait two days for it to air dry. The custard also left some type of white residue in my hair. I ended up giving a $30 bottle away for free to a friend who wanted to try it.


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We've been watching Hank Cherry demonstrate the fine art of skipping a lure under boat docks. James Overstreet, who has a lot more experience watching Elite Series anglers than I do, thinks Cherry ranks among the best he's seen.
There's not much air space under some of these floating docks, but Cherry can skip a jig from the front to the back of every dock he targets.
While the technique has been top-rank, the results have been nada. Cherry seems to be as relaxed as anyone can be in this situation, but you know he's feeling pressure.
It's one thing to need a big one to top off a limit. But Cherry has the big one, and can't find anymore keepers.


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By Catherine O'Hara

REVIEW STAFF

Should the City of Hamilton want to play host to a casino, a gaming complex featuring live horse racing should be located in Flamborough.

That's what the Ancaster-Dundas-Flamborough-Westdale MPP would like developed in his riding.

"I could see a facility in Flamborough, if that's what the city wants, with some expanded gaming opportunities but I sure don't want to see it in isolation of horse racing," said MPP Ted McMeekin, Minister of Agriculture, Food and Rural Affairs at the Flamborough Chamber of Commerce's Jan. 16 meeting at the Dutch Mill Country Market, where he was the guest speaker. "Apart from the horses, to be frank, all bets are off."

The future of horse racing in Ontario was the main topic discussed at last week's meeting, attended by roughly 50 individuals, who turned out to hear the Minister deliver a brief address about the state of the province before taking questions from guests in attendance.

"It wasn't that long ago that we made the controversial decision to end the Slots at Racetracks Program so we could devote more money – some $345 million a year – to education and health care," said McMeekin. "Now, I'll stand by that decision and at the same time note that I want to try and develop a vibrant future for the racing industry in Ontario."

In June, the Minister of Agriculture instructed a blue-ribbon panel to consult with the province's horse racing industry after his government decided to axe the profit-sharing agreement between the Ontario Lottery and Gaming Corporation and racetrack operators, effective March 31, 2013.

The panel, said McMeekin, has worked tirelessly to glean information from stakeholders and develop a model that would ensure the industry's viability in the future.

The group, which included former cabinet ministers John Wilkinson, John Snobelen and Elmer Buchanan, did just that, said McMeekin.

"They suggested that the SARP (Slots at Racetracks Program) was not good public policy. There were problems with it and that there needed to be some changes if the industry was to survive long-term," he said.

But little has been made public about the new model, including how much funding will be allocated to maintain the health of the industry, say horsemen, some of whom where in attendance at the Dutch Mill for the Flamborough Chamber of Commerce meeting.

According to Snobelen, who took to the mic last Wednesday morning, announcements about the future of horse racing in Ontario are expected soon as the panel continues to facilitate negotiations between the OLG and racetrack operators.

RELATED: Government signs funding agreement with Woodbine

"We have been pushing as hard as we can to make sure that decisions can be made across the board as quickly as possible," said the panellist, adding that locally, the three-member group has received the full co-operation of Great Canadian Gaming, the owner and operator of Flamboro and Georgian Downs, which recently voiced its commitment to horse racing.

"No one really expects really, really good news in the sense that it can't be what it was. These are not those times," said Snobelen following the meeting. "But I think there is every reason for optimism that there will be a healthy horse racing industry for my grandchildren."

Despite reassurances from McMeekin and members of the transition panel, horsemen and women remain concerned.

RELATED: Flamborough horsemen brace for the worse, hope for the best

"We don't believe that the government has a true understanding of the cost benefit of doing what they are doing," said Brian Tropea, general manager of the Ontario Harness Horse Association, following McMeekin's address. "We don't think, to this day, that they understand the impacts of putting these people out of work and people losing their farms and having their trucks and trailers repossessed and this type of thing."

And this, he noted, is a reality for many employed by the horse racing industry.

"Listening at what I heard today, you'd swear that there is nothing going on," said Donna Skelly, the Ancaster-Dundas-Flamborough-Westdale Progressive Conservative candidate, who attended the Jan. 16 meeting. "There's such a disconnect between the reality of the cancellation of this program, how it's going to affect the residents and farmers in this community and what is being said, politically."

While McMeekin and Snobelen spoke extensively about their work on the horse-racing portfolio, Hamilton Mayor Bob Bratina was invited to speak about the city's work on the casino file.

"The issue for Hamilton right now is the future site of a casino," noted Bratina.

Council has already voted in support of maintaining operations at Flamboro Downs in an attempt to ensure the success of the city's agricultural sector, namely the horse racing industry.

However, should a casino be located elsewhere in the city, Bratina suggested the possibility of re-allocating the funds Hamilton receives from the OLG – roughly $4.5 million annually – to support horse racing at Flamboro Downs.

"…I think it's fair to consider whether the city should apply some (or all) of those proceeds to sustain the horse racing industry," noted the mayor, stressing the municipality's strong rural and agricultural footprint – a $2 billion sector in Hamilton.

"Could we look at transferring the horse racing portion of the property to some organization that could write a charitable receipt, for instance, take the property and then operate it beside but separate from the actual casino?" It's a possibility, said Bratina, who has spoken with McMeekin about the city's options. "We are not finished discussing that around the council table."

However, the mayor's proposal came as a surprise to Flamborough councillors Robert Pasuta (Ward 14) and Judi Partridge (Ward 15), who say this has yet to be mentioned at city hall.

"I, certainly as a councillor for the area, have not been privy to any discussions that the mayor has had with regards to this new-found funding model," said Partridge. "It would have to be decided by council and, to my knowledge, it certainly has not been discussed with us as councillors."

Pasuta recalls the mayor mentioning this at a recent committee meeting. "He has mentioned it before but it's nothing that has been run by councillors; it's nothing that has come from staff," he said.

Should Bratina's suggestion of re-allocating the municipality's share of the slots revenues to bolster horse racing occur, the Ward 14 councillor said, "it wouldn't keep horse racing going" – not in their "wildest dreams."

Flamborough's elected municipal officials remain unwavering in their support of Flamboro Downs.

"The casino must be located at Flamboro Downs so we have a casino, slots and racetrack operations," said Partridge.

And while the decision of hosting a casino comes down to the municipality's willingness, there are three other parties involved in the decision-making process.

"We are at the end of the table waiting for the scraps," said Pasuta.