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Cancer is commonly associated with widespread disruption of DNA methylation, chromatin modification and miRNA expression. In this study, we established a robust discovery pipeline to identify epigenetically deregulated miRNAs in cancer.
Using an integrative approach that combines primary transcription, genome-wide DNA methylation and H3K9Ac marks with microRNA (miRNA) expression, we identified miRNA genes that were epigenetically modified in cancer. We find miR-205, miR-21, and miR-196b to be epigenetically repressed, and miR-615 epigenetically activated in prostate cancer cells.
We show that detecting changes in primary miRNA transcription levels is a valuable method for detection of local epigenetic modifications that are associated with changes in mature miRNA expression.
MiRNA genes are typically transcribed by RNA polymerase II into primary miRNA (pri-miRNA) transcripts, and transcription appears to be regulated in a similar process as traditional coding genes [1]. Pri-miRNA transcripts are long non-coding RNAs (ncRNA) with stem-loop secondary structures that contain precursor miRNAs (pre-miRNA). Pri-miRNA transcripts are cleaved co-transcriptionally by the enzyme Drosha into stem loop pre-miRNAs, which are exported to the cytoplasm by Exportin 5, and further processed by Dicer into mature miRNA that can be loaded in the RNA induced silencing complex (RISC). Pre-miRNA levels are low relative to pri- and mature miRNAs, suggesting that dicing is an efficient mechanism with little regulation [2]. During development, many pri-miRNAs are expressed but not efficiently processed to mature miRNA [3]. In healthy tissues, the ratio of pri- to mature miRNAs has been shown to be close to one, while in cancer cells, a large number of miRNA genes are transcribed but not processed to mature miRNA [4]. An expanding body of evidence supports a role for miRNAs in disease progression and the potential for epigenetic mechanisms to regulate miRNA expression [5,6]. Epigenetics is the heritable modification of gene expression without changes in the DNA sequence. The importance of regulated epigenetic information is highlighted by the disruption of multiple epigenetic marks in various disease states, including cancer, which is commonly associated with deregulation of DNA methylation, histone modifications and miRNA expression [7]. Cytosine methylation of CpG islands associated with gene promoters is a well-studied epigenetic mark, and aberrant DNA hypermethylation in combination with altered histone architecture is a common hallmark of neoplastic cells [8]. Histone acetylation at lysine 9 (H3K9Ac) is associated with an active chromatin state and changes in its global patterning are linked to poor prognosis in multiple cancers [9]. MiRNAs have been shown to function as tumor suppressors and oncogenes, through the targeting of essential elements of cellular growth, proliferation and apoptotic pathways [10]. Genomic loci encoding miRNAs can become epigenetically remodelled in cancer, however the extent and effect of these changes in cancer remains to be elucidated [6].
We argue that a minority of miRNA genes, critical to regulation of cellular growth, proliferation, or survival, gain epigenetic modifications to enable persistent enhancement or inhibition of their activity in order to maintain a neoplastic phenotype. Using an integrative approach combining primary transcription, genome-wide DNA methylation and H3K9Ac patterns with mature miRNA levels, we identified a number of miRNA genes that showed epigenetic remodeling in cancer in combination with de-regulation of both the primary and mature miRNA. This demonstrates that integration of multiple methods of analysis may provide a reliable method for the identification of epigenetically regulated miRNAs.
Results and Discussion
We proposed that epigenetically de-regulated miRNAs in cancer would show three characteristics: first, differential expression at the primary transcript level, second, differential mature miRNA expression, and finally an associated change in one or more epigenetic marks. To eliminate post-transcriptionally regulated miRNAs that showed accumulation of epigenetic marks independent of their transcription, an integrative pipeline that assessed all three criteria was developed (Figure 1).
Figure 1. Schematic displaying experimental discovery pipeline used for identification of miRNAs epigenetically deregulated in cancer. To identify epigenetically regulated miRNAs, we employed an integrative approach assessing changes in epigenetic marks, primary miRNA transcript, and mature miRNA. (A, B) Custom tiling arrays probing all miRNA loci (miRBase12.0, 668 miRNA loci) were used to assay primary miRNA transcription and epigenetic marks (H3K9Ac and DNA methylation) from PrEC and LNCaP cells. Example tiling array intensity plots are shown of the 2000 bp region spanning the miR-205 gene for both expression (A), and DNA methylation (B), showing the chromosomal coordinate (x-axis) and the hybridization intensity (y-axis), with regions displaying significant differences highlighted. (C) Global mature miRNA expression levels in PrEC and LNCaP cells +/- 5-Aza-CdR treatment (3 μM LNCaP, 1 μM PrEC; 72 hours) as determined by TLDA qRT-PCR (368 miRNAs).
Normal prostate epithelial (PrEC) and prostate cancer (LNCaP) cells were used as a model system to test this integrative pipeline in the discovery of epigenetically de-regulated miRNAs in cancer. Custom high-density NimbleGen tiling arrays covering miRNA loci from miRBase v12.0 were designed and used to probe total RNA to determine transcription levels, and DNA from methylated-DNA immunoprecipitation (MeDIP) and H3K9Ac immunoprecipitation to investigate epigenetic changes. The oligonucleotide probes of these arrays have an average length of 60nt, precluding interference of the mature 20-23nt miRNAs, thus assaying the full-length primary transcripts. Plotting the array probe intensities against chromosomal coordinates generated high-resolution maps of expression and epigenetic mark enrichment across each locus (plots for miR-205 are shown in Figure 1A & 1B as examples). To generate lists of loci significantly changed in LNCaP cells for each assay, we selected those that were consistently changed in the cancer cell line within 500 bp of the center of the miRNA gene. For our comparisons, we limited the number of miRNAs to those that were assayed by both TaqMan® Low Density Arrays (TLDA) (Figure 1C) and tiling array: 341 miRNAs in total. All changes are articulated as LNCaP with respect to PrEC.
Looking at those loci whose primary transcript repression or activation in LNCaP was associated with a significant change in the mature miRNA, we identified 29 upregulated and 33 downregulated miRNAs (Figure 2A,B). We then looked for concomitant epigenetic changes at these loci in the tiling array data. A high proportion of the upregulated (31%; 9/29) and downregulated loci (33%; 11/33) showed an overlap with at least one of these epigenetic marks (Figure 2C,D). The majority (18/20) of these miRNAs have previously been identified as deregulated in various cancers, and five with prior evidence for epigenetic regulation (Table 1). A concordant change in all assays; expression, DNA methylation, and H3K9Ac restricted the total number of candidate miRNAs to 2/29 up- and 4/33 downregulated (Figure 2C,D). Given the large number of possible epigenetic modifications (for a recent review see [6]), it is possible that many of the differentially expressed loci that show no change in DNA methylation or H3K9Ac could undergo changes in other epigenetic marks. Furthermore, miRNAs may be regulated by DNA methylation at CpG islands outside the 2000 bp regions interrogated in this analysis.
Figure 2. Progressive steps in the identification of epigenetically regulated miRNAs in prostate cells. Venn diagrams show the number of miRNA loci changed in LNCaP (L) with respect to PrEC (P) cells called significant by tiling arrays analysis using a t-statistic ≤-2 or ≥2 for regions +/-500 bp from the relevant miRNA. (A, B) Overlap of pri- and mature miRNA expression levels identified by tiling array and TLDA, respectively (TLDA miRNA expression data was normalized to RNU48, using a significance cutoff at p < 0.05 and ≥1.5 -fold change). Concomitant changes in active (H3K9Ac) or repressive (DNA methylation) epigenetic marks were then considered for miRNAs displaying significantly changed expression. (C, D) miRNA loci significantly changed in MeDIP-chip and H3K9Ac ChIP-chip tiling array analysis. (E, F) Four-way Venn diagrams showing the overlap of tiling array data with mature miRNA level upon 5-Aza-CdR treatment. MiR-615 lies at the four-way intersection indicating epigenetic up-regulation and miR-205, miR-196b and miR-21 are at the intersection of epigenetic down-regulation in LNCaP cells.
Table 1. Candidate epigenetically regulated miRNAs
In order to perform an independent test for miRNAs regulated by DNA methylation, we treated LNCaP and PrEC cells with the drug 5-Aza-CdR, an inhibitor of DNA methylation, and analysed their expression by TLDA. Restricting our list to miRNAs that were significantly deregulated between PrEC and LNCaP we found 30 upregulated by 5-Aza-CdR in PrEC and 16 in LNCaP. Of the miRNA loci showing epigenetic activation by the three criteria of primary transcript expression, DNA methylation loss and H3K9Ac gain, only miR-615 showed 5-Aza-CdR induced reactivation in the normal prostate PrEC cells (Figure 2E). MiR-205, miR-196b and miR-21 satisfied all three criteria for epigenetic repression, and LNCaP 5-Aza-CdR reactivation (Figure 2F).
MiRNA genes showing non-canonical epigenetic regulation were also observed. That is, miRNAs showing up regulation of primary and mature miRNA expression, with a concomitant gain of DNA methylation or loss of H3K9Ac, or vice versa. Twelve miRNAs showed activation and gain of repressive epigenetic marks and six (including the chromosome 14 miRNA cluster) showed repression in combination with active chromatin (Additional file 1, Table S1). None of these miRNAs showed significant up regulation upon treatment with 5-Aza-CdR. Several miRNAs were repressed in the presence of the demethylating drug, suggesting the repression of these loci may be dependent on genes that are themselves subject to regulation by DNA methylation.
Additional file 1. Table S1: MiRNAs showing non-canonical epigenetic regulation. Table listing miRNA loci and genes showing significant non-canonical epigenetic regulation. Values are expressed as changes in LNCaP relative to PrEC. Tiling array t-statistics represent the significance of the average difference for each miRNA, with increasing value indicating more significant change. Mature miRNA levels are given as fold change with the associated p-value. Chr: Chromosome, Str: Strand, meth: DNA-methylation, L: LNCaP, P: PrEC, 5Aza: treatment with 5-Aza-CdR, p *: p-value <0.05, Epi./Cancer: publication indicating epigenetic/cancer regulation. Chr14 cluster data includes: MIR127, MIR323, MIR376a-2, MIR432, MIR376a-1, MIR654, MIR382, MIR487b, MIR134, MIR487b, and MIR411.
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To validate the changes in primary miRNA expression, we performed pri-miRNA TaqMan® qPCR on the four miRNAs that met all our criteria. These assays confirmed the repression of pri-miR-205 and pri-miR-21, and over expression of pri-miR-615 in LNCaP cells (Figure 3A,B,D). There was no significant change in pri-miR-196b between LNCaP and PrEC, however treatment with 5-Aza-CdR led to overexpression in PrEC cells (Figure 3C). 5-Aza-CdR led to overexpression of pri-miR-21 and pri-miR-615 in LNCaP, as would be predicted by their DNA methylation status (Figure 3B,D). However, reactivation of pri-miR-205 by 5-Aza-CdR was not significant (Figure 3A).
Figure 3. Vailidation of pri-miRNA expression. Quantitative TaqMan® RT-PCR of pri-miRNA expression in LNCaP and PrEC cells +/- 5-Aza-CdR treatment (n = 6, +/-SD, *p <0.05). Pri-miRNA RT-PCR assays followed established TaqMan® workflows (Applied Biosystems). Briefly, total RNA was used for first-strand cDNA synthesis using 1 μg RNA Superscript reverse transcriptase (Invitrogen). cDNA was then subjected to 40 cycles of amplification using an ABI7900 instrument (Applied Biosystems). Data was normalized to 18S levels (2-dCt).
To verify changes in DNA methylation we analysed recently published methyl-CpG binding domain-based capture (MBDCap) data from LNCaP and PrEC cells [11]. MBDCap preferentially captures DNA methylated double-stranded DNA which is then eluted for high-throughput sequencing [12]. We examined the number of sequencing reads that mapped to within 300 bp of the selected miRNA loci. In agreement with the tiling array data, there was enrichment of sequencing reads at the MIR196b locus (Additional file 1, Figure S1). MIR205 and MIR21 had increased reads in LNCaP over PrEC, however there was also an increase in the input DNA controls. MIR615 showed a low number of sequencing events in both LNCaP and PrEC and input controls (Additional file 1, Figure S1).
The identification of miR-205 as an epigenetically repressed miRNA is consistent with a previous study that examined chromatin modifications in prostate cells using genome-wide promoter arrays, which revealed a gain of the repressive H3K27-trimethylation mark and loss of the active H3K4-trimethylation mark at the MIR205 locus in prostate cancer cells [13]. Furthermore, we recently reported that MIR205 locus is silenced and gains promoter hypermethylation and repressive chromatin marks in invasive bladder tumors and undifferentiated bladder cell lines [14]. Two expression array studies have also identified miR-205 as repressed in prostate cancer [15,16]. While the repression of pri-miR-205 was validated by qPCR, the lack of significant change in DNA methylation observed in the MBDCap high-throughput sequencing data could be due to differences between the techniques. Both MIR205 and MIR21 reside in regions of low CpG density, whereas MIR196b and MIR615 are located in regions of high CpG density, which may bias the purification efficiencies. Variations between pri-miRNA and array results, such as with pri-miR196b, could be accounted for by differences in assay design. An advantage of the tiling array approach is the high-density coverage of all loci. PCR typically provides improved dynamic range over array analysis, however, the efficiency of a reaction can be limited by location of the primers - as may be the case with pri-miRNA transcripts that have yet to be exhaustively mapped. MiR-21 shows significant epigenetic repression in our array and qPCR analyses, contradictory to its well-established role as an oncogenic miRNA that is overexpressed in cancer [17], however recent evidence has also shown that miR-21 knockdown elicited no change in proliferative or invasive properties in prostate cancer cells [18].
The potential for epigenetic regulation of MIR21 has previously been shown in ovarian cancer, where treatment with 5-Aza-CdR leads to miR-21 overexpression [19], consistent with hypermethylation of the locus. It is possible that epigenetic mechanisms may play a role in context dependent regulation of miR-21 expression, and it would be interesting to examine the prevalence of epigenetic marks at in a cohort of clinical prostate cancer samples. The epigenetic regulation of both MIR615 and MIR196b is particularly intriguing as both genes are encoded from CpG islands located within HOX gene clusters. MIR196b has recently been shown to be hypomethylated in acute lymphoblastic leukemia [20] and gastric cancer [21] but to the best of our knowledge, the epigenetic regulation of MIR615 is a novel discovery. MIR615 is located within the HOXC cluster on chromosome 13, which we previously have shown to be epigenetically deregulated in LNCaP cells in long range epigenetically silenced domains [22]. There is evidence that HOX associated miRNAs are essential for development and can function by targeting HOX messenger RNAs [23]. Furthermore, HOX genes have been reported to be involved in abnormal development and malignancy in numerous cancers via both genetic and epigenetic mechanisms [24].
There are inherent limitations to the various available global epigenetic discovery techniques [11]. Immunoprecipitation based techniques can be affected by antibody sensitivity and MeDIP can be biased towards regions of high CpG content, leading to increased false-negatives at GC-poor sites [25]. In addition, treatment with demethylating drugs such as 5-Aza-CdR can uncover false positives through activation or repression of downstream targets [26]. A multi-platform approach can help rule out many of these false positive results, but the problem of false negatives remains. Here we show that using miRNA primary transcript data enables leverage of the known mechanism of post-transcriptional regulation of miRNAs as an additional means of epigenetic marker discovery. Epigenetic marks such as DNA methylation possess several advantages as biomarkers; DNA is more robust than RNA, can be isolated from formalin fixed paraffin-embedded archival samples and DNA methylation provides a positive readout for genes or miRNAs that are downregulated [27].
We found that up to 33% of transcriptionally deregulated miRNA loci displayed concordant DNA methylation and H3K9 acetylation patterns. Extending the analysis to additional epigenetic marks, such as the methylation of H3K4, H3K9 and H3K27 and acetylation at H3K14 will increase the number of identified loci. The use of tiling arrays in this study enabled analysis within 2000 bp of a miRNA gene and the increasing availability of high-throughput sequencing technology will allow this approach to be extended genome wide. The prevalence of miRNAs that show contradicting expression and epigenetic marks suggests that there is a subset of genes that are subject to a complex pattern of regulation, where specific marks may recruit activators or repressors to a promoter. We propose that in cancer, a change in primary transcript expression at miRNA loci provides an important first step in the identification of those miRNAs that display variant epigenetic marks. Overlapping these loci with change in mature miRNA restricts analysis to miRNAs that are functionally relevant and that provide possible selective advantage to the cancer cell. Further discovery of DNA hyper- and hypo-methylated miRNAs in cancer using this type of integrative discovery pipeline should assist in identifying loci that may provide reliable novel diagnostic tools.
Cell Lines and Culture
LNCaP cells were propagated in T-media as described previously [28]. PrEC cells were propagated in PrEGM (Lonza, Switzerland). 5-Aza-2'-deoxycytidine (5-Aza-CdR) (Fluka, MO, USA) treatment was performed for 72 hours with fresh media and drug added at 24 and 48 hours. We optimized 5-Aza-CdR treatment of LNCaP and PrEC cells at 3 μM and 1 μM, respectively, based on reactivation of the imprinted gene IGF2 and the prostate cancer hypermethylated gene GSTP1 in response to treatment, as described previously [28]. Total RNA was isolated using Trizol reagent according to supplier's protocol (Invitrogen, CA, USA). Genomic DNA was extracted from untreated LNCaP and PrEC using the Mammalian Blood and Tissue Kit (Qiagen, Germany).
TaqMan® Low-Density Arrays (TLDA)
TLDA v1.0 Early Access (Applied Biosystems, CA, USA) assays for 368 miRNAs were performed as per manufacturer's protocols. In brief, 1 μg total RNA from 5-Aza-CdR treated and untreated LNCaP and PrEC biological duplicates was reverse transcribed with pools of miRNA specific RT-primers, and the resultant cDNA was subjected to real-time reverse transcription quantitative (qRT)-PCR using specific forward primers and TaqMan® fluorescent probes in an array format. RNU48 was selected as the most reliable control for expression normalization. Initial analyses were performed using SDS software (Applied Biosystems). Statistical tests for changes in expression (via Cts) were performed using moderated t-tests without multiple testing corrections using the R limma package [29]. Mature miRNAs were deemed differentially expressed in LNCaP compared to PrEC at p <0.05 and fold change of ≥1.5.
Expression Tiling Arrays
Total RNA (6 μg) from untreated LNCaP and PrEC duplicates was reverse transcribed with random primers and aminoallyl- conjugated dUTP. cDNA products were precipitated and resuspended with mono-reactive Cy3/5 dye (GE Healthcare, UK), and the products were purified with Fairplay columns (Stratagene, TX, USA). Labeling efficiency was determined spectrophotometrically. Hybridizations for untreated LNCaP and PrEC were repeated in two formats to test a method to minimize probe GC bias: competitive hybridization with fluorescently labeled gDNA in the Cy5 channel, and in a dye swap arrangement. The biological repeats were highly reproducible (Pearson correlations: LNCaP 0.94 and PrEC 0.96) and co-hybridization showed no correction to probe binding efficiency (data not shown).
Tiling Array MeDIP-chip/H3K9Ac-ChIP-chip
4 μg of genomic DNA from untreated LNCaP and PrEC biological duplicates was sonicated, then denatured followed by incubation with Anti-5-Methylcytosine antibody (Calibiochem) overnight at 4°C. The antibody/DNA complexes were affinity purified using protein A/G agarose beads (Santa Cruz Biotechnology). Products were washed, eluted and ethanol precipitated. Efficiency of immunoprecipitation was confirmed by PCR at three loci; centromeric chromosome 16, GSTP1 and GAPDH, and compared with no-antibody and input controls, as described previously [30]. Biological duplicates were highly reproducible (Pearson correlation: LNCaP and PrEC 0.96).
H3K9Ac ChIP
LNCaP and PrEC cells were formaldehyde fixed in duplicate prior to SDS lysis and sonication. Immunoprecipitation (IP) was performed overnight at 4°C with an anti-acetyl H3K9 antibody (Millipore, CA, USA). DNA was eluted with 0.1M NaHCO3, 1% SDS elution buffer and reverse cross-linked. IPs and input controls were purified by phenol chloroform extraction and ethanol precipitation, as described previously [31], duplicate IP reactions were then pooled to ensure sufficient material for array analysis. IP DNA from MeDIP and H3K9Ac were labeled as per the manufacturers protocol (NimbleGen, WI, USA). Briefly, 1 μg IP or input DNA was labeled with Cy-dye random nonamers (Trilink Biotechnologies, CA, USA) using Klenow fragment (New England Biolabs, MA, USA), precipitated, and resuspended in hybridization buffer.
Tiling Arrays
We designed probes for 384 K feature tiling arrays (NimbleGen) to cover the positive strand for the following genomic regions: all human miRNAs listed in mirBASE v12.0 +/-2000 bp; HOX A,B,C and D gene clusters; the chromosome 14 Dlk1 - Dio3 - miRNA cluster region; and hybridization control regions. Array hybridization was performed for 16 hours with 6 μg labeled cDNA with hybribization buffer and alignment oligonucleotides (NimbleGen). Arrays were washed as per the manufacturers protocol and scanned using a GenePix 4000B scanner (Molecular Devices, CA, USA). To screen for miRNA loci that were significantly changed between LNCaP and PrEC cells, we fitted a linear model to the quantile-normalized probe-level data for all probes within 500 bp of the center of a miRNA gene. From the linear model fit, the effect of interest is the average change between PrEC and LNCaP across all probes, after adjusting for probe-specific effects. The t-statistics shown in Table 1 represent the significance of this average difference for each miRNA. MiRNAs were deemed statistically significant using a t-statistic cutoff of ≤-2 or ≥2. Venn diagrams were drawn using Venny [32], and restricted to overlaps for those miRNAs analysed by TLDA. Results of all array experiments are listed in Additional file 2.
Additional file 2. Figure S1: Validation of DNA methylation in selected miRNA loci. Analysis of published DNA methylation enrichment data at selected miRNA loci from Robinson et al. [11]. Graph displays absolute number of MBDCap high throughput sequencing reads within 300 bp of the miRNA locus.
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Pri-miRNA Real-Time PCR
We used Applied Biosystems pri-miRNA stem-loop specific TaqMan assays to quantify pri-miRNAs, as per the manufacturers protocol. Briefly, first-strand cDNA was synthesized using 10 ng DNase (RNase free DNase, Qiagen) treated total RNA, or no-template controls, and Superscript reverse transcriptase (Invitrogen). Real-time PCR reactions for each miRNA (10 μl volume) included 4 μl of RT product, were performed in triplicate and included no-template and PCR controls using an ABI7900 PCR system (Applied Biosystems). Reactions performed under the following conditions: 95°C for 10 minutes, followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. Relative quantitative levels of individual miRNAs were determined using 18S for normalization, and statistically significant differences were analysed using the students t-test.
miRNA: microRNA; TLDA: TaqMan Low Density Array; ChIP: Chromatin Immunoprecipitation; bp: base pairs; 5-Aza-CdR: 5-Aza-2'-deoxycytidine; IP: immunoprecipitation.
Authors' contributions
TH and SJC conceived the project. TH wrote the manuscript, cultured cells, performed array experiments and analysis. TS cultured cells and performed ChIP, MeDIP and TLDA experiments. EDW performed tiling array experiments. SB performed MeDIP experiments. ALS, DS and MDR performed data analyses. All authors read and approved the final manuscript.
We thank Warren Kaplan, Mark Cowley and Bruce Tabor for bioinformatic support; Jenny Song and Clare Stirzaker for technical assistance; and Kate Patterson for critical reading of the manuscript. This work was supported by Cure Cancer Australia and New South Wales Cancer Institute Fellowships (to TH), NH&MRC project grants (SJC) and the SIROCCO small RNA EU consortium (EDW).
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Cicatiello L, Mutarelli M, Grober OM, Paris O, Ferraro L, Ravo M, Tarallo R, Luo S, Schroth GP, Seifert M, et al.: Estrogen receptor alpha controls a gene network in luminal-like breast cancer cells comprising multiple transcription factors and microRNAs.
The American journal of pathology 2010, 176:2113-2130. PubMed Abstract | Publisher Full Text
Zhang H, Luo XQ, Zhang P, Huang LB, Zheng YS, Wu J, Zhou H, Qu LH, Xu L, Chen YQ: MicroRNA patterns associated with clinical prognostic parameters and CNS relapse prediction in pediatric acute leukemia.
PLoS ONE 2009, 4:e7826. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Chen Y, Gorski DH: Regulation of angiogenesis through a microRNA (miR-130a) that down-regulates antiangiogenic homeobox genes GAX and HOXA5.
Blood 2008, 111:1217-1226. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Sarver AL, French AJ, Borralho PM, Thayanithy V, Oberg AL, Silverstein KA, Morlan BW, Riska SM, Boardman LA, Cunningham JM, et al.: Human colon cancer profiles show differential microRNA expression depending on mismatch repair status and are characteristic of undifferentiated proliferative states.
BMC Cancer 2009, 9:401. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Bandres E, Agirre X, Bitarte N, Ramirez N, Zarate R, Roman-Gomez J, Prosper F, Garcia-Foncillas J: Epigenetic regulation of microRNA expression in colorectal cancer.
International journal of cancer 2009, 125:2737-2743. Publisher Full Text
Popovic R, Riesbeck LE, Velu CS, Chaubey A, Zhang J, Achille NJ, Erfurth FE, Eaton K, Lu J, Grimes HL, et al.: Regulation of mir-196b by MLL and its overexpression by MLL fusions contributes to immortalization.
Blood 2009, 113:3314-3322. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Qin W, Shi Y, Zhao B, Yao C, Jin L, Ma J, Jin Y: miR-24 regulates apoptosis by targeting the open reading frame (ORF) region of FAF1 in cancer cells.
PLoS ONE 2010, 5:e9429. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Valastyan S, Reinhardt F, Benaich N, Calogrias D, Szasz AM, Wang ZC, Brock JE, Richardson AL, Weinberg RA: A pleiotropically acting microRNA, miR-31, inhibits breast cancer metastasis.
Cell 2009, 137:1032-1046. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Lee EJ, Gusev Y, Jiang J, Nuovo GJ, Lerner MR, Frankel WL, Morgan DL, Postier RG, Brackett DJ, Schmittgen TD: Expression profiling identifies microRNA signature in pancreatic cancer.
International journal of cancer 2007, 120:1046-1054. Publisher Full Text


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Photos: My Bloody Valentine, Savages, Parquet Courts, Pantha Du Prince, More at Berlin Festival
By Pitchfork on September 9, 2013 at 8:30 p.m. EDT
Photos by Tabea Malthern and Helge Mundt . Above photo by Tabea Malthern.
Berlin Festival took place last weekend at Berlin Tempelhof Airport. Pitchfork presented a stage at Hangar 2, which included performances from My Bloody Valentine, Savages, John Talabot, Tomahawk, Pantha du Prince, Parquet Courts, Villagers, Is Tropical, and more. Photographers Tabea Malthern and Helge Mundt were there.
Check out a selection of shots after the jump, then head over to our slideshow and Facebook page for even more photos.
Photos: Iceage, Simian Mobile Disco, Pantha du Prince, Xiu Xiu, Holly Herndon, Mykki Blanco at CTM
By Pitchfork on February 8, 2013 at 4:31 p.m. EST
Last weekend, Iceage, Simian Mobile Disco, Pantha du Prince (pictured above), Xiu Xiu, Holly Herndon, Mykki Blanco, and more played Berlin's emerging music festival CTM. Pitchfork contributor Erez Avissar was there. Head below to check out a selection of shots from the event and head to our gallery and Facebook page for more.
Watch: Pantha du Prince and the Bell Laboratory Perform "Spectral Split" From Elements of Light
By Evan Minsker on January 9, 2013 at 10:45 a.m. EST
On January 15, Berlin producer Pantha du Prince (Hendrik Weber) and Norwegian collective the Bell Laboratory will release Elements of Light via Rough Trade. The album prominently features marimbas, xylophones, cymbals, chimes, and a bell carillon (a three-ton instrument containing 50 bronze bells). Below, check out their performance of "Spectral Split". The clip, directed by German filmmaker Sandra Trostel, shows a close-up view of the bell carillon.
Pantha du Prince Announces New Album and Tour
By Evan Minsker on October 17, 2012 at 10:43 a.m. EDT
Photo by Sigurd Fandango
Berlin producer Pantha du Prince (Hendrik Weber) has collaborated with a group of Norwegian musicians called the Bell Laboratory for the album Elements of Light. The album features tubular bells, marimba, xylophone, cymbals, chimes, handclaps, finger snaps, and a bell carillon (a three-ton instrument containing 50 bronze bells). It's out January 15 via Rough Trade.
Listen to This Week's Animal Collective Radio
By Evan Minsker on August 5, 2012 at 9:00 p.m. EDT
Week two of Animal Collective's weekly radio show, Animal Collective Radio, is on right now (9pm Eastern) at radio.myanimalhome.net. Tonight's episode is curated by Deakin and will include guest DJ sets from Pantha du Prince, Turquoise Wisdom, and the DOODcast crew. Last week's episode featured new music by Animal Collective and Panda Bear, so be sure to tune in here.
Pantha du Prince Teams With Stephan Abry for LP
By Carrie Battan on March 29, 2012 at 10:04 a.m. EDT
Pantha du Prince has teamed up with fellow German producer Stephan Abry of Workshop, under the name Ursprung. Their self-titled debut is out May 21 in Europe and May 22 in North America via Dial.
Stream New Albums From Times New Viking, About Group, Pantha Du Prince, Thao & Mirah
By Tom Breihan on April 18, 2011 at 1:30 p.m. EDT
Dancer Equired is the new album from lo-fi Ohio bashers Times New Viking, and Merge will release it in the U.S. on April 26. (It's also coming in Europe from Wichita, in Australia from Pop Frenzy, and in Japan from Big Nothing.) Right now, you can stream the entire album over at NPR.
There's a whole pile of new albums streaming online right now, and we've got a bunch of them listed out below.
Animal Collective, Four Tet, Lots More Remix Pantha Du Prince for Black Noise Remix Album
By Tom Breihan on February 7, 2011 at 6:05 p.m. EST
Last year, German minimal techno master Pantha du Prince released his BNM'ed album Black Noise. And on April 19, Rough Trade will release a sequel of sorts, the remix album XI Versions of Black Noise.
Animal Collective contribute a remix of "Welt Am Draht"; Animal Collective member Panda Bear sang on the original version of "Stick to My Side". Other remixers include Four Tet, Moritz Von Oswald, Walls, Lawrence, and the Sight Below.
Blonde Redhead, Pantha du Prince Tour
By Tom Breihan on July 20, 2010 at 1:45 p.m. EDT
Blonde Redhead photo by Sebastian Mlynarski
New York art-punk veterans Blonde Redhead and German techno minimalist Pantha du Prince both specialize in sculpted waves of prettiness; they just use different means to make that music happen. And this fall, they'll share stages, as Pantha Du Prince will open the first half of Blonde Redhead's North American tour.
Listen: Animal Collective Remix Pantha du Prince's "Welt Am Draht"
By Larry Fitzmaurice on June 11, 2010 at 11:40 a.m. EDT
Photo by Asha Mines
Pantha du Prince: Welt Am Draht (Animal Collective Remix)
It's no secret that Animal Collective are bros with German minimal techno wiz Hendrik Weber, a.k.a. Pantha du Prince. Panda Bear lent his vocals to "Stick to My Side", from Pantha's BNM'd Black Noise, and Pantha remixed Animal Collective's "Peacebone".
Now, the whole AnCo gang have lent their remixing talents to another Black Noise track, "Welt Am Draht", and you can check out the results above.
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subset: commoncrawl


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An extraordinary wartime memoir, combining the best kind of adventure story with a coming-of-age testimony of unforgettable resonance and poignancy.
September 2011, Halkidiki, Northern Greece: A solitary 86-year-old man gazes across an Aegean headland, knowing that he must finally confront his past. He begins to write...September 1939, Nieppe, Northern France:14 year-old Stephen is living with his family, 25 kilometres from Ypres. His French mother battles with her encroaching blindness. Failing to escape the advancing German army, his English father can no longer look after the war graves that cast so heartbreaking a shadow across the region. Stephen and his friend Marcel embark upon their great adventure: collecting souvenirs from strafed convoys and crashed Messerschmitts.
But their world turns dark when arrested and imprisoned for sabotage and threatened with deportation or the firing squad. Upon his release, and still only 16, Stephen is recruited by the French Resistance. Growing up under the threat of imminent betrayal, he learns the arts of clandestine warfare, and - in a moment that haunts him still - how to kill....
Such was the impact of Stephen Grady's work for the French Resistance, (especially during the countdown to D-Day and its bloody aftermath) that he was awarded the Croix de Guerre and the American Medal of Freedom.
©2013 Stephen Grady and Michael Wright (P)2013 Hodder & Stoughton
54 years old. Love short stories, history, bios,
"great book, though with a technical problem"
The book is very good and interesting. There's a problem, which is probably caused by the editing of the recording.
In the chapter 25, minute 5,20 ( according to the recording order, not the book order ), suddenly you get the same part of the book that appeared some hours before.
This repetition lasts until the minute 14, which is the end of the chapter 25, and then the story continues correctly with chapter 26. The main problem is that you don't know if this mistake implies that you got the wrong recording during these 9 minutes and you are missing the good one, therefore missing a part of the book.
"The Best Yet"
I actually heard about this book during a radio book review and I sought it out. Honestly, I'm an audible vetran and this book is the Usain Bolt of audio book world, it's sheer gold.
It's such a straight forward, well narrated story of heroism and guts that it is quite simply, astounding. It's written in an almost modest, apologetic way. To all those people of that time involved in the French Resistance, I salute you. To a man and women you should all tell you tale.
"Excellent true story."
The narrators really brought it to life. Poignant and harrowing and well worth a listen. Just showed the bravery of ordinary citizens.
Report Inappropriate Content
If you find this review inappropriate and think it should be removed from our site, let us know. This report will be reviewed by Audible and we will take appropriate action.


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Social Media Shoes
37K views
Lina D.
BoredPanda staff
BoredPanda staff
Inspired by the colors of popular social media websites, Venezuelan graphic designer Lumen Bigott created a unique line of Social Media shoes. The collection includes Facebook, Twitter, flickr, Google, dribbble and Wikipedia sneakers.
Unfortunately, these shoes are in concept stage, but Lumen said that they may see the light of day. [Read more...]
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ha what
ha what
the music is what its all about, you love it just like i love it.
So many stupid people giving stupid opinions about a stupid list.
People not liking them doesn't even make sense anyways, because I would say the Germs are one of the best punk bands of all time, and fuck the Germs.
5/15-17/15 - Psycho CA @ Observatory // 5/22/15 - Laura Jane Grace @ Hollywood Forever Cemetary // 5/27/15 - Neutral Milk Hotel @ Warner Grand Theater
6/3/15 - Sufjan Stevens @ Dorothy Chandler // 7/15/15 - Shellac @ Regent // 7/31/15 - Melt Banana w/Torche @ Roxy
apparently, 80s hardcore is essential, while 90s hardcore is ridiculous.
20 years and still going strong.
The Ramones were the start of everything, as far as I can tell, and therefore deserve top honors. But Green Day are the most commercially successful punk band, I don't think there's any arguing that. I always thought it was kind of lame that the 924 Gilman crowd begrudged them their success. But when my wife and I went by there last year, it was pretty clear from the people we talked to that they're proud of their local hero's nowadays, as they should be, Green Day are a fucking great band and their shows have been a blast over the years.
They definitely rank right up there. I'd have Bad Religion on there too.
On where?
I love Ramones, but they'd never deny that most of their music was just amped up girl-group music and 50s rock, played fast. So if we wanna give top honors, how about Johnny Cash, Ronnie Spektor or Link Wray? Fuckin' punk rock right there.
On "the" list.
and the Pistols were just Chuck Berry played loud and fast. at least the riffs were. But I think in both the case of the Ramones and the Sex Pistols, the vocals and lyrics were such a departure that it made a whole new thing.
Trying to analyze, label, and rate punk rock is so oxymoronic.
I do not think that word means what you think it means.


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The Top 10 In Action Sports
01Brick By Brick: Camping Lego Style
02Booze and Bikes: 14 Tours That Let Combine Two of the Best Things in the World
03Who Needs Hollywood? These Incredible Videos Were All Filmed on the Simplest Cameras
04Sam Pilgrim vs. The World: The Champion Aiming To Reclaim His Crown In A New Era Of Biking
05Eccentric Adventures Episode 2: Britain's Most Brutal Illegal Event is All About Cheese
06A Journey Into Darkness: We Went Mountain Biking in the Abandoned Mines of Slovenia
07After the Apocalypse: Inside The World's Most Breathtaking Ghost Towns
08Debate: Let's Stop Hating On Parents Who Want To Raise Adventurous Kids
09Guns, Roses, And Eating Insects: Adventure Cycling in Vietnam
10From Intense Trails to Intensive Care: Dicing with Death In a Mountain Biking Paradise
Jon Olsson Invitational 2011: Finals
It had to end at some time but here's highlights of the racing finals and the big air finals as Elias Ambuhl takes the victory after one hell of an evening.
Oh, and there's the obligatory bath tub filled with champagne bottles...
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NEW YORK — The Washington Nationals will open the 2013 season at home, hosting the Miami Marlins at Nationals Park on Monday, April 1 to kick off a brand new type of season in baseball.
The full schedule, announced Wednesday afternoon, features 20 interleague games as MLB transitions to a state of constant interleague play with the Houston Astros moving from the National League Central to the American League West. The schedule league-wide becomes more equitable with all teams playing 76 intra-divisional games, 66 against the rest of the league and 20 interleague.
That means the Nationals will welcome the Chicago White Sox for their first interleague series from April 9-11, will host the Detroit Tigers for two on May 7-8, and the Minnesota Twins on June 7-9. The Twins, the original Washington Senators, will return to D.C. for the first time since 1971.
The Nationals will also visit Cleveland, Detroit and Kansas City in June, July and August, respectively.
The Beltway Series with the Baltimore Orioles remains, despite the Nationals playing the rest of their interleague slate against the American League Central, but it's in a modified format. The Nationals will host the Orioles to kick off a home-and-home series that begins May 27 with two games at Nationals Park. The teams will then head up the Beltway for two more May 29 and 30 at Camden Yards.
The Nationals will also see their divisional games heavily front and backloaded next season. They'll play five series against NL East opponents in April, but only eight from May through July. Then they'll play five more in August and six in September, including 21 straight divisional games to open the final month of play.
Nationals Park will play host on three holidays, Mother's Day (May 12), Memorial Day (May 27) and Independence Day (July 4), and will see its most action from June 1 through July 28, when the Nationals play 24 of 34 games at home. Sixteen of the team's final 26 games will be played on the road.
The Nationals will end the 2013 regular season on the road in Arizona after a three-game set in St. Louis. The regular season is scheduled to wrap up on Sept. 29.
© Copyright 2015 The Washington Times, LLC. Click here for reprint permission.


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|Web Site:
|3507 Breakwater Ave
Hayward, CA 94545
United States
|Prolog Ventures
Link up your social media account to automatically promote the positions at Singulex.
Get a job alert every time Singulex posts a job.
Please fill in the information below and use the navigational buttons to continue. Warning: Using your browser's Back and Forward buttons may produce undesirable results. Description: Accounts receivable follow-up with insurance carriers. Manage accounts receivable as6/2/2015 Alameda, CA Save This Job
Qualifications: BSc, MSc, or PhD in Biological Sciences (e.g. microbiology, immunology, cell biology, biochemistry). 3+ (PhD), 7+ (MSc), 10+ (BSc) years industry experience in a regulated environment (In-Vitro Diagnostic (IVD) and immunoassay development experience6/2/2015 Alameda, CA Save This Job
At Singulex, our mission is to improve lives by advancing medical diagnostics through leveraging the power of Single Molecule Counting (SMC) technology for medical diagnostics and scientific discovery. Our advanced cardiovascular disease test menu features proprietary6/2/2015 Alameda, CA Save This Job
Qualifications: BSc, MSc, or PhD in Physics, Biophysics, Electrical Engineering, Optical Engineering or a related field with a background in biomedical optics and laser hardware, firmware and software development Minimum of 2+ (PhD), 8+ (MSc), 11+ (BSc) years industry6/2/2015 Alameda, CA Save This Job
Summary: The Billing Customer Service Representative is responsible for assisting patients, clients, payers and sales representatives with activities surrounding billing. Qualifications: Knowledge in Medical Billing process (Diagnostic Lab) Previous customer service or call5/3/2015 Alameda, CA Save This Job
Warning: Using your browser's Back and Forward buttons may produce undesirable results. This is a hands-on, first line supervisory position for the P.M. shift in the Singulex Clinical Laboratory. This supervisor is responsible for supervising the P.M.shift clinical laboratory5/3/2015 Alameda, CA Save This Job
Essential Duties and Responsibilities: With consultation may design and conduct product development experiments, assay optimization and scale-up tasks as part of a group effort coordinated by senior members of the team. May also, with consultation, be involved in planning,5/3/2015 Alameda, CA Save This Job
Description: Perform all phases of testing with accuracy and precision. Utilize the LIS for accessioning, creation of work lists, entering and approving results, and other reports as they are developed. Perform quality control and Quality Assessment activities according to5/3/2015 Alameda, CA Save This Job
Responsibilities Assist VP, Operations with ensuring compliance in regards to key state and federal regulatory requirements, including that routine data is properly documented and stored, and remedial actions are completed and documented. Communicate and interface with5/3/2015 Alameda, CA Save This Job
Qualifications: BS or AA degree in a biological science, or minimum two years college education in a biological science with laboratory work. Previous experience with manipulating and interpreting large datasets. Demonstrated excellent teamwork. Demonstrated proficiency in MS5/3/2015 Alameda, CA Save This Job
Qualifications / Required Licensure: Current California CLS license; ASCP preferred. One year of experience in a licensed clinical laboratory as a licensed CLS preferred. Compile and use embedded formulas or automatic macros to accurately review large amount of laboratory data5/3/2015 Alameda, CA Save This Job
Summary: The In-Office Phlebotomist works as a Singulex employee, typically in a physician?s office, to collect specimen samples and collect and enter patient data into the Singulex LIS system and then processing and expediting delivery to the Singulex clinical5/3/2015 Alameda, CA Save This Job


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Camping Highlights Throwback 2006 & 2007
Song: Souvenirs - Switchfoot
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Jimmy Butler still second fiddle to Bulls' lead violin (you know who)
Column | Jul 2, 2015 | 9:12 PM
Jimmy Butler still second fiddle to Bulls' lead violin (you know who)
Column | Jul 2, 2015 | 9:12 PM
Love stays, Chandler moves, dollars fly in NBA free agency
Story | Jul 2, 2015 | 11:16 AM
Kevin Love has unfinished business in Cleveland and Tyson Chandler will be conducting his in Phoenix. The first full day of NBA free agency featured skyrocketing salaries and a flurry of moves, though no decision yet from LaMarcus Aldridge. Players and...
Cavaliers break the bank on Day 1 of free agency
Story | Jul 2, 2015 | 8:29 AM
Cleveland Cavaliers owner Dan Gilbert promised LeBron James he would do whatever it took financially to surround him with a team capable of ending the city's 51-year championship drought. On the first day of free agency, Gilbert spent nearly a quarter of...
Bob Ford: Okafor selection turns Sixers away from analytics, toward the past
Story | Jul 2, 2015 | 5:20 AM
The Golden State Warriors and Cleveland Cavaliers recently concluded an NBA championship series that was a model of the leaguewide trend toward pace-and-space basketball in which running the floor, slashing to the paint and kicking out for 3-point shots...


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I need a proxy for web development debugging. Preferably something standalone so I can use it with both IE and Firefox. The one I have used so far is CharlesProxy but I was hoping to find a free one. Anyone know of a good one?
Take the 2-minute tour ×
Stack Overflow is a question and answer site for professional and enthusiast programmers. It's 100% free, no registration required.
I suggest you: OWASP Zed Attack Proxy Very complete and easy to use.
fiddler perhaps? IE goes through it automatically if its running, you have to tell FF to go through 127.0.0.1:8888 (might be wrong about port num, but its in the docs)
Oh yes, and you can also use it to generate http requests :-)
Depends on what you need to do with it.
I found TrivialProxy to be awesome and very lightweight for monitoring what is going through.


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Hotel address5 rue Stella
How to reach the hotel
Contact the hotel
- Tel. (+33)4/78375050
- Fax. (+33)4/78423334
- email@example.com
Recently viewed hotels
Mercure Lyon Centre Plaza République Hotel - hotel description
Hotel code: 2951
The 4-star Mercure Lyon Centre Plaza République is at the heart of Lyons, close to the old town, and is perfect for combining work and pleasure during your stay in the City of Light. You will love the Haussmann style of the hotel while the enhanced comfort rooms will fully satisfy you. Renovation work will be carried out at the hotel until June 5, 2015. Work is being done floor by floor, on weekdays.
Extras of this hotel
Outside public parking, meeting rooms with natural light, free WiFi, and a bar with a contemporary atmosphere where you can enjoy the dishes on the room service menu on weekdays: Mercure Lyon Plaza is an ideal place to relax or work.
Message from the hotel manager
Mr Pierre MIQUET, the manager of the Mercure Lyon Centre Plaza République Hotel hotel, would like to welcome you.
Hotel - Mercure Lyon Centre Plaza République Hotel access map
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Work in this hotel
These prices may be based on different room types. Depending on the country, these prices may not include taxes, may include VAT only or may include all taxes (VAT and city tax). When prices do not include all taxes, the relevant taxes (VAT and/or city tax) will be stated in the following stages of the reservation process. The price is only guaranteed at the time of reservation.
Hotel classifications (1 to 5 stars for France) correspond to criteria laid down by local regulations.


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- BLACK GIRLS ROCK! INC.'s mission is to encourage positive, life-changing activity and critical thinking through the arts for "at risk" teenage women of color to help foster their dreams of a better tomorrow without comprising their integrity and self-worth today.
What We're Tweeting Now
- "Misty Copeland, whose openness about race in ballet helped to make her one of the most famous ballerinas in the... fb.me/41l8KkAqj 3 days ago
- "She departed with a dream and returned home a record-breaker. Inspired by the Tuskeegee Airmen, 15-year-old... fb.me/6wcfLKAg8 3 days ago
- Now this is civil disobedience at its finest. This sister geared-up, scaled the flag pole, and removed the... fb.me/6EpazEiVQ 6 days ago
- For reminding us that #SMARTGIRLSROCK (+ boys, too!) for decades. We salute you. "Collins' work at Westside... fb.me/4drirGbvv 1 week ago
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Monthly Archives: January 2010
Help Haiti This Thursday
Come out this Thursday January, 21 to help BLACK GIRLS ROCK! support Haiti. The proceeds will benefit Doctors without Borders & The Lambi Fund of Haiti.
Posted in Events Tagged event, Haiti Leave a comment


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The Belmont Nurses Christian Fellowship (NCF) has joined forces with Passion Partners, a nonprofit missions organization, to launch their annual Pad Project campaign that collects feminine hygiene products for Ugandan and Kenyan girls who would otherwise be unable to attend school. The donations are wrapped as gifts and distributed at monthly Purity Project meetings at high schools in Wakiso and Kampala, Uganda, where girls are taught about health, self-worth and the Bible. Continue reading
Category Archives: School of Nursing
Dean Taylor Contributes to Maternal and Child Health Navigator
Dean of the College of Health Sciences Cathy Taylor was part of the original design team of national experts, supported by the Maternal and Child Health Bureau (MCHB), who worked to develop and launch the MCH Navigator beginning in 2010.
The MCH Navigator is an online portal and clearinghouse for maternal and child health professionals, students and others working to improve the health of women, children, adolescents and families for training on key MCH and leadership topics. The project contains in-depth training portals on specific issues of importance to public health professionals and highlights learning opportunities focused on MCH topics.
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DNP students attend National Policy Summit in Washington DC
Four doctor of nursing practice (DNP) students attended the AACN Student Policy Summit held in Washington, DC earlier this week. The students were immersed in program sessions focused on the federal policy process and nursing's role in professional advocacy. Aspart of the summit they made visits to capitol hill with Associate Dean of Nursing Dr. Martha Buckner, meeting with legislative staff of the senate Health, Education, Labor and Pension committee. Student Jennifer Jaramillo said, "The policy summit was such an amazing opportunity to advocate for nurses in Tennessee and across the nation. Building a network with other nursing leaders was an invaluable experience that will enrich our future practices. Advocacy and policy play a major role in our nursing practice and the future of our profession."
Belmont MSN graduates achieve 100% first-time pass rate for 11th straight year
Since 2004, all graduates of Belmont's Masters of Science in Nursing (MSN) program for Family Nurse Practitioners (FNP), totaling 150 students, have passed the nursing certification exam on their first attempt. The most recent class of 28 graduates passed the exam this spring.
Dean of the College of Health Sciences Cathy Taylor said, "This is marvelous recognition for our outstanding students and a testimonial to the passionate dedication of an expert graduate nursing faculty team led by Dr. Leslie Higgins. We are so proud of this remarkable accomplishment."
The School of Nursing began offering its MSN degree 20 years ago and with the creation of the Doctorate of Nursing, the graduate programs have grown to a record enrollment of 87 students in the fall of 2014. Prepared to practice in a variety of settings, FNPs provide primary health care to families and individuals of all ages. Graduates from Belmont's program have gone on to practice in pediatrics, genetics, family practice and public health, among others.
Lucille Turmel, a 2012 Belmont graduate, found Belmont's post-master's certification program directly fit her needs and prepared her for a career as an FNP. Currently working in a private practice in Washington, Turmel said she enjoys working with all ages of patients and has recently developed a passion for teenagers and young adults.
While at Belmont, Turmel said the best part of her education was the "personal attention and guidance from Dr. Higgins and the faculty and staff…I felt very well prepared and was employed two months after leaving Belmont."
The advanced practice nursing examination for FNPs is administered by the American Credential Center (ANCC) and validates nursing skills, knowledge and abilities. Since 1990, more than a quarter million nurses have been certified by ANCC and over 80,000 advanced practice nurses are currently certified by the ANCC. The certification is accepted by governing boards throughout the U.S. as well as insurers and the military.
Nursing student completes military training programs
We missed this post several months ago, but the accomplishment is worth recognizing even a bit late.
As posted by Nashoba Publishing in Ayer, Massachusetts
Elizabeth M. Lochiatto, an Army Reserve Officers' Training Corps cadet at Belmont University, Nashville, Tenn., has graduated from the Leader Development and Assessment Course at Fort Knox, Ky.
The 29 days of training provide the best possible professional training and evaluation for all cadets in the aspects of military life, administration and logistical support. Although continued military training and leadership development is included in the curriculum, the primary focus of the course is to develop and evaluate each cadet's officer potential by exercising the cadet's intelligence, common sense, ingenuity and physical stamina. The cadet command assesses each cadet's performance and progress in officer traits, qualities and professionalism while attending the course.
Cadets usually attend LDAC between their junior and senior years of college, and they must complete the course to qualify for commissioning. Upon successful completion of the course, the ROTC program, and graduation from college, cadets are commissioned as second lieutenants in the U.S. Army, Army National Guard or Army Reserve.
Lochiatto has also completed the Nurse Summer Training Program.
This program assigns cadets who are nursing majors to Army medical facilities throughout the continental United States as well as in Germany, where they develop and practice leadership skills in a clinical environment. The cadets work side-by-side with an Army Nurse Corps officer preceptor.
Belmont's MSN program Included on U.S. News' 2016 Graduate School Rankings
Belmont University's Gordan E. Inman College of Health Sciences and Nursing was lauded last week when U.S. News and World Report released its 2016 rankings of Best Graduate Schools, a tool to help prospective graduate students better understand the graduate school landscape and identify potential programs. Belmont's Masters of Science in Nursing (MSN) ranked at No. 115, up from No. 234 in the 2011 rankings.
The MSN ranking is based on average peer assessment score, average undergraduate GPA, acceptance rate, faculty resources, student-faculty ratio and research activities, among others. In addition to its recent U.S. News ranking, Belmont's MSN program has seen great success through the first time pass rate of graduates. For the 11th consecutive year, graduates of the MSN program for Family Nurse Practitioners have achieved a 100 percent first time pass rate on the nursing certification exam totaling 150 student graduates since 2004.
2015 Mission to Guatemala: Final Day
Team Nursing/Pharmacy
from Pharmacy Faculty Members
Elisa Greene & Edgar Diaz-Cruz, and
Nursing Faculty Member Jamie Adam
Our team returned safely to Nashville just before midnight last night with exhausted, yet fulfilled students and faculty. This unique Springbreak experience gave students an incredible opportunity to be immersed not only as American students in the Guatemalan culture, but also as an inter-professional healthcare team. Nursing, OT, Pharmacy students and a student majoring in Communications, learned how to leverage each other's strengths to provide quality care to the people of Guatemala. Within the majors, students had various degrees of experience. Graduate students were mentoring undergraduates, seniors were mentoring freshman, and faculty were facilitating meaningful inter-professional learning experiences.
As most international trips go, students and faculty were challenged to be flexible about their own expectations and use the unexpected as "teachable moments." In addition, our team had to learn to manage the people's expectations of what we could provide. There was much we could offer, but in some cases, we had to acknowledge our own limitations. Regardless of whether we could identify a problem or a need, our patient might not be able to afford a physician or the medications needed. Continue reading
2015 Mission to Guatemala: Day 6
Two separate teams of health science students are in Guatemala over Spring Break this year. One team consists of nursing and pharmacy students. The other includes OT and PT students. Both team are writing about their experiences.
Team Nursing/Pharmacy
from Gabi Okoniewski, Danielle Marshall
& Tori Powers
A week ago, none of us knew what exactly we were getting into as we traveled to Antigua, Guatemala. Now a week later, our expectations of this trip have been far exceeded and our perspectives have changed. Not only have we learned about the culture here in Antigua, but we have also learned more about our own culture in America. Collaborating with the different professions this week has given us all a greater appreciation for the different disciplines in the healthcare field. This was a great experience that could not be matched by any other.
Top reasons why YOU should come on the Guatemala Spring Break Immersion Trip
-There is 75 degree weather everyday and there are NO mosquitos!
-The creation of new friendships.
-Cultural compentance within the city.
-Having the ability to interact with the kids at both the coffee plantation school and the God's Children School. Continue reading
2015 Mission to Guatemala: Day 5
Two separate teams of health science students are in Guatemala over Spring Break this year. One team consists of nursing and pharmacy students. The other includes OT and PT students. Both team are writing about their experiences.
Team Nursing/Pharmacy
from Erin Todd, Erin Oakley,
Kandice Squires, Noah Ploegman &
Justin Beasley
Today our group drove to San Luca, a small town ten minutes from Antigua, to visit a pharmaceutical manufacturer called PharmaDel. Here we had the unique opportunity to observe the medication manufacturing process first-hand. It was exciting for us as future pharmacists to follow a medication from its raw form into its final packaged product ready to be distributed across Central America. We were impressed with the level of dedication and integrity demonstrated by plant employees.
After observing these processes for both solid and liquid medication formulations, our tour then shifted focus from the manufacturing aspect towards PharmaDel`s quality assurance measures. The technology and lab techniques used to evaluate the purity of the medication produced are very similar to those which we use in our country. Special air pressurization, filtration systems, and room design functioned to enhance sterility and prevent contamination. PharmaDel is working towards becoming certified by the World Health Organization as meeting their highest standards for pharmaceutical manufacturing. It was surprising to our group that most of the drug development and quality assurance measures were performed by pharmacists, however, the guide explained to us that this is a common role for pharmacists in Guatemala. To become licensed, students complete 5 years of post-high school general and pharmacy education, plus 6 months each in a hospital and laboratory practice setting. Finally, they complete an internship in one of these areas, which often leads to employment. Continue reading
2015 Mission to Guatemala: Day 4
Two separate teams of health science students are in Guatemala over Spring Break this year. One team consists of nursing and pharmacy students. The other includes OT and PT students. Both team are writing about their experiences.
Team Nursing/Pharmacy
from Maddy Clarke Williams & Alexis Rhea-Anne Pendley
Language may be regional, but love is universal.
Today, we had no expectations of where we were going or what we would be doing. Upon arriving, we came to realize we were at a school that was tangible proof of how one man's work can establish a strong community for roughly 150 students in Antigua, Guatemala. Some twenty years ago, a man's heart was touched by God's radiating beauty that he experienced through the children he met on a mission trip. He was inspired to give back to the children who touched him. With the little money he had, he was able to purchase a Big Mac and split it twenty ways, so every child to which he was ministering was able to have some. The man went on to create a school for these orphans that has developed through the years and now provides a safe and empowering environment. We soon realized our purpose at the school differed from our previous experiences at the coffee plantation.
To begin our day, we taught the children the importance of nutrition and basic hygiene, such as washing hands and brushing teeth. To assist in our teachings, we taught the students a simple, yet catchy, song about washing their hands. After practicing this song with them several times, they requested more songs. With humble hearts and shaky voices, our group managed to quiet the area by singing Amazing Grace. Many of us later remarked on the power of the moment; though the students may not have understood the words we sang, they definitely appeared to share the same content feelings. Afterwards, the children were eager to share with us a few of the songs they had learned at school. Continue reading


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Kayak fishermen have a distinct advantage over their powerboating counterparts. The sleek, human-powered craft are silent as they slip across the grass flats. The fish don't stand a chance.
Kayak fishing has become the fastest growing segment of the recreational angling market, with the plastic, sit-on-top boats the vessel of choice for many inshore fishermen.
Some of the best paddle fishermen in the state will head to Tampa on Feb. 19-20 for the Whiskey Joe's Mammoth Slam Classic. This photo & release tournament will target redfish and trout. February is an ideal month to fish the grass flats of the Central Gulf Coast. Organizers expect anglers to come from all over the Southeast to compete in this kayak fishing season opener.
Anglers can register online. Participants will also earn points to qualify for the Invitational National Championship.


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I live in Albany with my best friend.
I love adventures, going to concerts, and impromptu road trips with friends. I like going out and meeting new people.
I'm a big fan of getting out as much as possible, but I'm open to being a homebody on weekends. Also, naps are amazing and cannot be overestimated.
Basically I'm down for trying new things, but I also like getting into a routine or having familiar things to fall back on, like favorite hot spots or restaurants. If you make me breakfast there's bonus points, since it's my favorite meal at ANY time of the day.
I like to run, hike, I'm learning to play tennis.


text:
Originally written on Fox Sports | Last updated 9/8/13
The Green Bay Packers found a way to keep Colin Kaepernick in the pocket and stop all those read-options runs that embarrassed them in the playoffs. They just had no answer for his arm. Or his newest receiver. Kaepernick threw for a career-high 412 yards and three touchdowns to outduel Aaron Rodgers, and the San Francisco 49ers held off the Packers 34-28 on Sunday in the season opener for both teams. ''It's disappointing when we play our best game on offense and we got to score more points,'' Rodgers said. Rodgers threw for 333 yards and three touchdowns with one interception. Jordy Nelson caught seven passes for 130 yards, and Randall Cobb had seven for 108 yards. The Packers lost to the 49ers for the third time in a year - and eight months after that 45-31 defeat in a playoff game at Candlestick Park. Anquan Boldin caught 13 passes for 208 yards and a touchdown in his 49ers debut after coming over in a trade from the Super Bowl champion Baltimore Ravens in the offseason. T...
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General Laboratory Techniques
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Mayuresh M Abhyankar1, Sarah M Haviland1, Carol A Gilchrist1, William A Petri, Jr.1
1Division of Infectious Disease and International Health, University of Virginia Health System
We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.
Published December 12, 2010. Keywords: Infectious Disease, Entamoeba, negative selectable marker, 5-fluorocytosine, gene knockout, Cytosine deaminase, UPRT CMFDA.