text:
- Research article
- Open Access
- Open Peer Review
Identification and validation of differentially expressed transcripts by RNA-sequencing of formalin-fixed, paraffin-embedded (FFPE) lung tissue from patients with Idiopathic Pulmonary Fibrosis
- Milica Vukmirovic†7Email authorView ORCID ID profile,
- Jose D. Herazo-Maya†7,
- John Blackmon1,
- Vesna Skodric-Trifunovic2, 3,
- Dragana Jovanovic2, 3,
- Sonja Pavlovic4,
- Jelena Stojsic5,
- Vesna Zeljkovic6,
- Xiting Yan7,
- Robert Homer8, 9,
- Branko Stefanovic†1 and
- Naftali Kaminski†7
© The Author(s). 2017
- Received: 14 April 2016
- Accepted: 20 December 2016
- Published: 12 January 2017
Idiopathic Pulmonary Fibrosis (IPF) is a lethal lung disease of unknown etiology. A major limitation in transcriptomic profiling of lung tissue in IPF has been a dependence on snap-frozen fresh tissues (FF). In this project we sought to determine whether genome scale transcript profiling using RNA Sequencing (RNA-Seq) could be applied to archived Formalin-Fixed Paraffin-Embedded (FFPE) IPF tissues.
We isolated total RNA from 7 IPF and 5 control FFPE lung tissues and performed 50 base pair paired-end sequencing on Illumina 2000 HiSeq. TopHat2 was used to map sequencing reads to the human genome. On average ~62 million reads (53.4% of ~116 million reads) were mapped per sample. 4,131 genes were differentially expressed between IPF and controls (1,920 increased and 2,211 decreased (FDR < 0.05). We compared our results to differentially expressed genes calculated from a previously published dataset generated from FF tissues analyzed on Agilent microarrays (GSE47460). The overlap of differentially expressed genes was very high (760 increased and 1,413 decreased, FDR < 0.05). Only 92 differentially expressed genes changed in opposite directions. Pathway enrichment analysis performed using MetaCore confirmed numerous IPF relevant genes and pathways including extracellular remodeling, TGF-beta, and WNT. Gene network analysis of MMP7, a highly differentially expressed gene in both datasets, revealed the same canonical pathways and gene network candidates in RNA-Seq and microarray data. For validation by NanoString nCounter® we selected 35 genes that had a fold change of 2 in at least one dataset (10 discordant, 10 significantly differentially expressed in one dataset only and 15 concordant genes). High concordance of fold change and FDR was observed for each type of the samples (FF vs FFPE) with both microarrays (r = 0.92) and RNA-Seq (r = 0.90) and the number of discordant genes was reduced to four.
Our results demonstrate that RNA sequencing of RNA obtained from archived FFPE lung tissues is feasible. The results obtained from FFPE tissue are highly comparable to FF tissues. The ability to perform RNA-Seq on archived FFPE IPF tissues should greatly enhance the availability of tissue biopsies for research in IPF.
- Idiopathic Pulmonary Fibrosis
- RNA-Seq
- Microarray
- DEGs
- Validation
- Pathways
- Network
- NanoString nCounter®
Idiopathic Pulmonary Fibrosis (IPF) is a chronic interstitial lung disease of unknown etiology associated with high mortality rates, and increased prevalence with age (14-42.7 cases per 100,000 population) [1]. IPF patients have an overall median survival of approximately 3.5 years from the onset of symptoms. The disease is characterized by progressive scaring of the lung parenchyma that leads to loss of lung function [2]. IPF is thought to result from repeated cycles of alveolar epithelial cell injury leading to fibroblast proliferation, exaggerated accumulation of extracellular matrix in the lung parenchyma and recapitulation of developmental pathways [3, 4].
Currently, IPF diagnosis is based on the exclusion of known causes of lung fibrosis, and the presence of a Usual Interstitial Pneumonia (UIP) pattern on High-Resolution Computed Tomography scan (HRCT) in patients who do not undergo lung biopsy or the combination of a permissive HRCT and the UIP pattern on surgical lung biopsy [5]. The UIP histology pattern is characterized by spatial and temporal heterogeneity, which refers to patchy distribution of dense parenchymal scar along with areas of fibroblast and myofibroblast accumulation and proliferation, known as fibroblastic foci, alternating with areas of less affected or normal lung parenchyma [5, 6]. While lung biopsies were frequently performed to identify the typical UIP pattern as part of the IPF diagnostic workup, the success of HRCT in demonstrating "UIP" radiological patterns has considerably limited the number of lung biopsies currently being performed [7]. That in turn has led to a decline in the availability of tissues for IPF research in general, and for transcriptomic profiling in particular.
Transcriptomic profiling in IPF has largely been performed by microarrays using RNA obtained from whole lung lysates obtained from fresh frozen tissues. These studies have provided significant mechanistic insights regarding IPF pathogenesis, and have largely impacted the field of lung fibrosis [4, 8]. However, these studies are limited because acquisition of fresh frozen tissues is only available in highly specialized academic centers with tissue banking facilities. Thus, the majority of studies contain mostly tissues explanted from patients with IPF at the time of biopsy, and studies containing tissues obtained from diagnostic biopsies are limited. Additionally, it is nearly impossible to assess the lung morphology on frozen tissue, thus the studies utilizing fresh frozen samples depend on histological assessment of adjacent tissue which may or may not contain the exact same pathology. While transcriptomic data generated from different dissections within a single lobe of the lung are highly correlated [9], and that transcriptomic data correlates well with UIP pattern itself [10, 11] the lack of visual confirmation of the histology of the region profiled is still considered a limitation [12].
RNA isolated from Formalin-Fixed Paraffin Embedded FFPE tissues is partially degraded, thus transcriptomic analysis of FFPE tissues was considered challenging [13, 14]. Several recent studies demonstrated that transcriptomic analysis of FFPE tissues using microarrays was possible, was nearly comparable to fresh frozen tissues but still had significant limitations [15–17]. In contrast to microarrays, next generation RNA Sequencing (RNA-Seq) allows for relatively unbiased measurements of expression levels across the entire length of a transcript and its level of expression [18], and therefore may be more suitable for sequencing of partially degraded FFPE RNA. Transcriptomic analysis of FFPE tissues by RNA-Seq demonstrates high concordance to RNA-Seq data produced from matching fresh frozen tissues [19–23]. Because formalin fixation and paraffin embedding is routinely done on all samples from clinically indicated lung biopsies, optimization of a method to perform genome scale transcript profiling of archived FFPE tissues will greatly enhance the access to IPF lungs.
FFPE Tissue specimens
Lung FFPE biopsies were obtained from departmental FFPE archives [Clinic for Pulmonology in Belgrade, n = 8, and the Lung Tissue Research Consortium (LTRC), n = 4] according to Institutional Review Board (IRB) approved study protocols. Informed consents to participate in the study were also obtained according to IRB. The median archival age of FFPE tissues was 6 years. IPF (n = 7) lung and control (n = 5) FFPE tissues were used for RNA-Seq. Clinical, demographic and histopathological features of the subjects in the study were analyzed by a multidisciplinary group of clinicians and pathologists to confirm IPF diagnosis [5].
FFPE RNA isolation and quality control
Five 10-μm slices of the whole lung tissue were cut from FFPE block, excess paraffin was trimmed and slices were treated twice with 1ml xylene for 30 min at 62°C, then washed twice with 100% ethanol as previously described [25]. Total RNA was isolated by using MasterPure kit (Epicentre Biotechnologies). The final RNA concentration and purity (A260/A280) was measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). RNA quality and RNA integrity (RIN) was assessed using a 2100 Bioanalyzer (Agilent). For each FFPE tissue block, two consecutive RNA isolations from the whole lung tissue slices (5x10-μm) were performed.
Fresh frozen tissue specimens
The Lung Genomic Research Consortium (LGRC) contains data for over 200 IPF patients and controls. To have similar size cohorts we picked 35 age and gender matching samples (19 IPF and 16 normal histology samples) and reanalyzed the data. The experiments were approved by the Institutional Review Board. These samples are publically available at GSE47460 and at the LGRC website - http://www.lung-genomics.org) and have been previously described at [24, 26].
RNA-Seq library preparation and paired-end RNA-Seq
Approximately 1.5μg of total RNA was isolated from each FFPE block (tissues size ~10mm x 7mm). FFPE RNA showed fragmentation in a range of ~100-150bp. To increase the depth of RNA-Seq sequencing and mapping rate of sequencing reads [19, 27], ribosomal RNA was removed by using the RiboZero rRNA removal kit (Epicentre) prior to cDNA library preparation. Double stranded cDNA library was prepared by using NEBNext® Ultra™ Directional RNA Library Prep kit for Illumina (New England Biolabs) following manufacturer's protocol from 1ug of total RNA. Kappa cDNA library quality control was performed prior to pooling libraries for flow cell amplification. All cDNA libraries were sequenced using Illumina HiSeq 2000 to produce 50 million reads, 50bp paired-end reads with multiplexing (4 samples/lane) (cDNA library preparation and RNA-Seq run was performed by Genomic Service Lab at Hudson Alpha).
RNA-Seq reads processing and alignment
TopHat2 [28] was used to map the sequencing reads to the human genome (UCSC hg19) by allowing multiple hits. Mapping rate was calculated as the percentage of read that were properly mapped. Samples with low mapping rates (<20%) were discarded from further analysis. Cufflinks 2.2.0 [29] was used to calculate FPKMs values as the estimated gene expression levels. FPKMs between IPF and controls were compared using Cuffdiff and genes with a false discovery rate (FDR) adjusted p < 0.05 were identified as differentially expressed genes (DEGs) [30]. Cuffdiff test assigns a status of genes as: OK - test successful, NOTEST - not enough alignments for testing, LOWDATA - too complex or shallowly sequenced, HIDATA - too many fragments in locus, or FAIL - an ill-conditioned covariance matrix or other numerical exception prevents testing [31]. In this study we report genes with OK status as genes with sufficient coverage (Additional file 1: Table S1). Multidimensional scaling (MDS) analysis was performed for data visualization [32].
Microarray data
We used previously generated microarray data (Agilent). Briefly, the normalization of the gProcessed signal was performed using cyclic-LOESS and bioconductor package. Complete datasets and protocols were previously published [24, 26], and deposited in data repository GEO (accession no. GSE47460), and are also available in the Lung Genomics Research Consortium's (LGRC) website (http://www.lung-genomics.org/). Fold change and FDR values were calculated using the Significance Analysis of Microarrays (SAM) tools. Microarray experiments were compliant with MIAME guidelines.
Comparison of IPF signatures obtained from FFPE RNA-Seq and from microarray analysis of fresh frozen tissue
Considering that nearly all of the transcriptomic data in IPF was generated on fresh frozen tissues we compared IPF signatures obtained with FFPE RNA-Seq (Illumina) with those obtained from Microarray (Agilent) gene expression data. Unique gene probes from microarrays (n = 16,741) were matched with genes having sufficient coverage by RNA-Seq (n = 15,149) by the gene symbol which provided a matched set of 13,304 genes. For analysis of differentially expressed genes, Significance Analysis of Microarrays (SAM) was used in the case of microarrays, and Cuffdiff was used in the case of RNA-Seq. Significance was defined as FDR adjusted p < 0.05. A discordant gene was defined as a significant gene that was increased in microarray but decreased in RNA-Seq and vice versa. The fold change (FC) of each gene was calculated by dividing the average mean value of IPF group by the average mean value of control group for both RNA-Seq and microarray. Log2FC is the log base 2 transformed FC. Log2FC of microarray were plotted against Log2FC of RNA-Seq values for matching genes between the two platforms.
Pathway enrichment analyses
Pathway enrichment analysis of the common Differentially Expressed Genes (DEGs) between the two techniques, RNA-Seq and Microarrays, was performed using MetaCore (Thomson Reuters). In this way, we identified the top 13 statistically significant enriched pathways with FDR adjusted p value <0.1 for pathways of common increased genes, and the top 50 statistically significant enriched pathways with FDR adjusted p < 0.1 for pathways of common decreased genes. Gene candidates with fold change > 1 from top pathways were presented in the heatmap to show the distribution of IPF relevant genes among the pathways. To analyze the interaction between members of the gene network in RNA-Seq and microarray data independently, networks were built around MMP7, one of the most-widely studied IPF-relevant genes [33]. When building the network, we used all RNA-Seq (4,131) and all microarray (5,859) differentially expressed genes. With auto-expand and canonical options, the network was built and drawn by plotting genes from datasets to pre-built MetaCore network for MMP7. Members of the gene networks with their expression values and interactions were compared.
NanoString nCounter® gene expression quantification and validation
100ng of RNA isolated from fresh frozen lung tissues and 250ng of RNA isolated from FFPE lung tissues, as suggested by the NanoString protocol, were used in experiment. 7 CTRL and 8 IPF FF samples, and 5 CTRL and 7 IPF FFPE samples were used for validation. Probe set for each gene were designed and synthesized by NanoString nCounter®. For validation, we focused on genes that had at least two-fold change in at least one dataset. Thus we selected 10 discordant genes, 10 genes that were significantly differentially expressed only in one dataset and 15 concordant genes (Additional file 2: Table S5). The discordant genes were: IFNG, BCL11B, PDPR, ADAM33, VPS13B, TNRC18, PPBP, PHYHIP, KRT14, ITLN1. The differentially expressed genes in only one dataset were: MMP1, COL1A1, SERPIND1, ADAM23, COL1A2, MMP9, WISP1, WNT2, FOS, FGG. The concordant genes were: SERPINE1, CXCL2, MMP19, CAV1, CTNNA1, WNT10A, MMP7, SPP1, CXCL13, PLA2G2A, POSTN, LAMA3, COL17A1, SLC6A4, ANKRD1. We used RNA isolated from 7 IPF and 5 CTRL FFPE tissues and RNA isolated from 8 IPF and 7 CTRL fresh frozen tissues from the LGRC cohort. We have followed a standard manufacturer protocol for sample preparation, hybridization and detection. Data were analyzed using nSolver 3.0 digital analyzer software.
Quality of RNA isolated from FFPE tissues
Consistent with previous reports [15, 25] the integrity of RNA isolated from FFPE tissues was decreased probably due to formalin fixation and archival time. All RNA isolations had OD260/280 > 1.9 confirming the high purity of RNA (data not shown). The RIN numbers were in the range of 2.1–2.6 and the most abundant RNA fragments were in the range of 100–150 ribonucleotides for all samples (Additional file 3: Figure S1) suggesting a similar degradation and quality regardless whether FFPE tissues were obtained from controls or IPF patients. Repeat isolations of RNA per FFPE block had very similar fragmentation and the same RIN number (Additional file 3: Figure S1). Archival time had no effect on RNA quality. We proceeded with one of the RNA isolation per FFPE block for cDNA library preparations and RNA-Seq analysis.
Mapping, transcript quantification and analysis of differentially expressed genes of FFPE RNA-Seq data
Summary of RNA-Seq (FFPE) gene mapping
RNA Seq
Orig Reads
QC failed reads
Unmapped Reads
Mapped Reads
Proper hits
Mapping Rate
Hits Rate
Proper hits Rate
Comparison of differentially expressed genes of FFPE RNA-Seq to Microarray analysis of fresh frozen tissues
FFPE RNA-Seq results contain IPF relevant biological information
NanoString nCounter® gene expression validation in fresh frozen and FFPE tissues
Our study demonstrates that transcriptomic analysis of RNA isolated from FFPE IPF lung biopsies by RNA-Seq is feasible and the results comparable to those obtained from gene expression microarrays. RNA-Seq resulted in an average of ~116 million 50 bp reads per sample with average of ~ 62 million mapped reads (Table 1). Our depth of sequencing, 62 million mapped reads, allowed for the detection of total of 15,149 genes with sufficient coverage, out of which 4,131 were differentially expressed between IPF and control (FDR adjusted p < 0.05). To validate the RNA-Seq FFPE results, we compared them to gene expression microarrays obtained from FF tissues and identified overlapping differentially expressed genes. The overlap was statistically significant. The common genes were enriched for signaling pathways relevant to IPF such as: ECM remodeling process, WNT, TGF-β, NFAT, IL-8 in angiogenesis, CCL2 signaling and PEDF signaling [8, 36, 38, 39] and network analyses of both datasets revealed similar networks suggesting that FFPE RNA-Seq generated information that was relevant to IPF and comparable if not perfectly identical to FF tissue. Validation by NanoString nCounter® of concordant, discordant and dataset specific genes largely confirmed the results. Taken together, these findings demonstrate the feasibility and validity of RNA-Seq FFPE data and its relevance to IPF.
Although we provide strong evidence about the validity of our transcriptome RNA-Seq analysis, there are several discrepancies present when comparing RNA-Seq and microarray data. In addition to detecting the commonly expressed genes, we also detected differentially expressed genes that are discordant or do not overlap between RNA-Seq and microarray (Fig. 3b, gray and white dots on Fig. 3a, Additional file 4: Table S4). Computationally, we used MMP7 as a model gene to build the gene network and assess the potential bias in detecting gene interacting candidates from two datasets due to the presence of discordant or non-overlapping genes. For network analysis, we took into consideration all differentially expressed genes in RNA-Seq (4,131) and in microarray (5,859) independently. Out of 15 differentially expressed genes from RNA-Seq and 21 differentially expressed genes from microarray that were found in the MMP7 network, 14 overlapped between datasets. 7 genes were only differentially expressed in microarray and one in RNA-Seq (Fig. 5). These 8 genes do not overlap between datasets (Fig. 3b, white dots on Fig. 3a) suggesting differences between datasets. To validate the results experimentally, and determine whether the results of comparison of different methodologies (microarrays vs RNA-Seq), or sample type (un-paired, FF vs FFPE) or other reasons we validated expression 35 genes: 10 discordant genes, 15 concordant genes, and 10 genes significant in only one dataset, using the NanoString nCounter®, a high precision system that measures gene expression based on digital color-coded barcode technology that provides significant accuracy and sensitivity and has been used successfully in partially degraded RNA samples. For most of the genes, directionality and significance of gene expression changes was confirmed. The number of discordant genes has been decreased with Nanostring nCounter® suggesting that differences in methodologies accounted for at least some of the differences.
The most significant limitations of our study are the small number of samples and the fact that we did not have paired FFPE and FF tissue from the same anatomical location in the same patient. Despite these limitations we found a significant overlap in differentially expressed genes in both tissue types, a significant overlap in functional annotations of the functional annotations of these genes, and good correlation between our Nanostring nCounter® validation with either microarray analysis of FF tissue or RNA-Seq analysis of FFPE tissue. Our results are in agreement with recent observations that RNA-Seq analysis of FFPE tissues can generate valid and comparable gene expression to FF tissues [16, 20, 27]. In cancer tissues high correlation was observed between RNA-Seq of paired FF and FFPE tissues [19–22, 27, 47]. Important to note, that in all of those studies authors mention that while the information derived from FF or FFPE tissue is comparable, comparisons should be limited within one type of tissue processing. Direct comparison of FFPE diseased tissues to control FF tissues for example, would be highly confounded and probably generate spurious results.
The RNA we isolated from FFPE tissues was partially degraded as previously observed [13–17, 19, 25]. However, while we used FFPE biopsies that had a wide range of archival times, and handling performed at different hospitals, we did not find systematic differences between the tissues. Of 12 FFPE biopsies, we experienced low number of original reads and low mapping rate of only one FFPE sample was indistinguishable from other samples. Archival age, RNA and cDNA quality were similar to the other samples. This could be the result of conditions that cannot be directly observed such as minor changes in fixation technique, or storage that could induce changes in the RNA structures [21]. Thus, in our relatively small study the technical success of RNA-Seq from FFPE tissue was 92%. It is plausible, that in prospectively designed studies and standardized fixation protocols the success rate would be even higher [48].
To the best of our knowledge, our study is the first to demonstrate the feasibility of RNA-Seq of FFPE IPF lung samples. We hope and believe that the availability of our protocols, as well as our results, will facilitate the use of FFPE tissue for genome scale transcript profiling of IPF. This will overcome the limitation on availability of FF tissues and increase the capacity for transcriptomic profiling of IPF [47, 49, 50].
Our study serves as a proof of principle, that RNA-Seq performed on RNA isolated from archival FFPE IPF lung tissues is feasible, and reveals a gene expression profile relevant for IPF. This study further shows that there is a high concordance between RNA-Seq (FFPE) and microarray (FF) expression profiles for biopsies performed on different patients, and at different hospitals encouraging the further usage of FFPE biopsies. Taking into consideration the great potential for transcriptomic research, FFPE tissues should be considered to overcome limitations in the availability of FF human lung tissues.
Differentially expressed gene
Fold change
Formalin fixed paraffin embedded
Fragments per kilobase of exon per million
High-resolution computed tomography
Idiopathic pulmonary fibrosis
Institutional Review Board
The lung genomic research consortium
Multidimensional scaling
Minimum information about a microarray experiment
Next generation sequencing
Optical density
RNA Integrity number
- RNA-Seq:
RNA sequencing
Ribonucleic acid
- rRNA:
Ribosomal RNA
Significance analysis of microarrays
Usual interstitial pneumonia
RNA quality assessment, cDNA library preparation and RNA-Seq run were performed by Genomic Service Lab at Hudson Alpha. We thank Cynthia Vied and Michelle Arbeitman from College of Medicine, Florida State University, for valuable discussions regarding the study design and RNA-Seq.
Work was supported in part by National Institutes of Health grant R01HL127349 to NK, Harold Amos Faculty development program of the Robert Wood Johnson Foundation Award and the Pulmonary Fibrosis Foundation to JHM and the FSU College of Medicine Internal Seed Grant to BS, JB, MV.
Availability of data and materials
The microarray datasets supporting the conclusions of this article are available in the GEO repository (accession no. GSE47460 and http://www.lung-genomics.org ).
The RNA-Seq dataset, representing normalized gene expression values (FPKM), and RNA-Seq vs Microarray dataset, representing comparison of gene expression values, supporting the conclusions of this article are included within the article supplement files and uploaded to GEO under GSE83717.
Authors' contributions
MV conceived the study, participated in study design and coordination, data collection and analysis, drafted the manuscript, JHM participated in study design, data collection and analysis, helped to draft the manuscript, JB helped conceive the study, participated in study design and histopathological evaluation of FFPE tissues, VST helped conceive the study, participated in FFPE tissues collection, clinical evaluation of IPF patients and study design, DJ participated in FFPE tissues collection, clinical evaluation of IPF patients and study design, VZ participated in literature review, helped with FFPE tissues collection, study design and coordination, SP participated in data analysis and valuable discussions, JS and RH participated in FFPE tissues collection and histopathological analysis, XY participated in RNA-seq data analysis, BS conceived the study, participated in study design, data collection and analysis and helped to draft the manuscript, NK participated in study design, generation of microarray and NanoString nCounter data, led data analysis and drafted the manuscript. All authors reviewed and approved the final manuscript.
Competing interests
NK consulted Biogen Idec, Boehringer Ingelheim, Third Rock, MMI, and Pliant and received a grant from Biogen Idec and non-financial support from with MiRagen, all outside the submitted work. In addition, NK has a patent New Therapies in Pulmonary Fibrosis licensed to Quitsa/SLI, and a patent on Peripheral Blood Gene Expression as diagnostic in Pulmonary Fibrosis. All of NK's competing interests are outside the submitted work. All other authors declare no competing interests.
Consent for publication
Not applicable.
Ethics approval and consent to participate
This study used Lung FFPE biopsies from the departmental FFPE archives of the Clinic for Pulmonology in Belgrade (n = 8), and the NHLBI funded Lung Tissue Research Consortium (n = 4) as previously described [51]. The use of samples was approved by the Institutional Review Boards (IRB) at the Clinical Center of Serbia (approval number 4072/54), University of Pittsburgh (approval number IRB0411036) and Yale School of Medicine (approval number 1409014689). Informed consents were obtained as appropriate according to IRB.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Authors' Affiliations
- Raghu G, Weycker D, Edelsberg J, Bradford WZ, Oster G. Incidence and Prevalence of Idiopathic Pulmonary Fibrosis. Am J Respir Crit Care Med. 2006;174(7):810–6.View ArticlePubMedGoogle Scholar
- King Jr TE, Pardo A, Selman M. Idiopathic pulmonary fibrosis. Lancet. 2011;378(9807):1949–61.View ArticlePubMedGoogle Scholar
- Selman M, Pardo A. Idiopathic pulmonary fibrosis: an epithelial/fibroblastic cross-talk disorder. Respir Res. 2002;3:3.View ArticlePubMedGoogle Scholar
- Kass DJ, Kaminski N. Evolving genomic approaches to idiopathic pulmonary fibrosis: moving beyond genes. Clin Transl Sci. 2011;4(5):372–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Raghu G, Collard HR, Egan JJ, Martinez FJ, Behr J, Brown KK, Colby TV, Cordier J-F, Flaherty KR, Lasky JA, et al. An Official ATS/ERS/JRS/ALAT Statement: Idiopathic Pulmonary Fibrosis: Evidence-based Guidelines for Diagnosis and Management. Am J Respir Crit Care Med. 2011;183(6):788–824.View ArticlePubMedGoogle Scholar
- Smith M, Dalurzo M, Panse P, Parish J, Leslie K. Usual interstitial pneumonia-pattern fibrosis in surgical lung biopsies. Clinical, radiological and histopathological clues to aetiology. J Clin Pathol. 2013;66(10):896–903.View ArticlePubMedPubMed CentralGoogle Scholar
- Kaarteenaho R. The current position of surgical lung biopsy in the diagnosis of idiopathic pulmonary fibrosis. Respir Res. 2013;14:43.View ArticlePubMedPubMed CentralGoogle Scholar
- Herazo-Maya JD, Kaminski N. Personalized medicine: applying 'omics' to lung fibrosis. Biomark Med. 2012;6(4):529–40.View ArticlePubMedPubMed CentralGoogle Scholar
- Kim SY, Diggans J, Pankratz D, Huang J, Pagan M, Sindy N, Tom E, Anderson J, Choi Y, Lynch DA, et al. Classification of usual interstitial pneumonia in patients with interstitial lung disease: assessment of a machine learning approach using high-dimensional transcriptional data. Lancet Res Med. 2015;3(6):473–82.View ArticleGoogle Scholar
- DePianto DJ, Chandriani S, Abbas AR, Jia G, N'Diaye EN, Caplazi P, Kauder SE, Biswas S, Karnik SK, Ha C, et al. Heterogeneous gene expression signatures correspond to distinct lung pathologies and biomarkers of disease severity in idiopathic pulmonary fibrosis. Thorax. 2015;70(1):48–56.View ArticlePubMedGoogle Scholar
- Yang IV, Coldren CD, Leach SM, Seibold MA, Murphy E, Lin J, Rosen R, Neidermyer AJ, McKean DF, Groshong SD, et al. Expression of cilium-associated genes defines novel molecular subtypes of idiopathic pulmonary fibrosis. Thorax. 2013;68(12):1114–21.View ArticlePubMedGoogle Scholar
- Maher TM. Transcriptional phenotyping of fibrotic lung disease: a new gold standard? Lancet Res Med. 2015;3(6):423–4.View ArticleGoogle Scholar
- Farragher SM, Tanney A, Kennedy RD, Paul Harkin D. RNA expression analysis from formalin fixed paraffin embedded tissues. Histochem Cell Biol. 2008;130(3):435–45.View ArticlePubMedGoogle Scholar
- Gilbert MT, Haselkorn T, Bunce M, Sanchez JJ, Lucas SB, Jewell LD, Van Marck E, Worobey M. The isolation of nucleic acids from fixed, paraffin-embedded tissues-which methods are useful when? PLoS One. 2007;2(6):e537.View ArticlePubMedPubMed CentralGoogle Scholar
- Bibikova M, Talantov D, Chudin E, Yeakley JM, Chen J, Doucet D, Wickham E, Atkins D, Barker D, Chee M, et al. Quantitative gene expression profiling in formalin-fixed, paraffin-embedded tissues using universal bead arrays. Am J Pathol. 2004;165(5):1799–807.View ArticlePubMedPubMed CentralGoogle Scholar
- Frank M, Döring C, Metzler D, Eckerle S, Hansmann M-L. Global gene expression profiling of formalin-fixed paraffin-embedded tumor samples: a comparison to snap-frozen material using oligonucleotide microarrays. Virchows Arch. 2007;450(6):699–711.View ArticlePubMedGoogle Scholar
- Hosey AM, Gorski JJ, Murray MM, Quinn JE, Chung WY, Stewart GE, James CR, Farragher SM, Mulligan JM, Scott AN, et al. Molecular basis for estrogen receptor alpha deficiency in BRCA1-linked breast cancer. J Natl Cancer Inst. 2007;99(22):1683–94.View ArticlePubMedGoogle Scholar
- Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet. 2009;10(1):57–63.View ArticlePubMedPubMed CentralGoogle Scholar
- Liu Y, Noon AP, Aguiar Cabeza E, Shen J, Kuk C, Ilczynski C, Ni R, Sukhu B, Chan K, Barbosa-Morais NL, et al. Next-generation RNA Sequencing of Archival Formalin-fixed Paraffin-embedded Urothelial Bladder Cancer. Eur Urol. 2014.Google Scholar
- Li P, Conley A, Zhang H, Kim H. Whole-Transcriptome profiling of formalin-fixed, paraffin-embedded renal cell carcinoma by RNA-seq. BMC Genomics. 2014;15(1):1087.View ArticlePubMedPubMed CentralGoogle Scholar
- Hedegaard J, Thorsen K, Lund MK, Hein AM, Hamilton-Dutoit SJ, Vang S, Nordentoft I, Birkenkamp-Demtroder K, Kruhoffer M, Hager H, et al. Next-generation sequencing of RNA and DNA isolated from paired fresh-frozen and formalin-fixed paraffin-embedded samples of human cancer and normal tissue. PLoS One. 2014;9(5):e98187.View ArticlePubMedPubMed CentralGoogle Scholar
- Xiao YL, Kash JC, Beres SB, Sheng ZM, Musser JM, Taubenberger JK. High-throughput RNA sequencing of a formalin-fixed, paraffin-embedded autopsy lung tissue sample from the 1918 influenza pandemic. J Pathol. 2013;229(4):535–45.View ArticlePubMedPubMed CentralGoogle Scholar
- Sinicropi D, Qu K, Collin F, Crager M, Liu M, Pelham R, Pho M, Dei Rossi A, Jeong J, Scott A, et al. Whole transcriptome RNA-Seq analysis of breast cancer recurrence risk using formalin-fixed paraffin-embedded tumor tissue. PLoS One. 2012;7:e40092.View ArticlePubMedPubMed CentralGoogle Scholar
- Bauer Y, Tedrow J, de Bernard S, Birker-Robaczewska M, Gibson KF, Guardela BJ, Hess P, Klenk A, Lindell KO, Poirey S, et al. A Novel Genomic Signature with Translational Significance for Human Idiopathic Pulmonary Fibrosis. Am J Respir Cell Mol Biol. 2014;52(2):217–31.View ArticleGoogle Scholar
- Glenn ST, Jones CA, Liang P, Kaushik D, Gross KW, Kim HL. Expression profiling of archival renal tumors by quantitative PCR to validate prognostic markers. Biotechniques. 2007;43(5):639–40. 642-633, 647.View ArticlePubMedGoogle Scholar
- Kim S, Herazo-Maya JD, Kang DD, Juan-Guardela BM, Tedrow J, Martinez FJ, Sciurba FC, Tseng GC, Kaminski N. Integrative phenotyping framework (iPF): integrative clustering of multiple omics data identifies novel lung disease subphenotypes. BMC Genomics. 2015;16:924.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhao W, He X, Hoadley KA, Parker JS, Hayes DN, Perou CM. Comparison of RNA-Seq by poly (A) capture, ribosomal RNA depletion, and DNA microarray for expression profiling. BMC Genomics. 2014;15(1):419.View ArticlePubMedPubMed CentralGoogle Scholar
- Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 2013;14(4):R36.View ArticlePubMedPubMed CentralGoogle Scholar
- Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010;28(5):511–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Storey JD. The positive false discovery rate: a Bayesian interpretation and the q -value. Ann Stat. 2003;31(6):2013–35.View ArticleGoogle Scholar
- Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc. 2012;7(3):562–78.View ArticlePubMedPubMed CentralGoogle Scholar
- Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics (Oxford, England). 2010;26(1):139–40.View ArticleGoogle Scholar
- Rosas IO, Richards TJ, Konishi K, Zhang Y, Gibson K, Lokshin AE, Lindell KO, Cisneros J, Macdonald SD, Pardo A, et al. MMP1 and MMP7 as potential peripheral blood biomarkers in idiopathic pulmonary fibrosis. PLoS Med. 2008;5(4).Google Scholar
- Kaminski N, Allard JD, Pittet JF, Zuo F, Griffiths MJD, Morris D, Huang X, Sheppard D, Heller RA. Global analysis of gene expression in pulmonary fibrosis reveals distinct programs regulating lung inflammation and fibrosis. Proc Natl Acad Sci. 2000;97(4):1778–83.View ArticlePubMedPubMed CentralGoogle Scholar
- Kaminski N. Microarray analysis of idiopathic pulmonary fibrosis. Am J Respir Cell Mol Biol. 2003;29(3 Suppl):S32–36.PubMedGoogle Scholar
- Selman M, Pardo A, Barrera L, Estrada A, Watson SR, Wilson K, Aziz N, Kaminski N, Zlotnik A. Gene Expression Profiles Distinguish Idiopathic Pulmonary Fibrosis from Hypersensitivity Pneumonitis. Am J Respir Crit Care Med. 2006;173(2):188–98.View ArticlePubMedGoogle Scholar
- Boon K, Bailey NW, Yang J, Steel MP, Groshong S, Kervitsky D, Brown KK, Schwarz MI, Schwartz DA. Molecular phenotypes distinguish patients with relatively stable from progressive idiopathic pulmonary fibrosis (IPF). PLoS One. 2009;4(4):e5134.View ArticlePubMedPubMed CentralGoogle Scholar
- Konishi K, Gibson KF, Lindell KO, Richards TJ, Zhang Y, Dhir R, Bisceglia M, Gilbert S, Yousem SA, Song JW, et al. Gene expression profiles of acute exacerbations of idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2009;180(2):167–75.View ArticlePubMedPubMed CentralGoogle Scholar
- Selman M, Pardo A, Kaminski N. Idiopathic pulmonary fibrosis: aberrant recapitulation of developmental programs? PLoS Med. 2008;5(3):e62.View ArticlePubMedPubMed CentralGoogle Scholar
- Konigshoff M, Balsara N, Pfaff EM, Kramer M, Chrobak I, Seeger W, Eickelberg O. Functional Wnt signaling is increased in idiopathic pulmonary fibrosis. PLoS One. 2008;3(5):e2142.View ArticlePubMedPubMed CentralGoogle Scholar
- Vuga LJ, Ben-Yehudah A, Kovkarova-Naumovski E, Oriss T, Gibson KF, Feghali-Bostwick C, Kaminski N. WNT5A is a regulator of fibroblast proliferation and resistance to apoptosis. Am J Respir Cell Mol Biol. 2009;41(5):583–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Liu RM. Oxidative stress, plasminogen activator inhibitor 1, and lung fibrosis. Antioxid Redox Signal. 2008;10(2):303–19.View ArticlePubMedPubMed CentralGoogle Scholar
- Wang XM, Zhang Y, Kim HP, Zhou Z, Feghali-Bostwick CA, Liu F, Ifedigbo E, Xu X, Oury TD, Kaminski N, et al. Caveolin-1: a critical regulator of lung fibrosis in idiopathic pulmonary fibrosis. J Exp Med. 2006;203(13):2895–906.View ArticlePubMedPubMed CentralGoogle Scholar
- Lappi-Blanco E, Lehtonen ST, Sormunen R, Merikallio HM, Soini Y, Kaarteenaho RL. Divergence of tight and adherens junction factors in alveolar epithelium in pulmonary fibrosis. Hum Pathol. 2013;44(5):895–907.View ArticlePubMedGoogle Scholar
- Zuo F, Kaminski N, Eugui E, Allard J, Yakhini Z, Ben-Dor A, Lollini L, Morris D, Kim Y, DeLustro B, et al. Gene expression analysis reveals matrilysin as a key regulator of pulmonary fibrosis in mice and humans. Proc Natl Acad Sci U S A. 2002;99(9):6292–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Reis PP, Waldron L, Goswami RS, Xu W, Xuan Y, Perez-Ordonez B, Gullane P, Irish J, Jurisica I, Kamel-Reid S. mRNA transcript quantification in archival samples using multiplexed, color-coded probes. BMC Biotechnol. 2011;11:46.View ArticlePubMedPubMed CentralGoogle Scholar
- Ley B, Brown KK, Collard HR. Molecular biomarkers in idiopathic pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol. 2014;307(9):L681–91.View ArticlePubMedPubMed CentralGoogle Scholar
- Waldron L, Simpson P, Parmigiani G, Huttenhower C. Report on emerging technologies for translational bioinformatics: a symposium on gene expression profiling for archival tissues. BMC Cancer. 2012;12:124.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhang Y, Kaminski N. Biomarkers in idiopathic pulmonary fibrosis. Curr Opin Pulm Med. 2012;18(5):441–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Borensztajn K, Crestani B, Kolb M. Idiopathic pulmonary fibrosis: from epithelial injury to biomarkers--insights from the bench side. Respiration. 2013;86(6):441–52.View ArticlePubMedGoogle Scholar
- Kusko RL, Brothers 2nd JF, Tedrow J, Pandit K, Huleihel L, Perdomo C, Liu G, Juan-Guardela B, Kass D, Zhang S, et al. Integrated Genomics Reveals Convergent Transcriptomic Networks Underlying Chronic Obstructive Pulmonary Disease and Idiopathic Pulmonary Fibrosis. Am J Respir Crit Care Med. 2016;194(8):948–60.View ArticlePubMedGoogle Scholar


text:
Providing quality and affordable PLR article re-writing service since March 2006!
How Much Patience Do You Have? | Article Ghostwriting | PLR Report Rewriting
PLR Report Writing | PLR Packs | Article Shop - Buy UNIQUE Niche Articles | Keyword Density Checker
Article Ghostwriting
Does your web site or blog need infused with fresh, high quality content? Are you looking to establish yourself as an expert in article directories like EzineArticles.com? Do you need informative, well-researched articles to use in your e-zines? Are you looking to start your own PLR article site and you need articles to sell?
Let me be your ghostwriter!
I've been a ghostwriter since 2003, working with only a handful of clients so I'd have time for my other writing projects. Over the past 2 years, however, many Custom-PLR-Articles.com clients and visitors have been inquiring if I offer article writing too (I do, but I wasn't advertising it). A number of clients who started out hiring me to re-write PLR articles ended up hiring me to craft original articles for them regularly. So I've decided to offer my ghostwriting services. My rates are reasonable and you can be sure you'll receive quality articles.
Quality matters a lot to me, and I'll work with you in nailing down your requirements before I start work. I'll give you a reasonable turn around time, but if you're thinking I can deliver 100 articles in 7 days, I'm telling you outright -- that's not going to happen. Quality work takes time; and I want to deliver articles to you that you'll be proud to attach your name to (or pass on to your customers).
And yes, you get ALL rights to the articles you hire me to write for you. This means you can edit them, sell them, publish them under your name, anything you want to do with them -- the sky's the limit. I'm not even going to include them in my portfolio. (I've got plenty of articles with my byline that I can use as samples anyway.)
My rates are reasonable. In fact, they are much lower than the rates I charge on an article brokerage site where I have sold over 400 articles ranging from $30 to $85 per piece. My ghostwriting rates vary depending on the length of articles, and they include research and Copyscaping. You can be sure that you'll receive 100% Copyscape and original articles. Delivery depends on how many articles you order, although my standard turn around time for 10 articles is 10 days.
Mini-articles/blog entries, 150 to 200 words
Rate: $5 each, minimum order of 10, delivery in 10 days or less
Orders between 25 and 50 articles, delivery within 30 days
Short articles, 250 to 350 words
Rate: $8 each, minimum order of 10, delivery in 10 days or less
Orders between 25 and 50 articles, delivery within 30 days
Medium length articles, 400 to 500 words
Rate: $10 each, minimum order of 10, delivery in 10 days or less
Orders between 25 and 50 articles, delivery within 30 days
Long articles, 550 to 700 words
Rate: $12 each, minimum order of 10, delivery in 10 days or less
Orders between 25 and 50 articles, delivery within 30 days
Very long articles, 750 to 1,000 words
Rate: $15 each, minimum order of 10, delivery in 10 days or less
Orders between 25 and 50 articles, delivery within 30 days
Bulk orders
Ask me about bulk orders (and discounts)
If you'd like to hire me as your ghostwriter, shoot me an e-mail! It's sheryruss AT gmail DOT com. Tell me your article topic and I'll let you know if I can write articles based on your topic. I'm not going to say I can write just about anything, because I can't!
There are topics I know I can't write (highly technical stuff like MySQL, programming languages, etc.) simply because I don't have the background and therefore I can't honestly write authoritatively about them. And then there are topics I won't write (adult industry, pornography, sex, racial or religious discrimination, witchcraft and paganism, astrology and horoscopes, warez, pirating, the trafficking of illegal substances, illicit drugs, guns or any other items, pyramid and get-rich-quick schemes, gambling and terrorism) because they go against my beliefs, ethics and values.
Once we've finalized your article needs and requirements, I'll send you a Paypal invoice. I'll get started on your articles as soon as I receive payment notification.
Before you hire me as your ghostwriter, I encourage you to look at samples of articles I've written first. These articles are representatives of the quality of the work you'll receive from me. Just send me an e-mail at sheryruss AT gmail DOT com and request my writing portfolio.
To your online success,
Shery Russ
Your Custom PLR Articles expert

subset: commoncrawl


text:
Louis Riel
A Comic-strip BiographyBook - 2006
From the critics
Community Activity
CommentAdd a Comment
Brown presents a fascinating way to engage with history. The story can seem over-simplified at times, but overall this is a neat way to learn about Riel.
"When he shall die,
Take him and cut him out in little stars,
And he will make the face of heaven so fine
That all the world will be in love with night
And pay no worship to the garish sun." — Will Shakespeare
Louis Riel: A Comic-Strip Biography by Chester Brown centres around Louis Riel, a Métis man who founded the province of Manitoba and fought for democratic recognition of the Métis people in the new government of Canada. The biography focuses primarily on his antagonistic relationship to the government of Canada and the Red River Rebellion.
While it only focuses on a small portion of Riel's life and often misses huge sections of history, I found this biography to be very accessible and provided excellent recommendations for further reading, many of which I have placed on hold at the library or already purchased.
If you're a Canadian high school or elementary school teacher I highly recommend reading this book as it provides a first-person perspective for Riel and makes some of the repetitive political history more readable and accessible. While I wouldn't recommend this tome for elementary school kids, I think some key pages could be used as a helpful discussion piece in any classroom.
… and oh my god, what a book this is. Sometimes, I had to close it and clutch it to my chest because Brown has written this so thoughtfully. This is well-researched and contains footnotes for many of the characters or historical instances in this biography, but that is not what makes it great. The artwork is simple, black and white, almost stark, but effective in its execution. Brown knows how to write silences, but also draw them. His empty or minimalist panels were some of my favourites and I fully support all his creative decisions. What makes it great is the quiet moment Brown takes with Riel, shows his uncertainty, his doubts, his concerns, his compassions.
I fell in love with the character Brown had crafted through his research, and I believed. If you know anything about Riel, you know how this story ends — but I believed in his cause, I believed in him.
I love this, so so much.
I think this is a great way of learning about the life of Louis RIel and not just him but more! And honestly it's better then read a large textbook.
Chester Brown's characters are reminiscent of Herge or Little Orphan Annie in this graphic version of Canadian folk hero Louis Riel's courageous struggle with the government of Upper Canada. Perfect to share with a high school student.
In the canon of historical graphic novels, this is a particular treat. Brown is a master of dialogue who moves the story forward without devoting pages to monologues and rants. The scope of Riel's life is equally impressive, and Brown is excellent at showing the reader his transformation.
Very worthwhile and a quick read.
A classic.
Quite simply: WOW. Mr. Brown, Thank You. This is ART! Every panel should be blown up and all displayed in the National Gallery of Canada. Nuff said.
Okay, one more thing: Why is this always found in the Children's section of the Central Library? Is it because of the "Oh, it has cartoons, it must be for children" mentality of library staff? DISRESPECTFUL.
Made history much more enjoyable until I read all the notes. Recommended read to all those non-history people. I learned so much about Canada.
SummaryAdd a Summary
The story of the charismatic nineteenth century Metis leader, whose struggle to win rights for his people led to violent rebellion on the Canadian frontier.
QuotesAdd a Quote
"He shall hang though every dog in Quebec bark in his favour." (pg.233)
Age SuitabilityAdd Age Suitability
There are no age suitabilities for this title yet.
NoticesAdd Notices
There are no notices for this title yet.
View originally-listed edition


text:
OK, kiddo! Here are all the fantastically amazing posts tagged with Jamaica
Check out this trailer for An Abominable Crime, a documentary about the rampant (and often ignored) homophobia problem in Jamaica. The filmmakers are currently fundraising to help get this film finished, and The Pulitzer Center has offered to match all Kickstarter donations. So go check it out and offer some support if you can.
iTunes Canada Pulls Homophobic Music
iTunes Canada has removed several songs by Jamaican artists Elephant Man, TOK, and Buju Banton because they call on listeners to murder gay men. Stop Murder Music Canada and Egale called on Apple to remove the songs, which contained lyrics translating to "Join our dance and let's burn the queer man" and "Boom Boom, queers must be killed."
Although removing the songs has caused some controversy about freedom of expression and censorship, one thing is clear: they're not legal. Speech calling for the murder and hatred of an identifiable group violates Canadian hate laws, and Stop Murder Music Canada has called on other music retailers—such as HMV, Amazon.ca, and Archambault Musique—to follow Apple's suit.
Jamaican dancehall music is notorious for its homophobic lyrics. Homophobia is rampant in Jamaica, with 43 lynch mob attacks on gay men reported in 2007 alone, resulting in the murder of at least 10 gay men.
Another Gay Jamaican Seeks Asylum In Canada
Michael Hayden, a gay police officer, is the latest refugee from Jamaica to seek asylum in Canada.
Michael received multiple death threats after he spoke out about Jamaica's deadly homophobia problem in a local newspaper this year. He now fears for his life and has gone into hiding, desperately seeking to escape to Canada:
I want to stay here [in Jamaica] and fight, but it's not safe for me. My life is in great, great jeopardy.
Despite Jamaica's image as an easygoing paradise for tourists, it is among the worlds most deadly places for gay people. Earlier this month, Gareth Henry, one of Jamaica's few gay activists, escaped to Canada after 13 of his friends were killed by lynch mobs.
The good news, however, is that with every new refugee, more attention is drawn to Jamaica's deadly secret. If Canada and other governments become aware enough of the issue to put out traveler warnings, Jamaica's tourism industry can be leveraged to stop this horrific violence.
- Gay Jamaican officer seeks asylum [Toronto Star]
- Gay policeman seeks asylum in Canada [Radio Jamaica]
Prize-Winning Zombie Basketballs
In my web travels, I often collect stories that I intend to share, but then become distracted by newer, shinier stories—or feel too lazy to illustrate them. This is what becomes of those stories: a Pile o' Slaps!
Dr. Brent Hawkes, a Toronto pastor and gay rights activist, has received the prestigious Order of Canada in recognition of his fight toward equal rights for gay people. I shall start the betting odds at 946341:¾ that an anti-gay lobbyist will burst a capillary over this within a week.
The rift between Canadian Anglicans is continuing to grow. A Vancouver congregation has voted to leave the country in favour of The Province of the Southern Cone in South America, which does not think of gay people as favourably. Authorities in The Province of the Southern Cone expressed overwhelming gratitude toward the Vancouver church for knowing that The Province of the Southern Cone exists.
The gay-targeting zombie lynch mobs in Jamaica are finally getting some media attention thanks in part to Gareth Henry's fight to stay in Canada. A New York Times feature on Jamaica describes some of the horror. (Hat tip to Montreal Simon)
Speaking of Canadian Refugees, Capital Xtra has an excellent article outlining the frustrating process that refugee claimants must go through to prove they're gay after years of effort to hide their sexuality in their home country. It's a great lesson for future refugees: If you truly desire to protect yourself, put yourself in as much visible danger as possible.
A sports photographer has angered and confused Kentuckians after a newspaper published a photo of two male basketball players hugging in celebration of their victory. Columnist Pam Platt, in a display of common sense, offers no apology to the scores of offended readers, who will move on to picket the guy that accidentally brushed up against them on the public transit.
Gay Jamaican Seeks Refuge in Canada
Jamaica is marketed as a vacation paradise, but for gay people, the danger can be shocking.
Gareth Henry, a Jamaican gay activist, has had 13 of his friends murdered by lynch mobs in the past four years, and has now fled to Canada to seek refugee status.
Sadly, Canada has an atrocious track record of protecting endangered gay refugees. Alvaro Orozco was ordered to be deported to Nicaragua in October because his adjudicator didn't believe he was gay. Leonardo Zuniga, a Mexican claimant, also had his refugee plea rejected last summer despite the threat of anti-gay violence in his home country. With Jamaica's popular image as an easy-going tourist spot, Gareth Henry might not have better luck.
Jamaica's perception needs to be challenged; the country's most popular musicians habitually call for the murder of gay people, and the public acts accordingly. Montreal Simon regularly reports on Jamaica's horror stories (I'm borrowing his recurring zombie island motif), but very little attention is paid by the mainstream media—and Jamaican police are often mob members themselves.
I wish Gareth the best of luck with his claim and hope his stories will gain the attention they deserve. If governments become aware enough of the issue to put out traveler warnings, Jamaica may finally be forced to stop the madness.
NDP Boycotts Homophobic Artists
The federal New Democrats have called for a boycott of Jamaican musicians Elephant Man and Sizzla. Both are currently touring in Canada, though several venues have canceled performances and refunded tickets after the nature of their lyrics, which calls for the murder of gays, was brought to their attention.
NDP MP Bill Siksay said that these sort of performances have no place in Canada:
I hope that fans of Jamaican dancehall music will appreciate that a vibrant musical tradition should not be used as a cover for the promotion of hatred. I hope that they will choose to boycott performances.
A spokesperson for Elephant Man offered a signed declaration stating that anti-gay songs would not be performed, but Stop Murder Music, a Jamaican activist group, said that past declarations aren't "worth the paper [they're] printed on," once the artist returns to Jamaica, where anti-gay violence is rampant.
- NDP calls for boycott of anti-gay & lesbian artists [NDP]
- Censors win: Elephant Man's Ottawa show canned because of past anti-gay lyrics [Capital Xtra]
- Controversial rapper heading for London [London Free Press]


text:
Cytomegalovirus is a member of the Herpesviridae family. It may be transmitted from person to person through urine, saliva, other bodily fluids, breast milk and transplanted organs. It is a common infection which mostly passes unnoticed. The infection may be dangerous to people who are immunocompromised such as those who are HIV positive or organ transplant recipients. The infection may also be dangerous to pre- and post-natal infants. Antiviral drugs are useful in the treatment of CMV infection.
Product Details
- Target Species
- Viral
- Product Form
- Inactivated Cytomegalovirus - liquid
- Preparation
- Cytomegalovirus strain AD-169, cultured in MRC-5, a diploid cell line of human lung origin. MRC-5 monolayers are infected in a manner which will ensure that the full host of viral proteins are present upon harvest. Optimally infected tissue is collected in glycine buffered saline. The cells are disrupted to release cellular CMV antigen and cellular debris is removed by centrifugation. The antigen preparation is inactivated using gamma radiation, which primarily damages viral genetic material.
- Buffer Solution
- Glycine buffered saline
- Preservative Stabilisers
- None present
- Activity
- This product has been rendered inactive by standard procedures. However this material should still be handled as infectious and should be disposed of appropriately.
- Approx. Protein Concentrations
- Total protein concentration 3.5 mg/ml
Storage Information
- Storage
- Store at -70oC.
Storage in frost-free freezers is not recommended.
This product should be stored undiluted. Avoid repeated freezing and thawing as this may denature the protein.
- Guarantee
- 18 months from date of despatch
More Information
- Regulatory
- For research purposes only
Applications of Cytomegalovirus
|Application Name
|Min Dilution
|Max Dilution
Where this product has not been tested for use in a particular technique this does not necessarily exclude its use in such procedures. Suggested working dilutions are given as a guide only. It is recommended that the user titrates the product for use in their own system using appropriate negative/positive controls.
- Instructions For Use
- PIP005 should be sonicated immediately before use to ensure the preparation is uniform. The product may be used in a variety of immunoassay formats or may be further purified to meet the requirements of a particular assay format.
Copyright © 2019 Bio-Rad Antibodies (formerly AbD Serotec)


text:
One of the most overlooked book marketing strategies is adequately preparing for your book launch.
I know it's tempting to give yourself some time to breath after the blood, sweat and tears you put in to getting the book ready for publication, but you really have to plan out your book marketing as well if you want the book to take off – and you want to avoid wasting precious time and losing out on those new release sales.
So first, download our free book marketing planner if you haven't already. It's really a great way to stay organized.
Then I want you to review the book launch cheat sheet I've outlined below.
For those of you who are thinking you'll hold off because your book is already out – don't fall into that trap of delaying your authorship education!
Having this knowledge and keeping these details on your radar for your next book will be invaluable, so start preparing yourself for a successful book launch today, whether your next release is next month, or next year. (If you'd like to know more about the best time to launch your book, read this.)
5-6 Months Out from Your Book Launch
- What's your launch plan? Don't wait till the last minute to create a solid launch plan to work from. (To learn more, read this article about when to start your book marketing.)
- Decide which magazines, media, bloggers or publications you'll target and do that early! Get your list ready and begin your research.
- Want endorsements? While they aren't mandatory, if you're interested in seeking blurbs from others in the industry, start on this early. Of all of the things I do when it comes to a campaign, this is often the trickiest because people get busy, or misplace the books I sent them. It's a long process. Accept that, and you'll be fine.
- Interested in doing speaking? Pitch yourself as early as you can and mention you have a book coming out. It's often more effective to pitch a speaker with a book because it's an easy way to prove you have content people need.
- Finish up your cover design. Ensure it aligns with what's topping the charts for the genre(s) you want to compete it – if the look is way off (even if you love it) – I suggest working with a designer to come up with something commercially sound. Covers sell.
- Don't have any social media set up? Now is the time! And start peppering them with teasers, special announcements, and help them get to know you.
3-4 Months Out from Your Book Launch
- Make sure your website is ready to go and you're creating blog content in advance.
- Pitching national magazines begins now, so do some smart research on which angles to use based on what's happening in your industry, in our country as a whole, if you can align yourself with current events people care about you're more likely to get picked up.
- Order your bookmarks, business cards, or any other collateral book marketing pieces you plan to use.
- Now might be a good time to refresh any branding on your social media sites, including header graphics, pinned posts, your about section, etc.
- If you want to do author events in bookstores, now would be a good time to start pitching them. If they're local, walk in and introduce yourself.
- What about local groups and associations that tie into your topic or are popular with your reader market? Pitch them in this window as well.
2 Months Out from Your Book Launch
- Your website is live and active!
- National broadcast media is being pitched.
- Newspapers around the country are being pitched.
- Indie bookstores are being pitched.
- Library pitching happens now too so walk in and introduce yourself and get involved in events, spending time around people who work with books can be key.
- Distribution should be confirmed by now or started ASAP.
- If you have an email list, now is a good time to warm it up.
- If you don't have one, create a basic landing page with a great giveaway offer or freebie to start collecting emails. A sample of the first few chapters, or a free worksheet, is perfect!
- Set up a giveaway on Goodreads for print copies.
- Claim your Bookbub profile and start making recommendations.
If your book is up for pre-order on Amazon, make sure you fine-tune your Amazon Author Central options.
1 Month Out from Your Book Launch
- Local media pitching begins.
- Planning a contest or special giveaway? If you are, it's good to get this out there early.
- If you're doing a cover reveal, this is a great time to share it with your networks.
- Blogger pitching starts now.
- How about that newsletter? Maybe it's time for another update to check in with people who have signed up.
- Consider scheduling a Facebook Live event on launch day.
- Do your Amazon keyword and category research and get those included with your book.
- Here are 13 solid pre-order strategies for selling more books.
Ideas For Book Launch Week
- Social, social, social, focus on engagement, teasers, giveaways.
- Try out a BOGO offer, and a few other ways to keep engagement up.
- Post comments on any positive reviews that start coming in, let readers know you appreciate their feedback.
- Get active in your Goodreads groups to gain some name recognition within your target markets.
- Run some Bookbub ads that let you target popular authors in your genre.
- Check in with any media or bloggers you sent books to, stay on their radar.
- Schedule a discount eBook promotion to happen in a couple weeks to rev up hits to your Amazon.
- Start filling out the next couple months of our free book marketing planner so you feel confident you're not leaving any opportunities on the table!
The ideas for your book launch week and that first month really are extensive.
We have so many great blog posts with free ideas, resources and tips for how to maximize on your DIY book promotion.
And of course I'm always happy to do coaching with you if you want to brainstorm some really unique and fantastic pre-book launch strategies or book marketing approaches that really dial in to your unique needs as a fiction author or business building goals as a non-fiction author.
And if you have a word of warning for new authors, please post in the comments the ONE thing you wish you would have been different with your last book launch, it's amazing what we can learn from each other!
Love this post? I've made it easy for you to share with these tweets below!
Click to Tweet: Here's a pre-publication cheat sheet with everything you'll need for your book launch via @bookgal #authortips #bookmarketing https://wp.me/p6TMt8-6OB
Click to Tweet: Looking for ideas in the months leading up to your next book launch? Here's what you need to know! via @bookgal #authortips #bookmarketing https://wp.me/p6TMt8-6OB


text:
The melt pot is out coz i found a brand new use for it..
It makes a fab little baking oven for your polymer clay, just set the heat to 110 and then leave your clay embellishments to cook for about half and hour, hay presto there done, its awesome.
I'll try and upload what i made in the next day or too.
Happy hopping peeps, You should know the drill by now, head over to Julia's blog to start the woyww fun HERE!


text:
Hi, my name is Nina. I am very new to SEO and would like to learn as much as I can about it to make money part time.
So I have watched a few videos already and have a few questions:
1- Creating content is very easy for me. If I do that, and create the same page with different headlines (that
include cities), will google penalize me? For example, let's say I write an article on the relationship between
gingivitis and obesity. And as the headline I put, "Alpine Obesity and Gingivitis" for a page, and for another I
put, "Jersey City Obesity and Gingivitis" and they have the SAME content. Will google penalize me for this?
The goal here is to be able to rank first on google for these terms and rent these sites out to local businesses.
2- What is the best way to make $500/month? I want to see if I can achieve this goal. From what I have gathered,
is that there are two ways: 1- copy and paste and rent sites out, and 2- create content and get visitors and
put affiliate links. What are your opinions?


text:
The East Franklin Fire Department would like to recognize Chief Dan Krushinski and congratulate him as he was recently asked to become a part of the Recruitment and Retention Review Board for FEMA and the National Volunteer Fire Council.
This board is a panel of experienced members of the Fire Service, who are looking to update the recruitment and retention process on a National Level, In order to better Volunteer Fire Departments across the country. Chief Krushinski along with several other key persons, will be brainstorming ideas to update the current FEMA Recruitment and Retention Manual which has not been changed since 2007.
Thank you to Deputy Fire Administrator Dr. Denis Onieal and Chairman Kevin Quinn of NVFC for inviting our Chief to be a part of bettering our Volunteer Fire Service.


text:
General Neurologist Jobs in Las Cruces, New Mexico
Found 1 jobs matching your criteria
Seeking a Neurologist Physician for Las Cruces, NM
General Neurology - Las Cruces, New Mexico MountainView Regional Medical Center Las Cruces, NM a Great Place for Families and Great Climate! Las Cruces is a sunny climate located close to the mountains and a lake for easy access to all kinds of outdoor activities. With four mildly distinct… View Job Details » Save to Dashboard


text:
How Do We Do What We Do...
Our Chiropractic
All chiropractors are unique. Although we all address the spine with our craft, we do not work all the same.
our techniques
Are you after a particular technique that your/friend's chiropractor used? Are you concerned about the thought of your bones clicking and popping? Are you aware that there is more than one way to adjust someone besides making their spine 'crack'?
Our Chiropractors utilise safe and gentle chiropractic techniques, and attend regular seminars and workshops to ensure that their knowledge and skills are current and up-to-date. That means that you receive the best chiropractic care possible.
Chiropractors use different ways of analysing spinal subluxations and delivering the chiropractic adjustment. We are often asked if we know a certain technique: whether for its method of analysis or mode of delivering the adjustment. Below are some of the chiropractic techniques that we employ in our office:
- Diversified: Traditional manual delivery of chiropractic adjustments using hands, specific positioning of the patient and the delivery of a short, sharp thrust. This is the most common method of delivering chiropractic adjustments in the world.
- Gonstead: The delivery method of the Gonstead technique is similar to Diversified. Our chiropractors limit the use of the Gonstead method of analysing the spine.
- Activator: Commonly referred to as 'the Clicker-gun', we use the Activator to provide gentle, low-force adjustments.
- Sacro-Occipital Technique (SOT): Both a method of analysing brain-body function and delivering chiropractic adjustments, SOT utilises gentle, low-force chiropractic adjustments using bio-mechanical blocks/wedges under the pelvis to help stabilise spinal function.
- Thompson Drop-piece: This gentle, low-force method of adjusting utilises the specially-designed drop sections of our leather-bound tables to restore stability and proper motion.
- Impulse Adjusting System: Commonly referred in our office as 'the adjusting gun', this instrument delivers short, rapid, controlled thrusts into the spine using rubber-tipped attachments. It can address both spinal subluxations and tight, tense muscles.
Our approach
Our approach is centred on the most important person in the world: YOU.
Every person who enters our office and seeks our help is unique. People come in to our office with differing ailments, backgrounds, histories, agendas and goals. Our Chiropractors' job is to:
- Help make sense of our patients' complaints and condition,
- Provide easy to understand explanations and analysis, without medical jargon,
- Advise on the care needed,
- Assist in setting meaningful goals and creating strategies to achieving them.
With this approach, we believe that we empower our clients to take control of their health and restore their faith in their body's ability to be healthy. At Point Cook Chiropractic, the ball is always in your court.
Ready to make an appointment? Call us on 9394 8888 or click here to book online!
Suite 807, 1st Floor, 5 Main Street, POINT COOK, VICTORIA, AUSTRALIA 3030
Ph: 03 9394 8888 | Email: firstname.lastname@example.com
© Copyright 2014 | Powered by Yola.com


text:
Birth, coming of age, weddings and unions, career changes, moves, coming into or leaving a relationship, spiritual practice or lifestyle changes, dying, crossing over, and all other crossroad moments in life can become more precious, magical, powerful and integrated into our psyches when we ritualize them.
Acknowledging the moments in our lives and the lives of others when things drastically change, those threshold moments in time when we cross from one way of being to another, these acknowledgments are ways we come more wholly into our selves and our right-sized sense of power. Sometimes, these rites are solitary; something we must do alone. At other times, we long for a community of witnesses encircling and supporting us. Regardless of our approach, these rituals can have a profound, healing and beautiful impact.
When we do these rites, we support our growth, build a legacy of rich community and re-weave a torn tapestry of connection to each other, the earth and ourselves. A group or individual can invite wholeness, support, meaning and unity when they undergo a ritualized rite of passage. When we do these rites, these rituals, something magic happens.
Whether you are celebrating, grieving, making a major life transition or seeking support, meaningful rituals can anchor focus, healing and power, restore and nourish those parts of yourself longing for deeper connection and create the memories of a lifetime.
I offer to help you, your partner or your group custom design the ideal rite of passage that ritualizes your intentions. I also offer to priestess your rite or to mentor you or your facilitators in leading this type of ritual.


text:
American Welding Society or AWS welding standards are shared with ANSI International. The AWS D1 requirements outline the requirements for welding metal and metal alloys. For example, ANSI/AWS D9.1 is the usual for sheet steel welding. ANSI/AWS D10.12 outlines the recommended procedures for welding low carbon metal, whereas ANSI/AWS D11.2 is the usual for welding iron castings. I don't and I never have. I am very aware of the fact that they do not educate Republican and Democrat as philosophies in any philosophy course at any university within the World. Republican and Democrat are NOT philosophies or ideolgies. Conservatism and Liberalism are each traced to their philosophic origins. Conservatism to Edmund Burke, and Liberalism to John Locke and the Enlightenment. King pointed to the Republican Social gathering ONLY as a result of they'd adopted a racist ideology that opposed Civil Rights and the positive aspects that have been remodeled years of blood and death.
K-12 lecturers make up a majority of the education jobs, with larger education academics coming in second. These educators plan for classes, instruct students, assess their understanding, grade papers and conduct other administrative duties. Between 2008 and 2018, the BLS initiatives that teaching jobs will enhance thirteen percent. Academics need to have the ability to lead by instance and talk clearly.
Freedom is the first aim of improvement; freedom can also be the principal means of improvement. It's the enhancement of freedoms that permit individuals to steer lives that they've cause to reside". Thus, growth is the method of increasing human freedom. It additionally means the removal of main sources of lack of freedoms resembling poverty, all forms of discrimination and inequalities, neglect of public facilities, lack of economic alternatives, social exclusion, state insurance policies that restrict freedom and so forth.
Nowadays, in schools, college students just depend on the janitors to clean the surroundings and worse they let their waste to throw all over the place. That's positively a nasty behavior. The mentioned challenge might actually help college students to appreciate how important the clean surroundings are. Additionally, by this project, college students might be responsible. Ought to we anticipate the Earth to develop into worst? Of course, not. This challenge possibly appears small thing to those who are usually not interested, but in reality, its definitely and precisely what we need. Having this sort of challenge in faculties can even develop the students consciousness in proper disposal of garbages comparable to separating the plastics, papers, biodegradable and the like.
In accordance with the Principal, the admission quotas for Faculties of Education remained the same for the past decade whereas the number of candidates kept soaring from 20,000 in 2003 to fifty four,000 in 2013. He revealed that the one two schools within the Higher East area – Boscos and Gbewaa have a complete admission quota of 500 students as against 3,000 qualified candidates and appealed to authorities to assist in increasing the consumption of applicants.


text:
Recently I found myself in a situation whereby I thought I'd have to change pretty much all of the photos in my house, which led me to panic that I have nothing to replace them with. Then I discovered that if you have photo syncing kept on with Facebook, it stores every photo that you've ever taken and they're super easy to resave and reuse! This lead to me getting all nostalgic and really, I just wanted to share possibly some of the most precious memories of my teenage life with you...
Yes, there are some questionable fashion choices as well as hair cuts in there but hey ho. I'm not going to explain who everyone is but they're all people who are very dear to me and who I'll always hold close to my heart no matter how far apart we seem to grow!
Love and kisses


text:
Sun, 6 July 2014
This week are re-posting our episode from December of 2012 with Mandy McFadden and Laura Zorch, two of the four authors of EatPGH. A lot of great stuff has happened with thier work since, but this was a fun conversation.
They sat down in the studio to talk about their continued success with their book, their tour, the blog that started it all, and to talk about the upcoming presentation at South by Southwest. That's right, the four brilliant young women from EatPGH are presenting at The SXSW digital conference. Amazing.
This is a fun episode with two really funny, awesome Pittsburghers who are following what they love and getting some much-deserved attention. They should serve as an example to anyone who has a passion for something to pursue it however possible. Check them out at EatPGH.com
Direct download: EPISODE_20_R_-_EatPGH_Part_II.mp3
Category:general -- posted at: 12:00pm EDT


text:
THE Andrew Lloyd Webber musical Sunset Boulevard comes to Plymouth's Theatre Royal from 12th to 17th March, with Swansea-born Ria Jones in the central role of Norma Desmond.
The award-winning musical is a compelling story of romance and obsession, based on Billy Wilder's film. The score includes the title track, Sunset Boulevard, as well as With One Look, As If We Never Said Goodbye, The Greatest Star Of All and The Perfect Year.
Norma, the faded, silent-screen goddess, lives in a fantasy world in her mansion on Sunset Boulevard. Her unwelcome peace is shattered by the arrival of impoverished screen writer Joe Gillis, on the run from debt collectors. Persuaded to work on Norma's "masterpiece" – a film script that she believes will put her back in front of the cameras – Joe is seduced by her luxurious lifestyle and trapped in a claustrophobic world.
When he falls in love with another woman, he tries to break free – with dramatic consequences.
In 2016, Ria Jones understudied Glenn Close in the role of Norma Desmond at the London Coliseum. She stood in when Glenn Close was indisposed, attracting rave reviews and standing ovations for her performance.
Since then Ria has starred as Dorothy Brock in 42nd Street in Paris. Her West End credits include Fantine in Les Miserables, Grizabella in Cats and Florence in Chess.
Ria has also played the iconic role of Mrs Overall in Acorn Antiques – The Musical.


text:
Premier League – United battle for points at Newcastle
Eurosport –
Manchester United came from a goal down to beat a spirited Newcastle United side 2-1 and extend their lead at the top of the Premier League to seven points.
More Stories
Peter Lovenkrands stunned the champions – and no doubt sparked excitement in certain parts of London and Merseyside – with a ninth-minute opener following an horrendous goalkeeping error by Edwin van der Sar.
But Wayne Rooney put the visitors back on level terms on 20 minutes – his ninth in nine games against the Magpies – before Dimitar Berbatov made United's increased pressure count with what proved to be the winner on 56 minutes.
It was far from United's most convincing performance of the season – for that Newcastle can take some credit – but nevertheless the result saw Alex Ferguson's side pull away again from their nearest rivals Chelsea and Liverpool, who had given their own title hopes a boost with victories 24 hours earlier.
United's big guns were recalled for the trip to Tyneside, with Van der Sar, Nemanja Vidic, Michael Carrick, Darren Fletcher, Park Ji-Sung, Berbatov and Rooney all returning after having missed the weekend's Carling Cup triumph.
But it was the men in black and white who first caught the eye as the assembled Toon Army were given a reminder of what it was like to follow Newcastle during the Nineties – the first half was full of cavalier attacking play from the hosts who had their illustrious guests rattled on a number of occasions.
From the first whistle, they got at Ferguson's side, Obafemi Martins in particular looking in the mood. The Nigerian signalled his intent in just the second minute with a run and shot that flew wide of the upright and again on 13 minutes with a low drive into the side netting.
In between, Lovenkrands broke the deadlock and by doing so became the first player to beat Van der Sar in 1,311 minutes of league football.
That record was an incredible testament to the Dutchman's form – and that of his defence – this season but the keeper was entirely to blame for failing to preserve it longer.
In an entirely uncharacteristic aberration, Van der Sar slipped a Jonas Gutierrez cross right into the path of Lovenkrands, who had the simple task of slotting home from close range. He will not score an easier goal all season.
Newcastle continued to press forward but in truth, the reigning champions looked just as likely to score and it came as no surprise when Rooney levelled 11 minutes later following a delightful piece of individual skill.
The England striker's first touch was exquisite, controlling and turning in one swift movement before firing past Steve Harper from the edge of the area, albeit with the aid of a slight deflection.
Soon after United had a big penalty shout turned down by referee Steve Bennett after Berbatov's cross clearly struck Sebastien Bassong's arm. 'Ball to hand' was the official's verdict.
The remainder of the first half was played out at an incredible pace and with an intensity that rarely dropped. Chances fell at both ends, most notably to Martins following a goalmouth scramble sparked by a poor Van der Sar punch, and then to Nemanja Vidic, who somehow conspired to direct a close-range header high and wide from Carrick's corner.
Almost inevitably, the pace slowed after the break as United began to assert their authority and take control of the game.
And the champions' increased pressure told before too long with Berbatov converting Park's cross after Steven Taylor had made a hash of an attempted chest back to his keeper.
The goal knocked the stuffing out of Newcastle and for the next half-hour United were irrepressible, Rooney firing over before he and Cristiano Ronaldo teed up Berbatov to force Harper into action from the edge of the box.
Rooney could have grabbed a second on 79 minutes when, with Harper off his line, he opted to try and find the top corner rather than square to a team-mate in a better position. It proved to be the wrong decision and his effort drifted over the bar.
For all United's dominance of possession, Newcastle still posed a threat on the break and Martins, who gave Vidic and Rio Ferdinand problems throughout, was only denied a late equaliser by a sharp Van der Sar stop.
That proved to be the last chance of the game, leaving Ferguson's side to edge closer to number three of five possible trophies this season.
As for stand-in Newcastle boss Chris Hughton, he will have to report back to Joe Kinnear on a performance that was full of spirit but one which ultimately left his team mired deep in relegation trouble, just a point above the drop zone.


text:
June 05, 2015
Steph Deidrick
Reptiles and amphibians on display at Hug a Herp Day at Prairie State Park June 20
Prairie State Park near Mindenmines will host Hug a Herp Day on Saturday, June 20, allowing guests to see and touch various reptiles and amphibians. Sponsored by Missouri State Parks, the event will run from 10 a.m.-3p.m.
Participants will see various reptiles and amphibians such as snakes, turtles, frogs, toads and salamanders. Visitors will also be able to make an amphibian or reptile craft as a part of the event.
Prairie State Park is located at 128 NW 150th Lane near Mindenmines. For more information, contact Prairie State Park at 417-843-6711. For information about state parks and historic sites, visit mostateparks.com. Missouri State Parks is a division of the Department of Natural Resources.


text:
All our samples are totally free, so it is possible to customize them according to your requirements. The samples in this article are simply a step to start writing. Just take a look at our IT sample to see some concrete examples on how to do this. You can receive samples of cover letters from all the categories mentioned above that could help you write a cover letter perfectly.
If you chose to quit a job, it is common for you to provide your employee with some specific details needed. Construction and labor needs practical experience using specific instruments and techniques to acquire the finished work. Many jobs in the construction and labor industry also need certain certifications and licenses, so be sure to highlight them as well. If you have to secure a part-time job to cover your education, or you just need to earn some money to spend, you will usually need to write a cover letter. The first thing you want to do is adapt it to the particular job you are requesting and, if possible, to the particular business. Now you can apply for a new job from the comfort of your home.
Reading through our sample student and internship cover letters will give you a clearer idea of ??how to compose yours. The best letters on the planet will not sell a terrible book. These letters show the format and details of the language that will help you capture the employer's attention and start conducting interviews with fantastic companies. As a consequence, your cover letter should verify that you take patient care seriously. While there is absolutely no cover letter that is appropriate for each position, these examples will offer you a good place to start.
Writing a cover letter can be remarkably quick and easy if you have the ideal tools to guide you at work. Simple cover letters are simple to write. Therefore, bank presentation letters should be equally dynamic and optimistic, which can offer a great opportunity to create a great impression. Letters of introduction in banking specialty should have few things in particular, since it is very important to show your experience and knowledge about things related to the banking sector.
In your cover letter, you must explain what makes you a well-qualified candidate for a particular position with a distinctive signature. Make sure your cover letter meets all these requirements and shows how it is measured. Sample job applications These sample job applications will offer you a concept of the information you will want to complete when you complete a job application. Actually, you should never make your document exactly like an example or template. So, your resignation letter format is not very simple. The templates also show you exactly what elements you need to have in your document. However, it is perfect if you use the template without altering the design.
Writing a cover letter for work in the field of education and teaching is about the details. It is possible to find more information on how to achieve this exactly in our samples of office workers. Be sure to include certain information about the organization in which you are presenting the application, along with mentioning the place of interest if there are several vacancies.
Cover letter examples, template, samples, covering letters, CV
simple cover letter samples
Cover letter examples, template, samples, covering letters, CV
simple cover letter samples
simple cover letter samples for resume Parlo.buenacocina.co
simple cover letter samples
Majestic Simple C Ideal Simple Cover Letter Samples For Resume
Cover letter examples, template, samples, covering letters, CV
Similar Posts:
- Simple Cover Letter Template
- Simple Cover Letter Format
- Simple Cover Letter
- Job Application Letter
- Free Cover Letter Samples
- Short Cover Letter
- Letter Of Application Sample
- Scholarship Essay Samples
- Cover Letter For Part Time Job
- Example Of Business Letter
- Example Of Reference Letter
- Cover Letter Format Template
- Investment Banking Cover Letter
- General Cover Letter
- Email Cover Letter Sample


text:
Includes a visit to downtown Victoria & Butchart Gardens
Victoria is called the City of Gardens and it truly lives up to its name with gorgeous Butchart Gardens a short distance away and over 1500 hanging baskets within the downtown area.
The city is quaint, friendly and full of early Canadian History. Stroll historic Government Street and Chinatown with their quaint shops. Enjoy a great seafood lunch along the waterfront, or high tea at the famous Fairmont Empress Hotel.
Victoria is a fabulous place to visit any time of year.
As this is a long day, I suggest that we depart early, however with long summer hours a later departure is also possible.
Early morning pick up at your hotel or the airport with arrival time on Vancouver Island at approximately 10:30am
During our time in Victoria we will:
- Drive through the University area, Beach Drive, Oak Bay and Beacon Hill
- Walk Government Street past the Parliament Buildings, the Fairmont Empress Hotel, Fisherman's Wharf, Market Square, Trounce Alley, Chinatown and famous Fan Tan Alley
- Lunch on the wharf front or High Tea at the Empress
- Visit to Butchart Gardens (I would recommend after 3pm as this is when it is quieter and the sun is at a great angle for photos!)
Return to your hotel will be around 8:00pm
Meeting Point + Tour Duration
Meeting Point Options: Rail or Bus Station, Cruise Ship Port, Airport, Hotel, Address or Intersection, Monument/Building
End Point Options: Rail or Bus Station, Cruise Ship Port, Airport, Hotel, Address or Intersection, Monument/Building
Duration: 11 hours
Transportation will be via 7 passenger SUV. Please note: Vehicle can only accommodate a maximum of 5 passengers with luggage.
What's Included
- Guiding Services
- Private Transportation
- Admission Tickets
Other: Includes:
My services as a tour guide on this 11-12 Hour tour
Transportation costs in a 7 passenger SUV
Return Ferry from Vancouver to Vancouver Island (1.5 hours sail each way)
Entrance to Butchart Gardens.
Estimated Local Cash Needed
40 CAD - Meals on the ferry as well as lunch in Victoria.
What's Extra
- Personal Expenses
- Souvenirs
- Food, Drinks, Snacks
Other: Meals, snacks and items of personal nature. Entrance fees to other attractions other than Butchart Gardens.
A Recent Review of this tour
Read More Reviews For This Tour
Lois was an outstanding guide. She went above and beyond in planning our day to Victoria. She arranged for us to dine at the legislative dining rooms in Parliament House - which was a highlight. She was a wealth of information and could explain things in an easy to follow manner. Butchart Gardens was a highlight and Lois tailored this part of the tour to maximise our time there. We really enjoyed Lois' company and the day flew past as we were having such a good time.
Stephanie Taylor
Share This Tour


text:
Cinnamon, sugar, and freshly baked goodies are all fall foodie favorites, but how do you enjoy these guilty pleasures without worrying about gaining the FALL 15? We have found a great recipe that is sure to satisfy your sweet tooth and keep your hand out of the cookie jar, well at least till christmas!
Candied Pecans are a great fall treat, both sweet and healthy, these little morsels are the perfect fall snack!
Pecans have many health benefits such as:
- Pecan nuts are rich source of energy
- rich in monounsaturated fatty acids
- an excellent source of vitamin-E
- very rich sources of several important B-complex groups of vitamins such as riboflavin, niacin, thiamin, pantothenic acid, vitamin B-6, and folates. Together, these vitamins work as co-factors for the enzyme metabolism inside the human body
- rich source of minerals like manganese, potassium, calcium, iron, magnesium, zinc, and selenium
Now that we have explained all the reasons Pecans are perfect, here is how to make them even better! This recipe was found at
All you need are 7 ingredients to make this delicious candied pecans recipe. They are perfect for topping salads, appetizers, main dishes, and desserts. And they're also perfect for gifting!
All you need to make these are 7 ingredients:
- pecan halves
- egg whites
- granulated sugar
- brown sugar
- ground cinnamon
- salt
- cayenne
There are many different ways that you can make candied nuts, but I still like the classic cinnamon-sugary version, with just a hint of cayenne. Trust me. It is the magic (and very subtle) ingredient that truly makes these nuts "pop"!
Whisk together your dry ingredients until they are well-combined. (I often toss them in a ziplock bag, and then smoosh it around with my fingers until the mixture is even. Quick and easy.)
Then in a separate bowl, add your egg whites and pecans, and toss until the pecans are evenly coated. Then add in your dry ingredients and carefully toss until combined.
Lay those babies out on a parchment-lined baking sheet, so that they don't stick to the pan.
And then bake them up to delicious perfection, and get ready for your home to start smelling amazing.
These guys are perfect for salads (stay tuned for a new recipe tomorrow!), yogurt parfaits, ice cream sundaes, or topping anything else that calls for sweet pecans. But I have to say, I still think they're best for snacking. And if you're feeling generous, they are perfect for gifting to friends and family this holiday season so that they can enjoy snacking on them as well. Because if you ask me, the more candied nuts in this world, the better it will smell be.


text:
Areti Camping & Bungalows
Areti Camping & Bungalows is located in one of the most unspoiled areas of Sithonia in the Chalkidiki peninsula, 12 km after Neos Marmaras and 132 km from Thessaloniki and the airport.
It is a quiet, family campsite equipped with all modern comforts. Seize the opportunity to enjoy water sports, tennis and volleyball games, indulge in flavors of the Mediterranean cuisine, and enjoy the relaxing vibe of the simplicity around you. Excursions to discover ancient and cultural monuments, walking tracks, wine tasting, boat trips, all make up a fulfilling holiday experience.
Set among olive groves, eucalyptus and pine trees, the stone built and wooden bungalows exist in harmony with the unspoiled nature around them. Just a few steps away from two private beaches where you can dive in the azure waters of the Mediterranean Sea, and sail to three small island, 'Spalathronisia', that frame the coastline and offer stunning views of the sunset.
The newly built, contemporary style stone built bungalows offer you a spacious 65m² accommodation of two bedrooms, living room (including light cooking facilities) and bathroom, equipped with A/C, and a private veranda and garden.
The picturesque wooden bungalows, equipped with facilities to ensure a relaxing stay (A/C, light cooking facility, WC, private veranda).
Also at a distance of 10km is located the 18 hole golf court protected by the warm Mediterranean climate and the area's micro-climate and meets all the criteria needed to host international tournaments all year round. Covering 77 hectares it is designed to make the game exciting for both professional and amateur players.
Areti Camping & Bungalows
Neos Marmaras, Chalkidiki, 63081, Greece
Tel: 00302375071430
Fax: 00302375071573
Winter Tel: 00302375071861
Email: email@example.com, firstname.lastname@example.com
Visit Areti Camping & Bungalows website for more information and reservations


text:
French, PDF, 463kb
En 2012, les plans de retraite privés dans l'OCDE, qui représentaient au total 32 100 milliards USD, étaient proposés par des fonds de pension (67.9%), des banques et des sociétés d'investissement (18.5%), des sociétés d'assurance (12.8%) ou des employeurs les provisionnant au bilan (0.8%).
This paper examines how institutional investors can access green infrastructure, the extent to which this is currently happening, and the barriers to scaling up these investment flows. Based on four case studies, broader lessons are drawn for governments on the policy settings which may support investment in green infrastructure by institutional investors.
This regional seminar in Montevideo, Uruguay, focused on the monitoring and transparency of insurance markets in Latin America through the provision of sound insurance statistics and indicators, regionally and globally.
This report provides a survey of disaster risk financing practices within APEC, including implementation challenges and key priorities for future work. It constitutes the first step towards promoting effective and widespread implementation of the G20/OECD Methodological Framework in the APEC region and beyond in the area of disaster risk financing.
At their meetings on 19-20 September 2013, APEC Finance Ministers welcomed a survey report prepared by the OECD on disaster risk financing practices in the Asia Pacific region.
Les chefs d'État et de gouvernement du Groupe des Vingt (G20) ont approuvé ce jour une nouvelle initiative mondiale destinée à inciter les investisseurs institutionnels à financer des actifs à long terme, en investissant par exemple dans des projets d'infrastructures ou liés aux énergies renouvelables, afin de renforcer l'économie mondiale et de favoriser une croissance plus durable.
This seminar took place in Palembang, Indonesia, with discussions focusing on institutional investors and long-term financing and policy measures and initiatives to address constraints to infrastructure investment identified by APEC under the Indonesian presidency.
At a time when many governments are looking to encourage private investment in infrastructure to bridge the infrastructure financing gap, this paper compares the experience of pension funds in Canada and Australia, looking at infrastructure policies, the pension system, investment strategies, asset allocation and governance of pension funds.
English, PDF, 1,817kb
Faced with the acute poverty of many Russian pensioners, the Russian government is engaged in wide-ranging systemic reforms. This report contributes to the policy discussion by identifying aspects of the system that may need to be reformed and describing the experience of other countries as a point of comparison.
Co-organised by the G20 Russian Presidency and the OECD, this roundtable focused on policy measures to address constraints in long-term investment. It was organised back-to-back with the 2013 OECD Forum and the OECD-Euromoney Infrastructure Summit.