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Methodology optimizing SAGE library tag-to-gene mapping: application to Leishmania
Sondos Smandi1,
Fatma Z Guerfali1,
Mohamed Farhat1,
Khadija Ben-Aissa1,
Dhafer Laouini1,
Lamia Guizani-Tabbane1,
Koussay Dellagi1 &
Alia Benkahla1
Leishmaniasis are widespread parasitic-diseases with an urgent need for more active and less toxic drugs and for effective vaccines. Understanding the biology of the parasite especially in the context of host parasite interaction is a crucial step towards such improvements in therapy and control. Several experimental approaches including SAGE (Serial analysis of gene expression) have been developed in order to investigate the parasite transcriptome organisation and plasticity. Usual SAGE tag-to-gene mapping techniques are inadequate because almost all tags are normally located in the 3'-UTR outside the CDS, whereas most information available for Leishmania transcripts is restricted to the CDS predictions. The aim of this work is to optimize a SAGE libraries tag-to-gene mapping technique and to show how this development improves the understanding of Leishmania transcriptome.
The in silico method implemented herein was based on mapping the tags to Leishmania genome using BLAST then mapping the tags to their gene using a data-driven probability distribution. This optimized tag-to-gene mappings improved the knowledge of Leishmania genome structure and transcription. It allowed analyzing the expression of a maximal number of Leishmania genes, the delimitation of the 3' UTR of 478 genes and the identification of biological processes that are differentially modulated during the promastigote to amastigote differentiation.
The developed method optimizes the assignment of SAGE tags in trypanosomatidae genomes as well as in any genome having polycistronic transcription and small intergenic regions.
Leishmania, the causative agent of leishmaniasis, is a protozoan parasite of the order Kinetoplastida. The Leishmania major genome is 33 Mb in size with a karyotype of 36 chromosomes. There are 911 RNA genes, 39 pseudogenes, 8272 protein coding genes of which 36% can be ascribed a putative function. The means of CDS and intergenic regions length are 1901 bp and 2045 bp, respectively [1].
Leishmania species exist in two distinct stages within the mammalian host they infect. Promastigotes, present in the sand fly insect vector, are inoculated to mammalian hosts, where they transform into amastigotes, a form adapted to survive within these mammalian host cells. The molecular events allowing the differentiation from promastigotes to amastigotes are still poorly understood. The post-transcriptional and/or post-translational regulation of genes involved in several biological processes is certainly important to adapt the parasite to survive in the harsh conditions of the parasitophorous vacuole and to circumvent the host's immune response. Hence, a systematic identification of these genes is necessary to understand the mechanisms underlying parasite intracellular survival.
Several gene expression experiments were performed on different Leishmania species using DNA, cDNA, oligonucleotides microarrays or SAGE technology [2–8]. Different studies performed at the transcriptomic level have focused, either on genes differentially expressed between promastigote and amastigote stages, or between distinct Leishmania species. Modulated genes encode for proteins with either hypothetical or unknown functions or for proteins with known function e.g. surface proteins, kinases, maintenance protein, metabolic enzymes, structural genes, transporters, and heat shock proteins.
SAGE is an approach that allows the rapid, quantitative, simultaneous and detailed analysis of thousands of transcripts [9], and is a powerful tool for the analysis of genome-wide gene expression without requiring knowledge of the gene content [10]. It was successfully used in a wide variety of organisms and applications including the elucidation of diseases [11], the detection of transcripts expressed at low levels [12] and the discovery of new genes [13]. Its output is a list of short sequence tags which size depend on the specific SAGE technology used. One of the critical steps using SAGE technique is the tag-to-gene mapping. Classical methods involve the mapping of any SAGE tag to the 3' most tag within each transcript. These methods either use known 3'UTR or artificially extend the 3'UTR of predicted genes or both. Unfortunately, these methods are not applicable for the study of organisms like Leishmania for which the full transcripts and the approximate length of their 3'UTR are unknown.
In a previous report [3] using SAGE, we have provided a large-scale gene expression profile of Leishmania major (L. major) promastigotes (Lm) and Leishmania infected Monocyte Derived Macrophages (MDM + Lm). The used SAGE technology produces 14nt cDNA tags. The tag-to-gene assignment technique was basic and the assignment restricted to the most abundant tags and to tags preferentially expressed by intra-macrophagic parasites. As a result several tags/genes were excluded from the analysis because they could not be assigned to their respective genes or because they had a steady expression. Considering this limitation, we present here a novel method that optimizes the tag-to-gene mapping process for all expressed tags. This method does not validate the SAGE libraries, it exploits the genomic sequence and gives different confidence values to each of the hits of a SAGE-tag in the genome. The confidence values were defined according to parameters obtained through the kernel density estimation (kde) [14] of a distribution of unambiguously assigned tags. A detailed workflow is shown in Figure 1.
Bioinformatics workflows: 1) Estimation of the maximum size of the 3'UTR. 2) Tags assignment. The three arrows mean that the probability derived from the estimation of the density of STOP-SMAT distances were used to evaluate the STOP-MMT distances.
The implemented technique allowed the assignment of a maximum number of SAGE-tags, and the evaluation of the expression of their transcripts and the delimitation of some 3'UTR. These results pushed a step forwards the initial analysis [3] by integrating the expression of a larger number of genes and extending the knowledge of Leishmania 3'UTR. Additionally, we systematically characterized the function of genes differentially expressed in Lm versus the library generated at amastigote stage (MDM + Lm) [3].
The main result of the present study is the development of a tag-to-gene assignment tool (R program available in the additional files). The developed program assigns a maximum number of SAGE-tags [3], to their respective genes. Multiple match tags (MMT) are the tags which assignment was the trickiest. The program uses a data-driven probability derived from the mapping of a set of single match assigned tags (SMAT), and evaluates the most probable tag-to-gene association of MMT. Results are available in Additional file 1. The limit 1.4 kb, maximal tolerated distance between the CDS and the end of the 3'UTR, was derived from the 800 mapped 3'ESTs available in Genbank on November 2010 (See Figure 2 and Materials and Methods - Estimation of the maximum size of the 3'UTR paragraph). Because 1.4 kb might be considered as being too short or too large, the authors have parameterized this length into the source R program leaving to the user the freedom to rerun the program with the distance that suits them best.
Histogram illustrating the size distribution of the 3'UTR of 800 3'ESTs mapped on the L. major genome. 573 ESTs overlapped with the CDS and with the 3'UTR of transcripts and 227 ESTs were in the 3'UTR of transcripts. 96% of the latter 3'UTR are less than 1.4 kb.
According to the method implemented for tags assignment, 7766 out of the 9530 unique tags were mapped on the parasite genome. Among these, 4168 tags were classified as single match tags (SMT) and 3598 as MMT. Among SMTs, 3171 were classified as SMAT and assigned to 2538 different genes. The 3598 MMT had 13617 mappings (on average 4 mappings/tag), and out of these, 908 tags were assigned to 832 different genes. In total, the implemented strategy was able to successfully assign 4079 tags (52.56% of total sequenced tags) to 3094 genes (37% of total genes) (Additional file 2).
Because gene expression is known to be driven from the 'sense' and 'antisense' strand (complementary) of DNA in Leishmania [15–17], we have used our SAGE data to predict the antisense gene transcription. From the 3171 SMT, 2449 were assigned to genes in the same direction ('sense') and 722 to genes in the opposite direction ('antisense'). From the 908 assigned MMT, 610 were sense and 298 antisense. In total, 2148 genes showed no antisense transcription and 470 no sense transcription, whereas 476 showed both a sense and an antisense transcription. This result estimates the rate of antisense genes as being higher than expected (~30%).
Among the 3094 identified genes, 772 were represented with different tags (275 with tags in the same direction, 21 with tags in the opposite direction and 476 with tags in both directions). 11 of these had significant expression changes (LmjF10.0090, LmjF15.0950, LmjF15.1203, LmjF24.2080, LmjF26.2220, LmjF29.1730, LmjF29.2370, LmjF34.2900, LmjF35.1890, LmjF36.3620 and LmjF36.6680); the direction of their expression change was the same according to the different tags counts. The list of these 11 genes is enriched in genes showing sense and antisense co-transcription (5/11) an observation that might reflect one of Leishmania post-transcriptional regulatory mechanisms.
Apart from the estimation of the expression of a maximum number of genes and the identification and the GO characterization of a maximum number of differentially expressed genes; the developed method allowed the delimitation of some 3'UTR.
Functional characterization of differentially expressed genes
A large proportion of L. major genes are differentially expressed between promastigote and amastigote stages [3]. Indeed, a total of 304 genes showed a significant differential expression between Lm and MDM + Lm, indicating that they might play a role in the parasite differentiation and/or in Leishmania virulence. Among these 304 genes, 189 and 115 genes were preferentially expressed by amastigotes and promastigotes respectively. The functional analysis of these genes using GOTermMapper and GOTermFinder revealed that 102 genes could be classified according to their biological processes, whereas 202 genes had no functional annotation (Additional file 3 and Additional file 4). KEGG pathways [18] associated to these genes (Additional file 4) revealed that 65 genes are Involved in known pathways. This result gives an idea about the Leishmania processes and pathways that are preferentially activated during the different stages.
3'UTR characterization
PRED-A-TERM allowed the prediction of 1581 3'UTR extremities (337 were located between the STOP and the tag; 452 were located between the tag and the following CATG; 648 between the following CATG and the start of the following gene; and 144 after the start of the following gene). Following the approach detailed in the paragraph 3'UTR characterization (in the Materials and Methods), we were able to validate the PRED-A-TERM predictions for 478 different L. major genes (Additional file 5): These were grouped into 452 predictions validated using the tags data and 26 using the EST data.
To assign the maximum number of tags to their respective genes in a SAGE library constructed from metacyclic Leishmania promastigotes [3], we developed a method that evaluates the most likely tag-to-gene association of MMT. The implemented method allowed the assignment of a maximum number of SAGE tags, without arbitrarily fixing the size of the 3'UTR as it was previously done for the assignment of Arabidopsis tags [19, 20].
Unlike the method presented by Malig and colleagues [21], the one developed here dealt with experimental tags and provided an estimation of the confidence on a tag-to-gene assignment. Malig and colleagues method [21] dealt with virtual SAGE tags and evaluated the tag-to-gene assignment intuitively. We also preferred to use what we have learned from the frequency distribution of the distance of SMAT from the STOP of the associated gene rather than extending the 3'UTR by a fixed number of nucleotides as in Pleasance and colleagues method [22].
SAGE technology is being routinely used to detect antisense transcription [23]. Such prediction has the same value as the prediction of sense transcription. The fact that antisense is being more common than expected (~30%) is presumed since gene regulation in Leishmania is driven through post-transcriptional mechanisms likely modulated through antisense transcription [24].
In our previous study [3], the purpose was to capture strong functional signals emitted during the differentiation from promastigotes to amastigotes. The tag-to-gene assignment was basic and not generalized to all tags. The assignment was restricted to the most abundant tags (1.1% of the total number of unique tags) and to tags preferentially expressed by intra-macrophagic parasites. The optimization of the tag-to-gene assignment procedure herein allowed the identification of a maximum number of expressed and differentially expressed genes. The consequence of the latter allowed the identification of 76 additional genes preferentially expressed by intra-macrophagic amastigotes [3].
Several statistical tests are available to evaluate differential expression in SAGE libraries. The comparison of the output of the used test [25] to that of other tests (Student's test [26] and Shapiro-Wilk test [27]) showed, as expected, no significant differences. Therefore, the used statistics should not impact on the interpretation of the results. However, it has to be noted that libraries of a size lower than 120000 (the size recommended for SAGE experiments [28]), could under-estimate the number of expressed and differentially expressed genes which could impact on the interpretation of the results. This might explain why some genes are not observed as being constitutively expressed as previously reported [6].
Compared to our previous study, the functional GO characterization was done for all differentially expressed tags. GOTermMapper, GoTermFinder and KEGG pathways annotation indicated that the list of modulated genes is enriched in genes which end products are involved in RNA 'translation', inferring that translation related processes are affected by the intracellular development of the parasite. This result agreed with those reported by McNicoll and colleagues [8] who suggested that protein translation is affected during promastigote to amastigote differentiation (a weak correlation was observed between the transcriptome and the proteome levels at the amastigote stage and no correlation at the promastigote stage) and that translational and post-translational mechanisms are important for controlling gene expression.
Clayton and Shapira show in 2008 [29] that the parasite uses a polycistronic transcriptional approach and that mRNA abundance is regulated by post-transcriptional mechanisms driven through elements located in the 3' untranslated region (3'UTR) [8, 29]. Therefore, to be properly interpreted, transcriptome data would need the development of tools that allow the study of the post-transcriptional regulatory elements located in the 3'UTR. The first step towards this development is a better delimitation of the 3'UTRs of known genes.
As the 478 3'UTR extremity predictions were supported by experimental data (i.e.: Leishmania 3'EST and/or L. major tags), they could be considered as true predictions and the predicted 3'UTR could be used, in our opinion, straight away for the investigation of post-transcriptional regulatory motifs. Additionally, the 3'UTR extremity predictions located between the CATG following a tag and the start of the following gene can be valid and would have to be validated through the sequencing of the corresponding transcript. Predictions located elsewhere should be considered as false positives unless they correspond to overlapping genes which existence is to be proven in Leishmania.
The presented method was not able to assign 5451 out of 9530 tags, among which 1764 were not mapped to L. major genome, which could be due to several reasons including tags sequencing errors (false positives), small size of the tags (14 nt), or polymorphic variations between the Friedlin strain used for the genome sequencing and the strain used to construct our SAGE library, or the presence of microbial component. Some unassigned tags could also belong to new protein or non-protein coding genes, absent from the GeneDB catalogue. These tags would gain to be further investigated to obtain an exhaustive L. major genes catalogue. It has also long been known that non-coding RNAs are relatively common in trypanosomatids, and represent relatively stable processing products from polycistronic transcripts [30]. Since the existence of non-protein coding genes was not considered because of the absence of a dedicated catalogue, this has probably contributed to the raising of the mis- and non-assigned tags rate and implied that a proportion of the remaining non-assigned tags (the SMT mapping between two genes and some MMT) could belong to this class of genes. The lack of exhaustive knowledge about the actual full transcripts catalogue could also generate erroneous tag-to-gene associations of multiple match tags. Polymorphism within the L. major genus and the tags sequencing errors were additional parameters that can increase these rates.
The Leishmania genome was characterized by the presence of duplicated segments containing large gene families [1]. MMT mappings into members of the same gene family were difficult to assign and often remained unassigned. A complementary approach would be considering the assignment of these tags to conserved gene families' rather than individual genes.
In the present study, we developed a novel approach for a better assignment of the SAGE tags to their genes for all organisms having polycistronic transcription and small intergenic regions. It expanded classical tag assignment methods to a method that does not require any extended knowledge concerning the 3'UTR. The implicit consequence of optimizing the assignment process was to approximate sense and antisense tag expression and to identify a maximum number of Leishmania genes that are differentially expressed. It should however worthy to note that all these assignments need to be validated experimentally.
The comparison of gene expression profile between promastigote and amastigote stage and the systematic GO classification of modulated proteins allowed the identification of the key biological processes that are modulated during differentiation from promastigotes to amastigotes. An exhaustive functional annotation of the genes involved in these processes would be helpful in understanding the mechanisms of intracellular parasite differentiation and in the identification of new drug target proteins.
In addition, a new approach indicating the most likely 3'UTR extremity of Leishmania genes is presented. This approach is based on the assessment of predicted Leishmania 3'UTR mRNA extremity by supporting biological evidences (Lm tags, Leishmania Expressed Sequence Tags and one prediction tool [31]).
Generation of SAGE libraries
SAGE libraries were previously generated by our group and corresponded to L. major GLC94 metacyclic extracellular promastigotes (Lm) and to L. major GLC94 in their intracellular form within infected macrophages (MDM + Lm). All details about the parasite culture and preparation, the RNA isolation, and the SAGE library construction and its computer-based analysis were described by Guerfali and colleagues [3]. Lm was obtained from purified metacyclic L. major promastigotes and contained a total of 33,906 SAGE-tags corresponding to a non-redundant set of 9,530 different tags. MDM + Lm was sequenced from L. major-infected macrophages and contains 57,514 tags corresponding to a non-redundant set of 24,418 tags. Both libraries associated each tag to its frequency.
Tags assignment
(a) Estimation of the maximum size of the 3'UTR
The 3'UTR of a given gene corresponds to the 3' sequence of a mature transcript that is not translated. The size of 3'UTR of Leishmania genes is relatively large and variable. Prior reports estimated, for example, the 3'UTR of HSP70 [32] and HSP83 [33] to 1063 and 886nt, respectively. Moreover, 12201 ESTs of Leishmania available on Genbank were blasted against L. major genome. The 800 3'ESTs having a single match and mapping in the 3'UTR of a known gene, with ≥ 80% identity and over ≥ 50% of their length, were identified and considered as ESTs with known 3'UTR. Out of these, 129 3'ESTs are L. major ESTs. The size varied from 1 to more than 2000 nt; 96% of the 3'UTRs being less than 1.4 kb (see Figure 2). This distance will be considered as the maximal tolerated distance between the CDS and the end of the 3'UTR. The list and the description of these 3'UTR is available in Additional file 6. 1.4 kb is used in the assignment approach paragraph as maximum length of a 3'UTR.
(b) Assignment approach
The non-redundant set of 9,530 promastigote SAGE-tags were mapped to L. major genome by blasting [34] the 14 nt tag sequences against the latest release of the parasite genome available and downloaded from GeneDB [35] on December 8, 2006. The distances between matching tags (100% of identity over 100% of the length) and the STOP codon of the nearest gene were retrieved.
Assigning SMT to the closest predicted CDS is quite natural. The closest CDS being the one located less than 1.4 kb (maximal tolerated distance between the CDS and the end of the 3'UTR), each SMT found within the coding sequence or 1.4 kb downstream the STOP codon of a given gene, was assigned to that particular gene. Accordingly, they were classified as SMAT. Tags not verifying these conditions (i.e. SMT within more than 1.4 kb away from any gene or MMT) were kept for further investigations.
A Gaussian Kernel Density Estimation approach [14] was then used to estimate the density of SMAT distances; the estimation function being:
f ^ h ( x ) = 1 N h ∑ i = 1 N K ( x - x i h )
where N is the size of SMAT dataset, x is the set of breakpoints, h is the bandwidth (smoothing parameter), and K is the standard Gaussian function with mean zero and variance 1:
K ( x ) = 1 2 p e - 1 2 x 2
The X-axis was later split into 50 classes, (x i ) i ≤ 50, each one corresponding to 41 nt (41 = (max of x - min of x)/50), and a probability P j was affected to each class j.
P j being equal to the difference between two consecutive Cumulative Density Functions (CDF):
P j = C D F j + 1 - C D F j j = 1 , … , 49
C D F ( j v ) = ∫ x x j f ^ h t d t
A plot of this probability is shown in Figure 3. To assess the most probable tag-to-gene association, these probabilities were attributed to the distances between the mappings of the MMT and the STOP of the nearest gene. The tag-to-gene association having a probability higher than 5%, were selected.
Gaussian kernel density estimation of the assigned tags. The x-axis represents the size of the segment STOP-tag. The right and left y-axis do not correspond to the same curve and have different scales. The right y-axis is for the red curve and represents the density of SMAT distances. The left y-axis is for the histogram and represents the tags count in the appropriate segment STOP-tag. Linear binning is used to obtain the bin counts (500) on the x-axis.
(c) Differentiate assigned 'sense' and 'antisense' tags
SAGE tags being directionally reliable short cDNA sequences [36], we have defined 'sense tags' as tags mapping to a given gene and 'antisense tags' as tags mapping to the reverse complement of a given gene.
The 697 tags listed by Guerfali and colleagues [3] (Additional file 7), present in the MDM + Lm and Lm and absent in other human libraries, and differentially expressed, were reloaded. Of these tags, 420 were found to be preferentially expressed by amastigotes, and 277 by promastigotes. Genes associated to these tags were considered as differentially expressed.
The biological process(es) to which the differentially expressed genes belong to were characterized using GOTermMapper (http://go.princeton.edu/cgi-bin/GOTermMapper) and GOTermFinder [37].
SAGE libraries were generated using polyA + RNA and converted to cDNA. The latter were cleaved with the NlaIII enzyme after the first CATG encountered and the 3'-terminal cDNA fragment were bound to streptavidin-coated beads. After concatemerization, these SAGE tags were sequenced. The sequenced tags should correspond to the first 14 nt fragment containing a CATG at the most 3'-end position of the mRNA (unless the mRNA contains a second polyA stretch). Departing from this observation, the true positive PRED-A-TERM polyA predictions [31] of all genes with an assigned tag (3094 genes) should be mapping in the 3'UTR of that gene and between the assigned tag and the following CATG.
As recommended by Smith and colleagues [31], PRED-A-TERM was run on the intergenic sequences of all genes with an assigned tag (3094 genes), plus the last 800 nt of the upstream gene, and the first 800 nt of the downstream gene. All predictions mapping in the 3'UTR of the upstream gene and between the corresponding tag and the following CATG or less than 100 nt away from the end of a mapped EST, were considered as a likely true polyA region prediction.
SAGE:
Serial analysis of gene expression
UTR:
UnTranslated Region
CDS:
Coding Sequence
DNA:
Deoxyribonucleic Acid
cDNA:
Complementary Deoxyribonucleic acid
Lm:
Promastigotes
MDM + Lm:
Leishmania infected Monocyte Derived Macrophages
kde:
Kernel Density Estimation
MMT:
Multiple Match Tags
SMAT:
Single Match Assigned Tags
SMT:
Single Match Tags
KEGG:
ORF:
Open Reading Frame
Expressed Sequence Tag
mRNA:
Messanger Ribonucleic Acid
nt:
Kb:
KiloBase
PolyA:
PolyAdenosine
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We would like to acknowledge Florence Ottones, Laurent Manchon, David Piquemal, Thérèse Commes, and Jacques Marti for participating to the generation of the SAGE libraries published in BMC genomics in 2008 [3]. We would like also to acknowledge Slimane Ben Miled, Karyn Megy, Oliver Hofmann and Abdeljelil Ghram for pre-reviewing this report. This work was done with the support of UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (the project identifier being A50967) and with the support of the EC Framework Program VII, Project: LEISHDRUG, Grant Agreement No 223414.
Laboratoire d'Immuno-Pathologie, Vaccinologie et Génétique Moléculaire (LIVGM), WHO Collaborating Center for Research and Training in Leishmaniasis, Institut Pasteur de Tunis, 13 place Pasteur, BP74 1002, Tunis, Tunisia
Sondos Smandi, Fatma Z Guerfali, Mohamed Farhat, Khadija Ben-Aissa, Dhafer Laouini, Lamia Guizani-Tabbane, Koussay Dellagi & Alia Benkahla
Sondos Smandi
Fatma Z Guerfali
Mohamed Farhat
Khadija Ben-Aissa
Dhafer Laouini
Lamia Guizani-Tabbane
Koussay Dellagi
Alia Benkahla
Correspondence to Alia Benkahla.
Sondos Smandi did all the analyses and participated in the writing. Mohamed Farhat did implement the statistical analysis. Alia Benkahla did coordinate all the analyses and the writing. Fatma Guerfali, Dhafer Laouini and Lamia Guizani-Tabbane did participate to the analysis of the results and the writing. Koussay Dellagi did initiate the project and participate to the writing. All authors have read and approved the final manuscript.
13104_2011_1375_MOESM1_ESM.XLSX
Additional file 1: The probability attributed to the distance between the mappings of the MMT and the stop of the nearest gene. (XLSX 941 KB)
Additional file 2: List of assigned tags from library Lm and their corresponding genes. (XLSX 305 KB)
13104_2011_1375_MOESM3_ESM.JPEG
Additional file 3: Distribution of L. major differentially expressed genes according to Genes Ontology (GO) biological process categories. The majority of genes code for proteins with no GO category. The metabolic process category is composed of genes involved in different processes (72 genes in primary metabolic process; 62 genes in protein metabolic process; 48 genes in biosynthetic process; 47 genes in the translation; 13 genes in transcription and nucleic acid metabolic process; 2 genes in lipid metabolic process). GOTermFinder estimates that the list of these genes is enriched in translation related proteins. (JPEG 109 KB)
Additional file 4: GoTermMapper biological process classification of modulated genes. (XLSX 25 KB)
Additional file 5: Genes' 3'UTR extremity predictions (452 using PRED-A-TERM and tags, and 26 using PRED-A-TERM and EST). (XLSX 51 KB)
Additional file 6: Description of the 3'UTR of 800 3'EST. (XLSX 76 KB)
Additional file 7: List of the modulated tags listed by Guerfali and colleagues (2008). (XLSX 38 KB)
Smandi, S., Guerfali, F.Z., Farhat, M. et al. Methodology optimizing SAGE library tag-to-gene mapping: application to Leishmania. BMC Res Notes 5, 74 (2012). https://doi.org/10.1186/1756-0500-5-74
Leishmania Species
Antisense Transcription
Major Genome
Sage Library
Amastigote Stage


I have a bunch of Stonehaven and other great tobaccos that I need (don't want) to get rid of. I see on eBay that it can be removed from listing. Happened to me too many times when I was selling some older Dunhill tobaccos and becomes very frustrating!! Where and how is the best way to sell the tobaccos?? I have another type of PAD.......Pistol AD!!!
Steve, if you get to the 30/30 requirement here on BoB, you can list them for sale in the Trader forum. And frankly that would be the best option for you, as Stony is one of the most sought after blends these days. You'd sell it out in a heatbeat here.
Just step up your post count (you obviously have the registration date), and perhaps provide some trader references from another site if you have it. Good luck!
I think he has that thirty day thingy; he joined in '08.
He best get busy posting, though.
What's 30 / 30 Rande mate?
One needs to have 30 posts and 30 days as a member.
Oh I see Rande. Reckon I might just sneak into that bracket now.
Stick around and make a few more posts...I'm sure once you've met the requirements to list things for sale here that Stony will be gone before you can blink.
This to me is really the best option. BoB is a way great site to discuss all things pipe/'baccy. Dunno why you dropped off the radar way back when, but if you're still into the pipe thing you could do a lot worse than to hang around here for awhile.
That said, if you're not into the chat board stuff anymore then pipestud is probably your best avenue.
Yuppers, get a hold of Pipestud..


text:
Q: Pass Excel Data "Date Range" to SQL Query I have a large 115 line SQL code that I have been tasked to put into Excel and allow the user with two cells to change the date range of the SQL code and show the results.  I have tried several of the other topics with no luck. I am using Microsoft 365. Each user has a ODBC connection on their PC named "Reports55"
In excel I have two tabs (Summary) and (SQLData).  on the (Summary) tab Cell A2 is the 1StartDate and Cell B2 is the 2EndDate. The (SQLDate) tab is where the SQL Query data will be shown. Any help will be greatly appreciated.
Skill level

*

*VBA: Beginner (Please dumb it down for this old guy)

*SQL: Intermediate

*Excel: Intermediate

DECLARE @1StartDate AS DATE
DECLARE @2EndDate AS DATE
SET @1StartDate = '2020-04-27'
SET @2EndDate = '2021-05-27'

SELECT
    'SHIP' AS RPT ,
    SO_Detail_Ext.LateReason ,
    (SO_Detail.ordnum_28 + SO_Detail.linnum_28 + SO_Detail.delnum_28) AS "Order",
    SO_Detail.custid_28 AS "CustID",
    SO_Detail.Prtnum_28 AS "Part",
    CONVERT(VARCHAR(10), SO_Master.ORDDTE_27, 101) AS "OrdDte",
    CONVERT(VARCHAR(10), SO_Detail.Shpdte_28, 101) AS "Shpdte",
    ExactMAX.dbo.NumShopDays(SO_Master.ORDDTE_27,SO_Detail.shpdte_28) AS "LT_ToShip",
    CONVERT(VARCHAR(10), SO_Detail.ORGDUE_28, 101) AS "OrgDue" ,
    CONVERT(VARCHAR(10), SO_Detail.Curdue_28, 101) AS "Curdue_PDSL",
    CONVERT(VARCHAR(10), SO_Detail.CUSDUE_28, 101) AS "Custdue_RDSL",
    SO_Detail.Price_28 AS "Price",
    SO_Detail.Shpqty_28 AS "ShpQty",
    Customer_Master.udfkey_23 AS "UDFKey",
    Customer_Master.Slster_23 AS "Territory",
    Account_Types.DESCRPTN_104 AS "AccType",
    Customer_Master.SLSTER_23 AS "WordArea" , --'Undefined World Area'
    Customer_Master_Ext.CustomerClass ,
    (SO_Detail.Price_28 * SO_Detail.Shpqty_28) AS "Extended",
    CAST('1' AS INTEGER) AS "Shipped",
    CASE WHEN SO_Detail.Shpdte_28 <= SO_Detail.Curdue_28  THEN 1 ELSE 0 END AS "PDSL_OnTime",
    CASE
    WHEN SO_Detail_Ext.LateReason = 'SSI' THEN '1' --Customer caused late order
    WHEN SO_Detail.shpdte_28 <= SO_Detail.Cusdue_28  THEN 1 ELSE 0 END AS "RDSL_OnTime",
    CASE WHEN Customer_Master_Ext.CustomerClass = 'TRADE'
    THEN 1 ELSE 0 END AS "Trade_Shipped", --01 Total Trade Lines Shipped
    CASE WHEN SO_Detail.Shpdte_28 <= SO_Detail.Curdue_28
    AND Customer_Master_Ext.CustomerClass = 'TRADE' THEN 1 ELSE 0 END AS "Trade_Ontime_PDSL",
    CASE WHEN SO_Detail.Shpdte_28 <= SO_Detail.CUSDUE_28
    AND Customer_Master_Ext.CustomerClass = 'TRADE' THEN 1 ELSE 0 END AS "Trade_Ontime_RDSL",
    CASE WHEN Customer_Master_Ext.CustomerClass = 'TRADE'
    AND isnull(Part_Master_Ext.ExpressShip, 0) = '1' THEN 1 ELSE 0 END AS "Trade_Exp_Shipped",
    CASE WHEN SO_Detail.Shpdte_28 <= SO_Detail.CUSDUE_28
    AND Customer_Master_Ext.CustomerClass = 'TRADE'
    AND isnull(Part_Master_Ext.ExpressShip, 0) = '1' THEN 1 ELSE 0 END AS "Trade_EXP_Ontime_RDSL",
    CASE WHEN SO_Detail.Shpdte_28 <= SO_Detail.Curdue_28
    AND Customer_Master_Ext.CustomerClass = 'TRADE'
    AND isnull(Part_Master_Ext.ExpressShip, 0) = '1' THEN 1 ELSE 0 END AS "Trade_EXP_Ontime_PDSL",
    CASE WHEN Customer_Master_Ext.CustomerClass = 'TRADE'
    AND isnull(Part_Master_Ext.ExpressShip, 0) <> '1' THEN 1 ELSE 0 END AS "Trade_MTO_Shipped",
    CASE WHEN SO_Detail.Shpdte_28 <= SO_Detail.CUSDUE_28
    AND Customer_Master_Ext.CustomerClass = 'TRADE'
    AND isnull(Part_Master_Ext.ExpressShip, 0) <> '1' THEN 1 ELSE 0 END AS "Trade_MTO_Ontime_RDSL",
    CASE WHEN SO_Detail.Shpdte_28 <= SO_Detail.Curdue_28
    AND Customer_Master_Ext.CustomerClass = 'TRADE'
    AND isnull(Part_Master_Ext.ExpressShip, 0) <> '1' THEN 1 ELSE 0 END AS "Trade_MTO_Ontime_PDSL",
    CASE WHEN Customer_Master_Ext.CustomerClass IN ('INTERCO', 'INTRA-CO')
    THEN 1 ELSE 0 END AS "Intraco_Shipped", -- 03 Intraco Lines Shipped
    CASE WHEN SO_Detail.Shpdte_28 <= SO_Detail.Curdue_28
    AND Customer_Master_Ext.CustomerClass IN ('INTERCO', 'INTRA-CO')
    THEN 1 ELSE 0 END AS "Intraco_OnTime_PDSL",
    CASE
    WHEN SO_Detail_Ext.LateReason = 'SSI' AND Customer_Master_Ext.CustomerClass IN ('INTERCO', 'INTRA-CO') THEN '1' --Customer caused late order
    WHEN SO_Detail.Shpdte_28 <= SO_Detail.CUSDUE_28
    AND Customer_Master_Ext.CustomerClass IN ('INTERCO', 'INTRA-CO')
    THEN 1 ELSE 0 END AS "Intraco_Ontime_RDSL",
    CASE WHEN isnull(Part_Master_Ext.ExpressShip, 0) = '1'
    THEN 1 ELSE 0 END AS "EXP_Shipped",
    CASE WHEN SO_Detail.Shpdte_28 <= SO_Detail.CURDUE_28
    AND isnull(Part_Master_Ext.ExpressShip, 0) = '1' THEN 1 ELSE 0 END AS "EXP_Ontime_PDSL",
    CASE
    WHEN SO_Detail_Ext.LateReason = 'SSI' AND isnull(Part_Master_Ext.ExpressShip, 0) = '1' THEN '1'
    WHEN SO_Detail.Shpdte_28 <= SO_Detail.CUSDUE_28
    AND isnull(Part_Master_Ext.ExpressShip, 0) = '1' THEN 1 ELSE 0 END AS "EXP_Ontime_RDSL" ,
ExactMAX.dbo.NumShopDays(SO_Master.ORDDTE_27,SO_Detail.shpdte_28) AS "AVG_LT_Days" ,
ExactMAX.dbo.NumShopDays(SO_Master.ORDDTE_27,SO_Detail.Cusdue_28) AS "RDSL_AVG_LT_Days" ,
ExactMAX.dbo.NumShopDays(SO_Master.ORDDTE_27,SO_Detail.Curdue_28) AS "PDSL_AVG_LT_Days" ,
CASE WHEN Customer_Master_Ext.CustomerClass = 'TRADE'
AND isnull(Part_Master_Ext.ExpressShip, 0) <> '1'
THEN ExactMAX.dbo.NumShopDays(SO_Master.ORDDTE_27,SO_Detail.shpdte_28)
ELSE NULL END AS "Trade_MTO_AVGLT" ,
CASE WHEN Customer_Master_Ext.CustomerClass IN ('INTERCO', 'INTRA-CO')
THEN ExactMAX.dbo.NumShopDays(SO_Master.ORDDTE_27,SO_Detail.shpdte_28)
ELSE NULL END AS "Intraco_AVG_LT" ,
CAST('0' AS FLOAT) AS "Trade_Backlog_PDSL" , --09 Trade $$ Past Due Backlog by PDSL
CAST('0' AS FLOAT) AS "Trade_Backlog_RDSL" ,  --10 Trade $$ Past Due Backlog by RDSL
CASE WHEN Customer_Master_Ext.CustomerClass = 'TRADE'
THEN (SO_Detail.Price_28 * SO_Detail.Shpqty_28)
ELSE NULL
END AS "Trade_Sales$$" ,
NULL AS "Total_PastDue_Dollars_RDSL" ,
NULL AS "Total_PastDue_Lines_RDSL" ,
NULL AS "Total_PastDue_Dollars_PDSL" ,
NULL AS "Total_PastDue_Lines_PDSL" ,
SO_Detail.CreatedBy ,
SO_Detail.CreationDate ,
Part_Sales.CRTLTO_29

FROM
    ExactMAX.dbo.SO_Detail LEFT JOIN
    ExactMAX.dbo.Part_Sales ON
    SO_Detail.PRTNUM_28 = Part_Sales.PRTNUM_29 LEFT JOIN
    ExactMAX.dbo.SO_Master ON
    SO_Detail.ORDNUM_28=SO_Master.ORDNUM_27 LEFT JOIN
    ExactMAX.dbo.part_Master ON
    SO_Detail.prtnum_28 = part_master.prtnum_01 LEFT JOIN
    ExactMAX.dbo.Account_Types ON
    Part_Master.ACTTYP_01=Account_Types.ACTTYP_104 LEFT JOIN
    ExactMAX.dbo.CUSTOMER_MASTER ON
    SO_Detail.CUSTID_28 = CUSTOMER_MASTER.CUSTID_23 LEFT JOIN
    ExactMAX.dbo.Part_Master_Ext ON
    SO_Detail.prtnum_28 = part_master_ext.prtnum_01 LEFT JOIN
    ExactMAX.dbo.Customer_Master_Ext ON
    Customer_Master.CUSTID_23 = Customer_Master_Ext.CUSTID_23 LEFT JOIN
    ExactMAX.dbo.SO_Detail_Ext ON
    SO_Detail_ext.ORDER_LIN_DEL = (SO_Detail.ORDNUM_28 + SO_Detail.LINNUM_28 + SO_Detail.DELNUM_28)

WHERE
    SO_Detail.Shpdte_28 BETWEEN @1StartDate AND @2EndDate
    AND SO_Detail.Status_28 IN ('4', '5')
    AND Part_Master.Acttyp_01 <> ''
    AND Part_Master.TYPE_01 IN ('A', 'C', 'P', 'S', 'X')

A: Consider the following steps for parameterized querying:
SQL

*

*Keep your long, intricate SQL in an .sql text file. Format with line breaks and indentation any way you need. Keep in same folder as workbook or centralized location for all users to read in VBA.

*Remove DECLARE and SET lines, keeping only the single SELECT command.

*In SELECT query replace every @ variable with qmarks ? for parameterization in VBA.
WHERE
    SO_Detail.Shpdte_28 BETWEEN ? AND ?

VBA

*

*Start a VBA Sub subroutine in Workbooks module or standalone module.

*Set up an ADO ODBC connection to your database. Many online examples and tutorials.

*Read in SQL query into a string variable:
' READ SQL QUERY FROM FILE INTO STRING
With CreateObject("Scripting.FileSystemObject")
      strSQL = .OpenTextFile("C:\path\to\my\SQL\Query.sql", 1).readall
End With

*Open an ADO Command object using connection object and query. Create and bind the two date parameters from wherever cells they are located in workbook. Then execute command to render an ADO recordset.
' DEFINE COMMAND OBJECT

Set cmd = New ADODB.Command
With cmd
    .ActiveConnection = conn   ' CONNECTION OBJECT
    .CommandType = adCmdText
    .CommandText = strSQL      ' SQL QUERY

    ' BIND DATE PARAMETERS FOR ? IN SQL, ASSUMING startDate AND endDate ARE VBA DATES
    .Parameters.Append .CreateParameter("dtparam1", adDate, adParamInput, , startDate)
    .Parameters.Append .CreateParameter("dtparam2", adDate, adParamInput, , endDate)

    ' BIND OUTPUT TO RECORDSET
    Set rs = .Execute
End With

*Use Range.CopyFromRecordset to output recordset to sheet starting at left most column. Do note: columns do not populate. Loop through recordset Fields for such column names.
ThisWorkbook.Worksheets("SQLData").Range("A2").CopyFromRecordset rs

(In entire VBA code, be sure to specify Dim variables with Option Explicit at very top and incorporate proper error handling that closes recordset and connection and release ASO resources by setting objects to Nothing with or without runtime errors.)

redpajama_set_name: RedPajamaStackExchange


And Thursday brought Little Frankie…he has pressure sores down to the bone. That's why his legs are bandaged….
Emaciated, covered in fleas and filth, rotting teeth..
Worst of all….the look of despair….
Her owner/trainer at the time was banned for drugging her…he has since been disqualified from the GBGB.
She has featured on more than one occasion for having drugs in her system.
By far in the best condition of them all..
He is emaciated, with rotting teeth and a rotting tail…he is filthy covered fleas and his own filth.


text:
The official travel site of the USA.
Destinations Trips Experiences
Trips Experiences
Shasta-Trinity National Forest
Sequoia and Kings Canyon National Parks
Cape Cod National Seashore
Pictured Rocks National Lakeshore
San Juan National Historic Site
San Juan Islands National Wildlife Refuge
National Mall and Monuments
Southwest Landscapes
Northern Atlantic Coast
Explore Northeast Beaches
Your Guide to 3 Big Cities: New York-Philadelphia-Washington, D.C.
The Yellowstone Loop: 9 Stops You Must Make on an Epic Western Road Trip
Culture and Canyons in Utah's San Juan County
By: Heiko Obermoeller
San Juan County, located in southeastern Utah, is known as the world's largest outdoor museum.
It was just over 100 years ago that the first Mormon settlers populated these mighty canyons. Today not only traces of these pioneers remain, but also of the indigenous peoples that lived here thousands of years earlier. San Juan's unique landscape, rich cultural heritage and monuments are certainly worth a visit.
The History of Pioneers at Bluff Fort
Begin your journey with a visit to the region's very first settlement. The first Mormons settled in Bluff, Utah, in the 1880s. Their dwellings have been reconstructed almost completely and the wooden houses have been painstakingly furnished. Audio tours tell interesting stories about the people who once lived here.
About an hour away is Natural Bridges National Monument, home to three of the biggest natural stone bridges in the world. In White Canyon you can see one of these bridges, the Sipapu Bridge, up close. This fun hike only takes around 45 minutes. Wooden ladders and stone staircases will lead you directly to impressive natural wonders. And, don't miss a visit to Owachomo Bridge, the largest in the park.
Plan to stay through the evening and at least part of the night. This is the first International Dark Sky Park, which means it's an area that prevents as much artificial light as possible from polluting the dark night skies.
Breathtaking Sights in Monument Valley
Continue your journey to the fascinating Monument Valley, one of the most famous natural landmarks in the U.S., known from countless films and commercials. Many of the buttes are named for what they resemble, such as The Mittens, Rooster Rock and Three Sisters. This area of iconic mesas is located on Navajo tribal territory, and locals offer guided tours. At John Ford's Point, a view known from the eponymous director's famous western movies, rent a horse and pose for a memorable picture!
Another place to visit for an incredible cultural experience is Hovenweep National Monument. Here, marvel at the towers that display the ability and resourcefulness of ancient civilizations. More than 7,000 years ago, what did they use to build the towers and houses on the edge of the cliffs? And how did they build them? A guide can give you all the answers.
Butler Wash Petroglyph
Next, visit the Butler Wash petroglyph. The lifelike pictures represented in the panel are impressive and perfect for a photo opportunity. Rent an all-terrain vehicle to experience the diversity of the landscape, hills and water. You can also rent a boat to ride along the San Juan River to visit Butler Wash.
Even More Iconic Places in San Juan County
The Four Corners Monument is where the borders of four U.S. states converge in one place. It is another fun spot to visit with the whole family. Come here and place your hands and feet in Colorado, New Mexico, Utah and Arizona all at once!
From here, drive around 2.5 hours to reach the Island in the Sky area of Canyonlands National Park, where you can enjoy breathtaking views onto wild canyon vistas.
As long as you have the time to explore, San Juan County will have time to welcome you. Travel in the off-season, from November through March, to enjoy travel savings and fewer crowds. San Juan County, Utah is definitely worth your visit!
More experiences nearby
An Insider's Guide to Monument Valley
How to See 5 Landscapes of Iconic Western Movies
Hole n' the Rock
Canyons of the Ancients
Spotlight: Mesa Verde National Park
Moab, Utah: Natural Landscapes Only Found Here
Getting the Shot: Mountain Biking in Moab, Utah
Explore Arches National Park
View more experiences
Salt Lake City: The Gems of Utah's Capital
Where to Ski in the USA for Families
Where to Stargaze in the USA
Romantic Adventures
Spotlight: Timpanogos Cave National Monument
7 Family-Friendly Outdoor Activities in Utah
Cedar City and Brian Head: Outdoor Family Fun in Utah
Kane County, Utah: Amazing Scenery and a Hollywood Past
Ski Utah: Winter Sports Heaven
Utah National Parks: The Mighty Five
Utah's Captivating Culture and Breathtaking Nature
5 Hikes Where You Have to Hold on Tight
10 Incredible Selfie Spots Across the USA
Getting the Shot: Climbing the Penguins in Arches National Park
National Parks Adventure: Rachel Pohl Shares Her Favorite Moments
12 Spectacular Images From National Parks Adventure
America Wild: National Parks Adventure: Interview With Director Greg MacGillivray
Where to See America Wild: National Parks Adventure
Visit USA (UK) Association

redpajama_set_name: RedPajamaCommonCrawl


for exploring behavioral issues across four primary dimensions.
The power of DiSC® Classic is now available on paper (see above) and online (web-based). Both options provide all the valuable feedback you'd expect from Wiley, the world's DiSC leader. You can also manage and administer multiple users within your company with an optional EPIC sub-account.
The DiSC Classic Facilitation System gives you the versatility to respond to a wide range of training demands, including sales, conflict management, teambuilding, or creating a DiSC culture in your organization. The DiSC Classic Facilitation System provides everything trainers need to administer the DiSC Classic in facilitated sessions. Modules are self-contained, can be mixed and matched to create a custom program and facilitate specific outcomes. The package includes more than 20 experiential learning activities for groups and individuals plus additional alternative activities. 250+ customizable PowerPoint® slides, integrated video into the PowerPoint so you don't have to toggle between one and the other with four sections (pure styles, meeting, effective communication, natural styles), course outlines (starter outlines for: communication, conflict, sales, interpersonal management, management, teambuilding) making it easy to organize modules into effective solutions.
"DiSC", "Everything DiSC", "Personal Profile System", and "QuikDiSC" are registered trademarks of John Wiley & Sons, Inc.


Did you catch these stamps? Ten stamps depicting British design classics. I love them - especially the routemaster bus.
I am known in my little neighbourhood post office for always asking for 'stamps with pictures' - well, they brighten up the mail don't you think?
i like the stamps .. i love the old red buses too and when i lived in London always prefered to take the bus not the tube hence i knew lots of numbers and routes. Hope you are no longer going round and round and that things have settled down.
I haven't seen those before... very classy! I like the penguin book.
I like 'stamps with pictures'! I am still using up my Christmas stamps!
I've been badgering my post office for them, but they're still running with the Xmas ones. And who wants those this time of year!
Yes I love them too.
I have bought some to stick on a postcard and display. The spitfire one especially, as the designer lived in a road just round the corner from where we live now and spitfires were made just down the road too, a bit of natioanl and local history.
What a good idea to ask for stamps with pictures. I must try that myself. Do you think they'll give me funny looks?
I haven't seen these - aren't they fab. I hardly ever buy stamps and still have a purse full of panto ones from Christmas.
I am visiting you via the wonderfule Pipany's site.
I am another fan of great stamps. Every time I get over to the UK I always pick up a bunch to see how the government is still supporting excellent graphic design.
Over here, I try to find good "commemorative" stamps, but often the selection is rather disma.
Still remember the Gee's Corner quilt stamps, and now we have some pretty exotic fruits decorating our stamps for postcards.
Oh, there was recently a very jazzy design based on the theme of Latin Jazz, and the 42 cent denomination was cleverly incorporated into the jaggedy, angled, yet graceful composition of that stamp's look.
Our Christmas, aka "holiday" stamps are not even on the same runway as yours.


The event sponsors for the 2018 iLINKS Innovations conference and exhibition have been announced as EMIS Health, InterSystems and Samsung.
Organised by NHS Informatics Merseyside, this year's event will be extra special, taking place on Thursday 5 July 2018 at Aintree Racecourse in Liverpool, which is also the 70th anniversary of our NHS.
Kindly supporting this year's event are EMIS Health, InterSystems and Samsung, who are our event sponsors for 2018.


text:
This Week in Health Care Reform - February 28th, 2014
CMS announces proposed changes to Medicare Advantage; while, both parties jockey for the political high ground on health care reform; most Americans are unaware of the open enrollment deadline; and, the promise of telehealth is explored.
Medicare Advantage on the Clock: As expected, last Friday, the Centers for Medicare & Medicaid Services (CMS) released its long-anticipated proposed changes to Medicare Advantage for 2015. And, also as expected, the announcement was met with more than a little displeasure…with equal parts confusion and anger mixed in for good measure. At dispute, just how large – or small – the proposed rate cuts to the popular program really are. With more than 15 million seniors and persons with disabilities – over a quarter of the total Medicare population – depending on Medicare Advantage, it's easy to see why supporters are so eager to step to its defense. For those that have been following along, already we've seen a who's who of trade associations, business organizations, and medical groups unite in opposition to any proposed cuts to Medicare Advantage. The issue has even drawn considerable support from lawmakers on both sides of the aisle, with 40 U.S. Senators – nearly half of whom were Democrats – sending a bipartisan letter to CMS earlier this month, urging the agency that oversees the program to maintain level funding for Medicare Advantage for 2015. (A similar letter is currently being circulated in the House.) In the wake of last week's release, Republican Senate leaders (who just saw their party edge out Democrats in a recent poll of seniors' views on how both parties handle Medicare issues), led by Senate Minority Leader Mitch McConnell (R-Kentucky), called upon the Administration this past Tuesday to suspend the proposed cuts. And, yesterday, a new analysis was released showing just what beneficiaries can expect should the proposed payment cuts go into effect. Prepared by management consulting firm, Oliver Wyman, their report estimates that CMS' proposed changes would result in a 5.9 percent cut to Medicare Advantage payments in 2015. That translates into those seniors and persons with disabilities that currently depend on their Medicare Advantage plans for their critical care having to face benefit reductions and premium increases of as much $75 per member per month next year. When combined with last year's 6 percent cut, the program now faces the very real possibility of being hit with a double-digit cut over just two years, leaving beneficiaries on the hook for as much as $1,740 over that same period. The report goes on to warn that, cuts of this magnitude could result in a "high risk of disruption" in the market, as well as the "potential for plan exits, reductions in service areas, reduced benefits, provider network changes, and (Medicare Advantage) plan disenrollment." With CMS expected to announce its final decision on the proposed changes in early April, supporters continue to raise their voices in support of this vital program. We will be looking to our Health Action Network members, like you, to join the effort, too. In the meantime, keep up with the latest by visiting the Coalition for Medicare Choices to learn how you can help.
The Politics of Reform: It's no secret that Washington finds itself locked in a seemingly endless game of political chicken. The hyper-partisanship that divides Capitol Hill's denizens into starkly red and blue camps has become more rule than exception. Perhaps nowhere is this more evident than in each party's approach to health care reform. Already, we've seen the GOP readying votes on a handful of issues (some relating to the health care law), seizing upon states' struggles with implementation, all the while, openly questioning the President's newfound preference to "sidestep Congress" to advance his agenda. Meanwhile, Democrats, believing the health care law's fortunes to have taken a turn for the better, have pledged to take the offensive on the issue heading into November's midterm elections, despite overtures of finding legislative comity amongst the parties. Regardless, it's clear that the politics of health care reform (not to mention the delays that have pockmarked the road to implementation) will continue to shape the national discussion.
What Deadline?: Earlier this week, CMS released the latest enrollment numbers for the state and federal exchange marketplaces. So far this month, approximately 700,000 people have signed up for coverage through the exchanges, bringing the total enrollment to roughly 4 million since October of last year. Whether the improved pace speaks to a vastly improved user experience on the troubled HealthCare.gov website or is more a result of the concerted outreach efforts currently underway, officials were encouraged by the "strong demand nationwide" that they are seeing. Surprising, some would say, given the recent Kaiser Family Foundation poll that found that the vast majority of the uninsured (76 percent) were completely unaware of the looming open enrollment deadline, just a month away, not to mention the recent revelation that enforcement of the controversial individual mandate by the IRS may be less than aggressive. Despite the uptick, many are left scratching their heads over the original projected target of 7 million enrollees, while others are more concerned with what the numbers behind those that have signed up aren't telling us.
Realizing Telehealth: With so much attention being paid to the numerous ways we can improve the nation's health care system, it's perhaps surprising to find that so little of that focus is being devoted to exploring the role that technology can play in helping us find common sense solutions. That was the focus of a briefing this past Wednesday held by the Information Technology & Innovation Foundation (ITIF) on Capitol Hill. The focus: Realizing the promise of telehealth. As health care coverage continues to expand under the health care law, new and greater demands will be placed on our already overtaxed health care system. As such, lawmakers need to leverage available technologies to increase access, lower costs, and improve the quality of care. One way this can be done is to deploy telehealth capabilities to connect patients and doctors in ways that encourage routine encounters across previously inconvenient divides. However, despite these technologies being available today, widespread adoption remains elusive, likely due to the variety of policy barriers that stand in the way. This week's ITIF event, featuring Reps. Doris Matsui (D-California) and Bill Johnson (R-Ohio) – cosponsors of the Telehealth Modernization Act – brought the issue into the legislative spotlight, discussing what Congress can do to help remove these barriers to adoption and ensure that all Americans can tap into the benefits of telehealth.
In this post-reform world, is it time to start thinking about reforming reform?


I have never claimed to be anyone but who I am. My point stands.
This is just nonsense and I'm not going to play this little game with you.
It's not a game, your logic is flawed, that is not my problem, it is yours.
Yep. might check out mike555 declaration that some believe in Jesus but don't really believe , then makes a thread where if you believe in Jesus your salvation is secure AND ANYTHING YOU DO DOES NOT MATTER.
Oh and don't forget that some will post that unbelievers are in hell but all will believe while in hell because they will understand what they missed out on.
Sure, everyone can be a bit inconsistant, but I think in matters of eternal damnation if it is true you all drop the ball big time because of what you believe is at stake. So lets talk about who's inconsistancies will do more damage, if your perspective is true, you guys really stink and probably won't even make it to heaven because you've bungled things up so badly.
If a person does not believe in the Jesus Christ of the Bible, but has instead placed his belief in some false Jesus, and anyone who denies that Jesus Christ is God has placed their faith in a false Jesus, that person is not saved. If a person has believed in the Jesus Christ of the Bible for salvation, then that person is eternally saved.
Yeah, hopefully since Jesus Christ of the bible is the only Christ there is, then there isn't gonna be much confusion there. But you create confusion by stating in a thread that some profess in Jesus Christ but really do not believe. There just isn't any other Jesus in the first place.
So more convoluted nonsense that only preoccupies Christians needlessly.
You interpret the bible to say so. That's all. As you saw in another thread, Paul did not teach that and none of you ET believers can figure out why he didn't.
No, I simply read the Bible because 100% of it is true. I don't need to try to justify certain parts in order for them to fit my belief. I don't need to talk about how the authors were ignorant savages and the words are just "written in ink." I don't need to talk about misinterpretations, mistranslations, or anything else to try to validate my beliefs. I'm not saying you do all of these things, but this is the kind of stuff that is posted on a daily basis by universalists.
Paul most certainly talked about it! Phil 3:18-19, Romans 9:22, 2 Thess 1:7-9, 1 Thes 5:3. Deny hell all you want, but Jesus spoke about it more than heaven!
Because their false belief doesn't hold water. They contradict themselves all the time. It's amazing to me that people can be so blind.
I agree with you, and also Mike555 has said that even if you believe in Jesus, if you think that being sorry for your sins somehow plays a role in your salvation, then you are not saved because you don't "understand" and have tried to add something to your own salvation. So, in that view, even though "understanding" is required, that somehow doesn't count as adding to your own salvation. So much confusion!
That's why it's a false Jesus. Such as the Jesus that the Jehovah's witnesses believe in. All who profess to believe in Christ, but deny His deity, have an improper object for their faith and are still under eternal condemnation and on their way to the lake of fire.
Many profess to believe in Jesus but have never entered into a saving relationship with Him.. As Matthew 7:21-23 makes clear.


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Home > Energy > Westerners Don't Believe Climate Change is Critical
Westerners Don't Believe Climate Change is Critical
by Martin Banks 2 years ago Nov 25, 2020 0 Comments
New research in Europe and the United States shows that large portions of the public still do not accept the urgency of the climate crisis, and only a minority believe it will impact them and their families severely over the next fifteen years.
The survey, which was commissioned by the Open Society and the European Policy Institute, forms part of a major new study of climate awareness. It charts attitudes on the existence, causes, and impacts of climate change in Germany, France, Italy, Spain, Sweden, Poland, Czech Republic, the UK and the US. It also examines public attitudes to a series of policies that the EU and national governments could harness to reduce the damage inflicted by human-made emissions.
The report finds that, though a clear majority of European and American respondents are aware that the climate is warming, and that it is likely to have negative impacts for humankind, there is a distorted public understanding of the scientific consensus in both Europe and America.
This, the report argues, has created a gap between public awareness and climate science, leaving the public underestimating the urgency of the crisis, and failing to appreciate the scale of the action required. All but a small minority accept that human activities have a role in climate change – with no more than 10% refusing to believe this in any country surveyed. However, while outright denial is rare, there is widespread confusion about the extent of human responsibility.
Large minorities – ranging from 17 per cent to 44 per cent across the surveyed countries – still believe that climate change is caused equally by humans and natural processes. This matters because those who do accept that climate change is the result of human action are twice as likely to believe it will cause negative consequences in their own lives. Significant minorities believe scientists are equally divided on the causes of global warming – including two thirds of voters in Czech Republic (67%) and nearly half in UK (46%).
In reality, 97 per cent of climate scientists agree that humans have caused recent global warming. A large majority of Europeans and US citizens in all nine countries polled agree that climate change requires a collective response, whether to mitigate climate change or adapt to its challenges. Majorities in Spain (80%) Italy (73%), Poland (64%), France (60%), the UK (58%) and the US (57%) agree with the statement that "we should do everything we can to stop climate change."
The report also finds that there is polarisation along party political lines on climate change – in Europe as well as the US. Those on the left tend to be more aware of the existence, causes and impact of climate change, and more in favour of action, than people on the right. These differences are more important than demographic variation in most countries. For example, in the US, those who identify as left in their political orientation are nearly three times as likely to expect a negative impact on their own lives (49%) compared to those who identify as more on the right (17%).
Polarisation is also marked in Sweden, France, Italy and the UK. The only country where there is balance across the spectrum is the Czech Republic. Majorities are willing to act on climate change, but the actions they favour tend to be consumer-focused rather than efforts to create collective social change. A majority of respondents in every country say they have already cut their plastic consumption (62%), their air travel (61%) or their car travel (55%). A majority also says they either already have or are planning to reduce their meat consumption, switch to a green energy supplier, vote for party because of their climate change programme, or buy more organic and locally produced food.
However, people are much less likely to support civil society engagement directly, with only small minorities having donated to an environmental organization (15% across the survey), joined an environmental organization, (8% across the survey), or joined an environmental protest (9% across the survey). Only a quarter (25%) of respondents across the survey say they have voted for a political party because of their climate change policies. Just 47 per cent of those surveyed believe they, as individuals, have very high responsibility for tackling climate change.
Only in the UK (66%), Germany (55%), the US (53%), Sweden, (52%), and Spain (50%) is there a majority who feel a high sense of responsibility themselves. In every country surveyed people are more likely to think that their national Government has a high responsibility for tackling climate change.
This ranges from 77% of those surveyed in Germany and the UK to 69% in the US, 69% in Sweden and 73% in Spain. In every EU country, respondents were slightly more likely to see the EU as having a high responsibility for reducing climate change than national Governments. The polling also finds that people prefer to be offered incentives to act on climate change rather than face bans or carbon taxes.
A small majority are willing to pay some more tax for greater action on climate change – apart from in France, Italy and the Czech Republic – but the percentage willing to pay more than a small amount (one hour's wage per month) is limited to at most a quarter – in Spain and the US. Increasing taxes on all flights, or introducing a levy for frequent flyers, garnered some support across the polled countries (between 18 per cent and 36 per cent, collectively). Although the preferred policy for tackling air travel emissions, by a clear margin, was improving ground infrastructure for buses and trains.
Heather Grabbe, director of Open Society European Policy Institute, said:"Many citizens across Europe and US still don't realise that scientific consensus on human responsibility for climate change is overwhelming. Though outright denialism is rare, there is a widespread false belief, promoted by vested interests opposed to emissions reductions, that scientists are split on whether humans are causing climate change – when in fact 97% of scientists know that.This soft denialism matters because it lulls the public into thinking that climate change won't affect their lives much over the next decades, and they don't realise how radically we need to change our economic system and habits to prevent ecological collapse. Our polling shows that the more convinced people are that climate change is the result of human activity, the more accurately they estimate its impact and the more they want action."
Jan Eichhorn, research director of d|part and lead author of the study, said: 'Publics in Europe and the US want to see action in response to climate change across all demographics. Politicians need to show leadership in responding to this desire in an ambitious way that enhances people's understanding of the severity of the crisis and the impact humans have – as this understanding is not developed enough so far. Relying on individual action is not enough. People see the state and international organisations at the EU in charge. People are principally open to being convinced to support more extensive action, but to achieve this urgently requires further work from political and civil society actors."
A sizeable majority of Europeans and Americans believe that climate change is happening. In all nine countries surveyed, an overwhelming majority of respondents say that the climate is probably or definitely changing – ranging from 83 per cent in the US to 95 per cent in Germany.
Outright climate change denial is scarce in all of the countries surveyed. The USA and Sweden have the largest group of people who either doubt climate change or are convinced it is not happening, and, even here, it only comprises just over 10 per cent of those surveyed.
However, over a third (35%) of those surveyed in the nine countries attribute climate change to a balance of natural and human processes – with this feeling most pronounced in France (44%), the Czech Republic (39%) and the US (38%). The plurality view among respondents is that it is caused "mainly by human activity".
A significant group of 'soft' attribution sceptics believe that, contrary to the scientific consensus, climate change is caused equally by human activities and natural processes: these constituencies range from 17 per cent in Spain to 44 per cent in France. When added to the "hard" attribution sceptics, who don't believe human activity is a contributing factor to climate change, these sceptics together make up the majority in France, Poland, the Czech Republic and the USA.
Majorities believe that climate change will have very negative consequences for life on earth in Spain (65%), Germany (64%), the UK (60%), Sweden (57%), the Czech Republic (56%) and Italy (51%). However, there is a significant minority of "impact sceptics" who believe the negative consequences will be outweighed by the positive – ranging from 17 per cent in the Czech Republic to 34 per cent in France. There is also a group in the middle who don't see global warming as harmless, but think that negative consequences will also be balanced by positive ones. This "middle group" ranges from 12 per cent in Spain to 43 per cent in France.
Most people don't think their own lives will be strongly affected by climate change in the next fifteen years. Only in Italy, Germany and France do more than a quarter of people think their lives will be strongly disrupted by climate change by 2035 if no additional action is taken. While the prevailing view is that there will be some change to their lives, a considerable minority believe their lives won't change at all as a result of unchecked climate change – with the largest group in the Czech Republic (26%) followed by Sweden (19%), the USA and Poland (18%), Germany (16%) and the UK (15%).
Age makes a difference to views on climate change, but only in certain countries. Overall, younger people tend to be more likely to expect negative impacts of climate change on their lives by 2035 if nothing is done to address the issues. This trend is particularly strong in Germany; where negative impacts are expected by 36 per cent of 18-34 year olds (compared to 30% of 55- 74 year olds), Italy; (46% of 18-34 year olds compared to 33% of 55-74-year olds), Spain; (43% of 18-34 year olds compared to 32% of 55-74 year olds) and the UK; (36% of 18-34 year olds compared to 22% of 55-74 year olds).
Imposing higher taxes on flights is only seen as the best option to reduce emissions from flights by a minority – ranging from 18 per cent in Spain to 30 per cent in the US and 36 per cent per cent in the UK. An outright ban on internal flights within countries is even less popular, enjoying most support in France (14%) and Germany (14%). The most popular policy for reducing emissions from plane travel is improving the train and bus networks, which is chosen as the best policy by a majority of respondents in Spain, Italy and Poland.
Majorities in most countries are willing to persuade their friends and family to behave in a more climate-friendly way – with only 11 per cent in Italy and 18 per cent in Spain not willing to do this. However, nearly 40 per cent of people in the Czech Republic, France, the US and the UK would not contemplate this idea at all.
There is widespread support for switching to a green energy firm to provide household energy. However, France and the US have large minorities (42% and 39% respectively) who would not consider a switch to green energy. This compares to just 14 per cent in Italy and 20 per cent in Spain who would not consider a change to green energy.
Majorities in Europe are willing to reduce their meat consumption, but figures vary widely. Only a quarter of people in Italy and Germany are not willing to reduce their meat consumption, compared to 58 per cent of people in the Czech Republic, 50 per cent people in the US, and around 40 per cent in the Spain, the UK, Sweden and Poland.
Martin Banks
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Well it's T-Mobile Tuesday once again and this marks the one week anniversary of me ditching AT&T after a decade of being a loyal customer.
Growing up unless you had a Sidekick, T-Mobile was looked at like a cut rate carrier. I'm happy to report that's not really the case anymore. And I don't think it's been that way for some time.
This was the perfect week to sample my new service as I was going to be traveling around all of my favorite spots, not only in Philadelphia, but New Jersey as well.
The most impressive was the 60Mbps when I was on the Market-Frankford Line (Philadelphia's underground subway).
I'd have to say one of my favorite things about making a switch to an Unlimited plan was being able to go into my iPhone settings and allow any and all apps to use data since I no longer was afraid of going over my limit. Also, as a big Spotify user I could now stream online music with the same lack of worry.
With that being said I used a whopping 4.2Gb of T-Mobile data in 7 days. Now keep in mind since I was traveling my Wifi usage was minimal.
Another interesting thing to note is the time the phone spent searching for a signal. Now the only time I've noticed it didn't have service is in my old basement office (where I didn't get AT&T either) plus I'm always on wifi so with wifi calling and iMessage I really went on without noticing. It is worth noting however that over the course of the last 7 days the phone spent 5% of it's battery life trying to find that signal.
I did not particularly notice the battery drain but that could have been in part of the Apple Smart Battery Case, I was using for some of the week.
The most important thing is that I finally feel like I'm using the phone in the way that Steve Jobs envisioned. Without limitations!
I hope this was helpful for you. If you're thinking about making the switch and have any questions please let me know in the comments below.


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Flood Barrier America, Inc. is committed to safeguarding your privacy. Contact us at privacy@floodbarrieramerica.com if you have any questions or problems regarding the use of your Personal Data and we will gladly assist you.


Carers and teachers need to know what to do when a child is injured or becomes suddenly ill. Most injuries that require first aid are not life-threatening. Usually, first aid involves simple, common sense procedures. However, first aid can sometimes mean the difference between life and death. All carers and teachers should have paediatric first aid training. Paediatric First Aid for Carers and Teachers (PaedFACTs) can be read either as a stand alone manual or as part of the very successful PaedFACTs course, designed to give carers and teachers the education and confidence that they need to effectively care for children. The PaedFACTs course and manual have been carefully written to align with the advice and guidance issued by prominent United Kingdom (UK) bodies. Endorsed by the College of Paramedics in the UK and the American Academy of Pediatrics internationally, any parent, carer, or teacher can be assured of up-to- date and relevant first aid information, approved by professional organisations.


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Q: node.js : rendering error with vash I'm getting a rendering error in node.js with vash. I'm really struggling to see what the issue is. Below is the error and the code causing the error.
TypeError: Problem while rendering template at line 4, character 2.
 Original message: object is not a function. Context: 2 |
 @html.block('body', function(model){ 3 |
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 (C:\Users\user009\Dropbox\cpp\node_modules\vash\build\vash.js:2213:4)
 at Object.vash.loadFile
 (C:\Users\user009\Dropbox\cpp\node_modules\vash\build\vash.js:2072:10)
 at helpers.extend
 (C:\Users\user009\Dropbox\cpp\node_modules\vash\build\vash.js:2190:8)
 at eval (eval at <anonymous>
 (C:\Users\user009\Dropbox\cpp\node_modules\vash\build\vash.js:1820:24),
 <anonymous>:7:21) at vash.link.linked
 (C:\Users\user009\Dropbox\cpp\node_modules\vash\build\vash.js:1860:12)
 at vash.renderFile
 (C:\Users\user009\Dropbox\cpp\node_modules\vash\build\vash.js:2082:21)
 at Object.vash.loadFile
 (C:\Users\user009\Dropbox\cpp\node_modules\vash\build\vash.js:2070:10)
 at View.vash.renderFile [as engine]
 (C:\Users\user009\Dropbox\cpp\node_modules\vash\build\vash.js:2078:8)
 at View.render
 (C:\Users\user009\Dropbox\cpp\node_modules\express\lib\view.js:76:8)
 at Function.app.render
 (C:\Users\user009\Dropbox\cpp\node_modules\express\lib\application.js:502:10)

router:
app.get("/", function (request, response) {
    response.render('index', {title: 'letsdoit'});
});

layout.vash
<!DOCTYPE html>

<link href="/css/site.css" rel="stylesheet" />
<title>@model.title</title>

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index.vash
@html.extend('layout', function(model){
    @html.block('body', function(model){

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    });
});

A: In your index.vash file, make sure the path to the layout.vash file is correct.
For example, if the views folder structure is views/home/layout.vash and views/home/index.vash, then:
@html.extend('layout', function(model){

should be:
@html.extend('home/layout', function(model){

hope it helps.


Certified sites offer prospective companies a host of advantages, including the opportunity to move quickly to the construction phase. Properties certified through Louisiana Economic Development Certified Sites Program have meet strict standards and are considered project ready.
North Louisiana offers 4,300 acres of certified sites - ready for development. In today's fast paced business environment, certified sites allow companies to fast-track their project by providing assurance the property holds no surprises that could delay development. A Louisiana Economic Development (LED) Certified Sites property is a project-ready industrial site that has completed a rigorous review process by Louisiana Economic Development and URS, an independent, third-party engineering firm.
Specific site details, such as zoning restrictions, title work, Phase I Environmental Assessments, geotechnical surveys and cultural resources surveys are reviewed for compliance and authenticity. The LED Certified Sites program provides prospective companies a peace of mind that the site is project-ready.
North Louisiana offers some of the largest certified sites in the state. Check out these certified properties.


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Here's a fabulous spring brunch dish for two. Simple and delicious.
Add 200-300ml/7-10fl oz water to a pan with the butter. Bring up to the boil, drop in the ham and warm through. Once the volume of liquid has reduced by half and has started to emulsify, add the asparagus and carefully remove the ham. Cook the asparagus for one minute in the emulsified liquor.
Add the chopped parsley at the end. Keep warm.
Cook the potato strips in boiling water until soft. Drain and dry the potatoes. Put the chips into the deep fat fryer and cook until crisp.
Meanwhile, heat a small frying pan and add the oil. Once hot, add the eggs and fry to your liking.
To serve, put the warm ham onto each of two serving plates, and pile the asparagus next the ham. Taste the ham cooking sauce and season with salt and pepper as necessary. Spoon the sauce over the ham and asparagus and top with a fried egg and a small pile of chips.


Home∕About Business∕definition of business∕What Is A Business Customer?
Recently, I've had an elevated number of inquiries on this very I figured, now could be as good as time as ever to briefly re-go to this concern for the benefit of my readership. Purchasers who have consistently had a great experience with your company shall be more than keen to spread the phrase to others who're in need of your product or service. Some individuals assume you don't need a marketing strategy except you are making an attempt to borrow money. Strategic human useful resource administration will be defined as the linking of human assets with strategic goals and aims with a view to enhance business efficiency and develop organizational tradition that foster innovation, flexibility and aggressive benefit.
Enterprise is an financial exercise , which is related with steady and common production and distribution of products and services for satisfying human desires. Currently organizations are beginning to see that information and content shouldn't be considered separate elements of information administration, but instead ought to be managed in an integrated enterprise strategy.
If enterprise is good, loads of services or products are being sold and if enterprise is bad , few of them are being sold. The distinction between internet sales revenues and cost of goods offered is known as gross income , for instance, whereas the online earnings from operations—before taxes and earlier than good points and losses from financial and extraordinary gadgets—is called operating income (or working earnings).
Culture influences management, selections and all enterprise capabilities from accounting to manufacturing. The value of business analysis is in realization of advantages, avoidance of value, identification of new opportunities, understanding of required capabilities and modeling the group. You may at all times find firms that offer services at very low prices.
These 4 tiers include planning strategically,working or business model evaluation, process definition and design and the technical business evaluation. Self-service Enterprise intelligence offers end-customers the ability to do extra with their information without necessarily having technical skills. In some cases, key parts of the Business Architecture may be performed in different activities; for instance, the enterprise mission, imaginative and prescient, technique, and goals may be documented as a part of some wider business technique or enterprise planning activity that has its personal lifecycle within the enterprise.


of spinal segments and correct posture.
*What Causes Spinal Pain after injury?
front to back or side to side of the vertebral column.
*Most commonly people in a vehicle that is suddenly and forcibly struck fromthe rear may sustain "whiplash" injuries. It also happens when workers slip and fall on the job, or experience some other type of injury. If not treated with due diligence, long term disability often occurs.
connective tissue - and sometimes other structures as well.
because they have been stretched beyond normal limits.
*Spinal vertebrae are also knocked painfully out of alignment.
involved in such an injury.


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In Sioux City, Trump says he's 'an outsider fighting for you.'
Jason Noble
jnoble2@dmreg.com
SIOUX CITY, Ia. — In his final visit to Iowa as a presidential candidate, Donald Trump cast himself as a political outsider and a force for change in Washington, D.C.
On both scores, the Republican nominee drew a sharp contrast with Democratic rival Hillary Clinton, whom he derided as the "candidate of yesterday."
"It's time for real leadership and it's time for change," Trump told a crowd of 4,300 at the downtown convention center here. "Hillary Clinton is not change. Hillary Clinton is four more years of Obama."
The visit to Sioux City — the population center of Iowa's conservative northwest corner — was the first of five rallies Trump held across the country on Sunday, less than 48 hours before the polls open on Election Day.
"We are going to give you the respect that you deserve," Trump told the crowd. "Hillary's an insider fighting only for herself and her special interests. I'm an outsider. I've become an outsider fighting for you."
The 35-minute speech hit all of Trump's familiar campaign promises — to build a wall along the southern border, to end illegal immigration and shut down refugee resettlement programs, and to renegotiate international trade deals — as well as the caustic attacks on Clinton, political "elites" and the news media that represent his most reliable applause lines.
But Trump also plainly and explicitly implored his supporters to get out and vote for him.
"In two days we will get the change you've been waiting for for your entire life," Trump said. "I hope you will consider it the greatest and most important vote that you will ever cast, because we will start winning again."
Rally attendee Jolaine Noreen of Sioux City said her support for Trump was rooted in her distrust and dislike for Clinton, but also a desire to return to the United States she knew as a child growing up in the 1950s. For her, making America "great again" means returning to that time, when she said everyone had stronger morals and jobs were more plentiful.
"It was just a much better country back then," the 66-year-old retiree and homemaker said. "And I'm hoping Donald Trump will take us back there again."
Trump's final Iowa visit comes as he appears headed toward victory in the state. He leads Clinton by 7 percentage points in a Des Moines Register/Mediacom Iowa Poll conducted last week, making Iowa perhaps the most Trump-friendly of any state that backed Obama in 2012 and which has been viewed as a swing state over the last few months.
In a statement on behalf of Clinton's campaign, former Gov. Tom Vilsack called Trump's vision for America "dark and divisive."
"He has shown us time and again that he is temperamentally unfit and unqualified to be president and commander in chief," Vilsack said. "Iowans deserve a president like Hillary Clinton who holds the optimistic view that Americans are stronger together, rather than one who could destroy the values that would tear our country apart."
Howie McGahey, a locomotive engineer from Urbandale who runs a train between Fort Dodge and Sioux City, said Trump's promises about improving the economy and ending corruption resonate with him.
"I just cannot see this country going through another four years of a Democrat in office," McGahey said. "There's been no growth in jobs. We're just kind of on a steady line, no growth, no nothing. And I do believe it's just been corrupt. Washington has been corrupt."
Key to Trump's rise in Iowa has been support from the state's Republican elected leaders, and that support was fully on display Sunday.
Preceding Trump on stage Sunday were Gov. Terry Branstad, Lt. Gov. Kim Reynolds, U.S. Sen. Joni Ernst and U.S. Rep. Steve King.
Branstad likened Trump's candidacy to that of Ronald Reagan in 1980, and echoed the candidate's rhetoric casting the presidential race as a contest between the American people and out-of-touch elites.
"When it comes right down to it, on Tuesday it's the American people, not the elite of America, that are going to determine the winner," Branstad said. He added, "We've got to have new leadership, we've got to have strong leadership, and we need to make America great again and Donald Trump will do it!"
Ernst, in turn, described Trump's candidacy as a continuation of the 2014 Republican wave that swept her into the Senate.
"Our country needs a change and it was all of you that led that way starting in 2014 when you sent me and a brand new class of freshman Republicans to the United States Senate," Ernst said. "You did that, folks, you did that, and now we need to finish what we started and take our White House back."
Trump returned the favor from the stage, complimenting all four of the Iowa Republicans and suggesting Branstad might have a role to play in his administration's relations with China.
"There is nobody that knows more about trade than him," Trump said. "He's one of the ones that dealing with China, he would be my prime candidate to take care of China."