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An organism can respond to changing environmental conditions by adjusting gene regulation and by forming alternative phenotypes. In nematodes, these mechanisms are coupled because many species will form dauer larvae, a stress-resistant and non-aging developmental stage, when exposed to unfavorable environmental conditions, and execute gene expression programs that have been selected for the survival of the animal in the wild. These dauer larvae represent an environmentally induced, homologous developmental stage across many nematode species, sharing conserved morphological and physiological properties. Hence it can be expected that some core components of the associated transcriptional program would be conserved across species, while others might diverge over the course of evolution. However, transcriptional and metabolic analysis of dauer development has been largely restricted to Caenorhabditis elegans. Here, we use a transcriptomic approach to compare the dauer stage in the evolutionary model system Pristionchus pacificus with the dauer stage in C. elegans.
We have employed Agilent microarrays, which represent 20,446 P. pacificus and 20,143 C. elegans genes to show an unexpected divergence in the expression profiles of these two nematodes in dauer and dauer exit samples. P. pacificus and C. elegans differ in the dynamics and function of genes that are differentially expressed. We find that only a small number of orthologous gene pairs show similar expression pattern in the dauers of the two species, while the non-orthologous fraction of genes is a major contributor to the active transcriptome in dauers. Interestingly, many of the genes acquired by horizontal gene transfer and orphan genes in P. pacificus, are differentially expressed suggesting that these genes are of evolutionary and functional importance.
Our data set provides a catalog for future functional investigations and indicates novel insight into evolutionary mechanisms. We discuss the limited conservation of core developmental and transcriptional programs as a common aspect of animal evolution.
Keywords:Dauer larvae; Developmental systems drift; Transcriptomics; Evolution of gene regulation; Horizontal gene transfer
The primary mechanisms of an organism to respond to changing environmental conditions are alterations of gene expression profiles and the formation of dormant or dormant-like developmental stages. Nematodes are found in great numbers and species richness in most ecosystems on earth. This omnipresence is attributed to the evolution of a potent life history strategy to respond to changing environments [1]. Under favorable conditions nematodes such as Caenorhabditis elegans and Pristionchus pacificus undergo direct development, which can be completed within 3–4 days. In contrast, under harsh environmental conditions such as food scarcity, high temperature and high population density, these nematodes form an arrested developmental stage, the so-called dauer larvae [2].
Dauer larvae are stress-resistant and "non-aging" and they facilitate the survival and dispersion of the organism [3]. Entry into the dauer stage represents the major life history response of nematodes to escape unfavorable environmental conditions [1]. This developmental switch is accompanied with remarkable transcriptional and metabolic changes as shown in several studies in C. elegans[4-8]. Since the dauer larva is an ecologically induced, conserved developmental stage found in many species of free-living nematodes, it can be expected that some genetic components involved in development might be conserved, while those facilitating ecological adaptation might diverge across species.
However, the similarity and divergence patterns of nematode transcriptomes have never been systematically investigated in the context of dauer larvae. While several transcriptional studies in C. elegans provide a platform for comparative studies [5,6,8-14], little is known in other free-living nematodes. Here we present a comparative transcriptomic approach to characterize the expression profiles of the dauer and dauer exit stages in C. elegans and P. pacificus. We measured transcriptome-wide expression changes with the help of the Agilent microarray platform, and use this data to identify the extent and nature of similarity or differences in the dauer-associated transcriptomes in the two nematode species.
C. elegans was the first metazoan to have its complete genome sequenced [15]. Ever since, C. elegans has been at the forefront of embracing novel "omics" technologies, including transcriptomics [16,17], proteomics [18,19] and RNAseq [20]. Comparative genomics is a powerful tool to address elementary questions in evolutionary developmental biology, such as what components of the regulatory "tool-kit" are conserved [21]. By now, the genome of six additional nematodes and many more transcriptomic studies have been reported (for review see [22]). Comparative transcriptomics opens up new vistas on how regulatory "tool-kit" components are employed to generate different morphologies across species [21,23]. In nematodes, comparative transcriptomic approaches provide a powerful way to analyze whole body responses to changes in the environment or the exposure to pathogens. The dauer stage represents a whole body response of the nematode to changing environmental conditions and is ideally suited for comparative transcriptomics studies.
We compare C. elegans to P. pacificus, an established model for comparative developmental biology, evolutionary biology and ecology [24]. Forward and reverse genetic as well as transgenic techniques have been established in P. pacificus, its genome has been sequenced [25] and its proteome has been analyzed by tandem mass spectrometry [26]. The sequencing of the P. pacificus genome revealed many important and unexpected features, such as a substantially larger size and a higher number of predicted protein-coding genes when compared to C. elegans, the presence of horizontal gene transfer and the duplication of genes encoding enzymes involved in the detoxification of xenobiotics [25]. Many of these features have been discussed in the context of a specific association with scarab beetles, in which P. pacificus is found in the wild [27]. Specifically, P. pacificus exists exclusively as dauer larvae on the living beetle and only reproduces after the beetle´s death by feeding on microbes that develop on the carcass [27-29]. This specific, so-called necromenic lifestyle shows the unique importance of the dauer stage and asks for a detailed functional investigation of the associated transcriptional programs.
Two developmental processes that have been studied in great detail at the genetic and molecular level in P. pacificus are the formation of the vulva, the egg-laying structure of nematode females and hermaphrodites [30-32] and the regulation of the dauer development [33,34]. Surprisingly, the regulation of vulva development in C. elegans and P. pacificus employs different signaling pathways to control the formation of this homologous morphological structure. In dauer formation, the two transcriptional regulators DAF-12, a nuclear hormone receptor, and DAF-16, a forkhead transcription factor, are well conserved between C. elegans and P. pacificus[33,34]. In P. pacificus, upstream factors consisting of insulin and TGF-beta signaling pathways in C. elegans await future analysis. Similarly, it is unclear whether downstream targets that are activated by DAF-12 and DAF-16 are also evolutionarily conserved. To address this question, we have taken a transcriptomic approach to directly compare expression profiles of the dauer stage (0 h) and dauer-exit stage (12 h post recovery-induction) in C. elegans and P. pacificus.
Here we show that P. pacificus has a more dynamic transcriptome during the dauer to dauer-exit transition as compared to that in C. elegans. The expression profiles from the two species look surprisingly different with limited overlap and weak correlation between orthologous genes that are differentially expressed during the dauer stage and the dauer-recovery process. Within the conserved genes, functionally different GO classes and protein domains are enriched in each profile, most striking differences being observed in regulation of metabolism related genes. These results highlight the importance of transcriptomic studies in revealing functional divergence in downstream effectors of homologous developmental processes, despite conservation of upstream regulatory factors.
Significant drop in RNA abundance in dauer larvae of C. Elegans and P. Pacificus
The dormant dauer stage in C. elegans is associated with a global repression of Pol-II based transcription, down to 11 - 17% of other stages [4]. We quantified the amount of total RNA per worm in the dauer and mix-stage samples and observed that dauers of C. elegans and P. pacificus contain approximately 20-fold less total RNA per worm as compared to the respective mix-stage sample. On average, ~100,000 dauer larvae yielded the same amount of total RNA as ~5,000 mixed stage worms. On top of this global transcriptional repression, we detected less mRNA in the total RNA of dauer larvae. The mRNA proportion is reduced to about half of the mix-stage levels in both species as measured by in vitro transcription (Additional file 1: Figure S1a). This global repression is expected to result in most of the genes being down regulated in a dauer versus mix-stage comparison. Our normalization strategy is based on differential weighing of spiked-in RNA probes (see Methods, Additional file 1: Figure S1b) and confirms this trend for both the species (Additional file 1: Figure S1c).
Additional file 1. Figure S1.The impact of different normalization strategies on expression fold change estimation. a) The general concept of using spike-ins to control for changes in total mRNA abundance relative to total RNA. See van de Peppel et al., 2003 for a detailed explanation. b) MA-plot of a Loess based normalization assuming a net mRNA expression fold change of 0 between the two samples. The M axis depicts the log2 fold change between the red and green microarray channels. The A axis is the average log2 intensity across both microarray channels. The position of the spike-in signals (colored dots) clearly indicate a deviation from this assumption. The corresponding legend in the upper left shows the expected fold change of the spike-ins relative to total RNA levels. Consequently, the location of the spike-ins corresponds to a down-shift of mRNA abundance levels within the total RNA population. c) Differentially expressed gene counts for the dauer vs. mixed stage comparison conditional on the normalization strategy. Figure S2. Explanation of a common reference design for microarrays (see Eisen and Brown 1999). For each species, RNA from biological replicates of mix-stage worms (M1 to M4) were combined together to generate one common reference pool. Labelled aRNA produced from independent biological replicates were then co-hybridized with labelled aRNA from the common reference pool, including two dye-swaps. Samples D1 to D4 represent Dauer samples and DE1 to DE4 represent dauer exit samples at 12 hour timepoint. The blue arrows indicate the direction of labelling in each co-hybridization (arrow head = Cy3 labelled, arrow tail = Cy5 labelled). This design allows comparison via the common reference pool, but at the same time remains flexible for adding more time-points to the study, if needed. Figure S3. Cumulative plot of log2 expression fold changes for genes with and without sequence conservation (pioneer genes). The two distributions are significantly different (two-sample Kolmogorov-Smirnov test; p-value < 10E- 16).
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The P. Pacificus transcriptome is more dynamic in the dauer to dauer-exit transition
The Agilent microarray technology enabled simultaneous expression profiling of 20,143 genes in C. elegans and 20,446 genes in P. pacificus. Our experimental setup contrasted gene expression in the dauer stage (time point 0 hour) with the dauer-exit samples (time point 12 hour) via a common reference sample (mixed stage) for each species. This so called "common reference" experimental design for microarray makes use of a species-specific pool of RNA as a common technical reference, and was chosen as it potentially facilitates an extension of this study to other time-points and conditions, if needed (see [35], Methods and Additional file 1: Figure S2 for details). We have chosen the 12-hour post induction time-point to enable comparisons with published expression profiling experiments [5]. We have chosen the same 12-hour time-point for the P. pacificus dauer exit samples after verifying that the post-dauer development follows similar kinetics in both species. We base this conclusion on the following morphological and developmental observations: First, most of the population (~90%) of the recovering worms from C. elegans as well as P. pacificus resume pharyngeal pumping within 3 hours after inducing dauer exit. Second, at the 12-hour time-point (the stage when we collect the dauer-exit samples), no discernible morphological differences are found between the recovered dauers from both species. Third, the recovering animals from both species enter a lethargus stage between 13 to 14 hours post-recovery [3]. Fourth, the worms that are allowed to develop further at 200 C reach the next moult (L4 for C. elegans, J4 for P. pacificus) between 22 to 23 hours after induction of dauer exit. Finally, recovered worms from both species started laying eggs between 42 to 45 hours post-induction. Based on all these criteria, we consider the 12-hour post recovery induction time-point to be developmentally equivalent and comparable across the two species.
The number of genes, which are differentially expressed in the dauer to dauer exit transition (Table 1), is much larger in P. pacificus (4942 genes, Additional file 2: Table S1) than that seen in C. elegans (917 genes, Additional file 3: Table S2), suggesting more dynamic transcriptional changes accompanying dauer recovery in P. pacificus. Also, the number of up-regulated versus down-regulated genes in the dauers to dauer-exit comparison is much larger in P. pacificus than that in C. elegans, where the number of up- and down- regulated genes is essentially the same (Table 1). Thus P. pacificus dauers potentially require the activity of more genes to survive in their ecological niche than the dauer recovery stage.
Additional file 2. Table S1. Differential expressed genes in the dauer versus dauer-exit comparison inP. pacificus.
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Additional file 3. Table S2. Differential expressed genes in the dauer versus dauer-exit comparison inC. elegans.
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Table 1. Differentially expressed genes in the dauer versus dauer-exit comparison
Signatures of conservation and divergence in the transcriptomes of the two species
Using pairwise best BLAST mapping, we identified 6,126 1:1 orthologous gene pairs in the two species, which are represented on both the arrays, while the remaining 14,212 P. pacificus genes and 13,099 C. elegans genes represent unresolved homology relations and lineage-specific genes. Within the 6,126 orthologs, we find that only 184 gene pairs are expressed at significantly different levels in the dauer versus dauer-exit comparisons of both species (FDR corrected p-value < 0.05) (Figure 1a). This small overlap is nonetheless statistically significant (Fisher's exact test p-value = 0.029) indicating some "conservation" between dauer related genes in the two species.
Figure 1. Limited conservation in the dauer related transcriptomes of the two species. (a) Overlap between genes differentially expressed in P. pacificus (green) and C. elegans dauers versus dauer-exit (12 hours) samples. The rectangular boxes represent the entire transcriptome on the array and their area of overlap represents the 1:1 orthologs between the two species, which are represented on both microarrays. The ovals represent the set of differentially expressed genes in each species. 184 orthologous gene pairs are called significantly differentially expressed in both the species, indicating limited conservation (Fisher's exact test p-value = 0.029). A substantial number of non-orthologous, species-specific genes are also differentially expressed in both the species. These gene sets are further analyzed by dividing them into distinct classes which are indicated in blue text (see main text for details) (b) Comparison of log2 expression fold changes for the set of 6,126 1:1 orthologs for the dauer versus dauer-exit comparison. Pearson's correlation coefficient over the entire set of 6,126 orthologs is r = 0.12, and increases to r = 0.29 for the 184 orthologs, which are significantly differentially expressed in both species (FDR corrected p-value < =0 0.05; purple boxes mark the significant orthologs, number per quadrant I - IV: 51 + 51 + 65 + 17 = 184).
The Pearson's correlation coefficient (r) between the fold changes of the 1:1 orthologs is r = 0.12 when calculated over all 6,126 orthologs, and increases to r = 0.29 when calculated on the 184 genes that are significantly differential in both species (Figure 1b). To further characterize the cross-species similarities and differences in transcriptomes, we group the set of 1:1 orthologs into the following classes:
(1) Class 1 = common orthologs that show a concordant pattern of differential regulation between the two species (up-regulated in both species, n = 51, quadrant 1, and down-regulated in both species n = 65, quadrant 3, Figure 1b. Total = 116 out of 184 common orthologs). These are expected to be the most conserved part of the transcriptomes across species (r = 0.88)
(2) Class 2 = orthologous genes that show a negative correlation in their direction of fold-change (n = 51, quadrant 2, and n = 17, quadrant 4, Figure 1b. Total = 68 out of total 184 common orthologs). This class of genes exhibits the most divergent expression pattern in the two transcriptomes (r = −0.80) and is comparable in size to class 1.
(3) Class 3 = Orthologs differentially expressed only in C. elegans dauers (n = 178 in C. elegans, Figure 1a)
(4) Class 4 = Orthologs differentially expressed only in P. pacificus expression profile (n = 2626 in P. pacificus, Figure 1a).
Gene ontology term enrichment
To better understand which functions are enriched in these gene sets, we made use of Gene Ontology (GO) term annotations from WormBase [36]. The set of 6,126 1:1 orthologs was used as the background set, and GO annotations were assigned to P. pacificus genes by directly mapping them from the corresponding C. elegans 1:1 orthologs. Enrichment statistics were calculated with the Bioconductor package topGO [37] (see Methods). We found the biological process of "neuropeptide-signaling pathway" to be the most significantly enriched (Table 2) in the class 1 gene set, which subsumes the most conserved expression pattern across the two species. We find six out of the 16 genes belonging to this GO class to be upregulated in dauers of both C. elegans and P. pacificus, and four of them are members of "FMRF-Like Peptide" family of neuropeptides, which also have a known role in regulating pharyngeal pumping [38]. GO enrichment analysis of class 2 genes that show an opposite fold change in their expression pattern between species yields "molting cycle, collagen and cuticulin-based cuticle" as the most significantly enriched term (Table 3). This suggests that the dauers of both species possibly use different genetic components for synthesis and/or shedding of the cuticle. This potential difference in molting related cuticle processing in the two species is further supported by the observation that this GO term is also the most significantly enriched in Class 3 genes - the set of orthologous genes differentially regulated exclusively in C. elegans (Table 4). For class 4 genes, the most significantly enriched GO terms are mostly development related but are too broad to point to any specific function (Table 5).
Table 2. GO terms enriched in orthologs with same direction of fold change in both species
Table 3. GO terms enriched in orthologs with opposite direction of fold change in both species
Table 4. GO terms enriched in orthologs, which are only differentially expressed inC. elegans
Table 5. GO terms enriched in orthologs, which are only differentially expressed inP. pacificus
Expression cluster analysis
Apart from analyzing enrichment for GO annotations, we also performed a functional analysis based on overlap of our data with gene expression clusters in C. elegans, which originate from other published microarray data sets. To this end, we retrieved predefined expression clusters from WormBase [36] and used them for a meta-level analysis. These expression clusters represent sets of genes reported to be co-expressed under various conditions. We restricted the analysis to the set of 1:1 orthologs, and assigned expression clusters to P. pacificus genes by mapping annotation from the corresponding C. elegans orthologs. The significance scores for enrichment of each pre-defined cluster was calculated separately for the significantly up- and down-regulated genes from the dauer versus dauer exit comparison in our data (see Methods for details), and are summarized in Table 6.
Table 6. Expression cluster enrichment analysis
These significantly enriched clusters enable us not only to identify system-wide trends in our data but also validate them against existing data-sets. For example, we see a highly significant overlap between our C. elegans data and previously reported expression profiles of dauers and dauer-exit stages [5,9]. Specifically, the cluster "Dauer enriched" identified by Wang and Kim in their time-course study [5] is enriched in genes called up-regulated in our data (Table 6, row 1, column "cel_dauer"). Furthermore, other clusters from later dauer-exit time points in the same study show a significant overlap with genes called over-expressed in our dauer-exit samples (Table 6, rows 2 to 4, column "cel_exit"), thus indicating good agreement between the two data-sets. Interestingly, the clusters corresponding to "early" and "climbing" genes in C. elegans (Table 6, rows 2 and 3) also show a significant overlap with genes over-expressed in our P. pacificus dauer-exit samples (column "ppa_exit" in Table 6), pointing towards a conservation of a part of the active transcriptome during dauer recovery in the two species. A similar trend is also seen for clusters obtained from a microarray study of dauers from daf-c/TGF-beta mutants [9] (Table 6, rows 5 and 6). We also see a significant overlap between genes regulated in response to heat shock, oxidative stress, osmotic stress and response to pathogens, highlighting the fact that activation of stress response pathways is a common feature of dauer stage in both C. elegans and P. pacificus. Further, genes activated in response to starvation and on resumption of feeding are also found to be enriched in dauer and dauer-exit stages respectively for both the species, in good agreement with non-feeding status of dauers and resumption of a feeding program during dauer-exit. We observe a significant overlap between the expression clusters that are enriched in the dauers of both the species (Fisher's exact test p-value = 0.0185), indicating a conserved signature of gene expression. This overlap of expression clusters is stronger for the dauer-exit stages of both the species (Fisher's exact test p-value = 6.833E-06), indicating that similar transcriptional programs are activated during dauer recovery in both the species. In summary, in spite of the low overlap and correlation between differentially expressed genes in the two species, functional analysis based on GO and expression cluster enrichment could identify conserved aspects of the transcriptional program associated with the dauer recovery process in the two species.
Apart from the differentially expressed genes in the limited set of 1:1 orthologous genes, there is a comparable number of differentially expressed genes for which either no orthologs exist in the other species or the orthology cannot be uniquely resolved (2,129 P. pacificus specific genes versus 2626 + 184 = 2810 1:1 orthologs in P. pacificus, 555 C. elegans specific genes versus 178 +184 = 362 1:1 orthologs in C. elegans data, Figure 1a). This indicates that the non-conserved part of the active transcriptome show a similar dynamic like the conserved part and might play a significant role in the biology of dauer larvae of the two species. Further functional studies would be required to understand the adaptive significance of these species-specific genes.
PFAM protein domain based analysis identifies potentially conserved and diverged functional gene classes
We further investigated the functional differences in the two transcriptomes by stratifying gene expression based on their protein domain annotations. This approach has the benefit of not being dependent on orthology relationships (like some of the analyses performed above), and can identify conserved functional signatures if the same gene function is carried out by different paralogs in the two species. For this analysis, we inferred possible gene functions by annotating 13,344 gene loci in C. elegans and 14,018 gene loci in P. pacificus with PFAM protein domains (p-value of domain match < 0.001). We then stratified the gene sets from the two species into gene families by PFAM domains with at least five members on each microarray. These 441 gene families may partially overlap and are represented by their median log2 fold expression changes. The Pearson's correlation coefficient of log2 fold changes is r = 0.52 for the dauer versus dauer exit comparison (Figure 2), an improvement over the aforementioned correlation values for 1:1 orthologs (Figure 1b). Some protein domains indeed show concordant expression in both the species, such as the HSP20 family of heat shock proteins that is induced in dauers of both the species (Figure 2) and potentially confers stress resistance. Similarly, the Peptidase_S28 family of proteins is repressed in the dauer stage of both species possibly because it contains proteins with lysosomal activity, which might not be required during the repressed metabolic state of dauers [7,39]. Interestingly, we also find some protein domains with totally opposite expression patterns, such as some of the proteasome subunit domains. The genes with domains "Proteasome" and "Proteasome_A_N" are induced strongly in P. pacificus dauers but are down regulated in C. elegans dauers. Since proteasome function has been implicated in regulation of longevity [40-42], up regulation of these specific proteasome domains in P. pacificus might be correlated with the increased longevity of P. pacificus dauers versus C. elegans dauers [43]. Thus, in summary, PFAM annotation based analysis of the two transcriptomes also identifies some conserved functional signatures as well as some divergent signatures. This analysis also provides an important catalog of gene function, and the role of genes corresponding to the protein families identified here can be studied in more detail in P. pacificus dauers in the future.
Figure 2. Log2 fold changes of median PFAM gene expression levels inP. pacificusandC. elegans. All genes on the microarrays were stratified by PFAM domain annotation into partially overlapping gene families with at least 5 members. A positive log2 fold change indicates that the corresponding genes were up-regulated in the dauer stages in comparison to the dauer exit time point.
Metabolic recovery during dauer exit displays different expression patterns of key enzymes in P. Pacificus
Dauer larvae of C. elegans are known to undergo a remarkable shift in their intermediary metabolism to survive long periods without actively feeding [7,39]. We therefore looked in more detail into expression changes in metabolism related genes using the KEGG database [44]. Gene-pathway assignments were obtained for C. elegans from KEGG and transferred to P. pacificus via the 6,126 orthologs identified through best pairwise BLAST mapping. For each KEGG pathway, we determined the number of genes from the 1:1 orthologous set that are upregulated, downregulated or show no differences in expression levels in the dauer versus dauer-exit comparisons in both species. We observed that the three key processes of central carbon metabolism, namely glycolysis, the Krebs (TCA) cycle and oxidative phosphorylation, show entirely different dynamics during the dauer-exit time course in the two species (see Figure 3). For C. elegans, mRNA abundance levels for most of the genes in the three pathways are found to be similar in the dauers and dauer-exit stage (category "no_change", Figure 3). Only a few genes are downregulated, while none of the genes is upregulated in C.elegans. In contrast, we see a more dynamic regulation of metabolic pathways in P. pacificus, with comparatively larger number of downregulated genes as well as a few upregulated genes. These differences in metabolic recovery in P. pacificus could have an obvious and trivial explanation in that P. pacificus might have a different developmental rate compared to C. elegans. However, as described above, several morphological and developmental traits suggest that the course of post-dauer development in P. pacificus and C. elegans is very similar. Hence, we hypothesize that metabolic regulation in P. pacificus dauers might itself be inherently different from that in C. elegans. This would also be consistent with the dauer recovery studies showing that P. pacificus dauers can survive much longer than C. elegans dauers [43], one of the possible reason being their ability to metabolize stored fats at a different rate and/or in a different way. Our genome-wide expression studies reflect this potential difference as divergence in pathway expression profiles between the two species. Since gene expression profiles are dynamic and can be sensitive to differences in developmental timing (e.g. [11]), more fine-grained and detailed functional studies will be needed to verify whether these differences are really due to inherent metabolic differences or an artifact of slight variations in developmental timing.
Figure 3. Differences in regulation of metabolism related genes during the dauer exit time course inC. elegansandP. pacificus. Gene-to-pathway assignments were retrieved for C. elegans from the KEGG database. Pathway assignments were transferred to P. pacificus by mapping 1:1 orthologs. All tables show genes that are up, down or at a similar expression level in a dauer vs. dauer exit (12 hours post induction) comparison.
Genes acquired by horizontal gene transfer and orphan genes in P. Pacificus are developmentally regulated
Previous studies have identified genes in P. pacificus that likely originate from lateral gene transfer events. For example, P. pacificus is the first nematode, which is not a plant parasitic species yet contains cellulase genes [25]. P. pacificus cellulase genes are most similar to genes found in the social amoeba of the Dictyostelid group [45]. These cellulases are fully functional and have been maintained over long evolutionary time periods [46]. We have identified seven cellulase genes in the current transcriptome annotation, six of which are represented on our P. pacificus microarrays. In a dauer versus mixed-stage comparison, four of these cellulases are significantly differentially expressed, all of them being down regulated in dauers (or equivalently, expressed predominantly in non-dauer mix-stages, Additional file 4: Table S3). This is in agreement with their hypothesized function of enabling feeding on novel food sources [46]. For example, one gene (Contig66-snapTAU.4) is already significantly up-regulated as early as 12 hours post induction (Additional file 4: Table S3, column logFC(D0/D12)).
Additional file 4. Table S3. Differential expression ofP. pacificuscellulase genes that were acquired by horizontal gene transfer. Table S4. Differential expression of P. pacificus diapausin genes that were acquired by horizontal gene transfer.
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Another interesting gene family, which was most likely acquired from beetles, is the diapausins. These genes encode small peptides, which are thought to protect the dormant beetle against microbial infections in diapause. Diapausins provide antifungal activity by acting as Ca2+ channel blockers [47]. Out of five members of this gene family in P. pacificus genome, three are represented on our microarrays. Two of them are significantly up-regulated in dauers compared to dauer exit (Additional file 4: Table S4, column logFC(D0/D12)) and might be important for antifungal immunity in the dauer stage on the beetle, while the third gene does not respond to the dauer exit program. This gene is in fact downregulated in a dauer versus mix-stage comparison (column logFC(D0/MixStage), Additional file 4: Table S4) suggesting potential anti-fungal role in other developmental stages. Thus, while previous studies in our lab have demonstrated that both gene families (cellulases and diapausins) originate from lateral gene transfer events, we are now able to show members of both gene families are developmentally regulated in agreement with their proposed functions: cellulase genes are implicated in feeding and are hence downregulated in dauers, and at least two diapausin gene appears to have a role in protecting hibernating stages. These hypotheses generated from our microarray data can further be investigated at a functional level.
One third of all P. pacificus genes are pioneer genes, which do not show any obvious sequence similarity to other organisms [26]. On average, pioneer genes are significantly higher expressed in the dauer stage in comparison to the remaining genes (p-value < 1.0E-06; Additional file 1: Figure S3). We speculate that pioneer genes could be especially important to the evolution of the dauer stage in P. pacificus, which is essential for its necromenic life style.
The dauer is an ecologically regulated developmental stage observed in many free-living nematode species and hence provides an interesting model to investigate how ecological adaptations are integrated into developmental pathways during evolution [1]. Extensive studies in C. elegans have uncovered the genetic regulators of dauer formation and comparative studies from other free living nematodes have begun to provide insights into evolution of dauer regulatory genes and pathways [33,34,48]. P. pacificus presents an ideal model for comparing dauer larvae of free living nematodes. In this study we provide a comprehensive comparison of gene expression in the dauer stage and dauer exit (12 hour post induction) of the two nematode model systems P. pacificus and C. elegans using the Agilent microarray platform. While we are aware of the available C. elegans expression data on dauer development (e.g. [5,6,8-14], we felt that it was necessary to generate C. elegans data de novo for two reasons. First, we wanted to use the same platform as that used for the analysis of P. pacificus in order to enhance the power of a direct comparison. Second, we wanted to benefit from technical advances in custom and long nucleotide microarrays. Based on cross-species comparison of transcriptomes of the ecologically important dauer stage, we draw four major conclusions from our studies.
First, we provide a list of similarities and differences between P. pacificus and C. elegans, which shows an unexpected level of divergence at transcriptome level, even though the dauer stage and recovery process appear to be developmentally conserved. While this comparison allows several evolutionary conclusions (see below), the P. pacificus data set on its own can be used as a starting point for a functional analysis of the dauer stage and dauer exit. This data set represents an invaluable resource given the importance of the dauer stage for the ecology of this nematode. The association of P. pacificus with scarab beetles is restricted to the dauer stage as long as the beetle is alive [27,28]. Only after the beetle´s death, the nematode exits from the dauer stage to feed on the microbes, which develop on the carcass of the insect. Thus, the P. pacificus dauer stage has a well-defined ecological niche and the expression profiles described in this study will serve as an entry point to future functional studies. For example, our transcriptomic data identifies many P. pacificus specific genes as upregulated in the dauer stage, which implies a potential function in adaptations enabling survival on beetles.
Second, we show that metabolic differences exist between both species, with different patterns of regulation of genes involved in the central carbon metabolism pathways (glycolysis, tricarboxylic acid cycle and oxidative phosphorylation) of P. pacificus upon dauer exit. This difference does not result in obvious phenotypic changes during dauer exit, but may be linked to the differences in life history traits. This is especially relevant given that P. pacificus is adapted for longevity [43]. Under experimental conditions, P. pacificus survives for up to one year in the dauer stage, whereas C. elegans N2 dauer larvae die after approximately 22 weeks. Further studies will reveal how much of the observed metabolic differences are explained by different life-history traits versus differences in rate of development.
Third, this study provides the first nematode evo-devo comparison looking at the downstream consequences of homologous developmental processes between species belonging to different nematode genera. While detailed studies between nematodes as distinct as C. elegans and P. pacificus have investigated the regulation of vulva and gonad development, sex determination and dauer formation [30,34,49,50], most of these studies are concerned with the regulatory mechanisms rather than the "executional programs" of the corresponding developmental processes. The divergence in the expression profiles of C. elegans and P. pacificus adds an important new finding to the growing literature of evo-devo. Previous studies have indicated the limited conservation in the genetic and molecular control of developmental processes in P. pacificus and C. elegans. For example, vulva induction relies on different signaling pathways, requires a novel regulatory linkage and the acquisition of novel protein domains in P. pacificus Wnt signaling [32]. This type of result has been discussed as an example for the theory of developmental systems drift, which proposes that conserved developmental and morphological structures can be regulated by largely diverse regulatory mechanisms [51]. Considering that gene regulatory networks are hierarchically structured, with possibly different rates of evolution at the top level regulatory genes and the most downstream level of effector genes [52], it can be argued that in principle, developmental systems may diverge due to differences/drift at any of these levels. Unfortunately, unlike the top-level regulatory network, the downstream effector programs of vulva development have largely escaped identification by developmental genetic approaches and have not been easily accessible to transcriptome studies as they are single-cell or small group of cell responses. Our study circumvents this limitation because dauer formation is a "whole body response" of the organism to harsh environmental conditions.
The dauer context is also interesting because in P. pacificus, the key transcription factors of the dauer regulatory network, DAF-16 and DAF-12, are conserved [33,34]. However, this in itself does not indicate the extent to which the downstream targets of the regulatory network are subject to evolutionary change. Herein, we could demonstrate for the first time that the core downstream execution program of a developmental stage can differ tremendously between P. pacificus and C. elegans, in spite of conservation of upstream regulators like DAF-16 and DAF-12. Thus, these observations make a case for extending the concept of developmental systems drift to the downstream molecular execution of specific developmental stages.
The fourth conclusion is also related to evolutionary theory. While future studies will have to reveal how much of the observed differences between P. pacificus and C. elegans is really of functional importance, it has often been assumed that such differences might simply be neutral [23,53]. Gene expression, which is neither strongly deleterious nor advantageous, previously termed "gratuitous expression" [53], will not be under selection and will be free to evolve by drift. Consequently, such expression is probably not functional. However, such arguments may not apply to the dauer stage since nematodes live off their internal limited energy resources and any random or neutral transcriptional activity would diminish these limited resources.
Comparative studies in developmental genetics have driven the studies on evolution of developmental mechanisms, and with whole genome sequencing of many animal species has now highlighted new facets of evolutionary dynamics through comparative genomic studies [21]. Since transcriptional regulation is a key building block in the genotype to phenotype translation, comparative transcriptomic studies add another dimension to the analysis of evolutionary processes [23]. Our work contributes to the growing set of results from comparative transcriptomics in diverse developmental systems (e.g. [54-58]). These studies together span a range of conclusions, from high transcriptomic conservation at one end, to relatively low conservation in others, suggesting inherent constraints as well as flexibility in the evolution of gene regulatory networks [23]. Future studies comparing transcriptomes of homologous biological processes in related species, will be important for understanding the role of transcriptome evolution in generating animal diversity. Ultimately, this will also reveal the extent to which the conserved or divergent expression changes are subject to adaptive and non-adaptive forces during evolution.
Worm strains and culture
We used wild-type strains of two distinct nematode species in all of our experiments. For Caenorhabditis elegans, we used the N2 (Bristol) strain. For Pristionchus pacificus, we used the RS2333 strain (formerly known as PS312). For mixed stage cultures, 10 to 15 early adults were spotted on 10 cm NGM plates and allowed to grow at 20°C for 5 days, and washed off with M9 for RNA extractions. The dauers for both species were obtained from liquid cultures grown at 25°C. For this, 15 to 20 mixed stage plates (see above) were washed off and suspended in a final volume of 500 ml of S-medium in a 3000 ml Erlenmayer flask. 200ul Nystatin (50 mg/ml stock in DMSO) and 500ul Kanamycin (20 mg/ml stock solution) was added to prevent fungal and bacterial contamination. On day 1, 5 and 8 of culture, 12.5 ml of OP50 (20% w/v stock in S-medium) was added as food source. The cultures were grown for 12 to 14 days in a shaker incubator @ 220 rpm, 25°C. Dauers were purified from liquid cultures between days 12 to 14. The culture was centrifuged to obtain a worm pellet, which was then incubated with 1% SDS for 30 minutes to kill any non-dauers. Live worms were separated by sucrose flotation, and a subsequent precipitation in 15% Ficoll400 resulted in a pure dauer pellet (confirmed under a stereo microscope). The washed dauers were incubated overnight in 0.1 M NaCl at 20°C, to let them recover from any possible stress induced during the harsh dauer purification process. The dauers were then collected by centrifugation, and used for RNA extraction and for starting dauer-exit cultures.
Dauer exit time course
For the dauer-exit 12-hour time-point samples, around 200 purified dauers were spotted on each 10 cm NGM plate pre-spotted with 2 ml OP50, and grown at 20°C for 12 hours. These conditions ensured abundant food supply and non-crowded conditions, favorable for inducing dauer exit [3]. Worms from 20 to 30 such plates were used for RNA extraction per sample. The 12-hour time-point was chosen for both the species to give them sufficient time to recover from dauer stage and manifest measurable transcriptional changes. The recovering worms from both species were monitored to ensure comparable developmental rates based on the following criteria: (1) Most of the population (~90%) of the recovering worms from C. elegans as well as P. pacificus resume pharyngeal pumping within 3 hours after inducing dauer exit. (2) At the 12 hour time-point (the stage of dauer-exit samples), no discernible morphological differences are found between the recovered dauers from both species. (3) The recovering animals from both the species enter a lethargus between 13 to 14 hours post-recovery [3]. (4) The worms that are allowed to develop further at 200 C reach the next moult (L4 for C. elegans, J4 for P. pacificus) between 22 to 23 hours after induction of dauer exit. (5) Finally, recovered worms from both species started laying eggs between 42 to 45 hours post-induction.
RNA extraction
Total RNA was isolated using TRIzol (Invitrogen) according to the manufacturer's instructions. Four biological replicates for each stage (mixed, dauers, dauer-exit at 12 hrs) were prepared from parallel cultures. The extracted RNA was purified by using phenol:chloroform:isoamyl alcohol precipitation, to remove any trace of TRIzol contamination which might interfere with subsequent reactions. The RNA pellet was suspended in RNAse free water, quantity and quality was assayed on a Nanodrop spectrophotometer, and stored at −80°C until the microarray reactions.
Microarray design and experiments
We used the Agilent C. elegans oligonucleotide microarrays (~ 43,000 probes for ~ 20,000 open reading frames, GEO accession: GPL10094) for all C. elegans experiments. For P. pacificus, we designed custom Agilent microarrays based on the most recent transcriptome annotation (~ 23,000 gene predictions, predicted with external evidence from 454 EST alignments). We could accommodate ~ 93,000 probe sequences on our custom P. pacificus microarrays (NCBI GEO platform accession GPL14372). This probe set was designed using the OligoWiz 2.0 software [59] and submitted for custom fabrication to Agilent Technologies via their eArray web tool. Out of 93,000 probes, 87,070 probes map uniquely to the latest genome assembly [26]. We restrict all subsequent analyses to a probe set of 69,916 high-confidence probes.
Microarray hybridizations were carried out in a two-color format, with 4 biological replicates per experiment including a pair of dye-flips. Two sets of hybridizations were carried out: (1) Dauers versus Mix-Stage, and (2) Dauer-Exit at 12 hr versus Mix-stage, thus making a total of 8 microarray hybridizations per species. Equal amounts of total RNA (500 ng to 1000 ng) for 4 biological replicates from each stage was used to prepare Cy5- or Cy3- labelled cRNA using Quick Amp Labelling Kit (Agilent Technologies, Inc, Santa Clara, CA, USA), as per manufacturer's instructions. Based on the starting amounts of total RNA, appropriate dilutions of positive controls (Spike Mix-A and Spike-Mix B from Agilent) had been added to the reaction-mix before the RT reaction, as per manufacturer instructions. The labelled cRNAs were hybridized either to Agilent C. elegans oligonucleotide microarrays or our custom P. pacificus microarrays from Agilent. The arrays were scanned using GenePix 4000B Microarray Scanner, and raw data extracted using GenePix Pro sofware (version 6).
Analysis of microarray expression data
The data was analyzed using the Bioconductor [60] package limma [61]. Briefly, the array quality was assessed by checking for uniform background and foreground intensities over the entire array. The signal was background corrected using the normexp method [62]. The arrays were lowess normalized individually ("normalizeWithinArrays" option), with differential weights assigned to probes and to positive control spike-ins, which are expected to show no fold change [63]. This differential weighing of probes is particularly necessary to account for changes in the relative proportion of mRNA versus total RNA. Without this differential weighing scheme, the fold change calculations can be erroneous [64]. The weight parameters were optimized based on MA-plots such that spike-in controls show their expected fold change values. lmFit function was used to fit a linear model to probe intensities across arrays, and differential expression was calculated by empirical Bayes method using the eBayes function [65]. Control of FDR was employed as correction for multiple testing. All the data from this publication have been deposited in a MIAME compliant format [66] at NCBIs Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/ webcite) and are accessible through GEO Series accession numbers GSE30977 and GSE31861.
Mapping of 1:1 orthology relations
We used a pairwise best BLASTP strategy to compute 1:1 orthologs. Briefly, we ran all protein sequences from C. elegans as query versus the database of P. pacificus gene predictions and vice versa. Only hits with a BLAST score > = 50 bits were retained. We define mutually best hits as 1:1 orthologs. We identified 7,176 ortholog pairs with this methodology.
Gene ontology enrichment analysis
GO ontologies for C. elegans were downloaded from wormmart WS200. Gene ontologies were assigned to P. pacificus genes based on the previously defined 1:1 orthologs. The topGO tool [37] (version 2.4.0) was used for computing significantly enriched GO terms. We used the "GOFisher" test statistic and a p-value cutoff of 0.05. The background set was limited to the 6,126 orthologs that are represented on both C. elegans and P. pacificus microarrays. We did not apply any multiple testing correction to the reported p-values. We followed this strategy to uncover all "trends" in the data to attain a comprehensive picture of the underlying biology.
Expression cluster enrichment analysis
WormBase [36] contains information on co-expressed gene groups in C. elegans. The list of microarray experiments where a given C. elegans gene is known to be differentially expressed can be extracted from the section "Expression Cluster" from the WormBase gene summary page for each gene. We retrieved all available expression clusters for C. elegans genes from the WormBase web site. We inferred expression clusters for P. pacificus based on the set of 1:1 orthologs. Only the clusters associated with the 6,126 1:1 orthologs were used as the background set. P-values for expression cluster enrichment in dauer-enriched or dauer exit-enriched gene sets were computed with a 2x2 Fisher exact test, and a multiple testing correction to control FDR was applied.
Pfam domain annotation
We annotated the proteome of C. elegans and P. pacificus with PFAM domain matches (PFAM release V23/4 [67])). Protein HMM searches were performed with HMMer 3.0 [68] using a p-value cutoff of 0.001.
KEGG pathway analysis
We retrieved the latest gene to pathway mapping for C. elegans from the KEGG SOAP server (see http://www.genome.jp/kegg/soap webcite for details). Again, pathway annotations were transferred to P. pacificus with the help of the 1:1 orthologs. Gene expression information and pathway information were overlaid to generate Figure 3.
Data access
Microarray data have been deposited in NCBIs Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/ webcite) and are accessible through GEO Series accession numbers GSE30977 and GSE31861. The gene set annotation for this manuscript may be obtained from http://www.pristionchus.org/download/ webcite . The list of 1:1 orthologs and PFAM domain annotations may be also obtained from there.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
AS, RJS and CD designed the experiments and wrote the manuscript. AS performed the experiments. AS and CD analyzed the data. All authors read and approved the final manuscript.
The authors want to thank various members of the labs for ongoing discussion. In particular, we thank Drs. G. Markov and C. Rödelsperger for bioinformatic support and suggestions.
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Nucleic Acids Research 2009, 38:D211-D222. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Eddy SR: A new generation of homology search tools based on probabilistic inference.
Genome Inform 2009, 23:205-211. PubMed Abstract


text:
Permanent HO layout with block & DCC track control that is open for State Fair. New members welcomed. Click for flyer (this is a PDF file)
Meeting: 7:00 -9:00 every Wednesday in southeast corner of the Agriculture Building west of Manual Lujan building.
President: Charles "Charlie" Joerg --- Email Address: firstname.lastname@example.com
Secretary/Treasurer: Mike Hadaway --- Email Address: email@example.com
Mailing Address: RGV Model RR Club, PO Box 13713, ABQ, NM, 87192
Link to other Albuquerque Clubs & Organizations(click here) -- For guide to model rairoading see: http://www.greatesthobby.com/
For some of our club cars, see below:
--NEW-- NEW MEXICO CENTENNIAL CAR FOR 2012 50' box car - $15 kit, $20 assembled
Click to see 2008 Convention 40' box car - $15 kit, $20 assembled (this is a PDF file)
2006 Golden Anniversary Car $15 as Kit, $20 built
CIMARRON CANYON MINE: Sante Fe engine squeezing through between mine and steep, rocky mountain.

subset: commoncrawl


text:
On June 20, the Concerned Veterans for America and The Weekly Standard are hosting a panel discussion in Washington: "Reforming Veterans Affairs: Preserving Promises to Those Who Serve." Peter Gaytan, executive director of The American Legion's Washington office, will be a featured panelist, as well as Darin Selnick of Concerned Veterans for America and Mark Flatten of The Washington Examiner.
"It's good to bring together various points of view on how to reform VA and make it better," Gaytan said. "The American Legion also brings some specific solutions to the table. For example, we've been working very closely with VA for several months on its fully developed claims (FDC) initiative. These are the kinds of disability claims that move through the VA system much faster, and we've been training our service officers nationwide to develop these FDCs for our veterans."
Bill Kristol, political analyst and commentator, will serve as moderator. And opening remarks will be made by Rep. Jeff Miller, R-Fla., chairman of the House Committee on Veterans' Affairs; Sen. Richard Burr, R-N.C., ranking member of the Senate Committee on Veterans' Affairs; and Anthony Principi, former Secretary of Veterans Affairs. The event will be streamed here [2].
Concerned Veterans for America is a non-partisan, non-profit organization that advocates for policies that preserve freedom and liberty. The event is the first of a five-part "Defend & Reform Breakfast Series" that will highlight the ways veterans — and all Americans — can contribute to defending freedom by advancing reform.
[1] http://www.legion.org/print/216239
[2] http://www.ConcernedVeteransForAmerica.org


text:
Written Answers. - Animal Feedstuffs.
Wednesday, 8 October 2003
Dáil Eireann Debate
104. Mr. Timmins asked the Minister for Agriculture and Food the amount of meat and bonemeal generated each week here; the total amount of this in storage to date; the cost implications of this to date; and if he will make a statement on the matter. [22602/03]
105. Mr. Timmins asked the Minister for Agriculture and Food his proposals to deal with the problem of the disposal of meat and bone meal; and if he will make a statement on the matter. [22603/03]
Minister for Agriculture and Food (Mr. Walsh): I propose to take Questions Nos. 104 and 105 together.
In 2002, a total amount of 144,000 tonnes of meat and bonemeal was produced in Ireland, which works out at an average of 2,769 tonnes per week. At present, the total amount of MBM in storage is 210,000 tonnes. Of this, my Department has responsibility for 172,000 tonnes which gives rise to an annual storage cost of €3.75 million. The balance, though stored under controlled conditions, is the responsibility of the private sector.
In the period January 2001 to date this year, close on €146 million will have been spent by the Exchequer on the production, storage and disposal of MBM. The subsidy ended on 31 May 2003 and any expenditure arising after that relates to a carry over of payments from before the ending of the subsidy and to the storage and disposal of product built up during the period of subsidy.
There are currently no domestic disposal facilities for MBM produced in Ireland. A number of proposals for the use of MBM in industry or energy generation in this country are at various stages of consideration by the relevant regulatory authorities including the Environmental Protection Agency.
I established an interdepartmental agency/ committee to examine the best available options for the use of MBM in industry and in non-feed uses here. I expect to have the report from that committee submitted to me in the near future.
|Last Updated: 10/09/2010 08:09:32
|Page of 118


text:
Connecticut College vs. Wesleyan
April 4, 2007
|Jesse Stevenson
|(2, 2)
|Sean Driscoll
|(3, 0)
|Mike Bruno
|(0, 2)
|Steve Dachille
|(2, 0)
|Robbie Logan
|(0, 1)
|Brendan Moses
|(0, 1)
|Mark Moran
|8 (0.500)
|Current Record
|Connecticut College
|go to Connecticut College page
|Grayson Connors
|(2, 1)
|Jason Ben-eliyahu
|(2, 0)
|Lonny Blumenthal
|(1, 1)
|Chris Jasinski
|(1, 1)
|Russell Follansbee
|(0, 1)
|Matt Burke
|(0, 1)
|Dan Latzman
|(1, 0)
|Mike Hines
|(1, 0)
|Charlie Congleton
|5 (0.417)
|Current Record
|go to Wesleyan page
|The Game Story:
|April 04, 2007
NEW LONDON, Conn.--Lonnie Blumenthal (Ballwin, Mo.) scored on a feed from Grayson Connors (Princeton, Junction, N.J.) with 3:01 remaining in the second overtime Wednesday, lifting fourth ranked Wesleyan University (8-1, 2-1) to an 8-7 victory at Connecticut College (0-7, 0-6). Connors and Jayson Ben-Eliyahu (East Setauket, N.Y.) tallied two goals apiece for Wesleyan. Chris Jasinski (Wilton, Conn.) added a goal and an assist in the NESCAC contest.
Freshman midfielder Sean Driscoll (Peabody, Mass.) tallied a hat-trick with three scores for the Camels. Jesse Stevenson (Marshfield, Mass.) had a four point afternoon with two goals and two assists for the hosts. Steve Dachille (Garden City, N.Y.) tallied two goals and Mike Bruno (Andover, Mass.) added two assists for Connecticut College.
The Cardinals jumped on the Camels early, building a 3-0 lead on Chris Jasinski's score with 8:40 left in the first quarter. But Driscoll scored the next two goals and the hosts took their first lead when Stevenson fed Dachille for a goal with 6:28 remaining in the half. Driscoll scored his third goal of the soggy afternoon with just 27 seconds to play and the Camels carried a 6-4 lead into the intermission.
The Cardinals rebounded with a productive third quarter, scoring three unanswered goals to regain the lead, 7-6. Ben-Eliyahu put in the go-ahead score, setting up a wild fourth quarter finish.
Wesleyan killed off two penalties at the start of the quarter. In the closing moments, the Camels gained possession and took a timeout with one minute to play. With 37 seconds remaining Stevenson spotted Dachille about 15 yards out in front of the left post and the freshman buried the equalizer, forcing extra play.
But with 3:01 remaining in the second overtime, Grayson Connors dished to Blumenthal who was coming across the middle for a one-timer and the Cardinals walked away with the 8-7 victory.
Charlie Congleton (Groton, Mass.) had five stops in net for the Cardinals. Mark Moran (Greenwich, Conn.) had eight saves for the Camels.
#4 Wesleyan University (8-1, 2-1) 3-1-3-0-0-1--8 Connecticut College (0-7, 0-6) 3-3-0-1-0-0--7
Scoring: Goals: WU: Grayson Connors 2, Jason Ben-Eliyahu 2, Chris Jasinski, Lonnie Blumenthal, Mike Hines, Dan Latzman. CC: Sean Driscoll 3, Steve Dachille 2, Jesse Stevenson 2. Assists: WU: Lonnie Blumenthal, Matt Burke, Grayson Connors, Russ Follansbee, Chris Jasinski. CC: Mike Bruno 2, Jesse Stevenson 2, Robbie Logan, Brendan Moses.
Saves: WU-Charlie Congleton, 5 saves. CC-Mark Moran, 8 saves. Shots: WU-26, CC-20 Extra Man Opportunities: WU-0-2, CC 0-3.
For more information contact: William Tomasian (860) 439-2501; firstname.lastname@example.com


text:
- Musician's Friend Best Selection, Price & Service. Guaranteed.
Call our Gear Heads
at: 800-449-9128
Call our Gear Heads
at: 800-449-9128
Find a similar product below or contact our experts for a recommendation of great alternatives. Call us at 800-449-9128.Similar Items:
Find a similar product below or contact our experts for a recommendation of great alternatives. Call us at 800-449-9128.Similar Items:
A time capsule with an ash body, maple neck and vintage-style pickups.
The Fender Custom Shop 1951 Nocaster Electric Guitar features an ash body with a lacquer finish given a NOS treatment. The body is fitted with a maple neck with a nitrocellulose, a '60s oval "C"-shape, tall narrow 6105 fretwire, and a 9.5" radius. The pickups include a hot-rodded Twisted Tele single-coil neck position pickup and a '51 Nocaster single-coil in the bridge position. The 1951 Nocaster electric guitar comes with a original Tele custom hardshell case and Custom Shop certificate.
Choose your color and step back in time. Order today.
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Comments about Fender Custom Shop '51 Nocaster Electric Guitar:
This by far the best Fender I have purchased bar none, nothing else be said.
The Guitar came complete, set up was great, no issues at all.
thank you
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text:
Inserting Flash into HTML
Click here to watch a video tutorial
For more information on streaming audio and video, please visit our other article:
Streaming Audio/Video on your Site
How to insert your Flash movies into your website
After creating a Flash movie you normally go to File ~>Save As from the top menu to save your movie. Save the file as "yourmovie.fla".
To embed the Flash movie into an HTML page, you should go back to your Flash progam and:
- choose file ~> Open the Flash movie
- choose file ~> Export Movie
- Name the file "yourmovie.swf". Choose the location where the file is to stored (in your Web folder) and click OK.
- Open the HTML page where you want to insert your Flash movie. Insert this code:
<object width="550" height="400">
<param name="movie" value="somefilename.swf">
<embed src="/support/somefilename.swf" width="550" height="400">
The width and height tags specify the frame of the movie, which you will need to adjust accordingly. This is the minimum code you need to embed a Flash movie in a browser. A broken icon will appear on the Web page if the user does not have the Flash plug-in installed.
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Commercial and Residential Buildings
Commercial and residential building construction projects are driven by a financial model that requires a high-quality product for the client (lessor or purchaser), under the umbrella of very tough cost constraints. It is imperative that well performing, warranted and cost-conscious solutions are used in these projects. Often the least cost available ground includes a component of poor soil. Menard's creative and economical ground improvement solutions have been implemented numerous times to help developers create a project that meets the required constraints.
- Control total and differential settlements
- Provide good bearing capacity under spread footings
- Allow very conventional and economical concrete foundations
- Eliminate any site soil disposal costs
Applications for:
Student Housing


text:
If you need Tractor Tires or Tires or even Tire in MS, look no further. www.hotfrog.com showcases more than 7 Tractor Tires businesses across MS. To find more related businesses and to filter by locality, use the left navigation menu.
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text:
Command™ Brand Home >
Watch amazing
Show off your photos strung across a line with Command™ Brushed Nickel Decorative Hooks.
Score style points with Command™ Utensil Hooks.
Hang your coolest posters with Command™ Poster Strips.
Don't throw in the towel. Keep it handy with a Command™ Double Hook.
They are a great product - easy on, easy off. They are excellent for a dorm room.
These new pick hooks are so cute and so fun to use. They will be great in a dorm room to hang keys on.
Must buy love this product!!!! I purchased a bunch of these when I moved into my dorm for college. Application was SUPER easy and they held PERFECTLY from August til May when I moved out. Thr removal is soooo clean and also super easy. They pulled right off. Amazing!
When they say no damage, they mean no damage! I used several of these last year for my dorm room. Super handy, and very sturdy. At the end of the year they came off super easily and I didn't have to worry about getting charged for wall damage. Overall I'm quite impressed.
Great! I love these Command Hooks. I use them all around my dorm room to hang my jewelry, calendar, umbrellas, jackets, etc. They are pretty strong for such small hooks. I would DEFINITELY recommend it to people!
© 3M 2015. All Rights Reserved.


text: Flickr User Stef Thomas [ What's the Going Rate for the Tooth Fairy? [POLL] By Deminski and Doyle February 28, 2013 In Oak Brook, Illinois a network of dental service corporations conducted a survey asking over a thousand primary caregivers what the Toothfairy had given their children. The results may make you consider vasectomies and tubal ligations. Read More|Comment Category: Deminski & Doyle, Polls, Talking About . . . Tags: Newsletter, rates, tooth fairy Send to a friend! Share on Facebook Share on Twitter Pin it! Reddit This! Share on Tumblr!


text:
Smithsonian Folklife Festival Entrance. Photo credit: Claire Fuller
After enjoying the cultural festivities of the Chicha! party last week, I decided to head over to the Smithsonian Folklife Festival for more immersion in Peruvian culture. I visited at around 11 AM, just when the festival opens for the day. Greeted by the welcoming scents of the Peruvian cuisine stands at the entrance of the festival, I eagerly entered the area – and it seems I wasn't alone. Even right at the festival's opening, there were many excited visitors spread across the enclosed section of the National Mall: both solitary travelers and large families of tourists wandered with curiosity through the large white tents lined along the perimeters of the space. The informational signs scattered along the grounds give visitors interesting and important background information about Peru and its culture, as well as introduce them to this year's Folklife. One of these signs deftly and concisely explained Perú: Pachamama:
Peru is one of the world's most biodiverse nations. Its distinctive cultural regions are not only connected by history but by rivers, roads, and pathways that existed long before the Inka Empire (15th to 16th centuries) and Spanish colonization (16th to 19th centuries). Across its different altitudes and climates, communities exchange commodities and customs, shaping historically rooted but constantly changing lifestyles.
The 2015 Smithsonian Folklife Festival program, Perú: Pachamama, features 12 projects, organizations, and groups whose distinctive traditions highlight creative social, cultural, and economic transformations. Sharing culture from the Andean highlands, Amazon River basin, northern coastal regions, and urban neighborhoods of Lima, the program demonstrates how networks of celebration and community, crops and markets, textiles and crafts, foodways and communications, and music, dance, and art forge the diverse cultural heritage of Peru.
The Pachamama, or Mother Earth, unifies the distinctiveness and diversity of the featured traditions into a national cultural identity. The Q'eswachaka Bridge at the center of the Festival represents connection between Peru's millennia-old traditions and their new meanings and uses given by today's cultural communities.
The arrangement of the demonstrations – separating the traditional and the new – encourages visitors to explore the complexities of the nation of Peru and visualize the transformation and growth of its culture.
I decided to first enter the traditional side of the demonstrations. Stepping through one of the two enormous orange and yellow portals, I was received by joyous Peruvian singing and the chattering of inquisitive people. I was immediately struck by the authenticity of the demonstrations – there was a complete absence of artificiality among the Peruvians offering their time and skills. I first followed the musical sounds and found a large crowd surrounding a square, wooden surface lying on top of the grass. This zone simulated La Plaza, a central space found in Latin American towns and cities used for celebrations and festivities. The artists performing at La Plaza when I came across it was a group of Afro-Peruvians presenting their traditional style of song and dance (see my video clip, below). The performance was enthusiastic and lively, brimming with cultural pride. After finishing their piece, the performers moved to a tent dedicated to children's education and warmly explained their traditions to dozens of small, transfixed faces.
Moving on from La Plaza, I was drawn to a tent labelled Textile Designs. Under the tent, a man dressed in customary Peruvian garb weaved vibrant fibers on a large awana, or loom. To his right was a yarn-dying station in which Peruvian women dipped spun fibers (usually from llamas, alpacas, and sheep) into boiling water combined with colorful spices and herbs. These spices, releasing a piquant aroma, were displayed in hand-crafted clay pots tabled on top of a hand-woven rug made from natural materials, which generated a satisfying crackle when crossed. The artist weaving at his loom was in the process of creating a floor mat; several of his finished pieces, all flawlessly designed and crafted with intricate and colorful patterns, were hung around the tent. Speaking only Spanish, the artist was able to communicate with non-Spanish speakers via a volunteer, with which each tent was equipped, able to translate and explain the history and process behind each demonstration. One visitor asked about the amount of time it took to create a single mat and discovered it took an entire month to finish a piece. I thought it was incredible to experience the artist constructing a work-in-progress and view its final form at the same time, revealing the thought and care that went into this demonstration.
Artist smiling while weaving on his loom. Photo credit: Claire Fuller
Artist's completed woven mats. Photo credit: Claire Fuller
Each tent was equally fascinating and brought new information to share with the community. One space featured an interactive dancer who pulled members from the audience to have fun and immerse themselves in the culture; another exhibited the creation of colorful Peruvian masks and ornate tapestries. I even ran into our friend MONKY, the talented screen printer and poster-maker from Lima (this time equipped with a much larger set-up than he had at the Chicha! party to create more fully-developed and personalized pieces). The modern tents on the opposite side shared modern Peru's delicious food and energetic singers. The Folklife Festival gives you very well-rounded experience – there's information about Peruvian art, music, geography, food, drinks, agriculture, etc. from the beginning of human life in the Andean region. This program managed to capture and condense the heart of Peruvian culture and offer it to the DC and larger metropolitan community.
Tent of tapestries and masks. Photo credit: Claire Fuller
Weavers enjoying their demonstration. Photo credit: Claire Fuller
I recommend allotting a significant amount of time to explore the festival – you'll want to fully experience every stand. Personally, I felt as though I only got a taste of the event, and I was there for a solid hour. By the time I left, the number of people had almost doubled, so it might be a good idea to head over early if you want to avoid crowds. The festival is open during the weeks of June 24th – June 28th and July 1st – July 5th. All of the demonstrations are free, and it's open daily from 11 AM to 5:30 PM. Something else to note – there is a stage centered between the traditional tents and the modern tents, which holds evening concerts at 7 PM. Check it out while you can!
Claire Fuller


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Don't settle for anything less than optimal performance in a photoconductor unit. It maximizes productivity, minimizes maintenance and delivers exceptional printing. Manufactured with stringent quality control standards. Global Product Type: Imaging Drums/Photoconductors; Imaging Device Type: N/A; Device Types: Printer; OEM/Compatible: OEM.
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Included with purchase ($20 value).
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|Color(s): Cyan
|Global Product Type: Imaging Drums/Photoconductors-Drum Unit
|Page-Yield: 30000
|Supply Type: Drum Unit
|Total Recycled Content Percent: 0 %
|Oem/Compatible: Oem
|Coverage Percent: 5.00 %
|Device Types: Printer
|Post-Consumer Recycled Content Percent: 0 %
|Pre-Consumer Recycled Content Percent: 0 %
|Remanufactured: No
|Country of Manufacture: China
About the ManufacturerDevelopment, manufacturing and sale of multi-functional peripherals (MFPs), printers, equipment for production print systems and graphic arts, equipment for healthcare systems, measuring instruments for industrial and healthcare applications, inkjet printheads and textile printers for industrial use, and related consumables and solution services
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great little machine
Great little machine. Our owners bought 4 buggy wheels for decoration. They were pretty beat up and needed some stripping of the old paint. This wander made quick work of the job and...Read complete review
Great little machine. Our owners bought 4 buggy wheels for decoration. They were pretty beat up and needed some stripping of the old paint. This wander made quick work of the job and they came out beatiful.
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Alot of Orbiting - Not Much Sanding
This model was the first orbital sander I have ever purchased. Easy to use but low power caused sander to stop spinning when even gently applied to stock. Exchanged first unit for another...Read complete review
This model was the first orbital sander I have ever purchased. Easy to use but low power caused sander to stop spinning when even gently applied to stock. Exchanged first unit for another one. Second unit started but didn't even spin at all. Exchanged second unit for a third... that one stopped spinning as soon as it touched stock. That was enough for me - back it went permanently. Purchased another well-known (red & white) brand sander with 3amp motor and love it. This is not a quality item and I was disappointed... and I am a huge Harbor Freight fan.
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Can't beat the price !
By MsAv8Tor
from Athens, GA.
About Me Avid Do-It-Yourselfer
Comments about Chicago Electric Power Tools 2 Amp 5 in. Random Orbital Heavy Duty Palm Sander:
This is the second unit I've purchased. The first lasted thousands of hours working on an auto restoration project I had. Sanding paper is available at Harbor Freight for a variety of projects and the price is right.
Bottom Line Yes, I would recommend this to a friend
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Great Orbital Sander
By Way Out There Brad
from Va
We bought two units at the same time. Used to sand an entire deck after power washing in an attempt to restore the wood after years of sitting unstainted by previous owners. 2 of us were using the units for hours on end in 95 degree heat. Worked great for the project. For what it cost it was worth every cent. No complaints or issues with either unit.
I am happy with Chicago Electric
By RAM
from Peoria, IL.
Works great
(0 of 1 customers found this review helpful)
performed like a champ
By DAVE
from MARYSVILLE CA. 95901
(1 of 1 customers found this review helpful)
By Dan the park manager
from Yuma, Arizona
Images shared by Dan the park manager
Awesome Sander for your money!!!!!
By Adam65Impala
from Hanford,CA
About Me Professional
Great sander. I've used it for a lot of projects sanding from wood to metal. You will not regret buying it. Bang for your money!!!! I've owned it for about 5-6 years and have never had any issues with it at all!!!
By kooler
from old fort, nc
works better than my yellow one ever has.. smooth running..
Plesantly suprised
By mcoge
from Topeka, Kansas
See all my reviews
Have been using this sander for 2 years. Have not changed brushed yet. Just as powerful as it was 2 years ago. I do a lot of woodworking, use this all the time. Would recommend to all. The only drawback is the dust control. Would like to see a vacuum set-up instead of the dust bag.
(2 of 2 customers found this review helpful)
5 in. Random Orbit Sander by Chicago Ele
By big daddy
from Eugene, OR
I purchased this sander after my Yellow one smoked in the middle of a job. The waranty was gone & so was my cash. So I went to Harbor Freight & found this one for just under $20. the plan: to "just get by untill I could get a better one." It's coming up on 5 years. I can vaccum up some dust when I saved around $40.Guess I could paint it yellow but why? I have had other people come into my shop & in the coarse of their work have had occasion to use this sander, all of them really liked it. It's not to heavy, could use a little more power but then so could the Yellow one. I WILL replace it with a like unit when the time comes, no hesitation.
Excellent Sander for light duty woodwork
By JMSetzler
from Valdese, NC
About Me Beginner
I purchased this sander to sand the boards I'm using to make a rolling table for my Kamado grill. I used this sander to smooth down quite a few 1 1/4 x 6, 2 x 4, 1 x 4, and 4 x 4 pieces of pressure treated lumber. The sander came with one 80-grit sanding disk. THAT particular disk didn't last long before chunks of the grit surface were gone from the disk. When I bought the sander, I got two 4-packs of the 100-grit disks and I switched to one of those to finish the job. The sander does a great job but the dust catcher bag is relatively useless. Most of the dust ended up elsewhere, but that is not a showstopper for me since I do my work outside on the patio. This is my first power sander of any kind and I'm extremely happy with it!
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