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Dynamic regulation of genome-wide pre-mRNA splicing and stress tolerance by the Sm-like protein LSm5 in Arabidopsis
DOI: 10.1186/gb-2014-15-1-r1
© Cui et al.; licensee BioMed Central Ltd. 2014
Received: 29 June 2013
Accepted: 7 January 2014
Published: 7 January 2014
Sm-like proteins are highly conserved proteins that form the core of the U6 ribonucleoprotein and function in several mRNA metabolism processes, including pre-mRNA splicing. Despite their wide occurrence in all eukaryotes, little is known about the roles of Sm-like proteins in the regulation of splicing.
Here, through comprehensive transcriptome analyses, we demonstrate that depletion of the Arabidopsis supersensitive to abscisic acid and drought 1 gene (SAD1), which encodes Sm-like protein 5 (LSm5), promotes an inaccurate selection of splice sites that leads to a genome-wide increase in alternative splicing. In contrast, overexpression of SAD1 strengthens the precision of splice-site recognition and globally inhibits alternative splicing. Further, SAD1 modulates the splicing of stress-responsive genes, particularly under salt-stress conditions. Finally, we find that overexpression of SAD1 in Arabidopsis improves salt tolerance in transgenic plants, which correlates with an increase in splicing accuracy and efficiency for stress-responsive genes.
We conclude that SAD1 dynamically controls splicing efficiency and splice-site recognition in Arabidopsis, and propose that this may contribute to SAD1-mediated stress tolerance through the metabolism of transcripts expressed from stress-responsive genes. Our study not only provides novel insights into the function of Sm-like proteins in splicing, but also uncovers new means to improve splicing efficiency and to enhance stress tolerance in a higher eukaryote.
Immediately following transcription, many eukaryotic precursor messenger RNAs (pre-mRNA) are subjected to a series of modifications that are essential for the maturation, nuclear export and subsequent translation of these transcripts. One such modification, the removal (splicing) of non-protein-coding sequences from pre-mRNA, is an important step in gene regulation that also contributes to increased protein diversity from a limited number of genes. The precision and efficiency of splicing are critical for gene function [1]. A non-precision splicing process would generate aberrant or non-functional mRNAs that are not only wasteful but can also lead to the production of unwanted or harmful proteins that may perturb normal cellular processes. Moreover, incorrectly spliced transcripts might also have a profound impact on other processes, including mRNA transcription, turnover, transport and translation. Accumulating evidence indicates that poor efficiency or defects in splicing can lead to diseases in humans [2, 3] and increase sensitivity to abiotic or biotic stresses in plants [4–6]. Although many molecular processes related to splicing have been well characterized, we still face a major challenge in understanding how precision and efficiency in splicing are regulated and how we could harness these regulations to enhance cellular functions.
Sm-like proteins (LSms) are a highly conserved family of proteins in eukaryotes both in terms of sequence and functions. LSms typically exist as heptameric complexes and play roles in multiple aspects of RNA metabolism [7–9]. The heptameric LSm1-7 cytoplasmic complex is located in discrete cytoplasmic structures called P-bodies, which are conserved in all eukaryotes and are thought to be involved in decapping and 5′ to 3′ RNA degradation [10, 11]. The LSm2-8 heptameric complex is located in the nucleus. This complex directly binds and stabilizes the 3′-terminal poly(U) tract of U6 small nuclear RNA, forms the core of the U6 small nuclear ribonucleoproteins (RNPs) and functions in pre-mRNA splicing [12, 13]. The Arabidopsis supersensitive to abscisic acid (ABA) and drought 1 (SAD1) gene locus encodes the LSm5 protein and was identified in a genetic screen for components that regulate the expression of stress-responsive genes in our previous work [14]. SAD1 directly interacts with two other subunits, LSm6 and LSm7, and is a component of the LSm2-8 nuclear complex [15]. Dysfunction of SAD1 increases the plant's sensitivity to salt stress and to the stress hormone ABA in seed germination and root growth; moreover, sad1 mutants are defective in the positive feedback regulation of ABA biosynthesis genes by ABA and are impaired in drought stress induction of ABA biosynthesis, although the detailed molecular bases for these defects have not been identified. Recent studies suggested that the depletion of SAD1 and the other LSm protein (LSm8) reduced the stability of U6 RNPs and resulted in defects in pre-mRNA splicing that lead to intron retention in Arabidopsis [15, 16]. However, it is still unclear if the depletion of SAD1 or other LSm proteins has any effect on the selection of splice sites and alternative splicing (AS), and whether overexpression of these LSm proteins could affect splicing efficiency or accuracy.
To investigate possible regulatory roles of SAD1 protein in pre-mRNA splicing, we performed RNA sequencing (RNA-seq) of the wild-type Arabidopsis (C24 ecotype), the sad1 mutant and the SAD1-overexpressing plants (SAD1-OE). We found that SAD1 could dynamically control splicing efficiency and splice-site recognition and selection in Arabidopsis. Additionally, we discovered that SAD1 is required for regulation of splicing efficiency of many stress-responsive genes under stress conditions. Whereas there are increased splicing defects in sad1 mutants under salt-stress conditions, overexpression of SAD1 increases the splicing efficiency of stress-related genes. SAD1-OE plants are also more salt-tolerant than wild-type plants. Our work not only provides novel insights into the regulatory role of SAD1 and LSm proteins in splicing, but also suggests a new way to improve splicing efficiency and to optimize cellular functions and generate stress-resistant plants.
RNA sequencing of wild-type, sad1 mutant and SAD1-overexpressing plants
The Arabidopsis sad1 mutant was isolated in our previous genetic screen for components that regulate the expression of stress-responsive genes [14]. The sad1 mutant was also more sensitive to stress and ABA inhibition of seed germination and seedling growth [14]. Since loss-of-function mutations in any single-copied core LSm genes are expected to be lethal, the recovery of this point-mutation sad1 mutant provided an invaluable opportunity to study the functions of this important group of proteins. To explore the role of SAD1 in gene expression and stress tolerance, we generated transgenic Arabidopsis plants over-expressing the wild-type SAD1 cDNA (under control of the cauliflower mosaic virus 35S promoter) both in the wild type (ecotype C24) and in the sad1 mutant background (SAD1-OE, see Methods). Although the transgenic plants in both backgrounds had similar physiological and molecular phenotypes, here we mainly focus on SAD1-OE in the sad1 mutant background (referred to as SAD1-OE hereafter).
We next performed RNA-seq using the Illumina HiSeq platform (Illumina Inc., San Diego, CA, USA) on two-week-old seedlings of C24 (wild type), sad1 and SAD1-OE. These seedlings were subjected to two treatments: control (H2O) and salt stress (300 mM NaCl, 3 h). The salt-stress treatment was based on our previous observations that stress-responsive genes were most obviously activated and that the mutant sad1 showed strong molecular phenotypes under these conditions [14, 17]. Based on six cDNA libraries (C24-control, sad1-control, SAD1-OE-control, C24-NaCl, sad1-NaCl and SAD1-OE-NaCl), we generated a total of 164 million reads (101 bp in length, except for the SAD1-OE control, whose reads were 85 bp in length), about 90% of which could be uniquely aligned to the TAIR10 reference genome sequence (version TAIR10; [18]) (Additional file 1). Comparison of the mapped reads to the gene model (version TAIR10) revealed that approximately 95% of the reads mapped to the exonic regions, whereas only about 3% mapped to intergenic regions (Additional file 2), which were consistent with the Arabidopsis gene annotation. Plotting the coverage of reads along each transcript exhibited a uniform distribution with no obvious 3′/5′ bias, which reflects the high quality of the cDNA libraries (Additional file 3). Furthermore, assessing the sequencing saturation demonstrated that as more reads were obtained, the number of new discovered genes plateaued (Additional file 4). This suggests that extensive coverage was achieved, which can also be seen when the read coverage was plotted by chromosome, demonstrating extensive transcriptional activity in the genome (Additional file 5).
We previously identified the sad1 mutation as a G-to-A change 34 bp from the putative translation start site and predicted that the mutation would change a glutamic acid (E) residue to lysine (K). In the RNA-seq data, the mutation of sad1 at the genomic position 19,813,556 of chromosome 5 was confirmed. However, it turned out that the mutation occurred at the 3′ splicing acceptor recognition site of the first intron, changing the invariant AG dinucleotide to AA. Consequently, all of sad1 mRNAs were aberrantly spliced in the mutants, as visualized with the Integrative Genomics Viewer (IGV) browser [19, 20] (Figure 1C). We identified three main mutant transcripts in sad1: two with obvious aberrant 3′ splice sites (3′SSs) that respectively occurred 2 bp and 20 bp downstream of the mutated splice site; and one with the retention of the first intron (Figure 1C). All of these transcripts were validated by RT-PCR using primers that span the alternative 3′SSs, in which the corresponding events were detected in the sad1 mutant, but not in C24 (Figure 1D). Sequence analysis suggested that the transcript with aberrant 3′SSs that occurred 20 bp downstream of the mutated splice site did not alter the coding frame. It was predicted to produce one novel protein with the deletion of seven amino acids compared to the normal SAD1 protein. It seems that this mutant protein could provide some of the wild-type's functions such that the sad1 mutation was not lethal. By contrast, the other alternative 3′SS and the intron retention led to a coding-frame shift that would generate a premature stop codon and thus would lead to truncated proteins. In the SAD1-OE plant, all these aberrantly spliced forms could be found, albeit at much lower levels than in sad1. However, normal SAD1 mRNA was overexpressed, with the transcript level more than 10-times higher than in C24, which was validated by quantitative RT-PCR (Figure 1E).
Identification of alternative splicing events in C24, sad1and SAD1-OE plants
To determine if there were any changes in pre-mRNA splicing upon the depletion or overexpression of SAD1, we first developed a pipeline to identify all AS events in C24, sad1 and SAD1-OE. The pipeline involved three steps: prediction of splice junctions, filtering of the false positive junctions and annotation of AS events. We randomly sampled 20 million uniquely mapped reads (estimated average approximately 57-times coverage on all the expressed transcripts) from each RNA-seq library for the identification or comparison of AS, respectively. This method ensured that the comparison of AS events would be performed at the same level.
To predict splice junctions, we mapped the RNA-seq reads onto the Arabidopsis genome using the software TopHat, which was designed to identify exon-exon splice junctions [21]. After the alignment, we identified 732,808 junctions from the six RNA-seq libraries. Comparison of these junctions to the gene annotation (TAIR10) revealed that about 83% of total junctions had previously been annotated, and the remaining 17% were assigned as novel junctions (Additional file 6A). However, when trying to characterize these novel and annotated junctions, we found that there was a large number of novel junctions that had short overhangs (that is, fewer than 20 bp) with the corresponding exons, while most of the annotated junctions had large overhangs, with the enrichment at approximately 90 bp (Additional file 6B). Moreover, the novel junctions had relatively low coverage compared with the annotated junctions (Additional file 6C). In general, the junctions with short overhangs and lower coverage were considered as false positives, which are often caused by non-specific or error alignment. Therefore, to distinguish between true splice junctions and false positives, we assessed the criteria based on simulated data of a set of randomly constituted junctions. To do this, we first generated a set of 80,000 splice junctions in which annotated exons from different chromosomes were randomly selected and spliced together in silico. We also constructed 119,618 annotated junctions from the gene annotation. Since the length of our sequencing reads was 101/85 bp, the splice junction sequences were determined to be 180/148 bp long (90/74 nucleotides on either side of the splice junction) to ensure an 11 bp overhang of the read mapping from one side of the junction onto the other. Alignments to the random splice junctions were considered to be false positives, because such junctions are thought to rarely exist when compared to annotated junctions. The alignment of the raw RNA-seq reads to the random junctions revealed that 99.90% of false positive junctions had an overhang size of fewer than 20 bp (Additional file 7A). In sharp contrast, the alignment to the annotated junctions indicated that most (98.60%) annotated junctions had larger overhang sizes. In addition, we estimated that 56.90% of false positive junctions had only one read spanning the junction, whereas the annotated junctions had higher read coverage (Additional file 7B). To minimize the false positive rate, we required that the overhang size must be more than 20 bp and that there be at least two reads spanning the junctions. Using these criteria, we filtered out almost all the false positive junctions (Additional file 7C). Finally, we obtained a junction data set of 52,599 confident novel junctions from the six RNA-seq libraries. Based on these junctions, we identified all the AS events including cassette exons, alternative 5′SSs, alternative 3′SSs, mutually exclusive exons, coordinate cassette exons, alternative first exons and alternative last exons (Additional file 8).
Depletion of SAD1 activates alternative splicing
Twenty-two selected events were further validated by RT-PCR using the splicing-site-flanking primers, in which the corresponding AS events were detected in sad1 mutants, but were weakly or not presented in C24 (Figure 2C and Additional file 16). Figure 2C highlights three representative examples visualized by the IGV junction browser and validated by RT-PCR. The SBI1 (AT1G02100) gene had alternative 5′SSs in the 10th intron in sad1, but not in C24, an observation validated by RT-PCR using the forward primer that covered the splice junction and the reverse one that was located at the 11th exon. One can see that the corresponding isoform was detected in the sad1 mutant, but was not present in C24 (Figure 2C). The HINT3 (AT5G48545) gene had alternative 3′SSs in the fifth exon in the mutant sad1, which was validated by RT-PCR using a forward primer in the first exon and a reverse primer that covered the splice junction (Figure 2C). The gene PAC (AT2G48120) exhibited exon-skipping between the third and fifth exons, which was validated by RT-PCR using primers at the third and sixth exon, which meant that two different products were amplified, representing exon inclusion and skipping isoforms, respectively (Figure 2C).
Sequence analysis of these over-represented alternative 5′SSs and alternative 3′SSs (in the NaCl-treated sad1 mutant) revealed that these activated splice sites were still associated with GU and AG dinucleotides (Figure 2D; Additional file 17A), suggesting that the depletion of SAD1 did not change the accuracy of the sequence recognition of the splicing sites. When investigating the distribution of these activated splice sites, we found that alternative 5′SSs and 3′SSs were enriched in the downstream or upstream approximately 10 bp region of the dominant 5′SSs and 3′SSs, respectively (Figure 2E; Additional file 17B). This indicates that the depletion of SAD1 leads to the activation of the 5′SSs and 3′SSs proximal to the respective dominant ones. These results suggest that SAD1, as a component of U6 RNPs, may play a regulatory role in the selection of splice sites.
Interestingly, exon-skipping events also increased in sad1 mutants. When correlating each exon-skipping event with alternative 5′SSs and 3′SSs, we found that about 20% of the skipped exons simultaneously had alternative 5′SSs or 3′SSs in the mutants. This chance of occurrence was significantly higher than that expected for random sampling of all annotated exons (the probability of random occurrence was 0.02%, Fisher's exact test, P <0.001). This result suggests a coordinated occurrence of exon-skipping and alternative splice site selection. Therefore, we considered that SAD1-depletion could simultaneously activate multiple alternative 5′SSs or 3′SSs that include not only the proximal ones, but also the distal ones, including those located at the next exons, albeit to a lesser extent. Nonetheless, the possibility that SAD1, probably as a splicing factor, may directly regulate exon-skipping in vivo could not be ruled out.
SAD1 depletion results in widespread intron retention
We next investigated if there is any influence of the splicing defects on the expression of the affected genes. Sequence analysis suggested that all of these intron retention events would generate premature stop codons in the intron-retained transcripts and, if translated, would produce truncated proteins. Although it is possible that some individual truncated proteins might still be functional, for our sequence analyses, we assumed that these intron-retained transcripts do not generate functional proteins. Through calculating the proportions of the intron-retained transcripts to the total transcripts for each gene with intron-retention in the mutant, we estimated that on average around 15% of total transcripts were with intron retention (Additional file 22). Moreover, when plotting the expression levels of the total and the functional transcripts (without intron) for each intron-retained gene between the wild type C24 and sad1 mutants (Additional files 23 and 24), we found that the expression levels of the total transcripts did not obviously change between C24 and sad1, but the functional transcripts tended to be down-regulated in the mutant. These results indicate that the splicing defects are associated with a global reduction of functional mRNAs, which might negatively affect the functions of these affected genes.
Genes with aberrant splicing in sad1are closely related to stress response and are activated by stress
Further analysis using Mapman [25] suggested that genes with aberrant splicing in sad1 mutants are involved in various stress response pathways, including hormone-signaling pathways, MAPK-signaling pathways and transcription regulation (Figure 4C; Additional file 29). Notably, some important genes (such as SnRK2.1 and 2.2, SOS2, DREB2A, NHX1, WRKY33, WRKY25, STT3A, CAX1 and RCI2A) involved in stress responses were identified to have splicing defects in the sad1 mutant. Among these genes, SnRK2.1 and 2.2 encode members of SNF1-related protein kinases activated by ionic (salt) and non-ionic (mannitol) osmotic stress that are required for osmotic stress tolerance [26]; SOS2 encodes a protein kinase essential for salt tolerance [27]; DREB2A encodes a transcription factor that activates drought and salt stress-responsive genes [28]; NHX1 encodes a vacuolar sodium/proton antiporter whose overexpression increases salt tolerance in several plant species including Arabidopsis[29]; WRKY33 and WRKY25 encode plant WRKY transcription factors involved in response to salt and other stresses [30, 31]; STT3A encodes an oligosaccharyl transferase whose knockout mutants are hypersensitive to high salt conditions [32]; CAX1 encodes a high affinity vacuolar calcium antiporter and can be activated by SOS2 to integrate Ca2+ transport and salt tolerance [33]; and RCI2A (Rare-cold inducible 2A), whose product plays a role in preventing over-accumulation of excess Na+ and contributes to salt tolerance [34]. These genes showed increased intron retention in the mutants, which were also validated by RT-PCR using intron-flanking primers where the corresponding intron-retained transcripts were more obviously identified in sad1, consistent with the RNA-seq data (Figure 4D). Above all, these results suggest that genes with aberrant splicing in sad1 are closely related to stress response, which could directly or indirectly contribute to the stress sensitivity of the sad1 mutant.
Overexpression of SAD1 rescues the splicing defects in the sad1mutant and strengthens splicing accuracy under salt stress
We further compared the expression levels of introns in SAD1-OE with those in C24 and sad1. We found that the expression of most introns in SAD1-OE was restored to normal levels (Figure 5F; Additional file 33), demonstrating that the intron retention indeed resulted from the sad1 mutation. Furthermore, using Fisher's exact test we identified 76 introns from 75 genes that were significantly absent in NaCl-treated SAD1-OE, but were over-represented in NaCl-treated C24 (Additional file 34). This result shows that SAD1-overexpression can increase splicing efficiency.
Overexpression of SAD1improves plant salt tolerance
Further analysis suggested that these genes are involved in various stress response pathways (Additional file 37). Some of the stress-responsive genes that were more effectively spliced in SAD1-OE included ABF3/ABF2, encoding ABRE binding factors that mediate ABA-dependent stress responses [35, 36]; CIPK3, encoding CBL-interacting serine/threonine-protein kinase 3 that is involved in the resistance to abiotic stresses (for example, high salt, hyperosmotic stress) by regulating the expression of several stress-inducible genes [37]; and DREB2A, that encodes a transcriptional factor mediating high salinity- and dehydration-inducible transcription [28]. These genes have been reported to be key regulators of ABA or salt-stress responses.
With the increased splicing efficiency in these key regulators of ABA or salt-stress responses, we were curious to know whether the SAD1-OE plants would have improved tolerance to salt stress. To test this, one-week-old seedlings of C24, sad1 and SAD1-OE grown on the regular Murashige and Skoog (MS) agar medium were transferred to MS agar plates supplemented with 0 (control), 50, 100 or 200 mM NaCl. We found that SAD1-OE seedlings showed enhanced tolerance to 100 mM NaCl on vertically placed plates (Figure 6C). At 200 mM NaCl, however, root elongation of all genotypes was inhibited and seedlings were not able to survive an extended period of the stress treatment (data not shown). Measuring the root growth of the seedlings showed that the roots of SAD1-OE were longer than those of C24 and sad1 at 100 mM NaCl (Additional file 38). We also tested salt tolerance of seedlings on horizontally placed agar medium plates. Two-week-old seedlings from ½ MS media were transferred onto 200 mM NaCl media and incubated for five days. The percentage of green leaf number over total leaf number was calculated for each seedling. The data indicated that SAD1-OE seedlings had a higher percentage of green leaves, suggesting that they were significantly less damaged by the salt stress than were the wild-type or sad1 seedlings (Figure 6D). To test further whether SAD1-OE plants were tolerant to salt stress at the adult stage and in soil, we grew these seedlings in soil and irrigated with either 50, 100, 150, 200 or 400 mM NaCl solutions at intervals of four days (see Methods). After two weeks of treatment, we found that sad1 plants were very sensitive to salt stress at concentrations above 150 mM and wild-type seedlings also exhibited signs of damages at higher salt concentrations as indicated by wilty inflorescence and damaged leaves, whereas the SAD1-OE plants were not obviously affected by the stress treatment and were also taller than the wild-type plants (Figure 6E; Additional file 39). These results indicate that SAD1-overexpression improves salt tolerance, which correlates with increased splicing accuracy and efficiency of stress-responsive genes.
Although studies in other eukaryotes, and more recently in plants, have demonstrated that LSm proteins 2-8, as the core of U6 RNPs, function in pre-mRNA splicing, whether or not these proteins have any roles in regulation of splicing efficiency and selection of splice sites has not yet been determined. In this study, through comprehensive transcriptome analysis of mutant and transgenic plants overexpressing the LSm5 gene SAD1, we demonstrated that SAD1 could dynamically regulate splicing efficiency and selection of splice sites in Arabidopsis. We also revealed that SAD1 modulates splicing of stress-responsive genes under salt-stress conditions. Finally, we showed that overexpression of SAD1 significantly improved splicing efficiency of the salt-responsive genes and resulted in enhanced salt tolerance in transgenic plants.
We found that SAD1 depletion activated the alternative 5′SSs and 3′SSs proximal to the dominant ones, suggesting that the wild-type SAD1 protein is necessary for precise splice-site recognition. To our surprise, relative to the wild-type plants, overexpression of SAD1 can strengthen the recognition accuracy and globally inhibit AS under salt-stress conditions. Therefore, we conclude that SAD1 can control selection of splice sites and splicing efficiency in a manner depending on SAD1's abundance. This kind of splicing regulation, which could be referred to as a dynamic model, differs from but complements the kinetic model of splicing regulation [1, 38, 39]. In the kinetic model, the elongation rate of RNA polymerase II (Pol II) affects splicing efficiency such that a slower Pol II would allow more time for the recognition and processing of weak splicing sites so that splicing efficiency is enhanced. In the dynamic model, we reasoned that the spliceosome or other complexes involved in splicing are under thermodynamic equilibrium between association (complex formation) and disassociation (complex breakdown) at any given condition. A higher dosage of certain key small nuclear RNPs or splicing factors may drive the reaction toward the formation of the complex to enhance splicing efficiency. This dosage-dependent control of splicing suggests an alternative splicing regulation and it may be particularly important for the splicing of particular group of genes such as stress-inducible genes as discussed below.
Whereas increased alternative 5′SS usage was seen in sad1 both under control and salt-stress conditions (Figures 2A,B; Additional file 9A,B), the increase of alternative 3′SSs caused by SAD1 depletion and the inhibition of AS caused by SAD1 overexpression were only observed under salt-stress conditions. These findings indicate that SAD1 depletion or overexpression appears to impact splicing under salt-stress conditions more than under normal conditions. We considered that this distinct impact of SAD1 on splicing under normal versus stress conditions might have to do with increased transcriptional activation of the stress-responsive genes. Under salt-stress or other abiotic-stress conditions, plants activate the expression of a large number of stress-responsive genes that are not expressed or are expressed at lower levels under normal non-stressful conditions [40, 41]. With the simultaneous production of a large amount of these stress-inducible pre-mRNAs, cells would need to immediately recruit a significant amount of splicing factors and other factors for their co-transcriptional or post-transcriptional processing. This imposes a huge burden on the splicing machinery and as a result a significant portion of these transcripts fail to be processed adequately when the splicing machinery is compromised. This may be the reason why most of the splicing defective genes in sad1 are stress-regulated (Figure 4). Conversely, a higher SAD1 dosage could play a dominant role in enhancing the splicing efficiency of these salt-responsive genes through the promotion of recruitment and assembly of the splicing machinery as discussed above. As a result, the change in the AS pattern in SAD1-OE plants was more obvious under salt-stress conditions than under control conditions. Thus, the expression of these (and other) highly inducible genes may be particularly subjected to the dynamic regulation by certain splicing factors, which to some extent is similar to the kinetic regulation of splicing, both reflecting the saturated capability of cellular machinery.
We thought that the decreased splicing efficiency of the stress-responsive genes might contribute to the stress-sensitivity of the sad1 mutant. The splicing defects in sad1 lead to widespread intron retention in many stress-responsive genes (317 genes, Additional file 40). These genes include those encoding known key determinants of salt tolerance such as SnRK2.1/2.2, SOS2, DREB2A, NHX1, WRKY33, WRKY25, STT3A, CAX1 and RCI2A. The expression level of the functional transcripts for many of these genes were also found to be down-regulated (Additional file 41), although the cause of this down-regulation is unclear. All of these intron-containing transcripts were predicted to generate premature stop codons and truncated proteins if translated. This large-scale 'hidden' change in pre-mRNA splicing efficiency or gene expression, although relatively small for some of the individual genes, may collectively undermine plant's readiness for the stress. However, it should be pointed that a direct relationship between the splicing defects and stress sensitivity in the sad1 mutant could not be established at this point.
Interestingly, an increase of splicing efficiency and expression of stress-responsive genes correlated with improved stress tolerance of the plants. Indeed, transgenic plants overexpressing SAD1 exhibited obviously increased tolerance to salt stress (Figure 6E), although the magnitude of the increase was moderate. Nonetheless, this finding is very significant for two reasons. First, it indicates that splicing efficiency may play an important role in regulating plant stress resistance. This is consistent with findings in several other genetic studies, where certain RNA processing factors were also found to be required for plant stress resistance. These factors include, for example, ABH1[42], LOS4[43] and RCF1[44], although the mechanisms involved were unclear. Secondly, our finding provides a new approach to improving plant stress resistance, namely, by regulating the splicing efficiency. Current methods to increase plant salt tolerance mostly involve the overexpression of structure genes such as ion transporter genes [29, 45, 46]. Constitutively expressing these structure genes may cause unwanted side effects that would result in reduced yield under normal growth conditions. However, enhancing splicing efficiency does not affect gene expression under normal conditions, as demonstrated in this study. Our finding may also be applicable to enhancing stress tolerance or other traits in other eukaryotic systems.
We demonstrated that SAD1 dynamically regulates splicing efficiency and plays a regulatory role in the selection of splice sites. Furthermore, we found that SAD1 specifically modulates splicing of the stress-responsive genes under stress conditions. Finally, we showed that overexpression of SAD1 improves salt tolerance of transgenic plant, which correlates with the increased splicing efficiency of the salt-stress-responsive genes. Our study provided novel insights into the regulatory role of SAD1 or LSm proteins in splicing and also suggested new strategies to improving splicing efficiency and bioengineering stress-resistant plants.
Materials and methods
Plant materials and growth conditions
The Arabidopsis sad1 mutant in the C24 background was described previously [14]. For overexpressing the SAD1 gene, the SAD1 cDNA, amplified from the wild-type plant, was cloned into pENTRY1A. The LR reaction was then performed between pGWB502 and the pENTRY1A-SAD1. The resulting plasmid (pGWB502-SAD1) was introduced into Agrobacterium tumefaciens GV3101 and transformed into sad1 mutant plants using the floral dipping method. The transformants were selected on a ½ MS medium supplemented with 25 μg/ml hygromycin. Positive transformants were further confirmed by genotyping using the primers 5-CACCGGATCCTGATGGCGAACAATCCTTCACAGC-3; 5-TAATGAATTCGATCATTCTCCATCTTCGGGAGACC-3 for SAD1 cDNA. The confirmed transgenic seedlings (referred to as SAD1-OE) were used for RNA sequencing and RT-PCR analyses.
Seeds of C24, sad1 and SAD1-OE plants were sterilized with 50% bleach and 0.01% Triton X-100. The sterilized seeds were sown on ½ MS plates supplemented with 3% sucrose. After four-day stratification at 4°C, the plates were placed under a 16 h-light and 8 h-dark cycle at 21°C for germination and seedling growth. Twelve days later, the seedlings were treated with H2O (control) or 300 mM NaCl for 3 h, and harvested for total RNA extraction.
RNA extraction, library construction and sequencing
Using the TRIzol Reagent (15596–026, Invitrogen Co., Carlsbad, CA, USA), total RNAs were extracted from 12-day-old seedlings of wild-type C24, sad1 and SAD1-OE. Polyadenylated RNAs were isolated using the Oligotex mRNA Midi Kit (70042, Qiagen Inc., Valencia, CA, USA). The RNA-seq libraries were constructed using Illumina Whole Transcriptome Analysis Kit following the standard protocol (Illumina, HiSeq system) and sequenced on the HiSeq platform to generate high-quality single-end reads of 101 nucleotides (some with 85 nucleotides due to machine failure) in length.
RNA-sequencing data analysis pipeline
To analyze RNA-seq data, a pipeline was developed, which involved five steps: read alignment and junction prediction, the filter of false positive junctions, annotation of AS events, global comparison of AS and the identification of differential AS events (for details, see Additional file 42).
Read alignment and junction prediction
TopHat [21] was used to align the reads against the Arabidopsis genome sequences and annotated gene models were downloaded from TAIR10 [18] allowing two nucleotide mismatches. Meanwhile, TopHat was also used to predict the splice junctions that did not permit any mismatches in the anchor region of a spliced alignment. The splice junctions were classified into known and novel splice junctions using the Perl script, which takes as input genome coordinates of all annotated exons and all predicted splice junctions. In addition, the expression levels of transcripts were measured by reads per kilo base per million values using the Cufflinks software [47].
The filter of false positive junctions
To estimate thresholds for filtering false positive junctions, two datasets of random and annotated splice junctions were first created. The dataset of 80,000 random splice junctions was created by joining each annotated 5′ donor sequence (90/74 bp from 5′SSs) and the annotated 3′ donor sequence (90/74 bp from 3′SSs) located on a different chromosome (Additional file 42). The 119,618 annotated splice junctions were created by joining each annotated 5′ donor sequence (90/74 bp from 5′SSs) and the annotated 3′ donor sequence (90/74 bp from 3′SSs) in order based on the gene annotation (Additional file 42). All splice junctions contained 90/74 nucleotides of exon sequence on either side of the junction to force an alignment overhang of at least 11 nucleotides from one side of the splice junction to the other. Then, the mapping software BWA [48] was used to align all reads to the random and annotated splice junctions that did not permit any mismatches. The alignments to random junctions were considered to be false positives, because such junctions are thought to rarely exist when compared to annotated junctions. We further characterized the false positive junctions, which generally have an overhang size of fewer than 20 bp and lower read coverage (Additional file 7A-B). To minimize the false positive rate, the overhang size with more than 20 bp and at least two reads spanning the junctions were required as cutoff value to filter the false positive junctions.
Annotation of alternative splicing events
JuncBASE [49] was used for annotating all AS events, including cassette exons, alternative 5′SSs, alternative 3′SSs, mutually exclusive exons, coordinate cassette exons, alternative first exons, alternative last exons and intron retention, which takes as input genome coordinates of all annotated exons and all confidently identified splice junctions. Notably, for identifying the events of intron retention, we required that at least five reads covered at least 50% of the region of one intron.
Global comparison of alternative splicing
The global comparison of AS among WT, sad1 and SAD1-OE was started with equally and randomly re-sampling uniquely-mapped reads to make sure that the comparison was at the same level. The comparison refers to the two facets: the absolute amount of each type of AS event and the number of junction reads that were assigned to each type of AS event, because both of them can be used to measure the global changes of AS. Meanwhile, Fisher's exact tests in R [50] were used to identify differential representation of each type of AS event, performed on the number of junction reads that were assigned to each type of AS event.
The identification of differential alternative splicing events
Fisher's exact tests were also used to identify differential representation of each AS event. For alternative 5′SSs and 3′SSs and exon-skipping events, Fisher's exact tests were performed on the comparison of the junction-read counts and the corresponding exon-read counts between C24 and sad1 or SAD1-OE. The events with P <0.01 were identified as significantly different events. In addition, for those AS events that were uniquely identified in C24, sad1 or SAD1-OE, we would consider them significant if there were at least five junction reads to support and the P value of these events was assigned to equal zero. Similarly, for intron retention, Fisher's exact tests were performed on the intron-read counts and the corresponding exon-read counts between C24 and sad1 or SAD1-OE. The events with P <0.001 were identified as significantly differential events. In addition, for those intron retention uniquely identified in C24 or the mutant, we would consider them significant if there was at least five-time coverage to support and the P value of these events was assigned to equal to zero.
RT-PCR validation
The selected AS and intron retention events were validated by RT-PCR using a set of primers (Additional file 43) that were designed based on each AS event. Total RNAs from the C24, sad1 and SAD1-OE plants were extracted using Trizol solution (Invitrogen; cat.10837-08), treated with DNAase I, and reverse-transcribed to cDNA (random priming) by using a standard protocol (SuperScript II reverse-transcriptase, Invitrogen).
Quantitative RT-PCR
For the RT reaction, we used 3 μg total RNAs from the control (H2O) and 300 mM NaCl-treated C24, sad1 and SAD1-OE seedlings. The RT reactions were done with the Invitrogen SuperScript® III First-Strand Synthesis SuperMix in a 20 μl reaction system; the random Hexamer was used for first strand synthesis. The RT-solution was diluted 10 times, and 1 μl of the solution was used as template in 10 μl reaction system with 2 × SYBR Green (Invitrogen) Supermix (ROX). The quantitative RT-PCRs were performed in triplicate using the ABI 7900HT Fast Real-Time PCR System (Applied Biosystems Inc., Foster City, CA, USA). The primers 5-AAGGAGATAAG-GAGCTCGTTGG-3 and 5-ATCTGATCAAGCTTTGTGACC-3 were used for detecting expression levels of SAD1.
Salt-stress tolerance assays
Surface-sterilized seeds of C24, sad1 and SAD1-OE were sown onto agar plates with ½ MS and 1.2% agar. The plates were then kept at 4°C for four days before being incubated at 21°C for germination. Four days after germination, the seedlings were transferred to ½ MS agar plates supplemented with 0, 50, 100 or 200 mM NaCl, respectively. The seedlings were then allowed to grow for four days, and seedlings were photographed. Root length of these seedlings was measured by using Image J [51]. For measuring leaf damage, two-week-old seedlings grown on ½ MS plates (0.6% agar) were transferred onto ½ MS medium plates supplemented with 200 mM NaCl and incubated for five days. The number of green leaves and yellowish or bleached leaves was counted for each seedling and percentage of green leaves among total leaves was calculated (leaves of the no-salt control treatment were all green and were not counted). To further test the tolerance to salt stress, seedlings grown on ½ MS agar plates were transferred to soil. One week after the transfer, the seedlings were irrigated with 50, 100 or 150 mM NaCl (in 1/8 MS salt), respectively [29]. At 24 days after the transfer, the plants were further irrigated with 400 mM NaCl (100 ml) and pictures were taken four days later.
Data availability
The RNA-seq data generated in this work has been submitted to the Sequence Read Archive database in NCBI. The accession number is SRP026082.
- 3′SSs:
3′ splice sites
- 5′SSs:
5′ splice sites
abscisic acid
alternative splicing
- bp:
base pairs
- LSm:
Sm-like protein
Murashige and Skoog
polymerase chain reaction
- RNA-seq:
reverse transcriptase.
This work was supported by King Abdullah University of Science and Technology (KAUST), Kingdom of Saudi Arabia.
Authors' Affiliations
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This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


text:
SMS-EB-BLK - STREET by 50 Professionally Tuned In Ear Earphones w/Apple 3-Button Remote/Mic - Black
Take it to the street. Bring your music with you in luxurious style with STREET by 50 In-Ear Headphones. In-line Apple control mic cord for tangle free streetwear and transport, plus a professionally tuned 11mm driver and 6 silicone passive noise cancelling tips ensure a comfortable ride no matter where your music takes you. The STREET by 50 wired in-ear headphones feature crystal clear sound,...
Plantronics BackBeat 906 Stereo Bluetooth headset
Plantronics BackBeat 906 Stereo Bluetooth headset with Bluetooth adapter 3.5mm audio ports. This wireless headphones feature Altec Lansing sound technology for rich, full-spectrum stereo music and dual-mic AudioIQ noisecanceling technology for exceptional call clarity. This provides up to 7 hours of talk time and up to 8 days of standby time. It is equipped with A2DP technology.
Earpiece Kit 6 Sizes STARSET/H31CD
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subset: commoncrawl


text:
mirkosp wrote:Qyot, Fate/kaleid liner Prisma Illya is just a spinoff with only characters in common with the other series, so if the rest of the franchise is what's keeping you, you're good.
It's more that I don't tend to go with spinoffs without being familiar with the main canon first, even if it's only tangentially related. Most of my exposure to Fate is via osmosis, despite having the VN and subsequent properties in the queue for years.


text:
Winter Jackets - Women'sSave on Women's Winter Jackets at Proboardshop.com! Great selection of street gear at unbelievably low prices. Proboardshop has been selling Winter Jackets and outdoor gear for over 30 years and we're proud of it.
Please refine here
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text:
Nothing to report in the Culver City area Targets, it seems strange that Target doesnt want to take advantage of the 30th Anniversary.
As for Celeb IV my son and I will be there every day, looking forward to meeting any salty dogs that make it.
TRU put out a case of VTAC and a case of Comic 2 packs today. D. Sanchez and I split the VTAC case. Best of all, the figures were only $9.99.
Found a lone Vos comic pack at TRU La Cienega... er, I mean... a certain toy store... somewhere in the LA area... Just kidding, I did stash it for any one who needs it. PM me.
Hide it, Post it, and PM it.
Hide it, Post it, and PM it.
I requested a badge yesterday, but have not received any response.


text:
A few weeks ago, as part of a booksprint on libre/open fonts, some of us started to update the French translation of FontForge, the libre software font editor. Fontforge uses GNU gettext and the UI translation documentation outlines the methodology for getting the messages updated and tested.
This was driven by the need to make the terminology coherent and to illustrate various chapters with screenshots in French for Fontes Libres a book written in French on libre/open fonts and related topics. This book effort has been made possible thanks to FLOSS Manuals fr and the financial support of Organisation Internationale de la Francophonie.
We built upon the previous translation work done by Pierre Hanser and Yannis Haralambous. Interestingly enough, much of the needed translation of
po/fr.po was done and tested using gobby in a hotel lobby over a wireless network. There are much better workflows out there but this ad-hoc method was helpful.
There is still a lot left to translate but things are on their way and I hope this can get finished soon and submitted for inclusion upstream.
One more way the wider community will benefit from this booksprint !


text:
In 2005, the Department began implementation of the Web Confidential Morbidity Report (WebCMR) and Electronic Laboratory Reporting (ELR) projects. When completed, these projects will provide the State of California with a secure, web-based system for use by health care providers, laboratories, local health departments, and CDPH for the reporting, surveillance, and case management of communicable diseases. WebCMR covers the clinical aspect of reportable diseases whereas ELR covers laboratory results, but both will be part of a single, integrated system of tracking communicable disease.
Initiated in 2005, the CalPHIN Laboratory Information Management System (LIMS) project will implement a common data structure and user interface across all six CDPH laboratories in order to centralize tracking of patient records and laboratory specimens. In addition, the Laboratory Data Interchange (LDI) project will facilitate exchange of laboratory data between clinical and local public health laboratories, CDPH laboratories, and the CDC or other federal partners. In conjunction with these activities, CDPH is one of seven state laboratories participating in the CDC-sponsored Public Health Laboratory Interoperability Project (PHLIP), which promotes the use of PHIN messaging standards.
The California Health Alert Network (CAHAN) is the State of California's web-based information and communications system available on a 24/7/365 basis for distribution of health alerts, dissemination of prevention guidelines, coordination of disease investigation efforts, preparedness planning, and other initiatives that strengthen state and local preparedness. CAHAN is managed by the Emergency Preparedness Office.
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Web Stories
'Streetcar Named Desire' to be staged at Berlind
Posted February 26, 2009; 01:58 p.m.
A student performance of Tennessee Williams' landmark play "A Streetcar Named Desire" is set for 8 p.m. Friday and Saturday, March 6-7, and Wednesday through Friday, March 11-13, at the Berlind Theatre.
The play is a senior thesis production by Shannon Lee Clair for the Program in Theater and Dance in the Lewis Center for the Arts.
The Pulitzer Prize-winning "Streetcar" centers on a culture clash between Blanche DuBois, a fading relic of the old South, and Stanley Kowalski, a rising member of the industrial, urban immigrant class. Clair stars as DuBois alongside senior Tyler Crosby as Kowalski. The production is directed by Tracy Bersley, a lecturer in theater and dance.
Clair said she chose "Streetcar" for a thesis project based on how she had been cast in campus productions in the past. "Being a fairly put-together, centered person, I often find myself cast in similar roles. But, while Blanche certainly lives on the principle of being put together, the tidal wave of life experience that crashes against her is so forceful that she is thrown far from her center," Clair said. "This part and my director, Tracy, push me out of all comfort zones I might ever try to live in as an actress."
Tickets are $10 for students and senior citizens, and $15 for others. For tickets, call the McCarter Theatre Center box office at (609) 258-2787 or University Ticketing at (609) 258-9220.


text:
I wonder how big this Galvatron is?
I want Cyclonus and Galvatron (secondly, Scourge) to pair up with my Masterpiece Rodimus Prime.
I would take a Voyager figure as well though - but not sure if it's worth $115 plus shipping.
However, I bought (kind of Voyager sized) Titanium Ultra Magnus because it's a one-piece and totally transforms instead of adds parts from his trailer and I paid about $100 for that. But it's also an item that's years old which I didn't buy back in the day when I had the chance.
So how big is this Galvatron?
Isn't there a 3rd party Cyclonus that's pretty good? How big is he?
JediTricks, do you own any personally? Which ones?
How do any here review their 3rd Party figures?
I'm thinking about the Constructicons and the Stunticons 3rd party pieces. Is there a Combaticon set? Bruticus is also an interest.


text:
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text:
For minor injury or illness of a child that requires immediate attention after hours.
If your child has an illness or injury that occurs after hours and is minor but requires immediate attention, your pediatrician may refer you to an Urgent Care Center. The Le Bonheur Urgent Care Centers are located throughout the city and are available on nights and weekends.
Discuss your child's needs with your pediatrician to determine the best options for your child's care.
For more information on urgent care for your child, visit Le Bonheur's website.


text:
Well-Rounded Debate
Dear Alibi,
I read Gene Grant's opinion piece ["Around and Around," Oct. 18-24] on the proposed roundabout at Rio Grande and Candelaria with great interest. I was pleased that he chose to reference my opinion piece previously published in the Journal. However, I'm sorry that Gene chose to ignore the main question that I have raised: Why is so much attention (and money) being showered on this particular intersection? Data given by roundabout proponents and the public relations company they have hired to sell this project to the public are misleading at best. It is totally insufficient to cite the number of accidents at an intersection in a given year and then say, "See how bad it is?" By contrast, professional transportation planners use what are known as comparative crash rates. These numbers reflect the ratios of accidents to traffic volumes at each intersection in the Albuquerque Metropolitan Planning Area over extended periods of time. This AMPA data is produced by UNM's Department of Government Research and provided under contract to the Mid-Region Council of Governments as the basis for all traffic planning in the metropolitan area.
This data clearly indicates that there are serious traffic issues in the North Valley. They simply don't happen to be at the Rio Grande and Candelaria intersection. In fact, three intersections in the area make the list of the top 20 most dangerous intersections in the entire metropolitan area. They are Rio Grande and Central, Rio Grande and I-40, and Candelaria and 12th Street. All three of these intersections have crash rates that are far higher than Rio Grande and Candelaria. If roundabouts are indeed as beneficial as their proponents claim, and if we are to spend millions of dollars in their construction, then obviously these are the intersections we should be focusing on.
If the proposed Rio Grande and Candelaria roundabout does nonetheless proceed as planned, then its proponents will simply confirm what already seems evident: namely, that public safety is not in fact their paramount concern. What the real considerations at play here may be I leave to the proponents to explain.
Jerry Ginsburg
Fooled Again
Dear Alibi,
In a speech in 1858, Abraham Lincoln said, "You can fool all of the people some of the time, and some of the people all of the time, but you cannot fool all the people all of the time." For the last 30-something years, about half the people have been fooled time and again, bringing us to the situation we find ourselves in today.
In 1980 Ronald Reagan was elected president on a platform of tax cuts, deregulation and smaller government. The economic plan was referred to as supply-side, trickle-down or voodoo economics. The people and the Congress bought it, and thus began the extreme acceleration of the wealth gap between the very rich and everyone else. Deficits and debt skyrocketed. Deregulation of the savings and loan industry resulted in a complete meltdown in only three years, and billions were spent to fix it. The great communicator had fooled most of the people, including himself, and was re-elected in a landslide.
Need we say much about President George W. Bush? A more inappropriate, incompetent, frat boy delinquent could not have come out of central casting. And about half of all voters were fooled. He starts an illegal and disastrous war based on false claims of WMDs, etc. and is re-elected. Fooled again. His tax cuts and war spending further accelerated the wealth gap and turned a surplus into a huge deficit. Then there was Hurricane Katrina and the financial collapse, among other faux pas. Foolishness has consequences.
Then there is the whole thing about climate change. For a while it was a settled issue. Then big industry—especially big oil, in an effort to avoid having to do anything that may diminish profits—began a PR campaign to cast doubt on the whole idea. Many millions, in this country at least, were fooled by the propaganda, and so nothing has been done about it. Being fooled about this will have very serious consequences, and parts of N.Y. and N.J. are underwater.
Now comes Mitt Romney, the poster boy for corporate raiders, predators, vultures and job off-shoring who is a whiz at extracting millions from ailing and not-so-ailing companies by dismantling them and/or shipping them to China. None of this is a secret. But this same Mr. Romney is running on a platform of jobs, jobs, jobs, tax cuts for the rich and spending cuts for the poor. And he's set to get about half the votes. Some people can be fooled all of the time.
Robert DiGiulio
Baby Bookworms
[Re: "Keeping Their Word," Nov. 1-7] As mentioned in your article, we don't give Kindles to 3-year-olds (at least not always, but the media capabilities of e-readers have opened a Pandora's box of options for children's book creators). And babies love books, so parents and relatives keep buying those books. When the music industry changed so dramatically with the easy entre of online ordering, the rapid change was facilitated in part by the near-unanimous agreement of consumers young and old that the new product was more desirable. That decision wasn't made by vote, by discussion, or by mandate. Consumers just began to use the new method of presentation and access en masse. We voted with our wallets.
As long as we raise babies in a tradition of books, actual ink on paper books, we create an early life memory love affair that preserves books in our hearts, and keeps us visiting the wonderlands that hold those treasures. Bookstores need to recognize their place in our psyche and actively court it. If they build engaging spaces filled with visual treasures, babies will come. They will come, and their parents will follow, wallets in hand.
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Results 1 to 10 of 11
Thread: Discus issues
Discus issues
I have four discus in a 65 gallon tank along with a ret. stingray, clown loach, and black ghost knife. The fish all get along with eachother from species to species, but my discus fight all the time. I feed them 2-3 times a day, once or twice with live black worms, hikari discus pellets, or freeze dried tubifex worms. I cannot get them to eat the frozen brine or bloodworms. Three of the discus are 3-4" and one is around 5" so they are quite young. Naturally the 5" is the boss. Is there anything I can do to get them to stop fighting? They chase the newest one in the corner all the time and bite at it whenever it tries to eat. Please help!
I'm no discus expert, but seems to me the issue may be not enough space. thats a TON of bioload for a 65g (and much too small for a ray). perhaps the discus experts will chime in soon...your friendly neighbourhood arowanaman!
All of the fish are quite small, the stingray is a pup, once he gets larger he will be moved. I read that discus require around 9 gallons/fish as adults, and that I could house 6 discus. The black ghost knife is 4" at best and the clown is 2-3" it is a fairly young tank.
The rule is 10 gals per fish and that is as small as one should go for adults - your young fish are fine in a 65 gal.
Having other species is asking for fighting between discus due to stress but worse, is having under six discus. You are doomed with four discus fighting - that is their nature and why people are always told to raise six or more. Other fish just add to the stress - especially large fish.
How often do you change all the tank water? What are the nitrate and phosphate levels? What is the pH and hardness? Young discus need near 100% water at least every two to three days (you want zero nitrates, and phosphates; if not, do the complete water changes more often.) Adults can handle higher numbers but not young if you want them to grow well and healthy. Having other fish just increases the bio-load.
Ghost fish are very aggressive at night and it is critical that discus not be bothered at night by either the fish's active nature towards discus or moving around the tank. That fish and the string ray are not proper companions for discus. Besides, ghost fish tend to get large like rays, do. The ray will need a far bigger tank, by the way.
Last edited by Cermet; 11-20-2012 at 01:01 PM.Knowledge is fun(damental)
A 75 gal with eight Discus, fake plants, and a lot of wood also with sand substrate. Clean up crew is down to just two Sterba's Corys. Filters: continuous new water flow; canister w/UV, in-tank algae scrubber!! Finally, junked the nitrate removal unit from hell.
For Fishless cycling:http://www.aquaticcommunity.com/aqua...ead.php?t=5640
My nitrates are >5 and I change the water frequently. The discus are definitely the bosses of the tank, they are the only ones that utilize the entire tank as the stingray usually sits on the bottom or the side of the tank, the clown sits on the bottom, and the knife hides most of the time due to the lights.
A+ Cermet
Discus have totally different requirements than the others in the tank and why Discus are generally kept as specie only tanks. Or with a few cardinals that can take the higher heat and softer water they require.
Atho you plan to move some of these fish later, they may keep the Discus stressed out until that time and the ray will need a huge tank as well as the BGK that grows to 20". Clown loaches are also big, bulky fish that should be in schools.
I can see no success coming from this tank as it stands now. You should not get fish until you have the tank for them. Thinking a bigger tank will be somewhere in the future just doesn't generally happen.
I'm sorry but I don't see how that setup could work... Discus are so skittish around large fish, and they really should be kept in groups of at least 6 for their own health... That the new one is being harassed is no surprise as it was introduced into some other already established discus territory. It will take time for them to set up their peking order but in a tank of only 4 you run the risk of having the runt of the pack being bullied to death. I suggest you trade in your ray, clown loach, and ghost knife for two more discus and plenty of plants (real or fake) to break up the visibility periphery of your fish and create more hiding spaces in your tank for your smaller discus.
Getting the right balance of things in your tank to create a better environment for your discus is essential if you want healthy and happy fish. It is not easy but it can be very rewarding...
11-24-2012, 11:42 AM #8Junior Member Guppy
- Join Date
- Nov 2012
- Location
- Cape Cod Ma
- Posts
- 2
Hey Bud; What works/Works. I would Change the Live Blck worm this is just to Much. And agree with the masses Change the Mix in this Tank.For sure in time you will Lessen the Growth. For all that Potien{Not enough balance in diet} will grow out those Discus Fast and the growth rate will Make them more Bullish.But Discus are Cichids but on the bottom rung of fighters but still.Find a Trade off with the other fish maybe for German Rams and or More Tetras. If you still carry and controll that Bio Load.... Do it with smaller fish in order to lessen the fight for food. The more you feed the more they Poop. And it will be a DAILY H2O changes and no time to enjoy and you will stun the growth rate and that will in time cause a new set of health issues. But as first said If it works!......
I have had bad experience with BGK in with discus in a previous tank i had. A very highly recommended local fish store told me that there would be no problem putting it in my tank then it happened... that knife fish started to stress my discus out so much they would hide all the time, and With that the BGK was returned to the store. Discus do tend to fight while establishing who is going to rule the tank...
thanks for the input everyone, I am looking into a larger tank for the ray, I am going to try and leave the clown with the discus because he doesn't seem to bother them at all, and sometimes a few black worms make it into the sand and he is very good at finding them and taking care of them. I also had a snail outbreak and he takes care of them as well!


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Finished the new NTFS Linux kernel driver (read-only)! Posted it and got some excellent feedback so far. Speedwise it definitely is a significant improvement. I am happy! (-8
Went to Richard M. Stallman's lecture on software patents this afternoon. Brilliant talk. RMS certainly is a very good speaker. And I fully agree with him, too. Software patents are evil full stop!


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Within the boundaries of these countries, the promise of having access to internet resources have been mostly to the ones who have the means. What has been considered a technical tool for technical people are now considered necessary resource. With all the information stored, be they scientific, economic or political in content, access to these resources must be provided at all levels."The information revolution has brought rapid transformation in the economies of many industrialized countries, and sweeping promises of a better quality of life for all. However, people living in the vast majority of other countries have been left untouched and unimpressed by this 'revolution' and its promises, since it has failed to improve their lives." [ 1 ]
One may ask: "What the heck does it have to do with me? Why should I be aware of it?"


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Ears Monitor Pro 4 (EMP4) In-Ear Style Earphone - Black
Anywhere you listen to music, this will create your own listening room. It cuts off most of outside noises, and recreates the source music faithfully. This product has gone through the aging process to optimize the quality. We wanted to create earphones equal to or better than the expensive models of other companies. We pursued the best quality without compromise. For example, the housing is...
MaxLife Easy Wrap Around Earphones
EarHugger ear phones are great for use while you are active; working, exercising, etc. The molded ear bands keep the ear phones in place so you can enjoy your music. Specifications: *Frequency response: 20-20,000 hz *Sensitivity: 112dB *Impedance: 32 ohm *Drivers: 15 mm
Earhugger Action A-1000 Headphone
Earhuggers stay put while you stay moving. The wrap around the ear design helps keep the earbud in place while you exercise or just do household chores. Comfortable for extended listening sessions. 1.2 meter cord and a limited lifetime warranty.


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Buy Item $6.99 Retail:
Save 42% ($5.00) $11.99
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In StockStock No: WW1195182
P Graham Dunn Inc Engravables / 2012 / Gift
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Contemporary graphic trim in neutral shades; order this gift for family and friends; affordable, personal, and easy to ship.
Personalization is done on the premises at Christianbook.com, and is included in the price of the plaque.
|Format: Gift
Vendor: P Graham Dunn Inc Engravables
|Publication Date: 2012
You can put your own photo on the plaque.


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Chapter 12: Rational Equations and Functions
Up until this point, you've dealt with non-fractional polynomials, but what if you were asked to solve a fractional function with a polynomial in the numerator and the denominator? Such functions are known as rational functions. The graphs of these functions grow closer and closer to certain values without ever reaching those values. This is called asymptotic behavior. In this chapter, you'll find the asymptotes of rational functions. You'll also perform operations on rational expressions and solve rational equations. In the real world, rational equations are often used to model electrical circuit and distance problems.
- 12.1.
Inverse Variation Models
- 12.2.
Graphs of Rational Functions
- 12.3.
Horizontal and Vertical Asymptotes
- 12.4.
Inverse Variation Problems
- 12.5.
Division of Polynomials
- 12.6.
Determining Asymptotes by Division
- 12.7.
Excluded Values for Rational Expressions
- 12.8.
Multiplication of Rational Expressions
- 12.9.
Division of Rational Expressions
- 12.10.
Addition and Subtraction of Rational Expressions
- 12.11.
Applications of Adding and Subtracting Rational Expressions
- 12.12.
Rational Equations Using Proportions
- 12.13.
Applications Using Rational Equations
Chapter Summary
This chapter begins by distinguishing between three variation models: direct variation, inverse variation, and joint variation. It then moves on to graphing and solving rational functions, with particular attention paid to asymptotes. Next, it addresses simplifying rational expressions by factoring and dividing. The four mathematical operations of multiplication, division, addition, and subtraction are then performed on rational expressions. The chapter concludes with real-life applications of rational equations and methods for solving them.


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Not Your Mother's Girl Scouts
Once an innocent, clean place for our daughters to experience life, grow character and learn about life, the girl scout organization has taken a dangerous new twist.
In an appalling effort to indoctrinate the youngest, most vulnerable and impressionable of society, Planned Parenthood has begun using the Girl Scout organization as a tool to promote its blatantly sexualized agenda.
See this from Catholic Family and Human Rights Institute:
Girl Scouts Distribute Planned Parenthood Sex Guide at UN Meeting
The World Association of Girl Scouts and Girl Guides hosted a no-adults-welcome panel at the United Nations this week where Planned Parenthood was allowed to distribute a brochure entitled "Healthy, Happy and Hot." The event was part of the annual United Nations Commission on the Status of Women (CSW) which concludes this week.
The brochure, aimed at young people living with HIV, contains explicit and graphic details on sex, as well as the promotion of casual sex in many forms.
The story goes on from there, and includes just a portion of the explicit details and practices promoted in "Healthy, Happy and Hot", including the admonition to "try everything", "there's nothing wrong" and encouraging the kids not to be afraid to "talk dirty".
Planned Parenthood is a disaster for our young people, and there are many at the United Nations who are also adamant about preaching immorality to young children.
The New York Times recently reported that UN Population Fund is promoting a very controversial curriculum with UNESCO, that includes teaching children as young as five to be sexually active and training adolescents to advocate for abortion.
"The main effort is to try to empower young people with knowledge that could actually save their lives," said Mr. Richmond, the Unesco H.I.V./AIDS coordinator. "We want to give them the opportunity for more informed choices than currently exist."
To empower? What does saving lives from AIDS have to do with promoting promiscuity???? It doesn't! There is no reason to encourage and promote the very promiscuity that results in millions of HIV, AIDS and other sexually transmitted diseases. These behaviors being pushed lead not only to loss of self respect, but have permanent lifelong consequences, in many cases, even death.
"Governments and NGOs should be aware of Planned Parenthood's insidious plan to work with UN agencies and girls' organizations in order to profit from encouraging kids to be sexually active." —Wendy Wright, President of Concerned Women of America
Parents Beware. They are promoting the very thing they claim to seek to educate against. With the kind of information being pushed in this guide, it's NO WONDER it's aimed at young people living with HIV. These kind of promiscuous lifestyles are not Healthy, Happy or Hot. They are Sad, Miserable and Sick. They have no place being promoted to anyone, at any level, much less vulnerable children at the United Nations.
How ironic that this would occur at a Conference on the Status of Women, a group meeting together with the goal of improving life among women. Teaching base promiscuity will only enslave our children to the sexual whims of those who would take advantage of their youth and lack of judgment.
Our daughters are more than the sum of their physical attributes. They are our daughters, they deserve to be taught that they have a right to expect better of themselves and others. They have unique blessings and gifts, including the ability to create life. They are exquisitely beautiful by design, and keepers of the next generation. There is more there than ability. There's a sacred responsibility, to themselves and to the potential they hold.
Government does not have our children's best interests at heart, neither apparently do well intentioned groups like the girl scouts. As parents we must fight against the march of immorality creeping into our society and lives. Teaching our children is our parental right, our responsibility— rights and responsibilities others are all too eager to assume.
—Beetle Blogger


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Elegant Intregrated PA System
The EMX2000 Powered Mixer is complete with a very versatile mixer, high-performance digital reverb and echo, a 7-band graphic equalizer, and high-efficiency 2-channel 200-watt power amplifier. All you need are your sources and a pair of speakers. More ...
Elegant Intregrated PA System
The EMX2000 Powered Mixer is complete with a very versatile mixer, high-performance digital reverb and echo, a 7-band graphic equalizer, and high-efficiency 2-channel 200-watt power amplifier. All you need are your sources and a pair of speakers. More importantly, it is designed to function perfectly as a system: ideal matching between all stages for low-noise, high-quality sound, and features that function flawlessly together for smooth, efficient control.
Mixer Capability: A Fine Line Between Flexibility and Frustration
The EMX2000 mixer offers the kind of signal-routing and patching flexibility you'd expect from a high-quality separate mixing console. In addition to the powered outputs it has independent stereo 1, stereo 2, mono, effect, monitor 1 and monitor 2 outputs to accommodate a wide range of system configurations. Its monitoring and effects send/return systems are also a powerful plus. Little extras like insertion patch points also let you process your sound in ways most powered mixers won't.
Clean, Efficient Power
The EMX2000 power stage puts out a solid 200 watts of exceptionally clean power per channel. The surprising thing is that it does so while drawing relatively little heat. In fact, that's why Yamaha was able to pack such a powerful amplifier into such a compact chassis. All this has been made possible by Yamaha's original EEEngine technology, using an innovative power control system to deliver high power when, and only when, it is really needed.
Built-in Effects, EQ to Polish Your Sound, For Plug-In-And-Play Performance
Built-in digital signal processing system offers a selection of 16 top-quality reverb and echo effects for enhancing your sound with just the right amount of ambiance. Built-in 7-band graphic equalizer is also an indispensable sound-reinforcement tool, offering critical feedback control as well as overall response shaping. Having both of these features built-in means less set-up time, less external wiring for maximum signal quality, and smoothly integrated overall system control.
Clean Signals Accepted
Many mixer circuits must handle low-level signals and must be designed with extra care or they can be a major source of noise and distortion. Other critical design criteria include inter-stage matching, internal impedence, circuit layout, grounding, component selection, and other factors that effect performance and the mixer's overall "sound". Whether you're using microphone or line input, Yamaha's high-performance circuitry gives you an exceptionally clean, quiet signal from input to output.
RF Rejection
RF (Radio Frequency) noise generated by motors, video monitors, and digital equipment of almost any kind can make the cleanest, quietest circuitry virtually worthless. The EMX2000 boasts outstanding RF noise rejection, so you can use it just about anywhere without picking up unwanted interference that can degrade your signal.
Power Mode Switching For Flexible Speaker Setup
You can configure your speaker system in a number of ways to meet a range of main speaker and monitoring requirements. A front-panel power mode selector provides three choices. Feed both power amps from the main stereo buss, driving speakers independently from the left and right channels. Feed one amp from the main stereo buss in mono mode, and one from the monitor buss, separately driving the main and monitor speaker systems. Or, feed both power amps from the main stereo buss in bridged mono.
Rugged and Reliable
From connectors to controls to chassis, the EMX2000 is built to last. Sound reinforcement equipment is generally destined to be bumped, dropped, and otherwise abused, so extra care was taken to ensure that the EMX2000 will stand up to even the roughest conditions on the road. Your sound depends on it.
Specs You Can Trust
Yamaha has never "tweaked" specifications. Never will. It's amazing how mediocre specs can be made to look impressive by simply optimizing test conditions. The electrical specifications we publish are brutally honest, measured under the stated and/or industry-standard conditions. When in doubt, we urge you to use the most sensitive sound measurement devices available; your own ears. They'll tell you who you can trust.
Channel Insertion
Insert send/return patch points are included on channels 1 through 4 for convenient insertion of compressor/limiters (a must for top-quality vocal sound), equalizers, or any other outboard equipment you might need to apply to individual channels.
Inputs and Level Matching
The EMX2000 has 8 mono input channels with both balanced XLR-type microphone inputs and balanced/unbalanced TRS phone jack line inputs. Switchable phantom power for all balanced inputs provides direct compatibility with high-performance phantom-powered condenser microphones and DI boxes.
Stereo Input
Stereo line sources can be directly connected to either of 2 stereo input channels (9/10 and 11/12) with unbalanced L/MONO and R phone jack inputs as well as RCA pin jacks. A/B input selectors switch between the phone and pin jack inputs. The L/MONO phone jack inputs allow the stereo channels to be used for mono signals.
Gain Trim Controls
Gain trim controls with a 44-dB range and LED peak indicators on all mono and stereo inputs facilitate optimum level matching with a wide range of sources.
3-Band Channel EQ
3-band equalization on each input channel can be used to bring out the best in the sound of individual instruments or voices. emphasize a channel in relation to others, blend the sound of several channels, create special effects, reduce noice, and much more.
Monitor and Effects Send
Two independent monitor sends and an effect send control on each channel provide considerable signal-routing flexibility. The effect send output can feed external effect processors as well as the EMX2000's internal digital signal processor system.
Pre-Fader Listen
All of the EMX2000 inputs - mono and stereo - feature PFL switches for isolated pre-fader monitoring of the corresponding signals.
Linear Channel Faders with Pan/Balance Controls
Smooth, noise-free linear faders make it easy to set up the optimism balance between channels, with pan controls on the mono input channels and balance controls on the stereo channels.
Stereo 1/2 and Mono Outputs
In addition to the powered outputs controlled by the stereo 1 fader, the EMX2000 has separate line-level stereo 1 outputs also fed by the stereo 1 fader, and stereo 2 outputs fed by a separate stereo 2 level control. An independent mono fader feeds the line-level mono output with the combined signal from the stereo buss.
Handy Rec Outputs and Tape Inputs
Rec outputs provide a line-level stereo output for convenient recording of the main program, while tape inputs with level control and PFL switch allow the output from a recorder or other line-level source to be added to the console's stereo signal.
Power Amp Inputs
Direct power amp inputs allow any external line-level signal to be directly applied to inputs of the EMX2000 power amplifier, prior to the power amp input level controls.
Power Amp Input Level Controls
These level controls independently adjust the left- and right-channel power amp input levels from +20 to +4 dB.
Convenient Monitoring and Metering
Convenient monitoring is provided by a control-room monitor and phones output jack with an independent level control. 12-segment peak-reading level meters provide accurate visual monitoring of the signal appearing at the stereo outputs.
Stereo Sub Inputs and Level Controls
Dual stereo "sub" inputs with independent level controls for the monitor 1, monitor 2, and stereo busses can be used as effect returns, or to add just about any line-level signal to the corresponding busses. Both inputs also feature PFL switches.
Built-In Digital Signal Processor (DSP)
For fuss-free convenience,Yamaha's professional DSP technology is packed into the EMX2000. Internal DSP provides a choice of 16 high-quality reverb and echo effects. No outboard signal processing equipment is required for most applications. Independent effect return level controls are provided for monitor 1, monitor 2, and stereo busses. Optimum amount of effect can be applied to each signal. A footswitch connected to the FOOT SW jack can be used to turn the internal effect system on or off.
Power Mode Selector
A front-panel power mode selector provides three choices. ST L/ST R Mode: Feed both power amps from the main stereo buss, driving speakers independently from the left and right channels. MONO/MONI Mode: Feed one amp from the main stereo buss in mono mode, and one from the monitor buss, separately driving the main and monitor speaker systems. MODO BRIDGE Mode: Feed both power amps from the main stereo buss in bridged mono configuration to deliver 400 watts to a single speaker system.
7-Band Graphic Equalizer
A feature that will be particularly appreciated in sound-reforcement applications is 7-band graphic equalization on the stereo outputs. Not only does this allow detailed overall response shaping, but it also makes it easy to identify and cut feedback frequencies for effective feedback control.
Master Monitor/Effect Send Faders
The master control section includes master monitor 1, monitor 2, and effect send faders feeding line-level phone plug outputs which can be used to drive external power amplifiers or effect units.
Audio & Video
|Maximum Output Power
|200W/4 ohm stereo @0.5% THD at 1Hz (400W BTL/MONO)
|Frequency Response
|20Hz~20kHz +1dB, -3dB @1W output into 8 ohm (SPEAKER OUT); 20Hz~20kHz +1dB, -3dB @+4dB output into 600 ohm (ST OUT, MONO OUT, EFFECT SEND, MONITOR OUT)
|Total Harmonic Distortion
|Less than 0.5% @20Hz~20kHz, 100W output into 4 ohm (SPEAKER OUT); Less than 0.2% @20Hz~20kHz, +14dB output into 600 ohm (ST OUT, MONO OUT, EFFECT SEND, MONITOR OUT)
|Hum and Noise
|[Average, Rs=150; with 20Hz~20kHz BPF]; -128dB equivalent input noise, -65dB residual output noise (SPEAKER OUT); -95dB residual output noise (ST OUT, MONO OUT, EFFECT SEND, MONITOR OUT)
|Crosstalk at 1kHz
|70dB adjacent input, 70dB input to output
|Input Channel Equalization
|HIGH: 10kHz shelving/MID:2.5Hz peaking/LOW:100Hz shelving
|Comp.:THD greather than or equal to 0.5%
|Graphic Equalizer
|7 bands (125, 250, 500, 1k, 2k, 4k, 8kHz), + and or - 12 dB Maximum
|Internal Digital Effect
|16 programs
|Foot Switch
|DIGITAL EFFECT MUTE: on/off
|Phantom Power
|+48V is supplied to electrically balanced inputs
|Power Requirements
|120V AC 60Hz
|Dimensions (W x H x D)
|480 x 107 x 442 mm (18-7/8" x 4-3/16" x 17-3/8")
|14kg (30.86 lbs.)


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Got word this morning that one of my pups from a litter 3 years ago run his first obedience trial Saturday and scored a perfect 200! Pups from this litter are all over the venues with one in Field Trail training, one running hunt tests(HRCH, SH, MH pass), one training for agility, two hunting homes and one being someones best friend! Grinning from ear to ear!!
FC Candlewood's Meet Joe Black X Bustin' Waters Amazing Grace SH


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Serve homemade soup in a flash thanks to canned beans and corn and ready-to-go salsa.
Store a bunch of fresh cilantro, stems down, in a glass of water and cover with a plastic bag. It will keep up to a week if you change the water every 2 or 3 days. When you're ready to use the cilantro, wash it, dry with paper towels, then chop.
Chopped fresh parsley can be substituted for the cilantro.
© 2017 ®/TM General Mills All Rights Reserved


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JavaScript Cookie Check
navigator.cookieEnabled flag works correctly in Chrome, Firefox, Opera and Safari but Internet Explorer is simply lying to us! It supports this flag (It's not
null or
undefined) but it is always set to
true! So we still need hacks for this browser. It's also strange that I wasn't able to find a working hack on the Internet. So I wrote my own:
/** * Checks if browser supports cookies. * * @return {boolean} * True if browser supports cookies, false if not. */ function supportsCookies() { var cookieEnabled = navigator.cookieEnabled; // When cookieEnabled flag is present and false then cookies are disabled. // Thanks to IE we can't trust the value "true". if (cookieEnabled === false) return false; // Internet Explorer is lying to us. So we have to set a test cookie // in this browser (We also do it for strange browsers not supporting // the cookieEnabled flag). But we only do this when no cookies are // already set (because that would mean cookies are enabled) if (!document.cookie && (cookieEnabled == null || /*@cc_on!@*/false)) { // Try to set a test cookie. If not set then cookies are disabled document.cookie = "testcookie=1"; if (!document.cookie) return false; // Remove test cookie document.cookie = "testcookie=; expires=" + new Date(0).toUTCString(); } // Well, at least we couldn't find out if cookies are disabled, so we // assume they are enabled return true; };
Let's explain what this code is doing:
- When the
navigator.cookiesEnabledflag is present and is
falsethen we definitely know that cookies are disabled. All major browsers except Internet Explorer will stop right here when cookies are blocked.
- When
navigator.cookiesEnabledflag is not present (maybe some really old browser) or when Internet Explorer is deteced (Checked with JScript conditional compilation) then a test cookie is set. But we only do this when there are no cookies already set (Because when there is already a cookie then cookies can't be disabled). When our test cookie can be read again then cookies are enabled, otherwise they are disabled.
The code works in IE7-10, Firefox 17, Chrome 23, Opera 12 and Safari 6.
What a shame that code like this is still needed in 2012...