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- © 2008 American Society of Plant Biologists
The signal transduction of the phytohormone cytokinin is mediated by a multistep histidine-to-aspartate phosphorelay system. One component of this system are B-type response regulators, transcription factors mediating at least part of the response to cytokinin. In planta functional analysis of this family is hampered by the high level of functional redundancy of its 11 members. We generated a dominant repressor version of the Arabidopsis (Arabidopsis thaliana) response regulator ARR1 (ARR1-SRDX) using chimeric repressor silencing technology in order to study the extent of the contribution of B-type response regulators to cytokinin activities. In a protoplast test system, ARR1-SRDX suppressed ARR6:β-glucuronidase reporter gene activation by different B-type ARRs. 35S:ARR1-SRDX transgenic Arabidopsis plants showed phenotypic changes reminiscent of plants with a reduced cytokinin status, such as a strongly reduced leaf size, an enhanced root system, and larger seeds. Several bioassays showed that 35S:ARR1-SRDX plants have an increased resistance toward cytokinin. The rapid induction of a large part of the cytokinin response genes was dampened. The transcript levels of more than 500 genes were more than 2.5-fold reduced in 35S:ARR1-SRDX transgenic seedlings, suggesting a broad function of B-type ARRs. Collectively, the suppression of pleiotropic cytokinin activities by a dominant repressor version of a B-type ARR indicates that this protein family is involved in mediating most, if not all, of the cytokinin activities in Arabidopsis. In addition, a role for B-type ARRs in mediating cross talk with other pathways is supported by the resistance of 35S:ARR1-SRDX seeds to phytochrome B-mediated inhibition of germination by far-red light. This study demonstrates the usefulness of chimeric repressor silencing technology to overcome redundancy in transcription factor families for functional studies.
The plant hormone cytokinin is involved in many developmental processes and plays a critical role in numerous physiological responses to changes in the environment (Mok and Mok, 2001). In recent years, significant progress has been made toward the understanding of how the cytokinin signal is perceived and transduced (Heyl and Schmülling, 2003; Kakimoto, 2003; Mizuno, 2004; Heyl et al., 2006; Hwang and Sakakibara, 2006; Müller and Sheen, 2007). In the current model, which has been developed mainly in Arabidopsis (Arabidopsis thaliana), the hormone is perceived by membrane-bound hybrid His-kinase receptors, which autophosphorylate upon binding of the hormone ligand. After transphosphorylation within the receptor, the phosphate group is transferred to His phosphotransfer proteins, which subsequently locate to the nucleus, where they activate B-type response regulators (ARRs) via phosphorylation. These transcription factors regulate the transcription of their target genes, one group of which codes for A-type response regulators. A negative feedback on the cytokinin signaling pathway was shown to be mediated by members of this protein class (Hwang and Sheen, 2001; To et al., 2004).
Arabidopsis encodes 11 B-type ARRs. They possess, in addition to the response regulator domain, a MYB-class DNA-binding domain (Riechmann et al., 2000). The DNA-binding domains share a high degree of sequence conservation, in particular in the nine amino acids that were identified in ARR10 to be most likely in direct contact with the DNA (Hosoda et al., 2002). Several B-type ARRs have been shown to bind to the same or very similar sequence motifs (Sakai et al., 2000; Hosoda et al., 2002; Imamura et al., 2003). B-type ARRs regulate the transcription of their target genes in response to cytokinin treatment (Sakai et al., 2000; Hwang and Sheen, 2001; Lohrmann et al., 2001; Imamura et al., 2003). Reverse transcription-PCR, microarray, and promoter-GUS fusion experiments have demonstrated that the members of the B-type ARR family have large and overlapping expression domains (Mason et al., 2004; Tajima et al., 2004; Zimmermann et al., 2004; summarized by Heyl et al., 2006). The analysis of B-type ARR mutants has revealed their involvement in cytokinin signaling but also a high degree of functional redundancy (Sakai et al., 2001; Horák et al., 2003; Hass et al., 2004; Mason et al., 2005). Loss-of-function mutants of single B-type ARR genes (ARR1, ARR2, ARR10, ARR11, and ARR18) caused no strong phenotypic alterations. However, different cytokinin response assays showed increasing cytokinin resistance for higher order mutants (Mason et al., 2005; Yokoyama et al., 2007). Surprisingly, besides quantitative changes of the root system and a defect of vascular development in the arr1 arr10 arr12 triple mutant (Mason et al., 2005; Yokoyama et al., 2007), no strong morphological alterations were described. This indicates that the degree of redundancy among B-type ARRs is very high and/or that other transcriptional regulators compensate for the loss of B-type ARRs, such as the cytokinin response factors of the ethylene response factor (ERF) transcription factor family (Rashotte et al., 2006).
In order to explore the functions of B-type response regulators, we took advantage of the chimeric repressor silencing technology (CRES-T; Hiratsu et al., 2003). This technology has been developed to study the consequences of silencing of the target genes of single transcription factors and has also been used to overcome the experimental limitations caused by functional redundancy of transcription factor families. In 2001, Ohta and colleagues mapped a repression motif of transcriptional repressors of the class II ERFs (Ohta et al., 2001). This ERF-associated amphiphilic repression motif or variations of it are found in numerous plant transcriptional repressors (Ohta et al., 2001; Tiwari et al., 2004; Kazan, 2006). Further improvement resulted in the SRDX motif (Hiratsu et al., 2003). Fusion of this motif to transcriptional activators converts them into dominant repressors, even in the presence of the original activator domain (Hiratsu et al., 2003). Interestingly, these dominant repressors may repress not only the transcription of their own target genes but also the expression of target genes of other members of their respective gene families and thus overcome functional redundancy (Hiratsu et al., 2003; Mitsuda et al., 2007).
In this study, the SRDX motif was fused to the C terminal end of ARR1 and expressed under the control of a 35S promoter in wild-type Arabidopsis plants. The resulting transgenic plants showed a cytokinin deficiency phenotype, including reduced shoot and enhanced root growth, altered reproductive organs, and increased resistance to cytokinin. Molecular analysis revealed the attenuation of early transcriptional responses to cytokinin. The implications for the function of this class of transcription factors are discussed.
ARR1-SRDX Suppresses the Cytokinin-Dependent Induction of a Cytokinin Primary Response Gene in Planta
A dominant repressor for the B-type ARRs was generated by introducing a 36-bp DNA sequence (5′-CTGGATCTGGATCTGGAACTGCGCCTGGGCTTTGCG-3′) encoding the SRDX domain (LDLDLELRLGFA; Hiratsu et al., 2003) replacing the last 20 nucleotides 5′ of the ARR1 stop codon (Supplemental Fig. S1). ARR1 was chosen as it is the best characterized B-type response regulator in Arabidopsis (Sakai et al., 2000, 2001, Mason et al., 2004, 2005; Taniguchi et al., 2007). The in planta effect of ARR1-SRDX was first examined in a protoplast transactivation assay (Ehlert et al., 2006) using a 1,000-bp promoter fragment upstream of the translational start of ARR6, a primary cytokinin response gene (Hwang and Sheen, 2001), fused to the GUS reporter gene. The addition of cytokinin resulted in a more than 3-fold induction of the reporter gene expression compared with the noninduced condition (Fig. 1A ). Cotransfection with ARR1 under the control of a 35S promoter led to a strong increase in GUS activity even in the absence of cytokinin. The addition of cytokinin caused a further 2-fold increase of the GUS activity, indicating that ARR1 mediates the cytokinin response in this assay. In contrast, the expression of ARR1-SRDX under the control of a 35S promoter effectively suppressed the cytokinin induction of GUS expression. Furthermore, ARR1-SRDX completely abolished the ARR1-caused expression of the reporter gene in the absence and presence of cytokinin (Fig. 1A). The expression of the GUS reporter gene under the control of a 35S promoter was not cytokinin inducible. In the next step, we tested in the same assay system whether other B-type ARRs activate the transcription of the ARR6:GUS reporter and whether this activation could be suppressed by ARR1-SRDX. For these experiments, ARR2 was chosen as this is the B-type ARR most closely related to ARR1. ARR10 and ARR12 were tested as well, as both have been shown to be at least partly functionally redundant to ARR1 (Yokoyama et al., 2007; Ishida et al., 2008). Furthermore, ARR19 and ARR21 were included in the analysis as representatives of the two pairs of more distantly related B-type ARRs. All of these B-type ARRs increased ARR6:GUS expression without cytokinin to a different extent (Fig. 1B). The addition of cytokinin in most cases increased the reporter gene activity further. The coexpression of ARR1-SRDX effectively repressed reporter gene activation by all of the B-type ARRs in the absence and presence of cytokinin (Fig. 1B). These results clearly demonstrate the potential of ARR1-SRDX as a dominant repressor of the primary transcriptional response to cytokinin in planta.
Transgenic Arabidopsis Plants Expressing a Dominant ARR1-SRDX Repressor
Next, we established transgenic lines by transforming the 35S:ARR1-SRDX gene into wild-type Columbia (Col-0) plants. Because B-type ARR expression was detected in virtually all tissues investigated (Heyl et al., 2006) and we aimed to study the effects of the repression of as many B-type ARRs as possible (not just ARR1 in the ARR1 expression domain), we purposefully chose the 35S promoter to drive gene expression. The 35S promoter has been previously used successfully in combination with CRES-T (Koyama et al., 2007). More than 10 independent transgenic lines with a similar phenotype, but distinct from the wild type, were recovered (Supplemental Fig. S2). Two of those lines, ARR1-S-8 and ARR1-S-10, which showed a characteristic phenotype with different expressivity, were characterized in more detail. Experiments were carried out with homozygote and phenotypically uniform progeny. Northern-blot analysis using a SRDX-specific probe showed the expression of the ARR1-SRDX transcript in both lines (Fig. 2A ). The steady-state transcript level was higher in ARR1-S-8 compared with ARR1-S-10 (Fig. 2A). In order to check whether the transcript level of the ARR1 gene itself was altered in the transgenic lines, we compared its level with the wild type using a probe that specifically recognizes the wild-type ARR1 but not ARR1-SRDX transcripts. This analysis revealed an increased ARR1 transcript level in both 35S:ARR1-SRDX transgenic lines, indicating a feedback regulation of ARR1 (Fig. 2B). This increase in transcript level was specific for ARR1 and did not affect four other B-type ARR genes, which were detectable in our microarray experiments (see below) and showed similar transcript levels in the transgenic lines and the wild type.
35S:ARR1-SRDX Transgenic Plants Display a Pleiotropic Shoot Phenotype
Transgenic plants expressing the 35S:ARR1-SRDX construct displayed a strong pleiotropic shoot phenotype, with a higher expressivity in line ARR1-S-8 than in line ARR1-S-10 (Fig. 3A ). Plants were generally smaller, and after flowering numerous individuals showed to a varying degree enhanced branching of the shoot. The leaves of the dominant repressors were strongly reduced in both size and number compared with wild-type plants. In the stronger expressing line ARR1-S-8, the true leaves were only about the size of the cotyledons in the wild type (Fig. 3B). Twenty days after germination (DAG), the wild-type plant had on average nine rosette leaves, while the ARR1-SRDX-expressing plants had only seven leaves at that time point (Fig. 3C).
35S:ARR1-SRDX Plants Develop an Enlarged Root System
In contrast to the reduced shoot size, 35S:ARR1-SRDX transgenic plants showed a generally enhanced root system compared with the wild type (Fig. 4A ). While the ARR1-S-8 plants had only a slightly longer primary root than the wild type at 10 DAG, the primary root of line ARR1-S-10 was more than 30% longer (34.9 ± 3.1 mm in the wild type compared with 51.9 ± 6.6 mm in line ARR1-S-10; Fig. 4B). The difference between the transgenic lines and the wild type was also marked in the number of lateral roots. At 10 DAG, plants of line ARR1-S-8 had developed about twice as many lateral roots as the wild type and line ARR1-S-10 had about three times more (13.2 ± 1.9 lateral roots compared with 4.8 ± 1.1 lateral roots in the wild type; Fig. 4C).
Reproductive Development of 35S:ARR1-SRDX Plants Is Altered
The 35S:ARR1-SRDX transgenic plants flowered earlier than wild-type plants (Fig. 5A ). In the wild type, the first flower buds opened at 19 DAG. In contrast, both transgenic lines flowered at around 14 DAG (Fig. 5B). In the 35S:ARR1-SRDX transgenic plants, all reproductive organs were reduced in size. This was more pronounced in line ARR1-S-8, in which the flowers were only half the size of the wild-type flowers (Fig. 5C). The smaller flowers of the 35S:ARR1-SRDX plants gave rise to smaller siliques. The length of the siliques of line ARR1-S-8 was about 30% of the length of the wild-type siliques. The shape differed also from the wild type, as it was twisted and crooked (Fig. 5D). The phenotype of line ARR1-S-10 was weaker, as the reduction of the silique size was not as dramatic as in line ARR1-S-8, and the shape resembled more that of the wild type (Fig. 5D). While the number of seeds obtained by selfing was considerably lower in the 35S:ARR1-SRDX plants (data not shown), the seeds themselves showed increased size (Fig. 5E). Their weight was 40% and 50% higher in the lines ARR1-S-8 and ARR1-S-10, respectively (Fig. 5F).
The Cytokinin Response of 35S:ARR1-SRDX Plants Is Altered in Shoots and Roots
As the phenotype of the 35S:ARR1-SRDX plants is reminiscent of plants with reduced cytokinin signaling (Higuchi et al., 2004; Nishimura et al., 2004; Riefler et al., 2006), we carried out several cytokinin sensitivity assays.
Cytokinin is known to delay the onset of leaf senescence and to increase the chlorophyll retention in detached leaves incubated in the dark (Richmond and Lang, 1957; Riefler et al., 2006). Detached wild-type leaves kept in the dark for 10 d lost more than 80% of their chlorophyll compared with fresh leaves (Fig. 6A ). The addition of increasing amounts of cytokinin reversed this effect. In wild-type leaves, 0.1 μm 6-benzyladenine (BA) strongly increased chlorophyll retention, and at 5 μm BA the chlorophyll level of dark-incubated detached leaves was similar to that in fresh leaves. In contrast, while in both 35S:ARR1-SRDX lines the loss of chlorophyll under dark conditions was similar to that in the wild type, the addition of cytokinin to the medium caused only a minor increase in chlorophyll retention, even at the highest concentration (Fig. 6A). This indicates that the cytokinin sensitivity in the leaves had been almost completely lost.
To investigate the cytokinin response in roots, seedlings of the wild type and the 35S:ARR1-SRDX lines were grown on medium containing increasing amounts of cytokinin. On control medium without cytokinin, the transgenic seedlings developed a longer primary root compared with the wild type (Fig. 4A). The relative difference in root length increased with increasing cytokinin concentrations (Fig. 6B). Seedlings of line ARR1-S-8 displayed a higher resistance to cytokinin than line ARR1-S-10. A 50% inhibition of root elongation was achieved by an approximately 5-fold higher cytokinin concentration. However, the sensitivity to cytokinin, while being clearly reduced, was not completely eliminated in either ARR1-SRDX line (Fig. 6B). Subsequently, we tested whether lateral root formation was altered in the presence of the hormone. In all seedlings, the addition of cytokinin to the medium led to a dramatic decrease in the number of lateral roots (Fig. 6C). But whereas the lateral root formation of the wild type was almost totally repressed at 0.01 μm BA, lateral root formation of both 35S:ARR1-SRDX lines was only halved at this cytokinin concentration. At 0.1 μm BA, the wild type was unable to produce lateral roots, while the dominant repressor lines still showed a significant number (Fig. 6C).
35S:ARR1-SRDX Plants Show a Reduced Induction of Cytokinin Response Genes
The phenotypic changes and cytokinin bioassays described above report altered long-term responses to the hormone. To analyze whether rapid cytokinin responses were also altered in the 35S:ARR1-SRDX transgenic lines, the expression of two known primary cytokinin response genes was tested. The transcript levels of ARR5 and ARR7, which both encode A-type ARRs, are direct targets of the B-type ARRs and rapidly induced by cytokinin (D'Agostino et al., 2000; Romanov et al., 2002; To et al., 2004). Both genes were rapidly and strongly induced in wild-type seedlings following the application of cytokinin. This induction was strongly diminished in both transgenic lines (Fig. 7A ).
Transcriptome Analysis Reveals Complex Changes in 35S:ARR1-SRDX Plants
Next, we investigated the changes occurring at 15 and 120 min following cytokinin treatment of 5-d-old seedlings in the transcriptome of the wild type and line ARR1-S-8 using the CATMA V2.4 array. Seedlings of both genotypes show only minor morphological differences at this stage. In all experiments, we regarded genes in which the expression was changed by ≥2.5-fold, the false discovery rate (FDR)-corrected P value (q value) was ≤0.05, and that had 50% or more spots above background in the higher expressed condition as significantly regulated. In total, 658 genes were found with a lower steady-state mRNA level and 505 genes were found with a higher steady-state mRNA level in seedlings of line ARR1-S-8 compared with the wild type (Table I ; Supplemental Table S1).
First we analyzed whether the expression of known ARR1 target genes was altered. Eleven of the 23 ARR1 target genes described by Taniguchi et al. (2007) were also on our array and could be detected. All of these were also induced in the wild type under our conditions, 10 showed a diminished and/or delayed induction in the 35S:ARR1-SRDX plants, and seven showed a lower basal expression level in the 35S:ARR1-SRDX plants compared with wild-type plants (Supplemental Table S2). Together, this shows a significant repressive influence of ARR1-SRDX at least on ARR1 target gene expression. Target genes of other B-type ARRs could not be analyzed as they are mostly not known.
We then studied broader cytokinin-induced changes in the transcript abundance, which should reveal whether there is an overall dampening of the cytokinin response or whether specific changes could be observed. In 35S:ARR1-SRDX transgenic seedlings, the total number of genes up-regulated by cytokinin treatment was about half the number observed in the wild type (Supplemental Table S1). The induction of the 10 strongest cytokinin-regulated genes in the wild type was reduced in line ARR1-S-8 to 6% and 30% after 15 and 120 min, respectively (Supplemental Table S1). Comparison of other subsets of up-regulated genes between the wild type and 35S:ARR1-SRDX transgenic seedlings yielded similar results. For example, the response to cytokinin of the majority of the seven A-type ARR genes that are represented in the CATMA V2.4 array was dampened and/or delayed (Fig. 7B), which is consistent with the result of the northern-blot analysis (Fig. 7A). However, the response curves show individual differences. For example, for ARR4 and ARR16, the early increase of transcript abundance seen in the wild type is completely missing in the 35S:ARR1-SRDX transgenic seedlings. Other genes, like ARR6 and ARR7, show a weaker response at the early time point (Fig. 7B). In total, 73% of the genes induced by cytokinin in the wild type were not induced in line ARR1-S-8.
We then checked whether genes that are down-regulated by cytokinin would show enhanced repression in 35S:ARR1-SRDX seedlings. Analysis of a set of 56 early or late cytokinin down-regulated genes (Brenner et al., 2005) revealed only five examples that show a stronger down-regulation following cytokinin treatment in the 35S:ARR1-SRDX seedlings compared with the wild type. In addition, this concerned only genes that were down-regulated at 2 h after cytokinin treatment but none of the genes that were rapidly (15 min) down-regulated. This result argues against the possibility that ARR1-SRDX enhances a putative repressor function of ARR1.
Analysis of the classes of genes and individual genes that are constitutively down-regulated in 35S:ARR1-SRDX plants should yield indications about the direct or indirect regulatory functions of B-type ARRs. There are numerous genes in line ARR1-S-8 that showed only one-tenth or even less of their wild-type transcript level, indicating that they are directly or indirectly regulated by B-type ARRs. Prominent examples are the nitrate reductase gene NR2 and the ammonium transporter gene AMT2, the nitrate transporter gene NTL1, two sugar transporter genes (STP1 and STP4), and the genes encoding the repressors of GA responses, RGA1 and RGA2 (Table I; Supplemental Table S1).
Among the individual genes that were very strongly up-regulated in ARR1-S-8 is a gene encoding an ELIP (for early light-induced protein) of the chlorophyll a/b-binding protein family and the cyclin-dependent kinase inhibitor gene ICK2. Among the cytokinin-related genes, IPT5 was 2.5-fold up-regulated (Table I; Supplemental Table S1).
The Ethylene Response of 35S:ARR1-SRDX Seedlings
The large number of differentially expressed genes in the 35S:ARR1-SRDX transgenic seedlings indicates the possibility that this family of transcription factors might not have a function solely in cytokinin signaling but also in other pathways. An involvement of the B-type ARR2 in ethylene signaling has been discussed (Hass et al., 2004; Mason et al., 2005). To test the possible involvement of B-type ARRs in mediating the ethylene response, the growth response of 35S:ARR1-SRDX seedlings on medium containing the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) was compared with that in wild-type seedlings. The hypocotyl lengths of wild-type and 35S:ARR1-SRDX seedlings decreased with increasing concentrations of ACC in the medium (Fig. 8A ). In contrast to the wild type, in which hypocotyl length decreased more with further increasing ACC concentrations, the 35S:ARR1-SRDX seedlings showed resistance to ACC concentrations greater than 1 μm, as their hypocotyl length did not decrease further (Fig. 8A). However, root shortening of wild-type and 35S:ARR1-SRDX seedlings was similar at low and high ethylene concentrations (Fig. 8B). Also, the bending of the apical hook did not differ between the genotypes (data not shown).
Germination of 35S:ARR1-SRDX Seeds Is Resistant to Inhibition by Far-Red Light
Several experiments have indicated an involvement of the two-component signaling system in red light signaling (Sweere et al., 2001; To et al., 2004; Riefler et al., 2006, Mira-Rodado et al., 2007). We used a seed germination assay to explore the possible role of B-type ARRs in response to red light. Far-red light inhibits seed germination, a response that is, under our experimental conditions, mediated by phytochrome B and reversible by red light (Shinomura, 1997; Sullivan and Deng, 2003). The germination behavior of 35S:ARR1-SRDX transgenic seeds and wild-type seeds in white light was similar. At 48 and 60 h after transfer from cold treatment to room temperature, a similar percentage of the seeds of all genotypes had germinated and eventually reached about 100%, indicating that there was no marked difference in the timing or capability of germination (Fig. 9A ). Similarly, about 100% of the seeds germinated after a white light pulse or a red light pulse of 5 min followed by a dark period of 7 d (Fig. 9B). However, when wild-type seeds were exposed to a 5-min far-red light pulse prior to dark incubation, only around 10% of the seeds germinated (Fig. 9B). In contrast, 35% and 60% of the seeds of lines ARR1-S-8 and ARR1-S-10, respectively, germinated following this treatment. A similar difference between the wild type and the transgenic lines was seen when the far-red light treatment was preceded by a 5-min red light pulse. Then, the germination rate of the transgenic lines reached about 80%, while it remained around 10% for wild-type seeds (Fig. 9B).
Application of the CRES-T Results in a Dominant Negative Effect and Thus Allows the Investigation of B-Type ARR Functions
Previous studies have shown the involvement of several members of the B-type ARRs in mediating cytokinin signaling (Imamura et al., 2003; Tajima et al., 2004; Mason et al., 2005; Yokoyama et al., 2007). However, because of a high degree of functional redundancy, the extent of their contribution was unclear. Here, we have shown that a dominant repressor form of a B-type ARR, ARR1-SRDX, causes a strong cytokinin deficiency phenotype, which is most likely due to the concerted repression of the activities of ARR1 and related transcription factors. This suggests that the activity of B-type ARRs is involved in most, if not all, of the cytokinin-regulated processes.
The results of the protoplast transactivation assay clearly demonstrate the dominant negative effect of the ARR1-SRDX protein on ARR1-dependent activation of a reporter gene as well as its induction by cytokinin (Fig. 1A). This dominant negative activity also suppressed the activity of all other B-type ARRs tested (Fig. 1B), further validating the chosen approach. The transcript level of the resident ARR1 gene was even enhanced in the 35S:ARR1-SRDX transgenic plants, indicating that the observed changes of gene expression and plant morphology were not due to cosuppression. The phenotypic changes described here were not observed in 35S:ARR1-overexpressing plants (Sakai et al., 2001; our unpublished data), making it unlikely that nonspecific negative effects such as transcriptional squelching (Kornberg, 2005) contribute to the phenotype. Importantly, the phenotypic changes occur in tissues expressing B-type ARR genes. Binding sites of several B-type ARRs are similar but not identical (Imamura et al., 2003). It is likely, therefore, that ARR1-SRDX suppresses the activity of most if not all B-type ARRs, which is supported by the results of the protoplast transactivation assay (Fig. 1B). However, we cannot completely exclude that part of the changes in phenotype and gene regulations caused by ARR1-SRDX are off-target effects due to interference with other related transcription factors. Candidates are the MYB domain-containing pseudo response regulators APRR2 and APRR4, which show in their DNA binding domains similarity to B-type ARRs, but nothing is known about the function of these APRRs (Makino et al., 2000). Recent experiments have indicated that the DNA motif necessary for ARR1 binding is actually longer than previously reported (Taniguchi et al., 2007), making it less likely that the effect of the ARR1-SRDX dominant repressor spreads far beyond the B-type ARRs. One also has to be aware that individual B-type ARRs are likely to have different functions and that some of the phenotypes seen in the 35S:ARR1-SRDX transgenic plants might be due to the repression of specific ARRs or combinations thereof and do not indicate functions for all members of this protein family. However, taken together, the phenotypic characteristics of the 35S:ARR1-SRDX plants clearly demonstrate that the CRES-T is useful to study larger transcription factor families and can be applied to study other regulatory pathways involving transcriptional regulators hampered by redundancy (Koyama et al., 2007).
The Morphological Phenotype of 35S:ARR1-SRDX Plants Is Consistent with a Broad Function of B-Type ARRs in Cytokinin Signaling
The phenotype of the 35S:ARR1-SRDX transgenic plants resembled in various aspects the phenotype of cytokinin receptor triple mutants and cytokinin-deficient plants (Werner et al., 2003; Yang et al., 2003; Higuchi et al., 2004; Nishimura et al., 2004; Riefler et al., 2006; Supplemental Fig. S3). As in those mutants, the shoot growth and in particular leaf size were strongly reduced, while seed size and the root system were enlarged. An exception to the similarities between 35S:ARR1-SRDX plants and plants with a reduced cytokinin status is the early-flowering phenotype, which contrasts with the retarded flowering reported for triple receptor mutants and cytokinin-deficient plants (Werner et al., 2003; Higuchi et al., 2004; Nishimura et al., 2004; Riefler et al., 2006). Earlier flowering in 35S:ARR1-SRDX plants was developmentally regulated, as fewer leaves had formed at the onset of flowering. This indicates that this function may be cytokinin-independent, as cytokinin deficiency causes only minor changes in leaf number at the onset of flower formation (Werner et al., 2003). A cytokinin-independent role in flowering has been suggested for the rice B-type response regulator gene Edh1 (Doi et al., 2004). Interestingly, arr2 mutant plants also flower earlier than wild-type plants, indicating a negative regulatory function of ARR2 in the induction of flowering (Hass et al., 2004). It could be that the early-flowering phenotype of the 35S:ARR1-SRDX plants is at least partially due to the suppression of ARR2 function, which was demonstrated in the protoplast transactivation assay (Fig. 1B).
35S:ARR1-SRDX Plants Show Specific Changes of Cytokinin Sensitivity
A decreased cytokinin sensitivity of the 35S:ARR1-SRDX transgenic plants was detected in several bioassays. 35S:ARR1-SRDX plants showed complete resistance to cytokinin in the chlorophyll retention assay, which is similar to the phenotype of ahk2 ahk3 receptor mutants (Riefler et al., 2006). This indicates that the cytokinin activities in regulating chlorophyll retention are entirely dependent on B-type ARRs. In particular, ARR2, the closest homolog of ARR1, has been shown to play a role in mediating cytokinin-dependent chlorophyll retention (Kim et al., 2006) and thus may be suppressed in leaves of 35S:ARR1-SRDX plants.
The reduction of cytokinin sensitivity was less pronounced in roots of 35S:ARR1-SRDX plants. One possibility to explain this distinction is that different sets of B-type ARRs act in shoots and roots and that the B-type ARRs in the roots are less affected by the dominant repressor activity of ARR1-SRDX. Alternatively, the affected B-type ARRs could play antagonistic roles in the root. The latter possibility is supported by the observation that the inclusion of an arr2 knockout allele in arr mutant combinations causes a reduction in the strength of the root phenotype, indicating that ARR2 might have an antagonistic function to the other B-type ARRs in regulating root elongation and root branching (Mason et al., 2005). Therefore, reduced activity of ARR2 in the dominant repression plants could explain the somewhat weaker root phenotype in the 35S:ARR1-SRDX transgenic plants.
Early Cytokinin Responses Are Attenuated in 35S:ARR1-SRDX Seedlings
The expression and inducibility of A-type ARR genes and other early cytokinin response genes, which have been shown to be direct target genes of B-type ARRs in the past (Hwang and Sheen, 2001; Taniguchi et al., 2007), were dampened in 35S:ARR1-SRDX transgenic seedlings (Fig. 7; Supplemental Table S1). This clearly shows that the ARR1-SRDX protein suppresses the activation of direct target genes of endogenous B-type ARRs. Interestingly, the response of the majority of the response genes was generally reduced, but each to a different extent. These differences may reflect the different contributions of ARR1 and related B-type ARRs to the cytokinin-dependent regulation of the respective genes. Furthermore, recent work has shown that the transcription of A-type ARRs is also regulated via a B-type ARR-independent pathway. The target genes of the cytokinin response factors of the ERF family of transcription factors partially overlap with those of the B-type ARRs (Rashotte et al., 2006).
We have identified large sets of genes that show significant changes in their transcript levels in association with the cytokinin deficiency syndrome of the 35S:ARR1-SRDX plants. The function of the deregulated genes can be linked, in many cases, to known functions of cytokinin in plant physiology and development, such as nitrogen metabolism (Sakakibara et al., 2006) and GA action (Brenner et al., 2005; Jasinski et al., 2005). The fact that the transcript levels of genes related to these processes (e.g. NR2, AMT2, NTL1, RGA1, and RGA2; Table I) change in plants with repressed B-type ARR functions confirms that cytokinin plays an important role in the control of these processes and that the B-type ARRs are at least partly involved in this regulation. This data set will be an important source to identify and study processes downstream of B-type ARRs.
B-Type ARRs Are Possible Nodes for the Integration of Different Signaling Pathways
As developmental programs are subject to fine-tuning by different factors, the integration of different signaling pathways is necessary. Evidence for a function for B-type ARRs in cross talk came from the red light experiments. Red light is a key regulator of seed germination, and its effect is mediated by phytochromes (Shinomura, 1997; Sullivan and Deng, 2003). Red light stimulates seed germination, while far-red light is inhibitory. The response under our light regime is mediated by phytochrome B, and the reversibility of red light-stimulated germination by far-red light is a hallmark of its activity (Shinomura, 1997). This reversibility was almost completely lost in 35S:ARR1-SRDX transgenic seeds (Fig. 9B). This result is consistent with the previously described reduced far-red light sensitivity of cytokinin receptor mutant seeds (Riefler et al., 2006) and seeds of cytokinin-deficient plants (data not shown). A plausible explanation is that cytokinin in an unknown way alters the Pfr-Pr ratio of phytochrome B (e.g. by stabilization of the active Pfr form). This may be mediated directly by downstream factors such as A-type ARRs, as was proposed for red light-dependent regulation of hypocotyl elongation (Sweere et al., 2001; Mira-Rodado et al., 2007). The dominant repressor effect of ARR1-SRDX could block the cytokinin signal involved in regulating this process. Alternatively, B-type ARRs may be involved in regulating red light responses independent of cytokinin (e.g. by acting downstream of phytochromes). In any case, there is now considerable evidence that the cytokinin status has a strong influence on the reaction to red light.
Using the CRES-T, we generated a dominant repressor of B-type ARR function and demonstrated that this approach is useful to overcome functional redundancy in this transcription factor family. Overall, we show that B-type ARRs are required for normal growth and development of the shoot and root, where they exert positive and negative regulatory functions. Comparisons with the phenotypes of cytokinin receptor mutants and cytokinin-deficient plants clearly show that the B-type ARRs, collectively, are involved in regulating most cytokinin-dependent processes in Arabidopsis. 35S:ARR1-SRDX transgenic plants and the data sets of deregulated genes will be a valuable tool for further investigation of the function of this class of transcription factors, in particular of their role in mediating the cross talk of the two-component signaling system with other pathways.
Plant Material and Growth Conditions
Plants of the Col-0 accession of Arabidopsis (Arabidopsis thaliana) were used as the wild type. The plants were grown in the greenhouse on soil at 22°C under long-day conditions (16 h of light/8 h of dark). For in vitro experiments, seeds were surface sterilized with saturated calcium hypochlorate solution. After sowing, they were kept at 4°C for 3 d in the dark and then exposed to white light (approximately 75 μE). Seedlings were grown at 22°C on medium containing 1× Murashige and Skoog (MS) salts, 3% Suc, 0.05% MES, and 0.9% agar (Merck) unless specified otherwise. For ethylene and red light experiments, Suc was omitted from the medium. For the flowering phenotype, the total number of rosette leaves was counted upon flower bud initiation.
Gene Cloning and Transformation
The protein-coding region of the ARR1 gene was amplified by PCR using a cDNA library from Arabidopsis C24 (Minet et al., 1992) and inserted in the plasmid pDONR201 (Invitrogen), yielding pDONR201-ARR1 (Dortay et al., 2006). The DNA fragment coding for the SRDX peptide (LDLDLELRLGFA) was synthesized with a TAA stop codon and a BsrGI restriction site at the 3′ end and an in-frame HhaI site at the 5′ end. The ARR1 gene was isolated from the plasmid pDONR201-ARR1 by restriction digestion with BsrGI, and the resulting fragment was further digested with HhaI. The DNA fragments were ligated and recloned into pDONR201. The resulting ARR1-SRDX gene was shuttled into the vector pB2GW7 (Karimi et al., 2002) for overexpression under the control of the 35S promoter. This construct was transformed using Agrobacterium tumefaciens-mediated transformation into Arabidopsis plants by the floral dip method (Clough and Bent, 1998).
For the protoplast assay, the reporter plasmid was generated by amplifying the 1,000-bp fragment directly upstream of the ARR6 gene (for primers used, see Supplemental Table S3). The resulting PCR product was digested with HindIII and XbaI and ligated into the pBT10-GUS vector (Sprenger-Haussels and Weisshaar, 2000). The B-type ARR overexpression constructs were generated by shuttling the respective genes from the pDONR vector into the pB2GW7 vector (Karimi et al., 2002) for expression under the control of the 35S promoter. All B-type ARR genes were cloned as cDNAs, except for ARR2, which was a genomic clone (for primers used, see Supplemental Table S3).
Protoplast Transformation and GUS Assays
Protoplast isolation and transformation were carried out according to the method described by Hwang and Sheen (2001). For isolation of mesophyll protoplasts, 4- to 5-week-old rosette leaves were used. Transformation of protoplasts was mediated by 40% polyethylene glycol solution. For cytokinin treatment, protoplasts were incubated overnight with 500 nm trans-zeatin. For the transactivation assays, 9 μg of the ARR6:GUS reporter plasmid and 14 μg of each effector plasmid carrying 35S:ARR1 or other B-type ARRs and 35S:ARR1-SRDX were used. For normalization, 3 μg of a plasmid harboring the 35S:NAN gene (Kirby and Kavanagh, 2002) was added. Both GUS and NAN enzyme assays were performed according to Kirby and Kavanagh (2002). The ratios of GUS and NAN activities were calculated as relative GUS/NAN activity units.
Gene Expression Analysis by Northern Blot
Total RNA was extracted from 3-week-old soil-grown plants (Fig. 2) or 5-d-old seedlings grown in 0.5× MS liquid medium (Fig. 7A). Seedlings were treated with 5 μm BA and incubated at 0, 15, 30, and 120 min prior to harvesting. Preparation of total RNA and northern-blot analysis were performed as described (Brenner et al., 2005). For northern-blot analysis, 20 μg of RNA was separated on a denaturing agarose-formaldehyde gel (1.2%) containing 10% of 10× MOPS and 3% of 37% formaldehyde, transferred to a Hybond-N+ nylon membrane (Amersham), and after fixation hybridized with radioactive [α-32P]dCTP-labeled DNA. Hybridization was performed at 68°C in a phosphate buffer containing 7% SDS and 1% bovine serum albumin. Washing was done with 2× SSC and 0.2× SSC, 0.1% SDS at 65°C. As a control for loading, blots were reprobed with an ACTIN2 probe. The different probes were generated by amplifying the respective cDNAs (for primers used, see Supplemental Table S3).
Root Growth Assay
Arabidopsis seeds were grown on vertical MS medium plates containing different concentrations of BA ranging from 0.01 to 1.0 μm. Dimethyl sulfoxide (DMSO; 0.1%) was included as a vehicle control. The primary root lengths were determined at 10 DAG (Fig. 4), between days 4 and 9 after germination (Fig. 6), or after 4 d of growth in the dark (Fig. 8). Photographs were taken with a digital camera (Nikon Coolpix 8800), and root lengths were determined using the Scion Image program version Beta 4.0.3 (www.scioncorp.com). The number of lateral roots emerging from the epidermis of the primary root was counted with a microscope at 10 DAG. The experiments were performed using three independent replicates and at least 15 seedlings in each replicate.
Chlorophyll Retention Assay
This assay was performed as described previously (Riefler et al., 2006). Either the sixth or the seventh leaf was detached from 24-d-old in vitro grown seedlings and floated on distilled water supplemented with 0, 0.01, 0.1, 0.5, 1.0, or 5.0 μm BA in 0.1% DMSO in small petri dishes for 10 d at room temperature in the dark. Three replicates of each genotype, each consisting of five leaves, were prepared and two chlorophyll measurements were taken per plate. Chlorophyll was extracted with methanol for 24 h in the dark. The amount of chlorophyll was measured with a spectrophotometer and normalized to fresh weight, and the chlorophyll content was calculated as described (Porra et al., 1989).
Ethylene Response Assay
Seeds were surface sterilized and placed on vertical plates containing sugar-free MS medium. Different concentrations of ACC (0.1, 1.0, 10, or 30 μM) were included in the medium. The seeds were grown in the dark at room temperature. After 4 d, the hypocotyl lengths were measured using the Scion Image program version Beta 4.0.3 (www.scioncorp.com). The experiment was done in two replicates, each with more than 15 seedlings. The root elongation assay was performed in the same way, but using only one ACC concentration (10 μm). The experiment was done with 15 seedlings per genotype in two replicates.
Determination of Seed Size and Weight
Determination of seed size and seed weight was carried out as described by Riefler et al. (2006) and Werner et al. (2003), respectively. Seed size of the wild type and ARR1-SRDX overexpressors was determined by measuring the length and width of 60 seeds harvested from two different plants. The volume was estimated by calculating with the formula for a spheroid (volume = 4/3 × π × length × width × depth). Biomass of the seed was weighed using a fine balance LE244S (Sartorius). The weight of one seed was calculated from the weight of pools of 200 seeds. The sample size for each genotype was 10.
Seed Germination Assay
Seeds of wild-type and 35S:ARR1-SRDX plants used for germination experiments had been harvested and stored under the same conditions. Seed batches of approximately 100 seeds were sown on petri dishes on four layers of filter paper and moistened with 5 mL of distilled water. Seeds were vernalized for 3 d at 4°C in the dark, followed by 6 h at 35°C to alleviate the seeds of hypersensitivity to red light (Fankhauser and Casal, 2004), and for 30 min at 25°C immediately prior to the irradiation. For the different light treatments, seeds were exposed for 5 min to white light (30 μE), red light (660 nm, 6 μE), or far-red light (724 nm, 15 μE) or to a sequential treatment of 5 min of red light followed by 5 min of far-red light. Seeds were stored at room temperature in a dark place for 7 d. Radicle protrusion was employed as the criterion for germination.
Microarray Experiments
For microarray experiments, surface-sterilized seedlings of the wild type and line ARR1-S-8 were grown in sterile 0.5× MS liquid medium (1 g L−1 Suc and 0.5 g L−1 MES, pH 5.7) in petri dishes and cultured under the conditions described above. At 5 DAG, the seedlings were treated with either 5 μm BA dissolved in 1.13 mm KCl for 15 or 120 min (BA15 and BA120, respectively) or with 1.13 mm KCl solution for 120 min as a control (BA0). Timing of the treatment was such that the plants were harvested within an interval of 6 to 8 h after the onset of light. The content of two petri dishes was pooled to yield one sample of biological material. Two biological samples were harvested for each experimental condition. RNA was extracted as described previously (Brenner et al., 2005). To reduce the number of false positives due to nonspecific changes in gene expression, we hybridized RNA samples extracted from uninduced wild-type seedlings (BA0) as a reference sample onto an additional set of microarrays. Genes with a significant expression change between this reference sample and the experimental Col-0 BA0 sample were excluded from the genotype comparison.
Preparation of the labeled samples for microarray hybridization was performed following the linear amplification protocol (Puskas et al., 2002) with modifications. A first-strand reaction was performed starting with 5 μg of total RNA using SuperScript II reverse transcriptase (Invitrogen). The whole product was used for second-strand synthesis with Escherichia coli DNA polymerase, E. coli DNA ligase, and E. coli RNase H (Invitrogen). After purification with the QiaQuick PCR purification kit (Qiagen), the whole second-strand product was used for in vitro transcription with the MEGAScript High Yield Transcription Kit (Ambion/Applera). The resulting copy RNA was purified with RNeasy mini columns (Qiagen). Five micrograms of the purified copy RNA was used for labeling with the SuperScript II reverse transcriptase (Invitrogen), 2 μg of random nonamers as primers, and 1.5 nmol Cy5-dCTP supplemented with a mixture of 2 nmol dCTP and 10 nmol dDTP. The labeled cDNA was purified with the help of QiaQuick PCR purification columns.
We followed the hybridization strategy used in the European Compendium of Arabidopsis Gene Expression project (http://www.cagecompendium.org), employing a Cy3-labeled mixture of oligonucleotides complementary to the secondary primers for gene-specific tag amplification as a common reference sample and Cy5 labeling for the biological samples. Forty picomoles of incorporated Cy5 and 1 pmol Cy3-labeled oligonucleotide reference were dissolved in a mixture of 25% Microarray Hybridization Buffer (Amersham), 50% formamide, and 33.3 ng μL−1 poly(dT35) DNA, applied onto CATMA V2.4 microarrays (ArrayExpress accession no. A-MEXP-274; http://www.ebi.ac.uk/aerep), covered with a coverslip, and incubated in hybridization chambers (Scienion) in a water bath at 42°C for 15 h. The arrays were washed once with 1× SSC, 0.2% SDS for 10 min at 55°C, twice with 0.1× SSC, 0.2% SDS for 10 min at 55°C, and once with 0.1× SSC at 37°C and finally dipped five times into distilled water at room temperature. After drying with an air stream, the microarrays were scanned with a Fuji FLA-5000 scanner at 5-μm resolution and photomultiplier tube gain settings of 93 and 83 for Cy5 and Cy3, respectively. The images were saved in TIFF format. Each biological sample was hybridized onto two microarrays, resulting in a total of four hybridizations per experimental condition. The raw data were submitted to the ArrayExpress database at the European Bioinformatics Institute (www.ebi.ac.uk/aerep) and normalized with a preprocessing pipeline (Allemeersch, 2006) written in R/Bioconductor (Gentleman et al., 2004; RDC Team, 2005). After normalization, general linear models were fitted for each gene (Kerr et al., 2000; Wolfinger et al., 2001). The resulting data were used to generate the interaction plots as well as a table of all expression values, ratios, and P values. FDR-corrected P values (q values) were also included. This table was exported into Microsoft Access, the filtering functions of which were used to identify significantly regulated genes and intersections between groups of genes. A gene was called significantly regulated if it had a q value of ≤0.05, a fold change of ≥2.5, and at least half of the spots above background.
Accession Numbers
The Arabidopsis Genome Initiative locus identifier for ARR1 is At3g16857. The raw data from the microarray experiment are accessible through the ArrayExpress database at the European Bioinformatics Institute (www.ebi.ac.uk/aerep).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Schematic representation of the ARR1-SRDX fusion protein.
Supplemental Figure S2. Phenotype of different 35S:ARR1-SRDX lines.
Supplemental Figure S3. Phenotype of the 35S:ARR1-SRDX transgenic plants compared with a triple cytokinin receptor mutant and a cytokinin-deficient plant.
Supplemental Table S1. Lists of all significantly regulated genes in the wild type and line ARR1-S-8.
Supplemental Table S2. Regulation of ARR1 target genes in 35S:ARR1-SRDX transgenic seedlings.
Supplemental Table S3. List of the primers used in this study.
We thank Cordula Braatz for skillful technical assistance and Klaus Harter and Virtudes Mira-Rodado for the use of and technical help with the red light chambers. We are grateful to Andrea Ehlert and Wolfgang Dröge-Laser for support with the protoplast assays. We thank also Yves Moreau for help in bioinformatics and Takeshi Mizuno for sending plasmids harboring cDNAs of ARR12, ARR19, and ARR21.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Thomas Schmülling (tschmue{at}zedat.fu-berlin.de).
↵1 This work was supported by the Deutsche Forschungsgemeinschaft (grant no. Schm 814/20–2) in the frame of the Arabidopsis Functional Genomics Network.
↵2 These authors contributed equally to the article.
↵3 Present address: Flanders Interuniversity Institute for Biotechnology, MicroArray Facility, B–3000 Leuven, Belgium.
↵[C] Some figures in this article are displayed in color online but in black and white in the print edition.
↵[W] The online version of this article contains Web-only data.
- Received December 21, 2007.
- Accepted May 16, 2008.
- Published May 23, 2008.


text:
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Victorian Women Artists,English 1832 - 1924.
He was a British watercolor artist. Her parents, Samuel Rayner and Anne Rayner (nee Manser) were both noted artists, the former Samuel having been accepted for exhibition at the Royal Academy when he was 15. Four of Louise's sisters - Ann ("Nancy"), Margaret, Rose and Frances - and her brother Richard were also artists. The family lived in Matlock Bath and Derby before moving to London in 1842. Louise studied painting from 15, with the guidance of her father and later with artist friends such as George Cattermole, Edmund Niemann, David Roberts and Frank Stone. Her first exhibited work was entitled The Interior of Haddon Chapel, shown at the Royal Academy in 1852, the first of a series of oils. From 1860, however, her medium was watercolour, which she exhibited for over 50 years via organisations including the Society of Lady Artists, Royal Academy, Royal Watercolour Society and the Royal Society of British Artists. She lived in Chester but travelled extensively, painting British scenes, during the summers in 1870s and 1880s. Her paintings are very detailed and highly picturesque populated street scenes capturing the "olde worlde" character of British towns and cities. Her paintings are very popular today as prints and on jigsaw puzzles. Around 1910 she moved with her sister to Tunbridge Wells, and later to St Leonards, where she died in 1924.
ID: 34752 The Cross,Eastgate,Chester
Louise Rayner
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text:
Most of our builds depend on dynamic versions of internal plugins that come from our internal Artifactory. This way we can easily update builds without having to change many repos, and it's all very nice. Except for caching.
The default cache timeout of 24 hours is messing with us a lot. It means that if I deploy a new version of an internal plugin, some people will get the update sometime later today, others only tomorrow. So I need to be on alert for the whole 24 hours to see if there was a problem, or ask everyone (there's always somebody not listening) to run a build with --refresh-dependencies.
It would make a lot more sense to me if the cache could be set to expire at a certain point in time, say, the next time the clock hits 5AM, so everybody would get the update in the morning.
I can do this with the current tools by calculating the number of seconds until the next 5AM, but it looks weird.
I was wondering if there was some best practice on how to do this better. Or if this is a good approach, would it make sense to have some DSL in Gradle to express "cache until 5AM" more easily?
Lóránt Pintér
Developer at Prezi
Have you investigated the impact of using a cache timeout of, say, 1 hour? Would this slow things down too much?
The extra directory listing time may not be significant.
On Wed, Aug 13, 2014 at 4:44 PM, Lóránt Pintér <[hidden email]> wrote:
Darrell (Daz) DeBoer
Lóránt, you asked for a best practice?Joachim
We have in our builds set the dynamic cache to 0 seconds, but only for our 3-4 own plugins in the buildscript section.
We have made sure that our plugin repository (Nexus in our case) is located on a machine 'close' to the developers and on a fast machine with an SSD disk.
I'm not sure if what we are doing is the best for you but it works very good for us.
On Thu, Aug 14, 2014 at 4:36 PM, Daz DeBoer <[hidden email]> wrote:
In reply to this post by Lóránt Pintér-2
On 14 Aug 2014, at 8:44 am, Lóránt Pintér <[hidden email]> wrote:
I think it would make sense to have a 'cache until' convenience for the 'cache for' stuff.
Adam Murdoch
Gradle Co-founder
CTO Gradleware Inc. - Gradle Training, Support, Consulting
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Lonnie Brown on William Barr, Ramsey Clark, and the necessity of independence
In light of AG William Barr's recent testimony before the House Judiciary Committee we present Lonnie T. Brown, Jr.'s January 2020 blog post for Defending the Public's Enemy ».
During his confirmation hearing, William Barr touted his ability to distance himself from politics and to safeguard the rule of law, declaring, under oath, that as attorney general he would be "truly independent." To many, Barr's words provided comfort or at least hope that he would be guided by law and principle, not the president's political agenda. However, since being confirmed, his actions have revealed the illusory nature of his sworn declaration.
Rather than exhibiting the fairmindedness and objectivity expected of the nation's chief law enforcement officer, Barr seems to have established a de facto attorney-client relationship with President Trump, serving as his strident advocate and partisan defender. For example, in March, he zealously made the president's case for "no collusion" by publicly recasting the substance and tenor of Robert Mueller's report on the investigation into Russian interference in the 2016 election.
More recently, in a similar vein, the attorney general sharply criticized the findings of the Justice Department's Inspector General's report on the FBI's investigation into Russian involvement in the president's 2016 campaign. That report concluded that the FBI had adequate grounds to open the investigation and was not politically motivated. In a rather transparent effort to parrot President Trump's "witch-hunt" narrative, Barr characterized the FBI's investigation as having been based on the "thinnest of suspicions" that he personally believed to be "insufficient to justify the steps taken."
My guess is that a large segment of the electorate, across the political spectrum, sees nothing wrong with or surprising about Attorney General Barr's alignment with the president, or any attorney general's close relationship with a president, for that matter. Such a rapport is by no means simply a Republican phenomenon. President Obama's attorney general Eric Holder, for instance, once referred to himself as the president's "wingman," connoting that his job was to have the chief executive's back.
Continue reading "What William Barr Can Learn from Ramsey Clark" »


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24" LED Monitor Acer K242HLbd will enchant you by an elegant slim design, which makes it the right highlight of your modern computer corners. With Full HD resolution and fast response time of 5 ms, this monitor enables you to watch movies and play the latest games. Due to the native aspect ratio of 16:9 you will not be bothered by side bars. The Modern LED backlight provides you with deeper black colour and a brighter white. This feature helps to achieve a high contrast ratio and a delineation of all image details.
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text:
W.E.B. Du Bois Biography Trifold Brochure
TpT Digital ActivityPDF (838 KB|2 pages)
- This set of trifold brochures features 37 black historical and modern figures that have contributed to history in various ways. These brochures are a perfect way to ensure your students are keeping their information organized and focusing on the most important aspects of the person they're studying.$27.00$54.00Save $27.00
- Product Description
- Standards
This brochure is a perfect way to ensure your students are keeping their information organized and focusing on the most important aspects of the person they're studying. This activity is great for biography projects and can easily serve as a note-taking tool for an essay or presentation. It can also serve as the biography project itself! The brochure is perfect for integrating literacy into other subjects, especially social studies. Students will not only benefit from the experience of learning about these diverse figures, but they'll practice their mapping and timeline skills as well. The brochure allows students to go beyond reading to really interact with the chosen text as they determine which facts are relevant. The biography brochure can be used year round, but it is also perfect for Black History Month.
Each trifold brochure has space for the following information about each figure:
•Important people in their lives
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I recommend printing the brochure using the "Actual Size" setting on your printer for maximum fit. You can also scale to fit if needed.
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I'm inviting you to join me on this trip, where you can meet the fearsome Harikos and Cifuros, the mysterious Leterlis, the evil Coque, and his sword, you will know how the Ocripsol Rocks will help our adventurers, and how the immense dragons will be able to travel through the magical portals of the three prisms, rooms that maintain the balance between good and evil.
SURPRISES: Books 3 and 4 are in the revision process to be released later and books 5 and 6 are in the process of being written. However, there is a mysterious book called the GOLDEN BOOK, and it is book 7
In the limits between the earthly and the magical, where the balance of good and evil is represented by a seal, formed by a ring that symbolizes the union of good (three prisms that maintain balance), in its center a scale that indicates the balance, and crisscrossed with it, a pair of wings that represents free will, is a cloudy and mysterious town, such a village bears the name of Nedvelyn; room of many families and among them the Collyn family (Sara, Roy, Etan, Eduart, grandfather Etan and …), who during a fun weekend, when they were camping in a cabin near Lake Londley, had a bad feeling, but ignoring, they continued with their plans…
Seal of The Three Prisms
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- Server 1
License to Wed
Newly engaged, Ben and Sadie can't wait to start their life together and live happily ever after. However Sadie's family church's Reverend Frank won't bless their union until they pass his patented, "foolproof" marriage prep course consisting of outrageous classes, outlandish homework assignments and some outright invasion of privacy.
Director: Ken Kwapis
Actors: Christine Taylor, DeRay Davis, Eric Christian Olsen, John Krasinski, Josh Flitter, Mandy Moore, Robin Williams
Duration: 91 min
Quality: HD
Release: 2007
IMDb: 5.3


text: The Women's Auxiliary of Washington DC 67th Annual Fashion Show Luncheon and Auction is just days away. We have an online preview just for you of the wonderful items up for auction at this year's show. Remember, all proceeds from the event will benefit young women with children and sending kids to Camp Happyland next summer! If you have not gotten your tickets, visit www.SAWADC.org today!


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Helping maintain Tech Fortress' existenceHello! As you might already know, it costs money to host a server. Being a supporter is more than just highly appreciated - your contributions help maintain this small community!
The easiest way to monetarily support Tech Fortress is to become a patron on Patreon. Every dollar matters, no matter how small! You can change your contribution amount at any time.
If you play on the Tech Fortress Minecraft servers, there are multiple ways you can obtain a "supporter rank":
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Quran Quotes: Asslam O Alikum! Welcome to all Muslims on my website. Alhamdulillah, I am here with you and today I am going to share such a beautiful Quran quotes and Verses. In sha Allah you people can easily understand. Ma Sha Allah Quran helps us in every moment of life. What is Quran Pak […]
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Jimmy Kimmel hosted the first-ever Celebrity Curse-Off on his show last night, featuring Sally Field vs. Julia Roberts in order to see who had the biggest potty mouth. It's pretty funny to watch the stars shock each other … Ultimately Sally Field won.
Watch: Julia Roberts and Sally Field shout filth at each other in 'Celebrity Curse Off' http://t.co/jkMeCXZxyc pic.twitter.com/oHNesJnbnc
— entertainment.ie (@entertainmentIE) May 6, 2014


text: Spring Special! Leave a reply See this item from the Poppy Store You don't see images and colors in this email? Click this link to view online Spring Special: Until May 21, 2018, purchase a golf shirt to receive a FREE matching ball cap! We thank you for your continued support. The Royal Canadian Legion Contact us at: email@example.com or toll free at 1-888-301-2268. The Poppy Store Website The Poppy Store newsletter © All rights reserved, The Poppy Store 2018 Share this:TwitterFacebookLike this:Like Loading... Related


text: David Bou, is an illustrator, who is creating our secret life moments, a life after midnight, after work. He is a fetish illustrator, who is inspired by visions and ideas seen in closed eyes at night. "I love colored lights, smokes, flashes!" See more of David's work here: FACEBOOK / INSTAGRAM SHARE THIS STORY!TwitterFacebookPinterestTumblrRedditWhatsAppEmail


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An Open Framework for Dynamic Big-data-driven Application Systems (DBDDAS) Development
Main article
TypeConference Paper
AuthorsDouglas, Craig
KAUST DepartmentComputer, Electrical and Mathematical Sciences and Engineering (CEMSE) Division
Numerical Porous Media SRI Center (NumPor)
SRI Uncertainty Quantification Center
Online Publication Date2014-06-06
Print Publication Date2014
Permanent link to this recordhttp://hdl.handle.net/10754/552353
MetadataShow full item record
AbstractIn this paper, we outline key features that dynamic data-driven application systems (DDDAS) have. A DDDAS is an application that has data assimilation that can change the models and/or scales of the computation and that the application controls the data collection based on the computational results. The term Big Data (BD) has come into being in recent years that is highly applicable to most DDDAS since most applications use networks of sensors that generate an overwhelming amount of data in the lifespan of the application runs. We describe what a dynamic big-data-driven application system (DBDDAS) toolkit must have in order to provide all of the essential building blocks that are necessary to easily create new DDDAS without re-inventing the building blocks.
CitationAn Open Framework for Dynamic Big-data-driven Application Systems (DBDDAS) Development 2014, 29:1246 Procedia Computer Science
PublisherElsevier BV
JournalProcedia Computer Science
Conference/Event name14th Annual International Conference on Computational Science, ICCS 2014
Additional Linkshttp://linkinghub.elsevier.com/retrieve/pii/S1877050914002890


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How to Locate the Right Swimming Pool Development Company
Development of a swimming pool for your home or business purposes can be a questioning errand. The space in your garden can be utilized well to make a stunning pool. With specially designed pools, it won't be necessary to go to an open or a private pool. But if you are making the pool for business purposes, both are necessary to hire a professional for the job. Nowadays, there are a considerable number of swimming building organization that offer perfect services at sensible costs. Building a swimming pool requires skill and experience. It is important to find a decent construction organization to make a brilliant swimming pool at sensible value rates. This article will give a few indications to think about when searching for the perfect company to build your pool.
First, hire a reputed company. It's reasonable to choose a respectable pool construction organization with enough involvement in this business. There are plenty of online stores that showcase a wide variety of services provided by various companies. Search for clients' audits before choosing any organization. It is similarly critical to look at other websites for extra data on the sort of administrations and costs cited by organizations. By choosing reputed companies, customers guarantee the quality of services offered.
In addition, consider how the pool looks. This is something else you have to consider for your swimming pool. Some people wish to include rocks and plants along the sides of the swimming pool which enhance the appearance of the pool while some prefer a big elegant pool. There are also various shapes of pools such as rectangular, round, square and many more. You should also consider the total space of the pool. Before enlisting a construction company, decide the amount of room expected to build the pool to know how huge the pool will be.
Third, hire an incorporated construction company. It is essential to hire the administrations of a registered construction company to guarantee dependability and quality. Different online portals feature different incorporated construction companies that offer quality administrations to thier clients. The other significant thing to consider about the pool building association, is their related involvement. This is a crucial factor to consider if you want to have a well-functioning swimming pool. There are noteworthy sites that offer excellent services in the pool development, however, be certain to verify their experience.
At long last, consider your budget. Monitoring the expense is a critical factor when building swimming pools. Construction companies have to consult the customer before purchasing construction materials to make sure that it is within the budget. The total price must be watched with the goal that the pool can be developed within the financial plan.
If You Think You Get Pools, Then Read This


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Prevent an Injury at the Florida State Fair
- Posted by bayshore
- Posted in Preventing Injuries
The Florida State Fair opens today. Although a state fair can be a lot of fun, there is always a risk of getting hurt. The personal injury attorneys at Perenich, Caulfield, Avril & Noyes want to provide the following tips to prevent injuries at this year's Florida State Fair:
Amusement Ride at State Fair Safety Tips
- Read all posted rules and listen to instructions given by ride operators.
- Watch the ride with children before boarding. Point out the operator and the entrance and exit locations prior to riding. Make sure they fully understand the instructions and warnings.
- Keep all body parts (hands, arms, legs, long hair, etc.) inside the ride at all times. If you have long hair, keep it pulled up with a hat or hair tie.
- If you can't count on a child to stay seated with hands and feet inside, don't let them ride.
- Always use the safety equipment provided (seat belt, shoulder harness, lap bar, etc.).
- Obey minimum height, age, and weight restrictions. Never sneak children onto rides if they are too small or too young. A smaller/younger child may not be physically or developmentally able to stay safely seated.
- Know your physical conditions and limitations. If you suspect that your health could be at risk for any reason, or you could aggravate a pre-existing condition of any kind, do not ride.
General State Fair Safety Tips
- We all know Florida's weather. Pay attention to weather forecasts and prepare appropriately.
- Be aware of your surroundings and know where you will go if you need to seek shelter from an unexpected thunderstorm.
- Wear closed toe shoes like tennis shoes, not flip-flops or sandals, to protect your feet.
- Wash your hands after petting animals, and always before eating.
- Read all instructions when viewing animals. Some animals should not be touched.
- Remember "If you see something, say something." If you see people or activities that seem suspicious, report your observations to security.
- Have an established family plan of a location where you will meet if separated.
Following these simple tips can make your trip to the Florida State Fair much more enjoyable and save!
Perenich, Caulfield, Avril & Noyes, P.A. is one of the oldest personal injury law firms in Tampa Bay. If you or a loved one is injured, contact us for a free case consultation. There is no attorneys fee or costs unless we win. Call today at 1-800-5FLALAW simply click here to schedule a free case consultation.


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At PlayStation, we're not afraid to show off our love of games. Sure, we have plenty of artwork from our own Sony Interactive Entertainment Studios splashed across our phones, desktops and just about any other device that'll let us showcase their incredible talent, but there are just as many amazing works of art from studios all across the industry that we're not shy about showing our love for too.
In an effort to help make it easier for you to do the same, we figured it might help to collect the various wallpapers and put them all in one place. We'll be updating this page with new wallpapers whenever we have more to share, so be sure to check back often, and thanks for gaming with PlayStation!
Click on a game title to jump to that section >


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A WONDERFULLY RARE & EARLY PRODUCTION SINGLE ROW CREAM LUSTER CULTURED NECKLACE, 9CT ROSE GOLD CLASP & SCREW BACK. EARRINGS, 9CT CLASPS BY CIRO – C1930S. ORIGINAL BOX & PAPERS. THIS IS A RARE. & DIFFICULT TO REPEAT! This necklace is complete with a pair of cultured pearl screw back earrings in 9ct yellow gold which complement the necklace beautifully. These items date from the 1930s and would have been made to order for a very special customer! These items are supplied with the original custom presentation case and a care leaflet. This is a custom made necklace, for a special customer and would have been very expensive when new! The cultured pearl necklaces were always supplied in the individual presentation boxes, supplied here. The 1970s were a boom time for the company and they led the way in the development of Zirconia, a diamond substitute. They produced a significant range of superb quality costume jewellery, marketed both under there own brand name and for major jewellers & dept. Much of the later output is unsigned, but all of their output is of enduring quality and is keenly sought after. Is set with white cultured pearls necklace which features the original early rose gold ingot 9ct clasp. The clasp is stamped Ciro & 9ct. The necklace measures 22 in length. This necklace has a wonderful period patina. Are of classic Ciro screw back type and match the necklace and feature 2 drop cultured pearls. The gold clasps are stamped Ciro & 9ct. They are in excellent undamaged order. Is the original custom supplied production item in cream chagrin, with a lovely satin interior. It is in vintage condition 2. The interior is foxed, but has good gold tone clasps. It is otherwise undamaged. It measures 9.5 long and is 3.5" wide. We are supplying an original care leaflet. These items are in vintage condition 1. There is no damage to any of the pearls. The clasp has age related wear and scratching to the faces and the original marks are clearly visible. The earrings are unmarked. The condition of the box is above. All of our jewellery is cleaned and checked before sale. We try to offer our buyers free P&P to U. It is unlikely that the post office will accept claims for goods lost in transit so buyers can upgrade service prior to check out. Larger items will be sent by myhermes and will be charged appropriately. This is an economy service and can take between 5 – 56 days to arrive, depending upon location. Please refer to listings for costing. This is clearly indicated in the listings. Items are described as honestly as possible and you should check that an item is suitable. Many of our items are unrepeatable and some of our items are offered through other channels and may be withdrawn ahead of the posted finish time. Large or high value item. Post code is CF81 8NW. We aim to respond within 24 hours. Please review our terms and conditions page for further information. Thank you, we appreciate your business. The item "CIRO CULTURED PEARL NECKLACE & EARRING SET, 9CT GOLD INGOT CLASP C1930'S" is in sale since Wednesday, October 18, 2017. This item is in the category "Jewellery & Watches\Vintage & Antique Jewellery\Vintage Fine Jewellery\Art Deco (1915-1935)". The seller is "t7nee" and is located in Lancashire. This item can be shipped worldwide.
- Metal: GOLD
- Type: NECKLACE & EARRING SET
- Gold Quality: 9CT
- Main Gemstone: Pearl
- Designer: CIRO
- Country/Region of Manufacture: United Kingdom


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This volume of "Les Cahiers" collection presents the history of the Anneau siège, Pierre Paulin's final creation for Ligne Roset, shortly before his death in June 2009.
Lace in Sèvres by designer Christian Biecher is an architectural creation in ceramic made by the Manufacture de Sèvres.
In the Donahue bar-set, a creation by designer Claude Bouchard, the Puiforcat silversmiths have renewed their lines.
Designer Vincent Dupont-Rougier created for the French Mobilier National the desk, shelving, and storage units destined for the French Ambassador in Romania.
We invite you to discover the poetic, artistic and highly technical universe of the Pleyel Manufacture which has opened its doors to the designer Michele De Lucchi.
Roland Daraspe, as a recognized Master of Art and goldsmith, shows us here his contemporary work of art inspired by submarine and vegetal life.
This book presents the work of 12 renowned textile designers that reveals a universe which is poetic, fantastic and full of humour.
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Ever Wonder Why Windows Takes So long to Boot?
Found an article on why Windows can take longer and longer to boot up. It describes the prefetch cache slowing it down because it gets clogged up with more and more programs, this is because Windows tries to load programs faster for you, so it loads parts of the programs you ran on your last session to make them load faster. It tells you how to make this faster, it also talks about the hard drive getting defragmented, network drives slowing you down, adware and spyware and file and printer sharing. Check it out, Why windows takes so long to start up. But, read on before you do.
Well, some of this info is incorrect, you should not clean out your prefetch folder, according to Ed Bott,
Bottom line: You will not improve Windows performance by cleaning out the Prefetch folder. You will, in fact, degrade Windows performance by cleaning out the Prefetch folder. I've done performance testing that establishes this definitively. In all the many sites that offer this bogus tip, I have yet to see a single piece of actual performance testing.
So, while the article is helpful in telling you to defragment your hard drive, clean spyware and adware, but, whatever you do, do not empty the prefecth folder. Some will say it will help if you test programs, or check out a program and never run it again, but Windows cleans up the folder and removes program that are no longer used.


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Oppo Reno Price in Pakistan - 62 results
Oppo Reno price in pakistan ranges from Rs. 16,750 to Rs. 86,999 with an estimated average price of Rs. 56,168
Oppo Reno Price in Pakistan - 62 results
Oppo Reno price in pakistan ranges from Rs. 16,750 to Rs. 86,999 with an estimated average price of Rs. 56,168
Oppo Reno Price List
|OPPO Reno 2F Lake Green
|Rs. 44,499
|OPPO Reno 2F Sky White
|Rs. 54,999
|OPPO Reno 2 Ocean Blue
|Rs. 69,999
|OPPO Reno 2 Luminous Black
|Rs. 69,999
|Oppo Reno 3 Pro (8GB,256GB) Dual Sim with Official Warranty
|Rs. 59,999
|Oppo Reno 3 Pro Octa Core 8 GB 256 GB 6.4"
|Rs. 61,500
|Oppo Reno 3 Octa Core 8 GB 128 GB 6.4"
|Rs. 47,200
|Oppo Reno 3 Pro 8GB 256GB Midnight Black
|Rs. 64,999
|Oppo Reno 2F 8GB Ram 256GB Rom White
|Rs. 46,990
|Oppo Reno 4 Pro 8GB 256GB
|Rs. 84,999
Oppo Reno Overview
The Oppo Reno 10X Zoom is great combination phone where system performance, camera quality, body design meet affordability. This is a great value for money smartphone with essential flagship features including a stunning large AMOLED display and tri lens camera setup of 48 MP. It is available online in Pakistan for a mid-range price. Other versions of the Reno series include the Reno 2, Reno 2Z and Reno 2F also available online.
Oppo Reno Pros and Cons
Fast processing
Limited storage capacity when compared to other versions with 500 plus GB internal storage in the same price range.
1080p can feel limited when compared to 4K while gaming
Oppo Reno Description
Oppo is a global electronics and technology service provider established in 2004. It entered into the mobile phone market with their Smile Phone model in 2008. With a rapid release philosophy for launching smartphones into the mainstream market, Oppo frequently updates its firmware improving the interface, pre-installed applications and their graphics display resolution.
Processor & Memory
Built with a high-performance ARM 64 BIT CPU processing system designed for smartphones is part of the Snapdragon 855 processor designed by Qualcomm. It has 8GB RAM enabling users to stream their favourite content and run high-intensity games without any lag or ugly choppy tearing of images.
Oppo Reno has smooth connectivity and can handle all social networking applications for chatting, texting and sharing content online with 128GB internal storage. Users can download thousands of applications from their Android Playstore, save all their photographs and videos in the Photo Gallery and also be able to download thousands of music files directly into their Media Player application.
Display & Design
Oppo Reno has a 6.6 inch panoramic AMOLED display. The 93.1% screen-to-body ratio and premium back cover give the Oppo Reno an ultra-modern body. The visuals and graphics on the Reno will be vivid and bright with 2340 x 1080 pixels resolution and a high 402 PPI pixel density. It is capable of the full 16.7M colour support spectrum giving users an immersive experience while watching videos on Youtube and Netflix.
It is available in colours Extreme Night Black, Fog Sea Green, Mist Powder, and Nebula Purple. These colour profiles are staying true to the signature OPPO gradient colour aesthetic as they look stylish and glamorous.
A 48MP camera is a 10x Zoom tri-lens mechanism that consists of 6 MP primary lens along with an advanced 13MP periscope lens and 8MP sub lens for wider angle. This is a powerful camera setup capable of delivering the highest quality resolution, with an impressive zooming capability that does not distort or pixelate on full zoom.
Their focal ranges together provide a bandwidth of 16-160mm that is 10 times more than an ultra-wide-angle lens. Called the Hybrid Zoom combines the optical and digital zoom to produce stable shots at maximum distance.
It has a Motorized pop-up 16 MP front-facing camera that takes hyper-realistic photographs in both day and low light settings. With camera features that include Panoramic, Portrait Mode, Night Mode, Time Lapse and Slo-Mo. The will give users the ability to process photos and videos without having to leave their smartphone. Ready to upload to your favourite social media profile, photographs can be further enhanced through editing tools on the camera application.
Android 9 (Pie)
The Oppo Reno runs on the Android Pie that is the 16th operating systems software released for smartphones. It has upgraded navigation and processing features such as Adaptive Battery and Brightness that can intuitively learn the user's preferences and customize power-saving processing accordingly. Users can connect to the Google Assistant Go edition increasing mobile productivity and seamless processing.
The App Action and gesture keys can easily predict user intent and behaviour shaping the dashboard and interface display according to individual users. With upgraded features to App Timers, Do Not Disturb and Wind Down settings, users will be able to take care of their overall wellbeing by not overburdening themselves with excessive digital screen time. Android P has better accessibility functionalities like Select to Speak that can read out loud selected text and Sound Amplifier with over 100 adjustable sound settings. The Reno can be now be upgraded to Android 10.
It has a 3765 MAH battery which is large enough to supply over 10 hours of uninterrupted power while web browsing with the WiFi turned on. For users with heavy processing needs such as video streaming, gaming and video chatting the VOOC Fast battery charging is 20W capable of charging the phone twice as fast as regular charging systems.
Connectivity Features
The Reno can automatically connect to the Wifi with Wi-Fi Direct and also supports mobile hotspot feature making it easy to share the internet connection with other users smartphones. The Bluetooth 5.0 is a fast and reliable connection along with a strong GPS tracking system for geo tagging posts and Google Maps.
Oppo Reno Specifications
- Released: 2019, April
- Dimensions: 156.6 x 74.3 x 9 mm (6.17 x 2.93 x 0.35 in)
- Weight: 185 g (6.53 oz)
- SIM: Dual SIM (Nano-SIM, dual stand-by)
- Display: AMOLED capacitive touchscreen, 16M colors
- Screen Size: 6.4 inches, 101.0 cm2 (~86.8% screen-to-body ratio)
- Resolution: 1080 x 2340 pixels, 19.5
- Screen Protection: Corning Gorilla Glass 6
- OS: Android 9.0 (Pie), upgradable to Android 10.0; ColorOS 6
- Chipset: Qualcomm SDM710 Snapdragon 710 (10 nm) Octa-core (2x2.2 GHz Kryo 360 Gold & 6x1.7 GHz Kryo 360 Silver)
- GPU: Adreno 616
- Memory Internal: 128GB 6GB RAM, 256GB 6GB RAM, 256GB 8GB RAM
- Rear Camera: Dual 48 MP, f/1.7, 26mm (wide), 1/2.0", 0.8µm, PDAF & 5 MP, f/2.4, depth sensor
- Camera Features: Dual-LED flash, HDR, panorama
- Video: [email protected], [email protected]/60/120fps, gyro-EIS
- Front Camera: Single Motorized pop-up 16 MP, f/2.0, 26mm (wide)
- Connectivity Features: Wi-Fi 802.11 a/b/g/n/ac, dual-band, Wi-Fi Direct, hotspot & Bluetooth 5.0, A2DP, EDR, LE, aptX HD & GPS, A-GPS, GLONASS, GALILEO, BDS & NFC, USB Type-C 1.0 reversible connector, USB On-The-Go
- Phone Features: Sensors Fingerprint (under display, optical), accelerometer, gyro, proximity, compass
- Battery: Non-removable Li-Po 3765 mAh battery
- Charging: Fast battery charging 20W (VOOC Flash Charge)
- Colours: Ocean Green, Jet Black, Nebula Purple, Pink Mist, Coral Orange, Inspiration Edition


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The Pendleton County Commission welcomes letters of interest from anyone who would be interested in serving on various community boards on a voluntary basis. Anyone wanting to find out more information about how to support your community, please contact Karen Pitsenbarger at (304) 358-7573 in the Commission Office. The following Board seats are open for interest: Board of Health, EDA, Farmland Protection Board, Library Board of Trustees, Public Service District.
- Post published:June 18, 2019
- Post category:News


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Inmate Can Sue Agents of Private Prison Operator
(CN) – Employees of a private company hired to run a federal prison can be sued for allegedly violating an inmate's constitutional rights, the 9th Circuit ruled on an issue that has yet to be "squarely addressed" by the Supreme Court.
Richard Lee Pollard, an inmate at a federal prison run by the private company GEO Group, slipped on a cart left in a doorway and injured both elbows.
As GEO employees were preparing to transport him to an outside orthopedic clinic, he said they made him wear a jumpsuit and a "black box" wrist restraint, despite his claim that both would cause him excruciating pain.
Pollard sued GEO and its employees for allegedly violating his Eighth Amendment protection against cruel and unusual punishment.
He cited the Supreme Court's landmark Bivens ruling, in which the justices held that a federal prisoner can recover damages from government agents for constitutional violations.
The district court dismissed Pollard's suit for two reasons. First, the court ruled that state law gave him other options for recovering damages, such as a negligence claim or a medical malpractice claim. Second, it ruled that although the private employees were under contract with the federal government, they were not acting under federal law.
On appeal, a three-judge panel for the 9th Circuit reviewed the Bivens ruling and concluded that the Supreme Court recognized an implied cause of action for injury caused by "a federal agent acting under his authority."
The panel said the threshold question presented in Pollard's case was whether the GEO employees can be considered federal agents acting professionally under the color of federal law.
"We conclude that they can," the 2-1 majority ruled.
"Neither the Supreme Court nor our court has squarely addressed whether employees of a private corporation operating a prison under contract with the federal government act under color of federal law," Judge Richard Paez wrote for the San Francisco-based panel. "That said, we have held that private defendants can be sued under Bivens if they engage in federal action."
In a partial dissent, Judge Jane Restani agreed with the decision to dismiss GEO from the case, but argued that a Bivens claim against private employees should likewise be dismissed.
She warned that the majority "overlooks the reality that the Supreme Court has recognized Bivens causes of action only where federal officials, by virtue of their position, enjoy impunity, if not immunity, from damages liability because of gaps or exemptions in statutes or in the common law."
"I would join other circuits in concluding that a Bivens cause of action is not available against employees of privately run prison corporations where, as here, state tort laws provide a remedy," Restani wrote.


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Limited Edition Print of Aphrodite
My take on the classic 'Birth of Venus' by Botticelli. Venus, known as the goddess of love and beauty or Aphrodite as she was known by the Greeks is such a powerful symbol because she represents beauty in the physical and spiritual sense, and contemplating her beauty brings you closer to the divine.
I painted this in a time of massive self growth, I actually felt like I was shedding the skin of my former self, around the time I turned 30. Around the time I could no longer stand the way I treated myself or let others treat me. I wanted to take the power of my life back, and own my story, own who I was, physically, emotionally and live my life completely authentically. Like any kind of growth it was painful and uncomfortable and I was filled with fear, so much so that when I came through the other side of it and made life changing decisions it was like some kind of rebirth.
To me this painting represents the awakening and owning of your feminine energy 💖 and I hope whoever needs to see this, sees it 🌸
I have 25 limited edition prints, available in A4 and A3 size


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Northwestern University (Evanston, Ill.)--Alumni and alumnae
Found in 1 Collection or Record:
Lloyd Thaxton Papers
Identifier: 31/6/123
Abstract Lloyd Eugene Thaxton (1927-2008) was a popular television host and producer, with a career spanning nearly fifty years. His musical variety show, titled The Lloyd Thaxton Show, ran from 1961-1968, and featured many popular musical acts of the era. Thaxton was also executive producer of Fight Back! With David Horowitz, a consumer advocacy show that ran 1976-1992. The collection includes biographical materials, photographs,...
Found in: Northwestern University Archives / Lloyd Thaxton Papers


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- Description
- Technical Specifications
- Support Documents
AvL's 1.2m Medium Class Manual Flyaway antenna System is ultra lightweight – packs into two airline carry-on compliant nylon backpacks with a robust carbon fiber reflector that's rugged enough for operations in harsh conditions. The antenna assembles in 5 minutes.
AvL's Manual FlyAway Antennas are lightweight, compact, portable, and robust with carbon fiber reflectors and easy-to-assemble tripods. AvL's Az/El positioners feature easy pointing as well as fine-tuning, and are easy and quick to assemble. As with all AvL antenna systems, the Manual FlyAway Antennas interface with any modem, RF electronics, or satellite service, and achieve pointing accuracy and pointing stability even in the most extreme conditions.
AvL Manual FlyAway Antenna Systems offer the following features and benefits:
Single- or multi-band options for worldwide operation
Prime focus offset optics with global mode-matched feed
Robust carbon fiber reflectors and tubes
Feed rotation for polarization
Lightweight, compact and portable two-case configuration
Manual full range of motion
Quick set-up – operational in minutes
Interface with all types of RF electronics and services
Numerous packaging options including backpacks and rugged cases
- Lightweight, compact and portabletwo-case configuration; total weight is75 lbs.withoutcases
- Fast interchangeable feed system with quad-band capability: Ku, Ka, and X; DBS capable
- Robust 1.2m segmented 4-piece carbon fiber reflector and tripod base for operations in harsh conditions
- Tool-less assembly in 5 minutes
- Feed rotation for polarization
- Easy coarse pointing and fine peakingAz/El positioner
- Manual full range of motion
- Interface with all types of RF electronics and services
- Tripod legs can be anchored to increase wind resistance
- Optional remote hand crank
- Optional BUC mounting
|Reflector Construction
|Carbon Fiber
|Azimuth - Coarse
|360° Continuous
|Azimuth - Fine
|360° Continuous
|Elevation - Coarse
|+5 - 95°
|Elevation - Fine
|+/ - 2°
|Polarization (Ku)
|± 95°
|RF Interface
|BUC Mounting
|Feed Boom (10 lbs max), or Remote
|Two connectors at Amplifiers
|Electrical Interface
|Connectors at Amplifiers
|Set-up Time
|Less than 5 minutes
|Assembled Weight
Note: BUC and LNB are CFE
|<75 lbs. (with Ku Precision Feed)
|Case Integration
SKB Injection Molded Resin – 2-case pack-up
Note: SKB cases are MIL-STD-810F and MIL-C-4150J compliant
|Reflector case: 33.7"x28.5"x16.3", 85 lbs. (packed); positioner case: 31.62"x20.5"x15.75, 62 lbs. (packed)
|Wind – Survival (anchored)
|80 mph in stowed position
|Wind - Operational
|Without anchoring
|20 mph
|With anchoring
|30 mph gusting to 45 mph
|Pointing Loss in Wind
|Ku-band Receive, Operational winds
|0.4 dB typical, 0.8 dB max
|-40° to 140° F (-40° to 60° C)
|-40° to 140° F (-40° to 60° C)
|Available Options, Upgrades & Services
|(View our PDF Datasheet for more information)


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Registered 2010-10-20 by master5o1
A mobile interface for bbPress forum software.
A mobile interface to bbPress forum software using the bbPress XML-RPC api and XML-RPC for PHP library. Created to meet the needs of Tartarus: The After Effect (http://
Support: http://
Example: http://
Project information
- Maintainer:
- master5o1
- Driver:
- Not yet selected
- Licence:
- GNU GPL v3
View full history Series and milestones
Loading graph...
trunk series is the current focus of development.
All code Code
- Version control system:
- Bazaar
- Programming languages:
- php, html
All bugs Latest bugs reported
Bug #677106: Authenticate First
Reported on 2010-11-18
Bug #675837: "Cannot modify header information" When loading mobile page
Reported on 2010-11-16
Bug #675139: tags
Reported on 2010-11-14
Bug #675136: Split XMLRPC functions into another file
Reported on 2010-11-14
Bug #670151: >>id and >>id|post linking doesn't link to the right page
Reported on 2010-11-02


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- The Admin Team
Hey LOOK! We Dropped a #2
Hello Battleborn!
As most of you know, clan participation has been at an all-time high. Pretty much everyone has been able to meet the participation criteria for the last several months. What some of you may not know is that over the summer we've had to maintain a waiting list for prospective members.
Between the release of Shadowkeep and the Destiny 2 base game becoming free with New Light, we are expecting the number of players seeking clans to increase dramatically. In order to meet this demand, your admin team has decided to create a second division of our clan: DoD Battleborn Deux!
This is great opportunity to add members to our roster and increase your ability to find a team on any given night. While xxCH3M1STxx will be moving to the new clan to serve as Founder, along with Bup89 as his backup, nothing else will be changing from an Admin perspective. Each Admin will still be serving in their current capacity in the overall clan.
Which brings up an important point: while we may now have two clans, Battleborn has been and will continue to be one unit. We will be sharing the same Discord, raiding and getting crushed at PvP the same way we are now: together. Members of this new clan are in no way to be considered second class. They are just as Battleborn as the rest of us.
With new members, come new personalities and levels of experience. This is not only an opportunity to fill out your fireteam.
This is a chance to make some new friends. To pass your skill and knowledge to other players that might be entirely new to the world of Destiny or significantly lapsed. Most importantly, it's a chance for us to demonstrate what Battleborn is all about: pictures of smoked meat and an appreciation for a good fart joke. Wait. That doesn't seem right. It's also a chance for to us demonstrate what Battleborn is all about: being a community that welcomes players of all skill levels and gives them a chance to find a home when they are fighting amongst the stars.
-the Admin Team