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Chromatin dynamics and stability are both required to control normal development of multicellular organisms. Chromatin assembly factor CAF-1 is a histone chaperone that facilitates chromatin formation and the maintenance of specific chromatin states. In plants and animals CAF-1 is essential for normal development, but it is poorly understood which developmental pathways require CAF-1 function.
Mutations in all three CAF-1 subunits affect Arabidopsis trichome morphology and lack of CAF-1 function results in formation of trichomes with supernumerary branches. This phenotype can be partially alleviated by external sucrose. In contrast, other aspects of the CAF-1 mutant phenotype, such as defective meristem function and organ formation, are aggravated by external sucrose. Double mutant analyses revealed epistatic interactions between CAF-1 mutants and stichel, but non-epistatic interactions between CAF-1 mutants and glabra3 and kaktus. In addition, mutations in CAF-1 could partly suppress the strong overbranching and polyploidization phenotype of kaktus mutants.
CAF-1 is required for cell differentiation and regulates trichome development together with STICHEL in an endoreduplication-independent pathway. This function of CAF-1 can be partially substituted by application of exogenous sucrose. Finally, CAF-1 is also needed for the high degree of endoreduplication in kaktus mutants and thus for the realization of kaktus' extreme overbranching phenotype.
Chromatin stability and dynamics have to be well balanced to guarantee normal development. While flexibility of the chromatin structure permits developmental transitions necessary during the life cycle of an organism, epigenetic as well as genetic information has to be reliably propagated within a certain developmental phase. Various protein complexes have been described to be involved in chromatin regulation [1-3]. One biochemically well characterized complex involved in chromatin replication is Chromatin Assembly Factor CAF-1, which deposits histones H3 and H4 in a replication-dependent manner onto DNA (for review see [4,5]. This complex was initially identified as a negative supercoiling-inducing factor in human cell extracts [6,7] and is conserved among all major eukaryotic lineages. Homologs have been found in yeast (subunits CAC1, CAC2, CAC3; [8], in mammals (p150, p60, p48; [9], in insects (p180, p105/75, p55; [10-12] and in plants (FASCIATA (FAS) 1, FAS2, MSI1; [13,14].
Yeast CAF-1 mutants have impaired maintenance of silencing at mating type loci and near the telomeres, and exhibit increased sensitivity towards ultraviolet radiation [8,15-20]. In higher eukaryotes, CAF-1 is specific for replication-coupled deposition of the H3.1 variant, while other histone chaperones deposit the H3.3 variant (called H3.2 in plants) in a replication-independent way [21,22]. Because mostly H3.3 and much less H3.1 is found in active chromatin [23], it has been proposed that CAF-1-mediated assembly of chromatin facilitates transcriptional repression through H3.1 deposition [24]. A recent report that H3.1-containing nucleosomes are more stable than H3.3-containing nucleosomes supports this model [25]. Replication-coupled deposition of H3.1 by CAF-1 is essential in metazoans, because loss of CAF-1 function causes severe defects in chromatin metabolism and eventual cell death in mouse and human cells [26-30]. Loss of CAF-1 causes developmental arrest in Xenopus laevis [31], Drosophila [32] and zebrafish [33].
Arabidopsis thaliana is the only higher eukaryote for which viable CAF-1 mutants are available (for review see [34]). Mutants deficient in FAS1 and FAS2, the two larger subunits of Arabidopsis CAF-1, were originally isolated for their altered phyllotaxis and their flattened and bifurcated stems [35,36], which is a phenotype known as fasciation [37]. Fasciation is associated with altered expression of WUSCHEL, which is a key regulatory gene that defines the stem cell niche in the shoot apical meristem (SAM) [13]. Misspecification of the WUSCHEL domain alters size and shape of the meristem, which subsequently changes primordia spacing and therefore causes distortion of phyllotaxis. In contrast to null mutants of FAS1 and FAS2 that are viable null mutants of the smallest CAF-1 subunit MSI1 are lethal [38]. This lethality is not caused by loss of CAF-1 function, however, but by loss of the FERTILIZATION INDEPENDENT SEED DEVELOPMENT (FIS) complex, of which MSI1 is a subunit as well [39].
Initial research with fas mutants focused on CAF-1 function in meristematic tissue [13,35,36] Recent studies showed, however, that CAF-1 is also needed for complete compaction of heterochromatin and maintenance of transcriptional gene silencing [40,41], homologous recombination [42,43], regulation of endoreduplication [34], and cell differentiation [44].
Trichomes or leaf hairs protrude from the leaf surface to protect the plant against adverse environmental conditions and herbivorous insects [45,46]. Depending on the plant species and function, trichomes are uni- or multicellular, metabolically active or inactive structures. In Arabidopsis thaliana, trichomes are single, living cells with a complex structure, which makes them well suited to study cell determination and differentiation. Trichomes originate from the epidermal cell layer and are evenly spaced by lateral inhibition (for an overview see: [47]). After determination, the trichome progenitor cell stops division and switches to endoreduplication. The cell enlarges and protrudes from the epidermal cell layer. On rosette leaves, two branching events give trichomes their characteristic three-ended morphology. Genetic analyses have revealed a complex regulatory network that controls trichome spacing and differentiation. Two major groups of genes control branching. Some of the genes influence branching directly, while others control branch number in an endoreduplication-dependent manner (reviewed by: [48]).
We have previously reported that trichome differentiation requires a functional CAF-1 complex, but it remained open in which genetic pathway CAF-1 acts during this process [44]. Here we provide evidence that CAF-1 and STICHEL (STI), which encodes a protein with similarity to ATP-binding eubacterial DNA-polymerase III-subunits [49], together control trichome differentiation in an endoreduplication-independent pathway.
Sucrose suppresses the CAF-1 mutant trichome phenotype
During the analysis of trichome development in CAF-1 mutants we observed that fas2-1 seedlings had fewer trichomes with supernumerary branches when grown on MS medium containing sucrose than on MS medium alone (data not shown). Carbohydrates control cell cycle activity and are known to influence plant development and organ formation (for review see: [50,51]), but a role in trichome development has not been reported. To test whether sucrose generally influences trichome development, wild type and CAF-1 mutant plants were grown on MS medium with 1% sucrose. Control plants were grown on MS medium containing 1% of the non-metabolizable sugar sorbitol. The number of trichome branches was recorded for the first and second rosette leaves (Fig. 1). In wild type plants of Columbia (Col), Enkheim (En) and Landsberg erecta (Ler) accessions, sucrose caused a small but consistent shift towards trichomes with fewer branches. This decrease in branch number was statistically significant (chi-squared test, p < 0.05) for Col, fas2-4, msi1-as, En and fas2-1. In CAF-1 mutants, sucrose suppressed, at least partially, the supernumerary branch phenotype. The effect was strongest in msi1-as, and weakest in fas1-4. Mutations of STI and GLABRA3 (GL3), which positively regulate trichome branching through the endoreduplication-independent and endoreduplication-dependent pathway, respectively, usually produce trichomes without branching (sti) or only a single branching event (gl3). Both mutants were unaffected by sucrose (Fig. 1). Thus, sucrose affects branching during trichome differentiation and can partially substitute for loss of CAF-1.
Figure 1. Sucrose influences trichome morphology in CAF-1 mutants. The overbranching of rosette leaf trichomes in CAF-1 mutants is reduced on medium containing sucrose. Trichome branch number was assessed on the first and second primary leaves of wild-types Col (294, 342), En (161, 136) and Ler (119, 449) and the mutants fas1-4 (223, 171), fas2-4 (164, 121), fas1-1 (123, 98), fas2-1 (66, 124),gl3-1 (42, 55) and sti-56 (129, 171). Figures in parentheses represent the number of trichomes analyzed on sorbitol and sucrose, respectively. Note that gl3-1 produces only a limited number of trichomes on the primary rosette leaves. Plants were grown on MS medium supplied with either 1% sorbitol (unmarked bars) or 1% sucrose (bars highlighted in yellow).
Sucrose does not generally attenuate CAF-1 mutant phenotypes
It is possible that sucrose generally suppresses CAF-1 mutant phenotypes. Detailed analysis of CAF1 mutants showed, however, that only trichome branching but not other aspects of the CAF-1 mutant phenotype were attenuated by sucrose. In fact, distortion of phyllotaxis was strongly enhanced in fas2-1 mutants grown on MS medium with sucrose (Fig. 2). The angles between successive leaves were highly irregular, and some primordia did not complete differentiation into leaves but showed weak radialization (data not shown). In addition, internodes elongated and the usual compact appearance of a rosette was lost (Table 1). Furthermore, even after fas2-1 seedlings were transferred from sucrose medium to soil, about 10% of the plants showed defects in flower development (Fig. 2F, G). These plants produced flowers with missing or severely malformed petals and stamens, and unfused carpels. Additionally, ectopic ovules were sometimes produced at the margin of cauline leaves. Such phenotypes were not observed in control plants. This strong enhancement of the mutant phenotype was not observed in the fas1-1, fas1-4 and fas2-4 CAF-1 mutant alleles, suggesting that Ler is especially sensitive to loss of CAF-1 function when additional factors such as sucrose perturb early development.
Figure 2. Sucrose influences the phenotype of fas2-1 mutants. Wild-type Ler and fas2-1 mutant seedlings were grown on medium supplied with 1% sorbitol or with 1% sucrose. A: Ler grown on sorbitol. B: Ler grown on sucrose. C: fas2-1 grown on sorbitol. D: fas2-1 grown on sucrose. Note the severely distorted phyllotaxis. E: fas2-1 grown on sucrose exhibiting internode elongation. F and G: Flowers of fas2-1 plants grown for 2.5 weeks on sucrose and later on soil. H: Flowers of a Ler plant grown for 2.5 weeks on sucrose and later on soil. Scale bars: 0.5 mm.
Table 1. Sucrose strongly alters the phenotype of fas2-1 but not of the other CAF-1 mutants or wild-type plants.
Mutations in CAF-1 partially suppress the kaktus supernumerary branching phenotype
We previously suggested that CAF-1 controls trichome branching via an endoreduplication-independent pathway [44]. To further test this hypothesis, we first analyzed fas2-1 kak-2 double mutants. KAKTUS (KAK) encodes a putative HECT-domain E3 ligase [52], and kak mutant trichomes have increased ploidy levels and highly supernumerary branches [53]. Characterization of the trichome morphology on rosette leaves of fas2-1 kak-2 double mutant plants revealed that the two alleles were not epistatic (Fig. 3A). This result is consistent with the hypothesis that CAF-1 controls trichome branching independent of the KAK-containing pathway. However, the branching phenotype of fas2-1 kak-2 trichomes was intermediate to the two single mutants rather than additive, suggesting that KAK and CAF-1 can influence each other.
Figure 3. Trichome phenotype in fas2-1 kak-2 double mutants. A: Trichome branching in Ler, fas2-1, kak-2 and fas2-1 kak-2. The double mutants have an intermediate number of branches per trichome compared to the single mutants. B: Nuclear DNA content of trichomes from Ler, fas2-1, kak-2 and fas2-1 kak-2 leaves. The DNA content of trichome nuclei of fas2-1 kak-2 mutants is in between the DNA content of the single mutants.
Loss of CAF1 function restricts DNA endoreduplication in kak-2 mutants
While fas2-1 mutants and wild-type plants have the same DNA content of trichome nuclei [44], mutations in KAK allow additional rounds of endoreduplication in leaf hair nuclei [53]. However, CAF-1 function is needed for chromatin integrity and has been suggested to be required during cell cycle progression [40]. It was therefore possible that loss of CAF-1 function in the fas2-1 kak-2 mutant restricts the kak endoreduplication potential and thus limits trichome branching in the fas2-1 kak-2 mutant. Analysis of the DNA content revealed that trichomes of fas2-1 kak-2 mutants had on average one third less nuclear DNA than trichomes of kak-2 single mutants (Fig. 3B). This level was between the numbers of endocycles observed in fas2-1 and kak-2. One possible explanation is that CAF-1 is needed for efficient progression through the endocycle in trichomes. Such a limitation would be consistent with the proposed slower progression through S-phase in CAF-1 mutants [40,54].
CAF-1 and STICHEL act together in the endoreduplication-independent pathway of trichome differentiation
Analysis of fas2-1 kak-2 (this work) and fas2-1 gl3-1 [44] double mutants suggested that FAS2 acts in a pathway parallel to KAK and GL3 and controls trichome branching in an endoreduplication-independent manner. STICHEL (STI), a protein with similarity to eubacterial DNA-polymerase III-subunits [49], also controls trichome branching in an endoreduplication-independent pathway. To test whether CAF-1 functions in the STI-pathway for trichome differentiation, fas2-1 was crossed with a strong and a weak sti allele. While sti-56 almost completely abolishes trichome branching, sti-40 develops many trichomes with one branching event [49,55]. Analysis of trichome morphology of the fas2 sti double mutants revealed strong, although not complete epistasis of the sti-56 null allele over fas2 (Fig. 4). Interestingly, fas2 fortifies the weak phenotype of the hypomorphic sti-40 allele. Together, these results suggest that FAS2 and STI function together in the same pathway for trichome differentiation.
Figure 4. Mutations in STI are epistatic over fas2. Trichome branching in Ler, fas2-1, sti-40, sti-56, fas2-1 sti-40 and fas2-1 sti-56. Double mutants between two sti alleles and fas2-1 exhibit the same branching phenotype as the two sti alleles alone.
We have reported earlier that FAS2 controls trichome branching in the context of the CAF-1 complex [44]. To test the hypothesis that CAF-1 and STI function in the same pathway, we generated double mutants of fas1 and gl3, kak and sti. The trichome branching phenotypes of the various double mutants with fas1 and fas2 were similar: fas1-4 gl3-1 exhibited intermediate phenotypes (Fig. 5A) compared with the single mutants, while fas1-4 sti-40 and fas1-4 sti-56 again showed strong epistasis of sti over fas1 (Fig. 5B). Furthermore, fas1-4 kak-2 double mutants had a similar partial suppression of the kak phenotype as did the fas2-1 kak-2 double mutants (Fig. 5B). These results are consistent with our view that CAF-1 and STI function in the same pathway of trichome differentiation.
Figure 5. Genetic interactions of fas1-4 with gl3-1, si-40, sti-56 and kak-2. A: Trichome branching on rosette leaves of fas1-4, gl3-1 and fas1-4 gl3-1. The double mutant is intermediate to the single mutants. B: Trichome branching on rosette leaves of fas1-4, sti-40, sti-56, fas1-4 sti-40 and fas1-4 sti-56. The two sti alleles are epistatic over fas1-4. C: Trichome branching on rosette leaves of fas1-4, kak-2 and fas1-4 kak-2. The strong overbranching phenotype of kak-2 is partially suppressed by fas1-4.
Because sti showed epistasis over CAF-1 mutant alleles, it is likely that STI acts downstream of CAF-1. One possibility is that CAF-1 is needed for correct STI expression during trichome differentiation. To test this hypothesis we measured STI mRNA levels in CAF-1 mutants by quantitative RT-PCR. However, STI transcript levels were not significantly increased in fas1 and fas2 trichomes (Fig. 6). Similar results were obtained for STI expression in fas1 and fas2 seedlings and apices (data not shown). These results suggest that CAF-1 affects STI function instead of modulating STI expression.
Figure 6. H3.2 but not STI expression is changed in fas1-1 and fas2-1 trichomes. A: STI transcript levels were measured by quantitative RT-PCR in En, Ler, fas1-1 and fas2-1. Expression is shown relative to the corresponding wild type. B: H3.2 transcript levels were measured by quantitative RT-PCR in En, Ler, Col, fas1 and fas2 mutants and msi1-as. Expression is shown relative to the corresponding wild type.
H3.2 is up-regulated in fasciata mutant trichomes
We previously showed that transcription of the gene for replacement histone variant H3.2 was upregulated in fas1-1, fas2-1 and msi1-as seedlings [40]. H3.2 is incorporated by a CAF-1 independent pathway into nucleosome of chromatin found mostly in transcriptionally active, less compact chromosome regions (reviewed by [4,56]). We asked whether altered trichome differentiation in CAF-1 mutants was correlated with increased expression of H3.2 in trichomes. RNA was extracted from trichomes of wild-type, CAF-1 mutants and msi1-as plants, and mRNA levels of the H3.2 gene At1g13370 were determined by quantitative RT-PCR. This analysis showed that H3.2 transcript levels were indeed increased by about 100-fold in trichomes of CAF-1 mutants and msi1-as plants (Fig. 6B). These results show that loss of CAF-1 function causes increased expression of H3.2 not only in whole seedlings but also in trichomes. Thus, it is likely that chromatin of CAF-1 mutant trichomes contains increased amounts of the H3.2 variant histone.
Trichome cell specification and maturation provide a good model system to study cell differentiation in Arabidopsis. Analysis of trichome differentiation has revealed a complex gene network that directs and controls the cell determination, specification and differentiation process [48,57,58]. Here, we report the effects of mutations in the chromatin remodeling complex CAF-1 on trichome development and the genetic interaction of CAF-1 mutant alleles with the trichome regulators GL3, STI and KAK.
Because CAF-1 mutants have increased trichome branching but normal endoreduplication [44], CAF-1 limits branching during trichome maturation independent of endoreduplication. Genetic evidence suggests that CAF-1 acts parallel to the GL3-KAK pathway (Fig. 7), which promotes trichome branching through the control of endoreduplication ([44,59], this work). Nevertheless, CAF-1 is needed for the GL3-KAK pathway to function normally, because the kak phenotype is partially suppressed in kak-2 fas2-1 double mutants. The kak-2 fas2-1 double mutants do not only have less trichome branching but also a lower DNA content than kak-2 single mutants. These results suggest that CAF-1 is needed for the increased endoreduplication cycles in kak-2 trichomes. One possible explanation for this observation is that the slower progression through the S phase in the mitotic cell cycle, which we proposed for CAF-1 mutants earlier [40], impedes the increased endoreduplication activity in kak-2 mutant trichomes. In seedlings and leaves, CAF-1 restricts endoreduplication [34,42-44], and it is possible that lack of CAF-1 triggers additional endocycles in certain cell types with low endoreduplication, but that CAF-1 is also needed to sustain multiple rounds of endocycles in cells types with high endoreduplication such as kak-2 trichomes.
Figure 7. Model of CAF-1 function in trichome branching. The initiation of branching is regulated by two independent pathways. In the first pathway, Gl3 is a postive regulator and KAK is a negative regulator. In this pathway, endoreduplication triggers trichome branching. In the second pathway, STI is a positive regulator and CAF-1 is a negative regulator. Exogenous sucrose can partly substitute the negative function of CAF-1. CAF-1 is also required for extensive endoreduplication such as in the kak-2 mutant. Images represent trichome phenotypes in the respective mutants.
Exogenous sucrose alleviates the CAF-1 mutant trichome branching phenotype and weakly suppresses trichome branching in wild-type plants. Since the branching phenotype of CAF-1 mutants grown on soil, which constitutes a less defined but rich medium, was much more similar to the trichome phenotype of CAF-1 mutants grown on sorbitol than on sucrose (data not shown), we suggest that the suppression of trichome branching results from sucrose signaling rather than a starvation effect. Sucrose is known as a potent signaling molecule that controls gene expression, cell cycle and development [50,51]. However, to our knowledge no effect of sucrose on trichome development has been reported before. Sucrose promotes cell cycle progression [60] and can induce endoreduplication [61], but these effects most likely do not explain the observed reduced trichome branching. More rapid progression through the cell cycle and faster growth on sucrose-containing medium could amplify defects associated with chromatin assembly during S-phase in CAF-1 mutants. We found that sucrose greatly enhances the organ development phenotype of fas2-1 in Ler, and mildly enhances this phenotype of other CAF-1 mutant alleles. We propose that specifically during trichome development, sucrose signals can partially substitute for the CAF-1 requirement by a currently unknown mechanism.
Together, we observed (i) that CAF-1 mutants in a wild-type background have increased trichome branching but no increased endoreduplication, (ii) that CAF-1 mutants and gl3 mutants (defective in the endoreduplication-dependent pathway) show an additive interaction, (iii) that CAF-1 mutants and sti-56 null mutants (defective in the endoreduplication-independent pathway) show an epistatic interaction, (iv) that CAF-1 mutants enhance the phenotype of the hypomorphic sti-40 allele (partially defective in the endoreduplication-independent pathway) and (v) that CAF-1 mutants and kak mutants (defective in the endoreduplication-dependent pathway) do not show an epistatic interaction. We conclude that the most parsimonious model to explain all results is that CAF-1 acts together with STI in an endoreduplication-independent pathway that is parallel to the endoreduplication-dependent pathway of GL3 and KAK (Fig. 7). In addition, while CAF-1 is not needed for the normal endoreduplication in WT trichomes, CAF-1 is needed for the extranumerous rounds of endoreduplication that occur in kak mutants. The genetic evidence places CAF-1 in the same pathway with STI, an activator of trichome branching that does not affect DNA content [49]. STI shares sequence similarity with the ATP-binding subunit of eubacterial DNA-polymerase III, but the functional relevance of this similarity has not yet been established, and it is not known if STI is a nuclear protein. CAF-1 does not affect trichome branching by modulating STI expression, but acts as a negative regulator in the STI pathway (Fig. 7). It is not known how CAF-1 can negatively regulate the STI pathway. One possibility is that CAF-1 mediated chromatin assembly and compaction [40] are directly needed for normal trichome maturation. Alternatively, it is possible that CAF-1 represses expression of other, limiting components of the STI pathway. CAF-1 mutants have increased expression of H3.2, which is incorporated into chromatin independently of CAF-1. If chromatin of other genes in the STI pathway was enriched in H3.2, the less stable nucleosomes that are formed as a result could facilitate increased transcription, eventually causing increased activity of the STI pathway. In summary, we conclude that CAF-1 is required to support the exceptionally high endoreduplication of kak-2 trichomes but not for normal endoreduplication of wild-type trichomes. In wild-type trichomes, CAF-1 restricts the activity of the STI pathway.
Plant material and growth conditions
Seeds of Columbia (Col), Landsberg erecta (Ler) and Enkheim (En) Arabidopsis thaliana wild-type accessions and of fas1-1 (accession En) [13,35], fas2-1 (accession Ler) [13,36], fas1-4 (accession Col) [44], fas2-4 (accession Col) [44] and gl3-1 (accession Ler) [62,63] mutants were obtained from the Nottingham Arabidopsis Stock Centre. Note that in addition to the used fas1-4 allele in Col and described first in [44], another fas1 allele was described under the same name (fas1-4) by Kirik and collaborators in accession C24 [43]. The msi1-as line has been described before [44]. The mutants kak-2 (accession Ler) [52], sti-40 (accession Ler) [55] and sti-56 (accession Ler) [49] were kindly provided by M. Hülskamp. Seeds were sown on sterile basal salts Murashige and Skoog (MS) medium (Duchefa, Brussels, Belgium), which was supplemented with 1% sucrose or 1% sorbitol when required. Plants were analyzed on plates or transferred to soil ("Einheitserde", H. Gilgen optima-Werke, Arlesheim, Switzerland) 10 days after germination. Alternatively, seeds were sown directly on soil. Plants were kept in Conviron growth chambers with mixed cold fluorescent and incandescent light (110 to 140 μmol/m2s, 21 ± 2°C) under long day (LD, 16 h light) photoperiods or were alternatively raised in green houses.
Analysis of trichome branching
To determine the branching pattern, all trichomes on the adaxial side of the first two leaves of an average of six plants were analyzed.
Ploidy analysis
Ploidy of trichome nuclei was determined as described [44,64]. Briefly, plant tissue was fixed in FAA (50% ethanol, 5% glacial acetic, 10% formaldehyde) and stained for 90 minutes with 130 μg/ml DAPI in McIlvaines buffer (60 mM citric acid, 80 mM sodium phosphate, pH 4.1). Samples were washed twice (15 minutes and 60 minutes) with McIlvaines buffer, and mounted in McIlvaines buffer with 50% glycerol. DAPI fluorescence was recorded with a MagnaFire CCD camera (Optronics, Goleta, CA), or with an Apogee Alta U32 CCD camera (Apogee Instruments, Roseville, CA). Images were quantified using ImageJ. Total fluorescence of at least 30 representative nuclei per experiment was determined and calibrated using guard cell nuclei (n ≥ 30), which are considered to be strictly diploid [64].
RNA isolation, RT-PCR and Real Time PCR
RNA was extracted from seedlings as previously described [65]. For RT-PCR analysis, 0.4–1 μg total RNA was treated with DNase I. The DNA-free RNA (0.2 – 1.0 μg) was reverse-transcribed using a RevertAid First Strand cDNA Synthesis Kit according to manufacturer's instructions (Fermentas, Nunningen, Switzerland). Trichomes for RNA extraction were harvested into a few microlitres RNA later (Ambion, Austin, TX) and then processed like the other samples. Aliquots of the generated cDNA were used as template for PCR with gene specific primers. For qPCR analysis, the Universal ProbeLibrary system (Roche Diagnostics, Rotkreuz, Switzerland) was used on a 7500 Fast Real-Time PCR instrument (Applied Biosystems, Lincoln, CA). Details of the assays used are in Table 2. Analysis of the results was performed according to the method described by Simon [66].
Table 2. qPCR assays.
Authors' contributions
VE performed plant analysis, carried out the molecular and genetic studies and drafted the manuscript. LH participated in the study design and helped to write the manuscript. WG helped to write the manuscript.
We thank Claudia Köhler and Cristina Alexandre for critical reading of the manuscript, and Bartosz Urbaniak for technical help with qPCR. Work in the authors' laboratory is supported by SNF project 3100AO-116060 and ETH project TH-16/05-2.
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Anurag Kashyap's 'Ugly', which is scheduled to hit the theaters in September, opened the fifth edition of the Jagran Film Festival here at Siri Fort Auditorium on Saturday.
'Udaan' helmer Vikramaditya Motwane and Rahul Bhat, who stars in 'Ugly', were present at the venue, along with film directors Subhash Ghai, Sudhir Mishra, Anubhav Sinha and National award winning director Hansal Mehta.
Model turned actor Bhat, who stars in the movie, explained to the viewers, "The beautiful thing about this movie is that it is made how it was supposed to be made as it is based on a true incident."
The movie, also starring Ronit Roy in the lead, is a psychological thriller, written and directed by Anurag Kashyap which will hit the screens later this year on September 19.
The film was also screened in the Directors' Fortnight section at the 2013 Cannes Film Festival. It also opened the 2014 New York Indian Film Festival.
Basant Rathore, Vice President, Jagran Group said, "This year, the fifth edition of the Jagran Film Festival presents a special section 'Cinema and Uprising' with a clutch of seven very special films. Freedom of human spirit has been a subject close to 'The Jagran Philosophy' always and when we were deciding on the festival content, we chose this subject and the films made around this theme."
The Jagran Film Festival will travel to 14 cities across the country including Lucknow, Ranchi, Kanpur, Bhopal, Agra and will conclude in Mumbai.

subset: commoncrawl


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'Crime and Justice' Archives
Credit Card Fraud Increasing in Larimer County
October 18, 2011 The Larimer County Sheriff's Office has experienced an increase in the number of reports of credit card fraud recently. It is not yet clear what is causing this increase but a formal investigation is underway. The Sheriff reminds citizens to be cautious when sharing personal information, especially when responding to emails or text messages. Banks and financial institutions usually will not request your personal information ... Full Story
Colorado Psychiatric Bed Shortage is Burden for
A severe shortage of psychiatric hospital beds in Colorado is putting a large burden on county jail facilities. When mentally ill inmates commit crimes, the issue of competency may be raised. The process of determining competency can take six months or longer. If the individual is deemed incompetent, the wait list for treatment at the hospital to provide restoration of competency can be longer. Someone with significant mental health concerns could be ... Full Story
Jail Deputies Discover Weapons
Date: October 5, 2011 Larimer County Jail deputies successfully ended a plot by inmates making sharpened metal pieces, commonly known as shanks. Yesterday, while on a routine check in one of the outdoor recreation areas, a Larimer County Jail corporal heard the distinct sound of metal scraping on concrete coming from another recreation area. Upon investigation, deputies discovered several inmates involved in trying to sharpen metal pieces approximately five to six inches in ... Full Story
Deputies Assaulted during Arrest Attempt
Date: October 4, 2011 Larimer County Sheriff's Office deputies have arrested Frank Ray Runyan Jr., DOB: 010478, of Fort Collins, on several felony counts including two counts of 1st Degree Assault on a Police Officer. Deputies were called to the 8100-block of Taylor Court in Fort Collins at approximately 5:00 p.m. yesterday on the report of a domestic disturbance. Upon arrival, deputies attempted to talk with Frank Runyan about the complaint. Mr. Runyan attempted to leave the ... Full Story
Lieutenant Andy Josey graduates from the FBI National
Lieutenant Andy Josey of the Larimer County Sheriff's Office Graduates from the FBI National Academy Larimer County Sheriff Justin Smith is pleased to announce that Lieutenant Andy Josey has graduated from the FBI's National Academy Program at Quantico, Virginia. The 246th session of the National Academy included participants from 46 states, the District of Columbia, 28 international countries, three military organizations and five federal civilian organizations. FBI Director ... Full Story
Marijuana Grow Operation Discovered in Livermore
Larimer County Sheriff's Office investigators have arrested two Livermore, Colorado residents after discovering a substantial marijuana grow at their rental home in the 1700 block of County Road 82E. Lacy Ann Patterson, DOB: 040988, and Anthony Michael Ryker, DOB: 093085 were arrested yesterday, September 1, 2011 after approximately 220 live marijuana plants, numerous baggies of packaged marijuana, drug smoking paraphernalia, psilocybin mushrooms, ... Full Story
Man arrested after chase in Fort Collins
Felony Eluding Date: May 10, 2011 Early this morning a Larimer County Sheriff's Office deputy observed a vehicle that was speeding westbound on Mulberry Street near 12th Street. As the deputy turned his patrol vehicle around, the vehicle continued speeding on Mulberry and ran a red light. As the deputy attempted to catch up with the vehicle it turned north on College Avenue. The deputy finally caught up to the speeding vehicle as it turned east onto Mountain Avenue. As the deputy ... Full Story
Lakewood man charged with embezzlement
Lakewood man charged with embezzlement from employee benefit plan DENVER – Mark F. Bishop, a 48 year old Lakewood man, made his initial appearance in U.S. District Court in Denver on Tuesday (May 3, 2011), where U.S. Magistrate Judge Craig B. Shaffer advised him of the pending charges, and the penalties associated with those charges. Bishop was also arraigned, where he entered the pro-forma not guilty plea. Bishop was indicted by a federal grand jury in Denver on April 18, 2011. He faces ... Full Story
Earl Albert Moore indicted on charges of arson
Earl Albert Moore indicted by federal grand jury on charges of arson of a building and use of a destructive device during a crime of violence DENVER – Earl Albert Moore, age 65, was indicted late yesterday by a federal grand jury in Denver for arson and use of a destructive device during a crime of violence, the U.S. Attorney's Office, the FBI's Joint Terrorism Task Force (JTTF), the Bureau of Alcohol, Tobacco, Firearms and Explosives (ATF), and the Jefferson County Sheriff's Office ... Full Story


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Azoteq Adds Coakley, Boyd and Abbett as Representative
Azoteq representative network expands to New England
Austin, TX (PRWEB) February 08, 2013
Azoteq, the world leader in capacitive proximity solutions, today announced that Coakley, Boyd and Abbett has been appointed to represent Azoteq in New England. Azoteq is represented in all the major US markets, central Europe and has extensive Asian coverage.
Azoteq´s ProxSense® offers the most sensitive capacitive sensing solutions with the highest signal to noise ratio in the market today. The high sensitivity enables long range proximity sensing and the ability to implement touch solutions that can work through thick cover materials.
"Azoteq partnered with Coakley, Boyd and Abbett because they have an excellent team with vast experience and access to key customers", said Kobus Marneweck, Azoteq´s VP of Marketing.
"Coakley, Boyd and Abbett is excited about our alliance with Azoteq. We believe there is a large untapped market for this technology, and we look forward to working with our key customers and Azoteq to bring leading edge proximity and touch solutions into real world consumer and industrial products" said Bob Walsh, President of CBA.
Azoteq´s ProxSense® offers the next generation of capacitive proximity and touch solutions. Proximity sensing enables new applications such as detecting when a user´s hand approaches the product. Features that can be implemented with proximity sensing include find-in-the-dark (enables backlight when hand approaches) and air gestures (wave hand to turn on/off, page and scroll). The combination of proximity and touch presents the next evolution in user interfaces.
About Azoteq
Azoteq is the world leader in next generation capacitive proximity and touch user interfaces that takes touch to the third dimension. Azoteq offers solutions for touch keys, sliders, scroll wheels, non-ITO touch screens, 3D touch screens and metal touch panels in consumer, medical and industrial applications. Azoteq has design and manufacturing centers in South Africa and China. Azoteq has sales offices and distributors in the USA, Europe, Asia and South Africa.
About Coakley, Boyd and Abbett:
Coakley, Boyd and Abbett, Inc. (CBA) is a leading electronics manufacturer's representative serving OEMs and distributors in New England. CBA is committed to long-term customer and principal relationships with the highest degree of customer satisfaction.
Editorial Contact & Interviews:
Kobus Marneweck
For the original version on PRWeb visit: http://www.prweb.com/releases/prweb2013/2/prweb10408023.htm


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Ok, thanks to this nice invention the pvp dailies for emblems. I now get to do my favorite game with people on both sides who just don't care. They are only there to get their emblem and it doesn't matter win or loose. I prefer to at least attempt to win. It's impossible to do that when your team thinks it's an arena match and tells you they are only there for the emblems, not the game. Kind of wrecks it for everyone else. I don't think anyone should get credit for throwing a match. If you don't win, you shouldn't get emblems.
Sorry to vent but it is frustrating! I'd like to pug up with more serious players. Maybe a ladder system so I won't have to be around the 'I think this is arena, who cares about flags'.
well join a fleet simples .
Personal i hate the cap & holds so if i do anything i treat it like an arena
So you dont wanna ever team with me as i hardly ever cap .;)
Partly, people are simple-minded and like to run toward things and attack them. This is an immutable constant of human behavior, and cannot be overcome by any gameplay or other design short of extended conditioning or severe trauma.
Partly, though, capturing and holding with the mechanic used in the space Salvage Operation map is just intolerably slow and boring. Who queues up for 3 PvP matches in a row just to "take one for the team" that doesn't care about you in the first place?
However you two do further my point that capture and hold is broken by it's participants and something should be done to help those who wish to play the game the way it was meant to be played. Possibly damage/healing scores could not be counted/displayed and instead the amount of captures?
I am also getting more and more annoyed by this. Just right now:
Another player who was sitting idle around and was just complaining how long that took till it was finally over. He had 0 - 0, neither damage nor healing. He was too busy doing other things, he gets the Emblems anyways and he complained why I would even bother fighting around.
So I have to agree:
Cryptic, please change this mission to 3x win instead of just taking part of the battle. I mean seriously, more and more players have absolutely zero interest to actual play Capture and Hold, they either idle around the starbases or they just fly around shooting instead of actually teamplaying.
If people actually need to work on the map to get their so much loved Emblems, they might even start thinking about playing this mode - else they'd lose and, oh my god, they won't get their reward and have to try again or avoid this mission. :eek:
What is PvP? An Elitist Club, or a game activity you want everyone to participate in?
It can be both. If you're tired of the 'rabble' messin up your precious, then go in only with a team of fleetmates. You can fix it. Really.
It's also nice when the other side can bring a good challenge. Just dominating the opponents isn't really that much fun.
I still don't think it should be winner take all. I'd rather see the dailies be universal. Instead of requiring you to complete a specific task they should be offered as they are now but with the option of completing them in PVP, PVE, or Fleet Actions. That way, PVEers could stick with their comfort area and just run expanses/clusters 9 times, while PVPers could do the type of missions they want. The point being, people who get to play the game that they want to play are less likely to just show up for the credit and leave.
You can do it with winner take all but consider how long a match can take when both sides are playing each other as hard as they can? I'm not saying that the challenge wouldn't be nice, but Cap and Hold could take hours to complete a single daily.
I'm in it for the win though its usaully turn into a pew-pew fest.
Just let it roll off your back, take the emblems and hope for a better team next match.
There is no way to spur those who don't care into caring.
Or ingonre those who only pew-pew and try to capture the points yourself. Eventually your will draw the attention of the other team who will move to stop your and the pew-pew'ers will follow.
Or if you are able - split your team into a capture group and a pew-pew group who keep the enemies too involved to do anything but fight back - thus giving your team more abilty to capture and hold.
I understand wanting a more competent group of players to team with. I have found, however, that if you free yourself from being too serious when playing a game, you can just laugh it off and start having fun with it. This is why I've started using T1, T2, and Runabout ships often in PvP maps. If my team can't pull it together (which they can't, 8 times out of 10) then why should I tie myself in knots over it? Just get some laughs and have some fun.
Its all a game.
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Need more detail as to what can be wrong with my 09 Camry SEI need more detail as to what can be wrong and how much would it cost to get it repaired.
I purchased extended warranty on my 2009 camry which is a certified toyota, purchased in June 2012.
I drove up the curb from driver side and flatten both the left tires, got the 2 new tires and got the wheel alignment done. It happened in July 2012.
The sterring wheel was so hard, went back to firestone, they did it again, still the same problem, went there 3rd time, they checked and said its not the alignment problem, may be rack or pump, they said the power steering fluid is enogh and doesn't look like a problem.
I am not sure if it will be covered under warranty and if I can claim in the insurance and How much would it cost to get it repaired in either a rack or a pump problem.
I would appreciate a reply.
Thanks and Have a Nice Day.
I would start with the power steering pump fuse and the belt and ECM codes and move to mechanical stuff like tie rod ends, suspension, steering column/arms, and ball joints.
If the hard-steering problem started right after the accident, it will probably become part of the accident claim and be excluded if you don't have accidental coverage on your warranty agreement.
If the warranty does cover the accident, find a reputable garage on the referral list that is specialized in suspension and steering would be my next move.
My guess is power stearing pump failure or belt failure. Probably not the belt because I am guessing it's one large serpentine belt that runs all of the accessories. You know when it fails because lots of stuff fails including the alternator. It could be a rack but many times those leak when they fail.
If it was me I would take it to where the extended warranty is from. At a minimum they just diagnose it and you can see if the warranty covers it. if it doesnt and the cost is too much you can take it to another well respected independent in your city.
Vague questions receive vague answers . . . . . .
If the rims, tie rod ends, and control arms aren't bent I would just take it to the dealer and complain about the hard steering and conveniently omit the part about hitting the curb. I would only do that if there were no evidence of hitting the curb though.
I don't see any physical signs of hitting the curb on the body, don't know if any physical damage to inner parts.
I would contact the dealer and see if they can fix it as part of the warranty, otherwise, will get estimates from different reputed mechanic shops.
I am not a DIY person for cars, so dont know much in detail for cars.
Thanks for all your replies.
Seems like a good time to go to the dealership and not bother with 3rd party auto shops.
I got it checked 3 times with firestone and y'day only they told me after checking 3rd time that its not an issue with alignment and may be with the rack or pump.
btw, I have got an appt. with Toyota Dealer for next friday, will let you guys know how it goes and what was it.
Thanks again for all your replies.
I couldn't get earlier appt then next friday.
As you said below, I am not a careless driver, but since I had a new job and I parked at a new place (Levittown Train Stations, PA), couldn't see a curb at night and it happened.
And not just at low speeds, even at 60-70, its not easy, and too tough for my wife since she drives slow.
Sorry, I took it that way, but I never thought that this small mistake could have made so much big deal for me and thought firestone didn't do their job correctly and they never said to me that there can be other issue.
It was only y'day that the manager pointed to me that there was no issue with the alignment and it could be something else, really, I do not understand and I think firestone should have pointed it to me before.
But thanks again for your concern and hopefully, will not cost too much to get it fixed.
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Matchup: Chicago Bears at Green Bay Packers
Date/Time: November 9th, 8:30 p.m. ET
NFL Betting Odds From BetDSI Sportsbook
Spread: Green Bay Packers -7.5
Total: 53
The Sunday night football game for week ten in the National Football League features one of the oldest and best rivalries in all of sports. The Green Bay Packers will host the Chicago Bears in this prime time affair with both teams well rested after a much needed bye week.
Both teams were very happy to have a bye week at this time, especially the Packers who are trying to recover from injuries. The Packers come back from the bye with four out of five games at home and are currently the favorites to win the NFC north.
In recent years, the Packers have used the bye week to get healthy and prepare for the rest of the season. Since Coach McCarthy took over in 2006, the Packers have posted a 7-1 mark in the game after the bye, which is tied for first in the NFL with three other teams. One of the teams they are tied with is the Bears, who are also coming off a bye, so the Sunday night matchup will be a statement game as both teams attempt to gain momentum heading into the second half of the season.
While the Packers just want to get healthy, the Bears just want to get back in the win column. The Bears are staggering after dropping four of five. They are 0-3 at home and in danger of missing the playoffs for the seventh time in eight years. They are having major concerns in the locker room as they are questioning the play of quarterback Jay Cutler as well as head coach Marc Trestman.
Marc Trestman is feeling the heat from passionate Bears fans after a 3-5 start. http://t.co/zZSYI11Axw pic.twitter.com/LUwXcWgE8E
— NBC Sports (@NBCSports) November 1, 2014
The hot seat will only get hotter for Trestman if he is not able to right the ship for this team. A win against the Packers would go a long way to soothe a frustrated fan base and could get the Bears right back into the hunt for the division. Although the offense gets scrutinized the Bears defense has been atrocious. Five-time Pro Bowl defensive end Jared Allen has just 1 1/2 sacks after signing a four-year deal that guaranteed $15.5 million. His string of seven straight seasons in double digits with Kansas City and Minnesota is in jeopardy. He is past his prime and that is allowing opposing offense to score at will.
The Bears need to score early and often against the Packers if the hope to take the pressure off of their defense. This rivalry game could be the turning point for the Bears if they win or they could be handing the Packers another title.
Check out this game and other rivalries like St. Louis at Arizona and Cleveland at Cincinnati in the BTB Betting Center, where we have a database of comprehensive stats and trends. Some basic content is available, but for the full benefit of what the BTB Betting Center has to offer, become a premium subscriber.
Cole Ryan
Cole started off as a part time handicapper and a full time computer programmer. After developing a computer program that consistently provided winners in NFL, NBA, MLB, and college sports Cole went into full time handicapping. The computer program and his knack for winners made him a rising star in the handicapping world. His complex computer program based on algorithms and analytical calculations have led to a 60% ATS rate.


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HOUSTON Charges have been filed against a man accused of holding four men against their will at a northwest Houston home, police said.
Walter Renard Jones, 31, is charged with two counts of injury to the elderly - serious bodily injury in the 263rd state District Court.
Investigators with the Houston Police Department said the four men were living in deplorable conditions while being forced to hand over their monthly incomes.
The victims told officers they were given only scraps to eat inside theresidencein the 8600 block of Whitecastle Lane.
Three of the men, who appeared to be suffering from malnutrition, were taken to LBJ Hospital.
The victims were discovered after a resident called police Friday morning after seeing a man at the window rubbing his stomach as if he was hungry.
When officers arrived at the duplex on White Castle, they were shocked. They described the living conditions as like a dungeon.
There was no bathroom and a chair was the only piece of furniture. The garage had burglar bars and locked doors.
Police were told the men were lured to the home with the promise of food, alcohol and cigarettes. Investigators are looking into whether they were homeless before.
They were also allegedly forced to turn over their Social Security and Veteran s benefits checks to their captor.
Steven Davis said he d been inside the house for about six months. Neighbors were heartbroken to learn what they men had been through.
Oh my God, it just touched my heart, Alberta Ewing said. I mean, I just feel for them. I don t know, it just really got me hurt.
She said that one of them had approached her asking for help just weeks earlier while she was cooking outside, but that she didn t take him seriously because he wasn t crying and she couldn t get him to explain further.
He said, Could you help me? Ewing recalled. I said, What s the problem?
Neighbors said they occasionally saw the men sitting outside.
Virginia Rogers, who lives five houses away, said she greeted them with a wave when she drove by.
I m shocked, Rogers said. I m baffled. I didn t have a clue.
Investigators said that Jones apparently did not live in the house.
Four women live in the other side of the duplex, three of whom appeared to have mental disabilities, police said. The fourth was a caretaker. Unlike the garage, the living conditions inside their home were relatively normal.
Harris County Adult Protective Services is also looking into this case. The agency will be checking into the mental capacities of the men held captive and the official residents, including the homeowner.
Court records showed Jones had previous charges beginning in 2002 for theft, marijuana possession and failing to register as a sex offender.
Police said the investigation into the case is ongoing.
The Associated Press contributed to this story.


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Amber & Sarah talk Netflix/Qwikster confusion, Facebook announcing music at F8, Erly's Colllections, Cloo for toilets on the go, & more!
Amber & Sarah have a plethora of services to try out this week, including Twylah, Codecademy, & Canvas. Plus YouTube founders resurrecting Delicious, Amazon book rentals, & more!
Amber & Sarah on Instragram dragging their Android feet, Pinterest for inspiration, what common threads founders share, why women click on more Facebook ads, & more!
Amber & Sarah chat with the founders of Buffer on scheduling our social lives, Jig.com to fill your needs, SimplyHired.com to make LinkedIn a job-finding machine, Talkoclock.com is Chatroulette for waking up, & more!
Amber & Sarah chat with Sonar founder Brett Martin on the future of geo-friending, Skype buys GroupMe, Google+ starts verifying accounts, Loosecubes for co-working, The Ice Break for couples, & more!
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Suspect killed after officer shot in leg in South Toledo
Authorities serving warrant anticipated resistance
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A shootout Saturday resulted in the death of a drug felon and the wounding of a Toledo police officer.
Dead at the South Toledo scene was Samuel T. Birch, 24, of 225 Linden Place, who was convicted of possession of cocaine and heroin last year in Lucas County Common Pleas Court and sentenced to community control, according to court records.
Officer Robert Orwig, 43, an 11-year veteran of the police department, was recovering at Mercy St. Vincent Medical Center from a leg injury. He was shot in the right calf, midway between the ankle and the knee, Police Chief Mike Navarre said.
The gunfire was exchanged about 1:30 a.m. while four members of the police SWAT team were at the Linden address trying to serve Birch an arrest warrant. Authorities said he violated his sentence by failing to finish a drug-treatment program.
Officer Orwig and Officer Neil Piasecki went to the front door of the house near Western Avenue and Broadway as two other officers, Richard Holland and Jeffrey Hood, went to the rear, Chief Navarre said.
Birch came to the door and was asked by the officers "if Sammy was there," the chief said.
Birch replied there was nobody at the house by that name, then pulled out a 9-mm semiautomatic handgun and fired two rounds.
LUCAS COUNTY JAIL Enlarge
On the second shot, Birch's firearm jammed. The two officers returned fire, with Officer Orwig firing three times and Officer Piasecki firing eight times, the chief said.
Birch was struck at least six times, Dr. Maneesha Pandey, a deputy Lucas County coroner, said after the autopsy yesterday. Dr. Pandey said the cause of death was multiple gunshot wounds with most injuries sustained in the chest and abdomen.
The slug striking Officer Orwig passed through his lower leg, fracturing the fibula, which is the smaller bone behind the shin. Officer Orwig also received a gash on his forehead when he dove over a porch rail. He was in considerable pain, Chief Navarre said.
The chief said the slug that passed through the officer's leg probably came from Birch's weapon, which was later found to contain six more live rounds.
Officer Piasecki was put on paid administrative leave pending an investigation by the Firearms Review Board. Such leaves are required after shootings, Chief Navarre said.
Officer Orwig served in Iraq as a member of Toledo's 323rd Military Police Company.
In the house at the time of the shooting were Chanel Scott, Birch's girlfriend, and her two children, 12 and 5, Chief Navarre said.
They did not witness the shooting, they said.
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Chief Navarre said Birch had served time in prison and had said he "would not be taken alive."
The chief said four officers were used because resistance was anticipated.
"Most warrants are served with a couple of officers. This was a dangerous situation," Chief Navarre said.
Neighbors recalled hearing shots and seeing police and fire crews swarm the area.
PHOTO GALLERY: Click to see images.
Officer Orwig was talking before he was transported, the chief said.
Ashley Cook of 229 Linden said she was watching TV with her boyfriend and 8-year-old son when the shooting occurred. She said she watched from her porch as police reinforcements arrived. The front of her house bears bullet holes from a drive-by shooting last July.
"My lease is up this year and I'm going to try to find something else," she said.
Beverly Venson, who said she was Birch's aunt, sobbed outside the Linden house. She said her nephew was a marijuana user but knew nothing about other drug use.
She described Birch as "hyper and real outspoken. He would speak his mind."
Michael Sifford of 242 Linden said he heard the shooting and watched as police taped off the scene and closed the street to traffic.
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"The neighborhood has its troubles, but most of the time it's OK. They've torn down a lot of the drug houses. I'm here for the duration," he said, pointing to two German shepherds, Wolf and Kennedy, he keeps for security. "Nobody is going to run me off."
Contact Carl Ryan at: firstname.lastname@example.com or 419-724-6050.


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Egypt protesters march on palace
CAIRO Egypt's political crisis spiraled deeper into bitterness and recrimination Friday as thousands of Islamist backers of the president vowed vengeance at a funeral for men killed in bloody clashes earlier this week and large crowds of the president's opponents marched on his palace to increase pressure after he rejected their demands.
The two camps in the country's divide appeared at a deadlock, after President Mohammed Morsi gave a fiery televised speech Thursday night denouncing his opponents and refusing to call off a referendum on a draft constitution promulgated by his allies, even as he appealed for dialogue. The opposition rejected talks, saying he must first cancel the referendum and meet other demands.
With Egypt's crisis now in its third week, anger was mounting in the streets, after the two camps clashed Wednesday in heavy battles outside the presidential palace that left at least six dead and more than 700 injured.
Each side is depicting the conflict as an all-out fight for Egypt's future and identity. The opposition accuses Morsi and his Islamist allies of turning increasingly dictatorial to force their agenda on the country, monopolize power and turn Egypt to a religious state. The Muslim Brotherhood, from which Morsi hails, and other Islamists say the opposition is trying to use the streets to overturn their victories in elections over the past year and stifle popular demands to implement Islamic Shariah law.
The tone was one of a battle cry as thousands of Islamists held funeral prayers Friday at Al-Azhar Mosque - the country's premier Islamic institution - for Morsi supporters killed in Wednesday's clashes. Seeking to rally their side, a series of speakers to the crowd portrayed the opposition as tools of the regime of ousted leader Hosni Mubarak - or as decadent and un-Islamic - and vowed to defend a constitution they say brings Islamic law to Egypt.
"Egypt is Islamic, it will not be secular, it will not be liberal," the crowd chanted in a funeral procession filling streets around the mosque. During the funeral, thousands chanted, "With blood and soul, we redeem Islam," pumping their fists in the air. Mourners yelled that opposition leaders were "murderers."
One hardline cleric speaking to the crowd denounced anti-Morsi protesters as "traitors." Another declared that they will not allow Egypt to become "a den of hash smokers."
"We march on this path in sacrifice for the nation and our martyrs," a leading Brotherhood figure, Mohammed el-Beltagy, told the crowd. "We will keep going even if we all become martyrs. We will avenge them or die like them.
"Bread! Freedom! Islamic Law!" the crowd chanted, twisting the revolutionary slogan of "Bread! Freedom! Social Justice!" used by leftists and secular activists in the 2011 uprising against Mubarak.
At the same time, thousands of protesters against Morsi streamed in several marches from different parts of Cairo toward his presidential palace in an upscale neighborhood for a third straight day. Many were furious over the president's speech the night before in which he accused "hired thugs" of attacking protesters outside the palace Wednesday, sparking the clashes. Most witnesses say the clashes began with Morsi supporters attacked a tent camp set up by anti-Morsi protesters.


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Mon-Fri: 7am-1am EST / Sat-Sun: 7am-11pm EST
ApplianceKitchenAid Dishwasher
Level of DifficultyReally easy
Time to do repairLess than 15 mins
Age of ApplianceMore than 10 years
0 out of 1 people found this instruction helpful
I removed the roll out top wire rack of the dishwasher, turned it over and pulled the old punctured water supply tube off the force fit connectors on each end. I put a small amount of vaseline lubricant immediately inside the mouth of the new tube at each end. I pressed the ends of the new tube over the force fit connectors and tested them by shaking the pipe. I replaced the rack and fastened it into place.
Help other customers find the most helpful instructions.
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Part Number: PS356311
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New Brockton Road Construction
Updated: 11/20/2006 - After 2-years of waiting, prep work has gotten underway on road repairs in central Coffee County.
Updated: 11/20/2006 - After 2-years of waiting, prep work has gotten underway on road repairs in central Coffee County.
Posted: 11/20/2006 - Police are searching for a motive in Monday's fatal shooting at a Jacksonville hospital pharmacy.
Posted: 11/20/2006 - The Florida NAACP is calling for a silent protest at the inauguration of Governor-elect Charlie Crist in January to demonstrate frustration with the length of the investigation into the death of Martin Lee Anderson.
Posted: 11/20/2006 - Members of the King family this morning will unveil the new crypt now shared by Coretta Scott King and Martin Luther King Junior.
Updated: 11/20/2006 - A Florida man has been jailed on a robbery charge in Dothan. Police Chief John Powell says James Paul Thomas of St. Petersburg robbed a man at gun-point and stole the victim's car.
Updated: 11/20/2006 - Gilland Mitsubishi and the Columbia Baptist Association have teamed up to help needy families this holiday season.
Posted: 11/20/2006 - Alabama state troopers estimate that 19 people will be killed on Alabama's highways in traffic crashes over the 102-hour Thanksgiving holiday travel period that begins at 6 pm Wednesday. It ends at midnight Sunday, November 26th.
Updated: 11/20/2006 - State troopers are reporting at least six fatal accidents on Alabama's rural roadways over the weekend.The fatalities include a Coffee County wreck that happened Saturday just after 12 noon.
Updated: 11/20/2006 - Authorities say a third teenager has died from injuries she suffered in this morning's horrific crash of a Huntsville city school bus.
Updated: 11/20/2006 - Dothan police just captured an inmate who escaped from the city jail Monday morning. Police say 19-year-old Cornelius Thomas walked away from the jail dressed only in a pair of hospital gowns.
Posted: 11/19/2006 - Local children are treated to their first drag strip experience.
Posted: 11/19/2006 - More than 30 soldiers volunteer to perform maintenance on a local girls' home
Posted: 11/18/2006 - Paramedics pronounce a Houston County inmate dead.
Updated: 11/18/2006 - Local students volunteer to help Habitat 4 Humanity.
Posted: 11/17/2006 - There were no takers Thursday night at an auction where one of downtown Dothan's premier buildings was up for sale.
Posted: 11/17/2006 - The city of Dothan's environmental services crews are delivering new garbage carts to some addresses.
Posted: 11/17/2006 - A mother now faces up to 10 years in prison for neglecting her disabled child. Police say this is one of the worst cases they've seen when it comes to neglect.
Posted: 11/17/2006 - The Dothan Police Department is taking a proactive stance to help prevent criminal activity this holiday season.
Posted: 11/17/2006 - Daleville woman has pled guilty to beating her four-year-old girl.
Posted: 11/17/2006 - A pet costume contest is being held as part of this year's Dothan Christmas Festivities.
Posted: 11/17/2006 - The National Weather Service confirms that eight tornadoes struck seven different counties in Alabama on Wednesday.
Updated: 11/17/2006 - Geneva County School Superintendent David Snell and the school board are weighing several options to keep teachers from migrating to surrounding districts.
Updated: 11/17/2006 - Auditors are still trying to determine how much is missing from a local Daleville grocery store, after a cashier admitted to stealing a large sum of money.
Posted: 11/17/2006 - We first told you Monday about the Wiregrass-area food bank being stonewalled when it came to their turkey supply. Officials feared there wouldn't be enough turkeys to give to families this Thanksgiving holiday, all because of a turkey production shortage. However, now there is good news!
Posted: 11/17/2006 - A downtown Dothan home became the scene of a SWAT team raid and now three men are behind bars for reportedly selling drugs. News 4's Alex Zequeira was with police when they made the arrests.


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Tech Helproom
It's free to register, to post a question or to start / join a discussion
Problem With MSVCP90.dll
Likes # 0
Posted May 27, 2011 at 6:19AM
I have a problem with MSVCP90.dll. I keep getting the following notice: "The procedure entry point ?_Xfunc@tr1@std@@YAXXZ could not be located in the dynamic library MSVCP90.dll" I get this message when I try to open a program called "Watchtower Library". I Googled this and found that others are having the same problem with Lavasoft's Ad-Aware program. I tried the following without success: reinstalling Watchtower Library, uninstalling Ad-Aware SE, using sfc /scannow, downloading Visual Studio 2010 Service Pack. Is any way I can get this problem resolved?
- Tags:
- msvcp90.dll
- procedure
- entry
- point
Likes # 0
Posted May 27, 2011 at 7:01AM
That message sounds to me that it's found the dll but it's the wrong version without the appropriate bit of code it's looking for.
All dll'd are common routines to save a programmer having to continually repeat the same code. the code snippets are placed into these dll's and referenced in the program.
All I can suggest is see if there's newer one available.
I haven't got that dll and I'm running Visual Studio 2010 Premium so I don't think it comes with that.
Likes # 0
Posted May 27, 2011 at 7:24AM
KrummenUK, Thanks for this information. I have just one question: Where will I locate the file? I looked in the add/remove programs area and found that I have the following on my PC: Microsoft Visual C++ 2008 Redistributable -.86 9.0.30729.17 and 9.0.21022.218, as well as Microsoft Visual C++ 2010 x86 Redistributable 10.0.30219. Should I try to find an updated version of the latter one? I downloaded the Microsoft Studio 2010 Service Pack, which I take isn't the one I should have downloaded? Please let me know which file or program I should look for.
Likes # 0
Posted May 27, 2011 at 8:41AM
Give Hitman Pro a run.
Adaware is not as good as some others.
Worth a try just to be in the safe side.
Or give C Cleaner a run to see if that fixes the problem.
Likes # 0
Posted May 27, 2011 at 8:48AM
Run the system file checker to replace a missing or corrupt dll
Start -Run (Search) - type sfc /scannow (note the space between c and /) - press OK windows will find and replace missing or corrupt system files, it may take a while to complete.
Likes # 0
Posted May 27, 2011 at 8:49AM
MSVCP90.dll should be in C:\windows\system32 folder a copy should be in the dll cache or driver cache folders.
Likes # 0
Posted May 27, 2011 at 9:03AM
If the problem persists Download Revo Uninstaller put it to setting 4 which removes all and remove Adaware.
Only delete things in bold black and leave the rest.When nearly finished it may give you a list of Ad-aware details still left on your computer those will not be in bold black but just tick remove all.
Then try a search to see if any bits have been left behind and remove them.
Then Maybe try using SuperantiSpyware instead of Ad-aware
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Daniel Bullocks is entering his third season as an assistant coach at the University of Northern Iowa. Bullocks works primarily with the Panther secondary.
Bullocks helped coach a young secondary and safety group during the 2013 season, seeing newcomers Tate Omli and Ray Mitchell shine. Both Omli and Mitchell earned All-MVFC Newcomer team recognition with Mitchell also picking up All-MVFC honorable mention status. The Panthers finished ranked 19th in the FCS in total defense.
Mitchell and Omli were both named National Defensive Player of the Week during the 2013 season under the direction of Bullocks, seeing Mitchell break the UNI-Dome record for interception return yards against Western Illinois (11/25). Bullocks also oversaw the punt return unit in 2013, which was nationally ranked second in the FCS with three blocked punts during the season.
Bullocks, who worked with the cornerbacks last season, helped the defensive corps to 767 total tackles on the season with senior Wilmot Wellington racking up a team-high 85 tackles and helped coach Varmah Sonie to an All-MVFC second team performance. Bullocks also led the team to 10 interceptions on the season for 102 return yards.
Bullocks came to UNI after playing safety in the National Football League for the Detroit Lions for five years. Bullocks was drafted in the second round to the Lions in 2006 and played with the franchise until 2011. He finished his career with 168 tackles. In 2008, he was voted for the Ed Block Courage Award for the Lions and was the Chuck Hughes Most Improved Player.
Bullocks played college football for the University of Nebraska where he played in 48 games with 33 starts. He finished his career with 226 tackles, 22 pass breakups and eight interceptions. He was named to the second team All-Big 12, Big 12 Fall Commissioner's Academic Roll, the Lott Trophy Watch List and the Jim Thorpe Trophy Award Watch List in 2005. Bullocks is the founder of the Bullocks Foundation, which mentors and establishes scholarships for student-athletes to further their education in Chattanooga, Tenn. Bullocks, a native of Chattanooga, Tenn., earned his degree from Nebraska in sociology in 2005.
Bullocks has a wife, Vanessa, and three daughters, Bella, Thailia and Starlet.


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Barkley L. Hendricks
Barkley L. Hendricks (born 1945, Philadelphia, Pennsylvania) is a contemporary American painter who has made pioneering contributions to black portraiture and conceptualism. While he has worked in a variety of media and genres throughout his career (from photography to landscape painting), Hendricks' best known work takes the form of life-sized painted oil portraits. In these portraits, he attempts to imbue a proud, dignified presence upon his subjects, most frequently urban people of color. Hendricks' work has been noted as unique for its matrimony of both American realism and post-modernism.
Hendricks earned his certificate at the Pennsylvania Academy of the Fine Arts, and received both his Bachelor's and Master's degrees from Yale University. Hendricks is Professor Emeritus of Studio Art at Connecticut College.[1]
Hendricks' work can be viewed in many public institutions, including the National Gallery of Art, the Brooklyn Museum, the Chrysler Museum of Art, the Nasher Museum of Art at Duke University, and the Philadelphia Museum of Art, among others. Hendricks' first career painting retrospective, titled Barkley L. Hendricks: Birth of the Cool, with works dating from 1964 to present, was organized by Trevor Schoonmaker at the Nasher Museum of Art at Duke University in spring 2008, traveled to the Studio Museum in Harlem, the Santa Monica Museum of Art, the Pennsylvania Academy of the Fine Arts, and the Contemporary Arts Museum Houston.[2] Hendricks's work was featured on the cover of the April 2009 issue of Artforum Magazine, with an extensive review of Barkley L. Hendricks: Birth of the Cool. Hendricks is represented by Jack Shainman Gallery in New York.[3]
Public collections[edit]
Hendricks's work is represented in a number of major museum collections, including the National Gallery of Art, the Philadelphia Museum of Art, the Studio Museum in Harlem, and the National Endowment for the Arts.[4]
- ^ "Connecticut College: Barkley Hendricks". Connecticut College.
- ^ "Barkley Hendricks: Birth of the Cool". Nasher Museum of Art.
- ^ "Jack Shainman: Barkley Hendricks". Jack Shainman.
- ^ "30 Americans: Barkley Hendricks". Corcoran Gallery of Art.
External links[edit]
- Hendricks Faculty Homepage, Connecticut College
- Retrospective exhibition and catalogue at the Nasher Museum of Art – "Barkley L. Hendricks: Birth of the Cool"
- Hendricks Birth of the Cool interview on YouTube
- Hendricks interview on WUNC / NPR
- Pop Art Meets Photorealism (NY Times)
- Hendricks Birth of the Cool review in NY Times
- Hendricks Birth of the Cool review in Time Out New York
- Hendricks on the cover of Artforum


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Advocacy Group Promotes Fire Sprinklers With Introduction of PSAs Featuring People Who Lost Family Members in Fires
PLEASANT VIEW, Tenn., Nov. 1, 2010 /PRNewswire/ — Common Voices, an advocates' coalition determined to create a Fire Safe America, released a series of six new Public Service Announcements (PSA) that tell the stories of women who have lost family members in fires and explain the need for fire sprinklers in homes. Each PSA has a personal message from Common Voices advocates, but many with a twist.
Advocate Vina Drennan's husband was a firefighter who lost his life fighting a house fire. She stresses how fire sprinklers can be "like having firefighters in your home that work 24/7, 365 days a year." Drennan's goal is to get people talking about fire sprinklers in homes.
"Vina is a different kind of advocate, working tirelessly to raise awareness about firefighter safety since her husband's death in 1994," explains Vickie Pritchett, facilitator of the coalition. "Her somber reminder about losing her husband highlights the important role that firefighter safety has on the nation's fire service."
Donna Henson lost her son in an off-campus house fire. She relates fire sprinklers in homes to air bags in cars. The main point being that once technology is proven to save lives, it must be incorporated into governing codes and manufacturing requirements.
Justina Page, who lost her son in a house fire and who is a burn survivor, shows her emotion as she gazes out a window, watching neighborhood children playing and discussing how she will never again be able to see her son play outside. Her direct plea to the viewer to install fire sprinklers and her genuine account of "if I had only known…" makes the issue and solution obvious and simple.
Amy Acton, Executive Director of The Phoenix Society for Burn Survivors, reminds viewers that fire does not discriminate. "It happens to the rich, it happens to the poor, it could happen to you," she says. Acton, who is also a burn survivor, notes that many times we focus only on those who die, forgetting the impact to society and to the survivors themselves. Burn injuries occur every 57 minutes, and are a significant part of our nation's fire problem.
Gail Minger explains how her son went away to college, but was never able to graduate due to a deadly fire that took his life. The "deadly mistake" that she made in the numerous safety checks before her son left for college was that she did not check if his campus residence had fire sprinklers.
Bonnie Woodruff tells the story of her son bringing home a turtle when he was a child. She tells viewers that the turtle is still alive while her son is not. He died in a fraternity house fire on Mother's Day. She describes it as a senseless death that could have been prevented with fire sprinklers. Woodruff highlights her current efforts as an advocate challenging those who work to defeat code requirements that include fire sprinklers.
According to Pritchett, state and local battles are currently occurring across the nation. Through these women's advocacy efforts, they hope that their stories and the statistics proving the need for residential fire sprinklers can make a difference and encourage people to get involved with residential fire sprinkler advocacy.
The complete media kit, which is free thanks to the Department of Homeland Security Assistance to Firefighters Act fire prevention grant, is available by visiting www.fireadvocates.org and will also be mailed to fire safety advocacy organizations nationwide to help promote the use of residential fire sprinklers.
SOURCE Common Voices