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Differential Regulation of Saccharomyces cerevisiae Phospholipase D in Sporulation and Sec14-Independent Secretion
Simon A. Rudge, Chun Zhou and JoAnne Engebrecht
Genetics April 1, 2002 vol. 160 no. 4 1353-1361
Simon A. Rudge
Department of Pharmacological Sciences, State University of New York, Stony Brook, New York 11794-8651
Chun Zhou
JoAnne Engebrecht
For correspondence: joanne@pharm.sunysb.edu
Saccharomyces cerevisiae Spo14, a phosphatidylcholine-specific, phosphatidylinositol (4,5) bisphosphate-activated phospholipase D (PLD), is essential for meiosis and spore formation. Spo14 is also required for secretion in the absence of the phosphatidylinositol/phosphatidylcholine transfer protein Sec14 (i.e., Sec14-independent secretion). In sporulating cells Spo14 is phosphorylated and relocalized within the cell. In contrast, Spo14 does not relocalize and is not phosphorylated in Sec14-independent secretion. Analysis of a partially phosphatidylinositol (4,5) bisphosphate-activated Spo14 mutant, spo14R894G, revealed that Spo14 function in Sec14-independent secretion, unlike the situation in meiosis, requires fully stimulated PLD activity. Consistent with the differential regulation of Spo14 function during sporulation and secretion, we isolated a mutant allele, spo14-S251P, the product of which is improperly phosphorylated and fails to relocalize and rescue the sporulation phenotype of homozygous spo14 diploids, but supports Sec14-independent secretion. Furthermore, we show that the N-terminal domain of Spo14 is both phosphorylated and sufficient for prospore membrane localization during sporulation. These data indicate that Spo14 phosphorylation and relocalization are essential for the process of sporulation, but dispensable for Sec14-independent secretion. Finally, we demonstrate that Spo14 phosphorylation and relocalization are initiated by nitrogen and glucose limitation and occur independently of the process of meiosis.
THE Saccharomyces cerevisiae SPO14 gene product encodes a phosphatidylinositol (4,5) bisphosphate [PtdIns(4,5)P2]-activated, phosphatidylcholine-specific phospholipase D (PLD) whose localized enzymatic activity is required for the successful completion of the meiotic divisions and is essential for spore formation (Roseet al. 1995; Rudgeet al. 1998b; Sciorraet al. 1999). Genetic analyses indicate that Spo14 is dispensable for mitotically dividing wild-type yeast cells (Honigberget al. 1992; Roseet al. 1995; Ellaet al. 1996; Waksmanet al. 1996). However, Spo14 enzymatic activity is essential for mutant yeast cells to bypass the normally essential requirement for Sec14 in growth and secretion (Sreenivaset al. 1998; Xieet al. 1998).
Sec14, a phosphatidylinositol/phosphatidylcholine transfer protein, is required for Golgi secretory function (Bankaitiset al. 1990). However, yeast cells mutated in at least one of seven genes are able to produce Golgi vesicles independently of Sec14, a process referred to as Sec14-independent secretion (Liet al. 2000b). These genes include KES1 (a yeast homolog of a human oxysterol binding protein; Fanget al. 1996), SAC1 (a phosphoinositide phosphatase; Cleveset al. 1989), and genes encoding for enzymes of the cytidine 5′-diphosphate-choline route of phosphatidylcholine synthesis (CKI1, PCT1, and CPT1; Cleveset al. 1991). The dependency of Sec14-independent secretion on Spo14 most probably reflects the requirement of PLD activity to compensate for the role of Sec14 in regulating phosphatidylcholine and phosphatidic acid levels required for Golgi function.
During sporulation Spo14 function is dependent on cellular relocation. Failure of Spo14 to relocalize to the sites of prospore membrane formation, at the yeast spindle pole bodies, results in the complete absence of spores (Rudgeet al. 1998b). However, the dependency of Sec14-independent secretion on Spo14 intracellular relocalization is not known. We previously proposed that protein phosphorylation drives Spo14 movement during sporulation (Rudgeet al. 1998b); consequently, we were interested in determining whether Spo14 was regulated by these same mechanisms when required for secretion. In this report we demonstrate that Spo14 neither is phosphorylated nor undergoes relocalization in Sec14-independent secretion. Analyses of mutant alleles suggest that PtdIns(4,5)P2 activation is the primary regulator of Spo14 PLD activity in secretion, while phosphorylation and relocalization, in addition to PtdIns(4,5)P2, regulate the enzyme during meiosis.
Strains, genetic manipulation, and media: Genotypes of yeast strains are listed in Table 1. Yeast manipulations were performed and media were prepared using standard procedures (Roseet al. 1990). Yeast transformations were carried out by the lithium acetate procedure (Itoet al. 1983).
Cells were initiated to sporulate in 2% potassium acetate after transfer from synthetic complete medium without uracil (SC-ura). Sporulation frequencies were determined after 48 hr by examining a minimum of 600 cells. Strains were grown in SD(-N) (0.17% yeast nitrogen base without ammonium sulfate or amino acids containing 2% glucose; Takeshigeet al. 1992) for 15 hr after transfer from SC-ura for analysis of Spo14 phosphorylation.
Plasmid construction: spo14-S251P was generated by amplifying the nucleotide sequence corresponding to the phosphorylation domain of SPO14 open reading frame (Rudgeet al. 1998b), using the polymerase chain reaction (PCR) and in vivo recombination with a gapped plasmid (Muhlradet al. 1992). PCR was performed using standard conditions with SPO14 centromere plasmid ME325 as template (Roseet al. 1995). In vivo recombination was achieved by cotransformation of the PCR product and the EcoRI-gapped spo14ΔN centromere plasmid ME419 (Roseet al. 1995).
The construction and characterization of fully functional triple hemagglutin (HA)-tagged and green fluorescent protein (GFP)-tagged Spo14 proteins have previously been described in full detail elsewhere (Rudgeet al. 1998b; Sciorraet al. 1999). HA- and GFP-tagged derivatives of spo14313 (Roseet al. 1995) and spo14S251P were constructed by insertion of HA and GFP sequences (excised from HA- and GFP-SPO14 using SphI; Rudgeet al. 1998b) into the unique SphI site of spo14-313 (pME1686 and pME1759, respectively) and spo14-S251P (pME1153 and pME1163, respectively).
Immunoprecipitation of HA-Spo14 and HA-spo14S251P: HA-Spo14 proteins were immunoprecipitated using mAb 12CA5 (BabCO, Richmond, CA) as previously described in detail by Rudge et al. (1998b). After 15 hr in sporulation medium at 25° or 33.5°, homozygous spo14 diploids containing HA-SPO14 or HA-spo14-S251P in low-copy CEN vectors were collected by centrifugation and Nonidet P-40-soluble fractions prepared for immunoprecipitation as previously described (Rudgeet al. 1998b).
Treatment of immunoprecipitated HA-Spo14 with shrimp alkaline phosphatase: Immune complexes of HA-Spo14 and HA-spo14S251P subjected to treatment with shrimp alkaline phosphatase (SAP) were suspended in 15 μl phosphatase buffer (40 mm HEPES, pH 8.0, 10 mm magnesium chloride) and incubated for 30 min at 30° with 1.5 units of SAP (United States Biochemical, Cleveland) in the presence or absence of phosphatase inhibitors (10 mm sodium pyrophosphate, 5 mm EGTA, 5 mm EDTA; Rudgeet al. 1998b). For immunoblot analysis, the phosphatase reaction was terminated with the addition of 5 μl of 4× Laemmli buffer, and the beads were boiled for 5 min.
Preparation of whole cell extracts: Extracts of yeast proteins were prepared from frozen cell pellets by glass-bead homogenization (Rudgeet al. 1998a). The homogenates were assayed for protein content (Bradford 1976) and an appropriate volume of 4× Laemmli buffer was added. The samples were then boiled for 3 min and centrifuged at 16,000 × g for 10 min prior to SDS-PAGE analysis.
Immunoblot analysis: Immunoblot analysis was conducted essentially as described (Rudge et al. 1998a,b, 2001). Proteins were electrophoretically transferred from SDS-polyacrylamide gels onto nitrocellulose membranes (pore size: 0.45 μm; Bio-Rad Laboratories, Hercules, CA) for 2 hr and HA-Spo14 proteins were detected using mAb 12CA5 and horseradish peroxidase-conjugated anti-mouse antiserum (Amersham International, Buckinghamshire, England) and visualized by enhanced chemiluminescence detection on Kodak BIOMAX MR film (Eastman Kodak, Rochester, NY; Rudge et al. 1998a,b, 2001).
Cytology: For the visualization of GFP-Spo14 and variants, yeast cells were fixed for 15 min at 25° with 3.7% formaldehyde as described (Rudgeet al. 1998b). Samples were mounted in Prolong mounting media (Molecular Probes, Eugene, OR) and viewed and images were captured with a Nikon Eclipse E600 confocal microscope.
PLD-mediated Sec14-independent secretion is more sensitive to changes in the specific activity of Spo14: To better understand the physiological roles of PLD in cells, we sought to differentiate the regulatory properties of Spo14 in yeast meiosis and Sec14-independent Golgi function. The demonstration that Spo14 drives Sec14-independent secretion (Sreenivaset al. 1998; Xieet al. 1998) prompted us to examine how Spo14 is activated to generate phosphatidic acid under these conditions. In theory, the increase in Spo14 activity observed in Sec14-independent secretion (Sreenivaset al. 1998; Xieet al. 1998) could be achieved through several nonexclusive mechanisms: increased protein expression, phosphorylation, relocalization, and increased exposure to substrate (i.e., phosphatidylcholine) and/or activator [PtdIns(4,5)P2]. Analyses of Spo14 protein in Sec14-independent secretion revealed that protein expression, phosphorylation, and localization are not significantly altered in the absence of Sec14 (data not shown). Thus, changes in these properties cannot account for the increase in PLD activity observed in Sec14-independent secretion.
To address whether changes in enzymatic activity contributed to activation in Sec14-independent secretion, we examined in more detail its regulation by PtdIns(4,5)P2. We previously demonstrated that PtdIns(4,5)P2-mediated activation of Spo14 is essential for both sporulation and Sec14-independent secretion (Sciorraet al. 1999). This was achieved through mutagenesis of a newly identified PtdIns(4,5)2-activation domain in eukaryotic PLDs (Sciorraet al. 1999). One mutation in particular, spo14R894G, resulted in a partial loss of Spo14 PtdIns(4,5)P2 activation (Sciorraet al. 1999) and was only partially functional for sporulation and secretion when expressed from a high-copy vector. Surprisingly, spo14-R894G expressed from a low-copy vector is incapable of supporting secretion in sec14-1 kes1 spo14 mutants as measured by growth at 33.5° or 37° (Figure 1). However, the same construct enables homozygous spo14 deletion mutants to sporulate to a similar degree as those cells expressing spo14-R894G on a high-copy vector (spo14-R894G CEN 35 ± 3%, spo14-R894G 2μ 36 ± 5%). Since spo14R894G is not activated to the extent to which PtdIns(4,5)P2 activates wild-type Spo14 (Sciorraet al. 1999), Spo14 function in Sec14-independent secretion appears more sensitive to loss of PLD activity. Moreover, this result reiterates our previous conclusion that PtdIns(4,5)P2 activation of Spo14 drives Sec14-independent secretion (Sciorraet al. 1999).
Sec14-independent secretion is more sensitive to changes in the specific activity of Spo14. spo14-R894G CEN fails to support secretion in sec14-1 kes1 spo14 mutants at the restrictive temperatures of 33.5° and 37°.
The failure of spo14R894G to sustain Sec14-independent secretion at 33.5° and 37° (Figure 1) could simply reflect that this mutation rendered the protein temperature sensitive. However, spo14R894G retains its ability to partially rescue the sporulation defect of homozygous spo14 diploids at elevated temperatures of up to 35° (the maximal permissible temperature for sporulation in the SK-1 strain background). At 25° the sporulation frequencies were 90 ± 4% for SPO14 vs. 61 ± 5% for spo14-R894G, and at 35° the frequencies were 53 ± 6% for wild type vs. 34 ± 4% for spo14-R894G. Therefore, spo14R894G is not a temperature-sensitive protein.
The N terminus of Spo14 is phosphorylated during meiosis and is sufficient to localize PLD protein to the prospore membrane. (A) Wild-type homozygous diploids containing HA-spo14-313 2μ were grown in SC-ura or sporulation medium for 15 hr at 30°. Whole cell lysates were prepared and the mobility of HA-spo14313 was determined by SDS-PAGE and immunoblotting. (B) Cellular fluorescence of GFP-spo14313 within sporulating cells.
The N terminus of Spo14 is phosphorylated during meiosis and is sufficient to localize PLD protein to the prospore membrane: Fungal members of the PLD gene family are unique in that they have an extended N terminus (Morriset al. 1996), which is adjacent to the conserved PX and PH domains (Ponting 1996; Steedet al. 1998). We previously showed that the Spo14 N-terminal domain is the major site of phosphorylation and is necessary for localization of the enzyme to the prospore membrane during sporulation (Rudgeet al. 1998b). To determine if this region alone is sufficient for mediating localization, a truncated mutant, spo14-313, which encodes for the first 313 amino acids but does not include the PX and PH domains of Spo14 (Roseet al. 1995), was epitope tagged separately with both HA and GFP. The phosphorylation state and localization of the corresponding proteins were examined.
Wild-type diploid cells containing HA-spo14-313 on a high-copy vector were grown vegetatively and induced to sporulate. Protein extracts were made, and the mobility of HA-spo14313 was determined by SDS-PAGE. The immunoblot shown in Figure 2A reveals that additional slower-migrating species of HA-spo14313 were detected only in samples prepared from sporulating cells. This result suggests that HA-spo14313 is phosphorylated during meiosis and reaffirms our previous data that demonstrated that the N terminus of Spo14 is the major site of phosphorylation (Rudgeet al. 1998b).
To investigate whether the N terminus of Spo14 is sufficient to localize Spo14 to the prospore membrane, GFP-spo14-313 on a high-copy vector was introduced into wild-type diploid cells. Using confocal microscopy, the cellular localization of GFP-spo14313 was determined in formaldehyde-fixed sporulating cells. Figure 2B shows that, in common with GFP-Spo14 (Figure 5C and Rudge et al. 1998a,b, 2001), GFP-spo14313 localizes to the prospore membrane. This result indicates that the N terminus of Spo14 is sufficient for localizing PLD activity to the prospore membrane during meiosis.
Spo14 phosphorylation and relocalization are essential for sporulation, but dispensable for Sec14-independent secretion: The essential role for Spo14 in Sec14-independent secretion is dependent on both PLD catalytic activity and the extended N-terminal domain (Xieet al. 1998). However, analysis of Spo14 in the absence of Sec14 revealed that it is neither phosphorylated nor relocalized, while this domain alone is sufficient for these events (see above). To differentiate the role of the N-terminal extension in Sec14-independent secretion and meiosis, we isolated conditional mutations within this domain of SPO14 by targeted PCR mutagenesis (see materials and methods). In vivo recombination (Muhlradet al. 1992) generated a library of replicating plasmids harboring mutant spo14 sequences; the resultant transformants were screened for their ability to sporulate at both 25° and 33.5°. Two mutants were identified that exhibited the conditional sporulation phenotype. DNA sequencing revealed that both contained three base changes within a single codon in the N-terminal domain at nucleotides 751–753 (TCT mutated to CTC), generating a serine-to-proline change at position 251. The resulting protein is designated spo14S251P.
At the permissive temperature (25°) spo14S251P expressed from a low-copy (CEN) yeast plasmid allowed homozygous spo14 diploids to sporulate at near wild-type frequency (spo14-S251P 50 ± 4% vs. SPO14 62 ± 3%). However, at the restrictive temperature (33.5°) no spores were formed in spo14-S251P mutants (<0.1%). In contrast, homozygous spo14 mutants containing SPO14 sporulated at reasonable efficiency at 33.5° (28 ± 2%). Elevated temperatures are known to impede the process of meiosis and sporulation (Byers and Goetsch 1982); consequently, the efficiency to which wild-type diploids can sporulate is affected at 33.5°.
Although Sec14-independent secretion is normally assayed by examining growth at 37°, the report that sec14-1 kes1 spo14 mutants are unable to grow at 33.5° (Rivaset al. 1999) enabled us to test the ability of spo14S251P grown at the nonpermissive temperature to support secretion. Interestingly, spo14S251P restored growth of the sec14-1 kes1 spo14 mutants to the same degree as the wild-type protein at 33.5° (Figure 3).
To examine protein phosphorylation and localization of spo14S251P at the permissive and restrictive temperatures, HA- and GFP-tagged derivatives were constructed and introduced into yeast cells. SAP treatment of immunoprecipitated HA-Spo14 results in the removal of phosphate groups from Spo14 and a decrease in the apparent molecular weight of the protein on SDS-PAGE (Rudgeet al. 1998b). To determine whether HA-spo14S251P was phosphorylated correctly, both HA-Spo14 and HA-spo14S251P were immunoprecipitated from extracts prepared from homozygous spo14 diploids sporulated at 25° and 33.5°. At both temperatures HA-Spo14 is detected exclusively as a single phosphorylated species, whose electrophoretic mobility is sensitive to treatment with SAP (Figure 4, A and B; Rudgeet al. 1998b). In contrast, immunoprecipitated HA-spo14S251P was found to exist in three states at 25° (Figure 4A). The electrophoretic mobility of the two least-mobile species of HA-spo14S251P was sensitive to SAP treatment (Figure 4A). However, the electrophoretic mobility of the third, and most migratory and prominent, species of HA-spo14S251P was insensitive to treatment with SAP (Figure 4, A and B). Interestingly, only this third species of HA-spo14S251P was detected at 33.5° (Figure 4B), a temperature at which Sec14-independent secretion occurs but not sporulation (Figure 3 and above). Moreover, since HA-spo14S251P and HA-Spo14 are both expressed to the same levels at 25° and 33.5° (Figure 4, A and B), we can eliminate the possibility that the sporulation phenotype associated with HA-spo14S251P is due to this protein being uniquely labile at high temperature.
Spo14 phosphorylation is essential for sporulation, but dispensable for Sec14-independent secretion. A sec14-1 kes1 spo14 strain harboring no SPO14 sequences (CEN), HA-SPO14 (HA-SPO14 CEN), or HA-spo14-S251P (HA-spo14-S251P CEN) was incubated on solid SC-ura medium at 25° and 33.5° for 48 hr.
One possible explanation for this data is that the serine-to-proline mutation perturbs the folding of the N terminus such that the SDS-PAGE mobility of spo14S251P is altered independently of protein phosphorylation. To test this, the electrophoretic mobility of HA-spo14S251P isolated from mitotically dividing cells was determined. During vegetative growth Spo14 is not phosphorylated to the extent to which the protein is phosphorylated during sporulation (Rudge and Engebrecht 1999). Figure 4C shows that the electrophoretic mobility of HA-spo14S251P is significantly diminished during vegetative growth both at 25° and 33.5°. Furthermore, only one species of HA-spo14S251P is detected in mitotically dividing cells, consistent with the notion that the mobility of the mutant protein is altered independently of phosphorylation. Taken together, these results suggest that a fraction of the protein is phosphorylated at the permissive temperature, as monitored by the slower-mobility species; however, at the restrictive temperature the enzyme is not phosphorylated and hence fails to support sporulation.
When expressed from a high-copy yeast (2μ) plasmid, GFP-spo14S251P remains incapable of rescuing the sporulation phenotype of homozygous spo14 diploids at 33.5° (<0.1% sporulation vs. 30% ± 4% for SPO14). Thus, we examined the intracellular localization of GFP-spo14S251P 2μ within sporulating spo14 and SPO14 diploids. In spo14 cells propagated at the permissive temperature, GFP-spo14S251P localized to the prospore membrane (Figure 5A); however, at the restrictive temperature GFP-spo14S251P was unable to localize to sites of prospore membrane synthesis (Figure 5B). GFP-Spo14 staining looked normal at 33.5° in this strain background (Figure 5C), indicating that the temperature did not affect GFP-Spo14 localization and subsequent PLD-mediated prospore membrane synthesis and spore formation. Furthermore, in SPO14 cells where the prospore membrane is formed, GFP-spo14S251P failed to localize (Figure 5D). These results demonstrate that spo14S251P perturbs both phosphorylation and relocalization of the enzyme to sites of prospore membrane assembly during sporulation.
Spo14 phosphorylation does not influence in vitro PLD activity: spo14S251P is capable of supporting at least one process absolutely dependent on Spo14 PLD activity (i.e., Sec14-independent secretion; Xieet al. 1998; Rivaset al. 1999), but not sporulation. These results are consistent with our previous observation that Spo14 phosphorylation does not influence the protein's in vitro PLD catalytic activity (Rudgeet al. 1998b). To further test this prediction, HA-Spo14 and HA-spo14S251P were immunoprecipitated from homozygous spo14 diploids cultured in sporulation medium at 33.5°. Quantification of PLD assays conducted with immunoprecipitated HA-Spo14 and HA-spo14S251P revealed that the catalytic activity of Spo14 was not significantly altered by the introduction of the serine-to-proline mutation in the N terminus. The specific activity of immunoprecipitated HA-Spo14 was 410 ± 22 pmol/min compared with 373 ± 31 pmol/min for HA-spo14S251P. Thus, perturbation of phosphorylation and localization does not influence PLD activity per se.
Spo14 is phosphorylated independently of meiosis in response to nitrogen and carbon starvation: As a first step toward identifying the kinase(s) responsible for Spo14 phosphorylation during sporulation, the external and genetic requirements for Spo14 phosphorylation were examined. Since catalytically deficient mutants of Spo14 are phosphorylated even when meiosis and sporulation are impaired (Rudgeet al. 1998a; Sciorraet al. 1999), we hypothesized that the kinase(s) would not be one whose activity was intimately regulated by the proficiency of the meiotic divisions and/or spore packaging. Instead, we investigated the idea that the kinase(s) responsible for Spo14 phosphorylation would be activated prior to the meiotic divisions in response to the nutrient conditions that initiate yeast sporulation, i.e., nitrogen limitation and the presence of a nonfermentable carbon source.
Spo14S251P is improperly phosphorylated. Homozygous spo14 diploids containing either HA-SPO14 CEN or HA-spo14-S251P CEN were grown in sporulation medium for 15 hr at (A) 25° and (B) 33.5°. HA-Spo14 and HA-spo14S251P were subsequently immunoprecipitated from Nonidet-P40-solubilized cell extracts, and immunecomplexes were subjected to SAP treatment. The electrophoretic mobility of HA-Spo14 was determined by SDS-PAGE and immunoblot analysis. Arrows denote discrete Spo14 phosphorylated species. (C) Homozygous spo14 diploids containing either HA-SPO14 CEN or HA-spo14-S251P CEN were grown in SC-ura for 15 hr at 25° and 33.5°. Whole cell extracts were prepared and the mobility of the proteins determined by SDS-PAGE and immunoblotting.
The immunoblot displayed in Figure 6A shows the apparent molecular weight of immunoprecipitated HA-Spo14 proteins extracted from yeast cell cultures grown in either sporulation medium (2% potassium acetate) or SD(-N). SD(-N) does not contain a nitrogen source; however, the presence of glucose in the medium prevents the initiation of sporulation in diploid yeast (Kupiecet al. 1997). The extent of HA-Spo14 phosphorylation was detectable but reduced when cells were placed in SD(-N) medium (compare the electrophoretic mobility of HA-Spo14 isolated from cells grown in SD (-N) vs. 2% potassium acetate; Figure 6A). Treatment with SAP confirmed that HA-Spo14 was phosphorylated during exposure to SD(-N) (Figure 6A). These results demonstrate that Spo14 phosphorylation is acutely sensitive to both extracellular nitrogen and glucose levels and suggests that the kinase (or kinases) responsible for Spo14 phosphorylation during sporulation are activated by nutrient limitation.
Only diploids of opposite mating types (a/α) are capable of sporulating, while diploids of the same mating type (a/a or α/α) are incapable of initiating the meiotic program (Kupiecet al. 1997). Taking advantage of this genetic requirement for meiosis and sporulation, the phosphorylation state of Spo14 was determined in diploids homozygous at the mating-type locus. The electrophoretic mobility of HA-Spo14 extracted from a/a diploids exposed to SD(-N) and 2% potassium acetate was similarly altered as HA-Spo14 extracted from a/α diploid cells exposed to the same medium (Figure 6B). The only noticeable difference between the two diploid strains was a decrease in the total levels of HA-Spo14 detected in the a/a diploids. This most probably reflects the fact that diploids homozygous at the MAT locus are unable to initiate meiosis and sporulation and SPO14 is not transcriptionally induced (Roseet al. 1995). Similar to what is observed in meiosis (Rudgeet al. 1998a), HA-Spo14 is solubilized by detergent in a/a cells propagated in sporulation medium (data not shown), indicating that phosphorylation and relocalization are coupled under these conditions. Thus, Spo14 is phosphorylated and relocalizes independently of meiosis.
Spo14S251P fails to relocalize to the prospore membrane at the restrictive temperature. Cellular fluorescence of GFP-spo14S251P within fixed homozygous spo14 diploid cells initiated to sporulate at (A) 25° and (B) 33.5°. (C) Cellular fluorescence of GFP-Spo14 in spo14 cells sporulated at 33.5°. (D) GFP-spo14S251P fluorescence in wild-type cells sporulated at 33.5°.
Spo14 regulation in Sec14-independent secretion: Spo14 function during spore formation and Sec14-independent secretion are absolutely dependent on PtdIns (4,5)P2 activation of PLD catalytic activity (Rudgeet al. 1998b; Sciorraet al. 1999). Moreover, the correct intracellular location of Spo14 is also essential for PLD function (Rudgeet al. 1998b; Xieet al. 1998). In this article we provide further evidence that Spo14 function during sporulation is dependent on protein phosphorylation and movement within the cell. In contrast, Spo14 phosphorylation and relocalization do not occur and are indeed both dispensable for Sec14-independent secretion. These results argue against the idea that Spo14 is activated in the absence of Sec14 by relocalizing to regions within the cell necessary for Sec14-independent secretion or that are perhaps rich in PLD substrate (phosphatidylcholine) or activator [PtdIns(4,5)P2] and suggest that Spo14 executes its function within a cellular compartment, presumably endosomal membranes, normally occupied by the protein during Sec14-dependent growth (Liet al. 2000a). However, we cannot eliminate the possibility that a fraction of Spo14 resides in another compartment such as the Golgi and that production of phosphatidic acid there drives Sec14-independent secretion. As we discuss below, PtdIns(4,5)P2 activation of PLD activity does play a major role in regulating Spo14 in secretion.
Further characterization of a partially PtdIns(4,5)P2-responsive allele of SPO14, spo14-R894G (Sciorraet al. 1999), indicates that Sec14-independent secretion is more acutely sensitive to loss of maximal Spo14 PLD activity. Moreover, we conclude that PtdIns(4,5)P2 activation of PLD catalytic activity is perhaps a major regulatory mechanism of Spo14 function in Sec14-independent secretion. In support of this idea, Li et al. (2000a) have shown that Spo14 function in secretion is dependent on the presence of members of a novel family of phosphatidylinositol transfer proteins (SFH proteins). One of these proteins, Sfh2, co-localizes with Spo14 in the endosome (Liet al. 2000a) and is presumably responsible for maintaining phosphoinositide levels at sites of PLD-mediated phosphatidylcholine hydrolysis (Liet al. 2000b; Rudgeet al. 2001).
Sreenivas et al. (1998) suggested that Sec14 is a negative regulator of Spo14 catalytic activity and that loss of Sec14 results in activation of Spo14. However, overexpression of SPO14 fails to suppress sec14-1 in an otherwise wild-type background (Xieet al. 1998). In contrast, increased gene dosage of SPO14 improves Sec14-independent growth (Rudgeet al. 2001). To accommodate these findings we propose that Spo14 is activated in Sec14-independent secretion as a consequence of simultaneous loss of Sec14 inhibition and maximal PtdIns(4,5)P2 activation. However, the precise mechanism of how and where Spo14-mediated hydrolysis of phosphatidylcholine drives Sec14-independent secretion remains elusive.
The role of the N-terminal extension in PLD function: Analysis of spo14S251P indicates that phosphorylation and relocalization are essential for PLD function in meiosis. These events are tightly coupled, raising the possibility that phosphorylation triggers relocalization of the entranslocation (Keranenet al. 1995). However, our data are also consistent with the idea that phosphorylation is a consequence of relocalization.
Spo14 phosphorylation is regulated by nitrogen and glucose limitation and occurs independently of the meiotic divisions. (A) Immunoblot of immunoprecipitated HA-Spo14 from extracts of a/α diploid wild-type yeast grown in 2% potassium acetate and SD(-N) and treated with SAP. (B) Immunoblot of HA-Spo14 from whole cell extracts of a/a and a/α diploids grown in SC-ura, 2% potassium acetate, and SD(-N).
The finding that spo14S251P but not spo14ΔN, which harbors a deletion encompassing S251 (Rudgeet al. 1998b; Xieet al. 1998), supports Sec14-independent secretion indicates that the N terminus is important for more than phosphorylation and relocalization of the enzyme. We propose that the N-terminal domain plays a role in targeting the enzyme to endosomal membranes in vegetative cells and early in meiosis and its absence from endosomes results in loss of PLD activity in both secretion and sporulation. In contrast to deletion of this domain, mutation of serine 251 enables the enzyme to be targeted to endosomal membranes in vegetative cells and early in meiosis; however, introduction of a proline residue prevents phosphorylation and relocalization during meiosis. The inability to phosphorylate serine 251 in spo14S251P alone is unlikely to account for the phenotypes associated with spo14-S251P, since this potentially phosphorylated residue cannot account for the multiple phosphorylation sites on Spo14 (Rudgeet al. 1998b). Thus, we favor the hypothesis that introduction of proline at 251 induces a conformational change in the N terminus of Spo14 such that the mobility of the protein is altered during SDS-PAGE analysis and the N terminus is not phosphorylated and fails to relocalize at elevated temperatures of ⩾33.5°. We believe this is a relatively specific effect on phosphorylation and relocalization as the enzyme functions in Sec14-independent secretion and displays wild-type PLD activity. However, we cannot eliminate the possibility that introduction of a proline leads to gross alteration in the secondary structure of the protein and perturbs additional properties of the enzyme.
Alignment of the three fungal PLDs sequenced to date—Spo14, CaPLD (Candida albicans PLD; Kanohet al. 1998), and SPAC2F7.16c (Schizosaccharomyces pombe PLD; Rudge and Engebrecht 1999)—reveals a single region within the N terminus of Spo14 (amino acids 230–246; Kanohet al. 1998) that shares significant homology to CaPLD (Kanohet al. 1998) and SPAC2F7.16c (Rudge and Engebrecht 1999). From results presented here, we hypothesize that the extended N termini of CaPLD and SPAC2F7.16C are also phosphorylated and serve to regulate the localization of these PLD enzymes to phospholipid membranes necessary for cellular function.
Spo14 kinases: Phosphorylation appears essential for Spo14 function during meiosis and sporulation. Here we report that the kinase(s) responsible for Spo14 phosphorylation are responsive to both nitrogen and glucose limitation. Since it is the absence of extracellular nitrogen and the presence of a nonfermentable carbon source that provokes S. cerevisiae to form spores, budding yeast appear to have harnessed a nutritional cue to activate a kinase (or kinases) responsible for regulating the localization of a PLD activity essential for meiosis and sporulation. The identity of the intracellular signaling pathways that respond to nitrogen and carbon source availability and the kinases responsible for phosphorylating Spo14 merit further study.
We have previously noted that overexpression of Spo14 during sporulation does not result in a concurrent increase in the number of asci formed (Rudgeet al. 2001). However, increased gene dosage of SPO14 significantly improves Sec14-independent secretion (Rudgeet al. 2001). These data suggest that there exists a rate-limiting step of Spo14 function in sporulation, but not in secretion. When expressed from a low-copy vector, HA-Spo14 is detected exclusively as a single phosphorylated protein in meiotic cell extracts (Rudge et al. 1998a,b, 2001; this report). Interestingly, expression of HA-Spo14 from a high-copy vector during sporulation results in detection of both a phosphorylated and faster-migrating species (Rudgeet al. 1998a). However, in Sec14-independent secretion only the faster-migrating species of Spo14 is detected. These observations indicate that Spo14 phosphorylation may be the rate-limiting step of PLD function during yeast sporulation.
In summary, Spo14 is differentially regulated during sporulation and Sec14-independent secretion. This result is in keeping with our recent work demonstrating that the function of Spo14 is different during sporulation and secretion (Rudgeet al. 2001). In Sec14-independent secretion the total cellular levels of phosphatidic acid are sensitive to Spo14 inactivation. In contrast, the total levels of phosphatidic acid within homozygous spo14 mutants are equivalent to those levels measured in wild-type cells (Rudgeet al. 2001). Thus, Spo14 mediates small changes, which we propose are highly localized, of a specific molecular species of phosphatidic acid essential for sporulation (Rudgeet al. 2001). Taken together, our data indicate that Spo14 is regulated and functions differently during the processes of sporulation and secretion.
We thank Dr. Vytas Bankaitis (State University of North Carolina at Chapel Hill) for sec14 strains. We acknowledge Drs. Vicki Sciorra (University of California, San Diego), Aaron Neiman, and James Trimmer (State University of New York at Stony Brook) for helpful comments during the writing of this article. Michelle Iwamoto is also gratefully acknowledged for facilitating the completion of this work. This work was supported by an American Cancer Society grant (RPG-99-122-01-MBC) to J. Engebrecht.
Communicating editor: A. P. Mitchell
Received November 8, 2001.
Accepted January 15, 2002.
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, 1996 Characterization of Saccharomyces cerevisiae deficient in expression of phospholipase D. Biochem. J. 314: 15–19.
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Kearns B. G.,
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, 1996 Kes1p shares homology with human oxysterol binding protein and participates in a novel regulatory pathway for yeast Golgi-derived transport vesicle biogenesis. EMBO J. 15: 6447–6459.
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, 1995 MSH5, a novel MutS homolog, facilitates meiotic reciprocal recombination between homologs in Saccharomyces cerevisiae but not mismatch repair. Genes Dev. 9: 1728–1739.
Honigberg S. M.,
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, 1992 Commitment to meiosis in Saccharomyces cerevisiae: involvement of the SPO14 gene. Genetics 130: 703–716.
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, 1983 Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153: 163–168.
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Kitajima Y.,
, 1998 Molecular cloning of a gene encoding phospholipase D from the pathogenic and dimorphic fungus, Candida albicans. Biochim. Biophys. Acta 1398: 359–364.
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Dutil E. M.,
Newton A. C.
, 1995 Protein kinase C is regulated in vivo by three functionally distinct phosphorylations. Curr. Biol. 5: 1394–1403.
Kupiec M.,
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Mitchell A. P.
, 1997 Meiosis and sporulation in Saccharomyces cerevisiae, pp. 889–1036 in The Molecular and Cellular Biology of the Yeast Saccharomyces, Cell Cycle and Cell Biology, Vol. 3, edited by Pringle J. R., Broach J. R., Jones E. W.. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
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, 2000a Identification of a novel family of nonclassic yeast phosphatidylinositol transfer proteins whose function modulates phospholipase D activity and Sec14-independent cell growth. Mol. Biol. Cell 11: 1989–2005.
, 2000b Phosphatidylinositol/phosphatidylcholine transfer proteins in yeast. Biochim. Biophys. Acta 1486: 55–71.
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Engebrecht J.,
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, 1996 Structure and regulation of phospholipase D. Trends Pharmacol. Sci. 17: 182–185.
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, 1992 A rapid method for localized mutagenesis of yeast genes. Yeast 8: 79–82.
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, 1996 Novel domains in NADPH oxidase subunits, sorting nexins, and PtdIns 3-kinases: Binding partners of SH3 domains? Protein Sci. 5: 2353–2357.
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, 1999 Pleiotropic alterations in lipid metabolism in yeast sac1 mutants: relationship to "bypass Sec14p" and inositol auxotrophy. Mol. Biol. Cell 10: 2235–2250.
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Winston F.,
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, 1990 Methods in Yeast Genetics: Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
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Engebrecht J.
, 1995 Phospholipase D signaling is essential for meiosis. Proc. Natl. Acad. Sci. USA 92: 12151–12155.
, 1999 Regulation and function of PLDs in yeast. Biochim. Biophys. Acta 1439: 167–174.
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, 1998a ADP-ribosylation factors do not activate yeast phospholipase Ds but are required for sporulation. Mol. Biol. Cell 9: 2025–2036.
, 1998b Relocalization of phospholipase D mediates membrane formation during meiosis. J. Cell Biol. 140: 81–90.
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, 2001 SPO14 separation-of-function mutations define unique roles for PLD in secretion and cellular differentiation in Saccharomyces cerevisiae. Genetics 158: 1431–1444.
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, 1999 Identification of a phosphoinositide binding motif that mediates activation of mammalian and yeast phospholipase D isoenzymes. EMBO J. 18: 5911–5921.
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Volume 160 Issue 4, April 2002
You are going to email the following Differential Regulation of Saccharomyces cerevisiae Phospholipase D in Sporulation and Sec14-Independent Secretion


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This introductory course does two things at once. First, there's the technologies of geography: maps and mapping, GIS, GPS, remote sensing, etc. We also consider the geographies of technology: where is the cloud? Can you map the Internet? We explore political, population, and urban geography as ways to understand the connections between people, place, and technology.
Region by region, this course looks at urbanization processes around the world. What's the spatial structure of cities in different parts of the world, and what difference does that structure make to what it's like to live in those cities? We include elements of culture, environment, and health alongside politics and economics.
This is a new course for Spring 2019 at UIUC, and as far as I know, a unique geography course anywhere. It's a regional geography of the U.S. through a two-part landscape lens: memory and commemoration, and environmental justice. The course meets our U.S. Minority Cultures requirement by emphasizing the role of Native Americans and African-Americans in particular in producing cultural and socio-environmental landscapes across the U.S.
This course begins with global systems of logistics and air travel and works its way down to national systems of road and rail. Then we move to the metropolitan scale to talk about transportation planning, followed by the scale of the body: how do individual people experience transportation and mobility? Our field trip to the Joliet region considers how multiple layers of transportation, past and present, shape the landscape. I am currently writing a textbook for this course for Rowman & Littlefield; contact me if you'd like to hear more or have ideas!
I occasionally teach a graduate seminar on urban environments and/or urban sustainability. I've also taught a seminar on mobilities, as well as our qualitative methods course for graduate students. I have also taught GEOG 412, Geospatial Technologies & Society, which covers the topics of GEOG 105 in more detail.


Troublesome emotions unchecked Anger deepens, rises and overflows Where does this deepening depression lead? Meanwhile the stars dance and twinkle Uncaring, unsharing shards of light A smile widens; teeth shining bright white Happiness is a viral happening at times Like Julia Roberts in Notting Hill No need to swallow any bitter pill Laughter tickles in the back of my throat A sneeze of exquisite joy explodes Those little drops of laughter burst out Infecting those around me It's simply now a cacophony Infectious, riotous and bold As global laughter takes a hold.
Bobbing along the stream Sometimes fighting Other times alighting For a longer look Checking out scenery But no real choices Except to go with the flow I go where I need to go Can only slow The inevitable As I start at the beginning I must end at the end That's the way it really is Life is like a stream Winding its lonely way Always just passing through Never sitting still Ripples speak to movement Movement is my destiny One day, I will reach the Sea And that, my friends Will be the end of me.


text:
Q: Display JTable in JPanel I've been trying to add JTable to my JPanel, yet it doesn't appear there. I've searched through other questions, but solutions proposed there didn't help me.
The function below is a part of a class which extends JFrame. "repaint" is a BufferedImage, "imageLabel" is JLabel and "image" is ImageIcon.
public void showTable() {
    try {
        repaint = ImageIO.read(new File("filename.jpg"));
    } catch (IOException e) { }

    Graphics g = repaint.createGraphics();
    g.setFont(font);
    g.setColor(black);
    String[] columnsName = {"id","text"};
    Object[][] data = {{new Integer(1),"text one"},{new Integer(2),"text two"}};
    JTable table = new JTable(data, columnsName);
    JScrollPane tableContainer = new JScrollPane(table);
    image = new ImageIcon(repaint.getScaledInstance(sizeX,sizeY, Image.SCALE_SMOOTH));
    imageLabel.setIcon(image);
    imageLabel.add(tableContainer,BorderLayout.CENTER);
    getContentPane().add(imageLabel);

    pack();
    setVisible(true);
    repaint();
    revalidate();
}

What I want to achieve is to display table over loaded image.
Thanks in advance for Your help :)

A: *

*Give your JLabel a layout manager, here BorderLayout via setLayout(new BorderLayout()).

*Don't get your Graphics object via getGraphics() as it returns an object that is short-lived. To see for yourself, minimize your program and then restore it and watch your drawing disappear. Instead, draw in the paintComponent(...) override method.

*Read the Swing tutorials for greater detail on how to do these things, especially the layout manager tutorial.

A: I'm not sure if I understood correctly, but you want to put a table and an image below it, right?
I would create a JPanel with BorderLayout, put the image (JLabel) on the bottom (Page end) and the table (that scrollPanel you created) on the center.
If you need help with adding items to JPanel with BorderLayout, see how to do it on this Tutorial:
http://docs.oracle.com/javase/tutorial/uiswing/layout/border.html
I hope it helps.

A: Thanks for all the hints so far :) They were really useful. I know that I might be doing some things in a not elegant way, but I'm still learning, will try to improve :)
The given hints and some more searching lead me to such code :
    public void showTable()
    {
    try
    {
        repaint = ImageIO.read(new File("filename.jpg"));
    }catch (IOException e) {
    }
    Graphics g = repaint.createGraphics();
    g.setFont(font);
    g.setColor(black);
    String[] columnsName = {"id","text"};
    Object[][] data = {{new Integer(1),"text one"},{new Integer(2),"text two"}};
    JTable table = new JTable(data, columnsName);
    table.setOpaque(false);
    JScrollPane tableContainer = new JScrollPane(table);
    tableContainer.setBorder(BorderFactory.createEmptyBorder());
    tableContainer.setOpaque(false);
    tableContainer.getViewport().setOpaque(false);
    image = new ImageIcon(repaint.getScaledInstance(sizeX,sizeY, Image.SCALE_SMOOTH));
    imageLabel.setIcon(image);
    imageLabel.setLayout(new BorderLayout());
    imageLabel.add(tableContainer, BorderLayout.SOUTH);
    repaint();
    revalidate();
    }

Which works fine for me :)
Once again thanks all for the help :)

redpajama_set_name: RedPajamaStackExchange


text:
Walter Aller
Detroit, Wayne, Michigan, United States
The Life of Walter
When Walter Aller was born on 4 July 1903, in Detroit, Wayne, Michigan, United States, his father, Louis Aller, was 36 and his mother, Anna Grasier, was 31. He had at least 1 son with Margaret Gagnon. He lived in Detroit Ward 21, Detroit, Wayne, Michigan, United States in 1920. He died on 6 November 1980, in Fraser, Macomb, Michigan, United States, at the age of 77.
Learn about Walter's homeland.
Put your face in a costume from Walter's homelands.
Louis Aller
Anna Grasier
Christine Aller
Fred Aller
Edward R. Aller
St. Louis, Missouri, United States hosts Summer Olympic Games.
1904 · Belle Isle Aquarium
The Belle Isle Aquarium is a public aquarium located in Belle Isle Park in Detroit. It opened on August 18, 1904, and was the oldest continually operating public aquarium in North America when it closed on April 3, 2005. The aquarium reopened to the public on August 18, 2012, and is now run entirely by volunteers
Charles Lindbergh makes the first solo nonstop transatlantic flight in his plane The Spirit of St. Louis.
German: variant of Ahler .
Walter W Aller in household of Louis Aller, "United States Census, 1930"
Walter Aller in household of Louis Aller, "United States Census, 1920"

redpajama_set_name: RedPajamaCommonCrawl


text:
connect / 2014
SPEAK UP ACROSS LA!
Idea submitted in the My LA2050 Maker Challenge by Youth Speak Collective
We will share the stories of 50 youth across LA to help turn their ideas in action!
Please describe yourself.
Proposed collaboration (we want to work with partners!)
In one sentence, please describe your idea or project.
Does your project impact Los Angeles County?
Yes (benefits a region of LA County)
Which area(s) of LA does your project benefit?
Central LA
East LA
What is your idea/project in more detail?
We want to present the voices of our future leaders. By 2050, the youth of today will be the generation that inherits Los Angeles. One of them may even be the mayor in 2050. So why are we not listening to them?
We plan to film 50 young people across LA and develop 50 solution stories. These short films will be shown in nontraditional and traditional settings in communities throughout Los Angeles. These stories will inspire others to follow their passion and be the difference they want to see in their community. Viewers of these short films can learn from these stories and in turn new partnerships will form because of this candid and powerful form of sharing solutions.
What will you do to implement this idea/project?
We plan to work with our team of students and mentors to film these stories across Los Angeles. We have broken up the yearlong project into four stages, outlined below.
Story Outreach (2 months) • Brainstorm with team, mentors and partners. • Create outreach strategy and implement outreach campaign and story website to collect stories across Los Angeles. • Create criteria for story selection and clear format for filming, editing and the interview process. • Visit elected offices, neighborhood councils, nonprofits and community centers to present on our mission and vision to document inspiring stories from our young people. • Select 50 individuals evenly spread out across the five main regions of Los Angeles (South LA, East LA, Central LA, West Side and Valley)
Filming (6 months) • Arrange film times with the young people to be interviewed. • Partner with local nonprofits to schedule interview locations. • Film stories
Editing (2 months) • Edit all footage • Meet with interviewees again if necessary • Complete all 50 two minute films
Community Outreach (2 months) • Arrange film screenings as conversations in local community locations. • Arrange larger film screening in an open location (like Grand Park) in Central Los Angeles. • Film screenings would allow youth and participants the opportunity to speak up after each film screening. We plan for break out conversations around the topics the stories evoke.
How will your idea/project help make LA the best place to CONNECT today? In 2050?
It is our belief that young people have a need to give input and speak up. In the last years we have seen this in the students we work with and the staff on our team. When given the opportunity, they have the drive and ability to surprise us with amazing ideas. Through genuine listening and authentic conversation we can tap into the fresh outlook and wisdom of youth in our currently estranged communities. The guidance and mentorship of invested, caring adults can help transform Los Angeles into a socially connected, civically active community.
The program enables students to share a powerful story, an example of how they plan to be the change they see in their community. Young people will be able share how they give back and inspire others to step up in volunteer roles in their community, schools and city. Sharing stories among the community, residents create the type of human connections that play an important role in civic engagement, emotional intelligence and self-confidence we need to succeed and create a more connected LA.
Our project will create a more engaged generation across Los Angeles and develop a line of stories that bind our experiences and commitment to give back. Once we present the stories in the communities they were born, the stories will be connected to others and potential partnerships will form.
By the simple act of documenting stories and listening to each other we also see our project impacting our society at 2050. These films will remind one another of our shared humanity, strengthen and build the connections between people and creating a culture of understanding in our higher risk communities in Los Angeles.
Every movement of change needs a spark. These videos will create the spark. The film screenings will fan this energy and create conversations between people that will spread and continue for years. In the long run impacting the individual telling the story and the people who heard their story.
Each story will answer the questions: What legacy do they want to leave in their community? How do they see LA in 2050 if we all volunteered our time? How do they currently make an impact in their community? These videos could be the first step towards creating the impact that we need to transform our community.
Whom will your project benefit?
Our project will benefit a variety of groups. First, the individual. The person who tells the story will be valued and listened to. Their story and ideas will be viewed by thousands. Through this message their own experiences will be honored and they will be empowered to continue their efforts in the community. These short videos will give young students a platform not only to find their voice, but to let that voice be heard across LA. By sharing their story, these students will become more confident in themselves, more empathetic towards their peers, and more committed to their community. These students will inspire others by the simple act of telling their story and sharing their solution ideas for an issue in their community.
Second, the individuals who hear the story will be engaged with the message and will be encouraged to find a way to partner with the storyteller. Through our film screening sessions we hope to inspire new partnerships and collaborative efforts through these stories and films.
Our Youth Speak Media Solutions team of students and mentors will also benefit from this project as they will experience the stories first hand during the time of production. Our team of students also become inspired to give back to their communities after capturing such powerful stories and witnessing the positive work organizations are doing across Los Angeles.
Please identify any partners or collaborators who will work with you on this project.
We would love to partner with you! As a nonprofit we have worked with groups across Los Angeles, and we would love to hear from new young people and groups making an impact in LA.
Currently we plan to work with and actively collaborate with the following nonprofit and business partners: Youth Justice Coalition, The Hub LA, New Leaders Council, Proyecto Pastoral, Boys and Girls Club of Venice, and Unusual Suspects Theatre Company. We plan to reach out to other established and grassroots organizations throughout Los Angeles in our target communities once the campaign is funded.
We hope to partner also with various marketing firms, public relation organizations and media outlets to communicate these stories to as broad of an audience as possible. Some of which include, MTA that could showcase these short films in bus routes across Los Angeles. We currently have media partnerships with LATV, CNN, NBC, Junker Wagner Marketing, Media Image Public Relations and AK Design Strategies.
Once funded we will also partner with civic leaders in our target communities along with local neighborhood councils and the elected council members in that district. We know that by partnering with elected leaders in local, city and state levels we can create conversations both at the community and policy levels to really affect change and give an avenue for the students solutions and voice.
How will your project impact the LA2050 "Connect" metrics?
Rates of volunteerism
Attendance at cultural events
Participation in neighborhood councils
Percentage of Angelenos that volunteer informally (Dream Metric)
Government responsiveness to residents' needs (Dream Metric)
Attendance at public/open street gatherings (Dream Metric)
Please elaborate on how your project will impact the above metrics.
Our project has both an immediate and long-term impact in Los Angeles. The 50 individuals initially involved in the program will feel an immediate social connectedness to each other and their community.
The 50 films that are developed through the power of social media and community film screenings can have an impact that we estimate to hit 100,000 people. This inter-generational relationship building will make a powerful impact across generations. The young people sharing their stories will empower professionals to connect themselves to local nonprofits, become civically active and volunteer with community groups. The students through the filming and screening process will find adult allies that actually listen and care about what they have to say and contribute. To give voice is to give a stake in the public discourse, and that is what our project with Youth Speak Media Solutions intends to do.
The participants in the film screenings and discussion will be inspired to volunteer more or become more involved in local government. As young stakeholders, students will become more civic-minded, attend more volunteer events and be motivated to simply vote in order to get their voice heard. With involvement of many community partners throughout the process, we are breathing new life into a city where citizen's care and neighbors talk to each other.
The students we interview are the individuals that others follow. If they set a good example and affect change in their social spheres, a domino effect of social awareness, civic engagement, and increased volunteerism among Los Angelenos city-wide is possible.
Please explain how you will evaluate your project.
Outcome #1: The majority of students involved in the filming of their stories will have reported increased self-awareness and social awareness. These young people (ages 16-24) will have expressed greater ambition and motivation to succeed, specifically in their community in the City of Los Angeles.
Outcome #2: Eighty percent of participants in the community screening should feel a desire to be more civic-minded. Through the viewing of these impact stories, participants will be engaged and inspired through the films and facilitated conversation after film screenings.
Outcome #3: In the course of the year following the creation of the films and screenings, the community that was specifically engaged (example: Boyle Heights, not entire East Los Angeles) will see a 20% increase in civic engagement. This will be measured by voting rates at neighborhood council elections, participants in major volunteer events in community and intern/volunteer rates at local nonprofits.
Outcome #4: A coalition of individuals and nonprofits across Los Angeles would be created to tackle some of the issues that have been heard through the videos. This network would start to meet on a regular basis soon after the screenings.
What two lessons have informed your solution or project?
The first lesson we have learned in the process of becoming a youth-driven nonprofit, is to listen. This has been an acquired skill that we continue to grow in as staff and mentors to the students in our programs. As adults we can easily dismiss the youth in our community, but we have learned to listen and take their ideas and help put them into action. Many of our programs are an example of the youth speaking up about issues in their community and creating a solution that is supporting by civic leaders.
The second lesson we have learned is about the power of media and the way in which today's youth utilize it. Young people today incorporate media into their lives in almost a minute to minute basis. It is immediately accessible 24 hours a day, seven days a week. It is the medium in which our youth of today inform one another and themselves. Media, movies and phones have taken over our modes of communication and we have learned to utilize the strengths of our students and staff.
Explain how implementing your project within the next twelve months is an achievable goal.
We believe we have laid out achievable goals. We have a team of 20 students and 5 mentors that can juggle an average of 6 jobs a week that would allow us to engage 50 individuals within the time allowed. We have learned as creative professionals to be organized with our work load, having priorities each week and setting benchmarks for our progress.
As a program of the nonprofit, Youth Speak Collective we have an additional team of staff, boardmembers and advisory council members that will assist in our planning, outreach and development of our program goals.
Please list at least two major barriers/challenges you anticipate. What is your strategy for ensuring a successful implementation?
We anticipate two main challenges within this proposal. The first is the amount of individuals that we will be working with and the scheduling of time and the details that this will encompass. With so many stories to tell we will be working with our students schedules, our mentors work schedules and the individual's schedules. We plan to allocate a portion of the funding to be able to hire and designate one point person for this project. In film terms this would be our Producer. Our strategy for making a smoother storytelling and editing process we plan on having one specific person do the communication with the young people telling their stories.
The second challenge that we foresee is related to the concise telling of stories. In order to make an effective film that people would watch online we believe that all stories should be under two minutes each. In order to do this more effectively we plan to reach out to our public relations and marketing partners who can work with us pro bono to assist in some branding and outreach techniques to help people tell their story more efficiently and really explain their solution in a manner that makes an impact.
What resources does your project need?
Network/relationship support
Money (financial capital)
Volunteers/staff (human capital)
Publicity/awareness (social capital)


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text:
BLOG June 13, 2017
O'Reilly's Takeaways From Comey Testimony: 'He Destroyed Any Sense of His Nobility'
Bill O'Reilly joined Glenn on radio Tuesday to analyze the headlines of the day. Top on the list was James Comey's testimony in front of the Senate Intelligence Committee.
"What did you pull out of Comey's testimony last week? Give me two highlights that you thought people didn't really understand, or the news media didn't get right," Glenn asked.
O'Reilly offered his opinion, including one bombshell.
"The amazing thing is when he dropped the bomb --- and it certainly was that --- that he is a leaker, that he took notes that he wrote on an FBI computer and gave them to The New York Times," O'Reilly said.
O'Reilly, who live tweeted with more than three million followers during the testimony, took note.
"My head bolted up, and I immediately tweeted, 'That's huge. That's enormous. He just admitted that he's a leaker.' The head of the FBI, going to The New York Times, a paper that hates the president? So there's got to be vitriol there. He didn't go to some neutral place," O'Reilly said.
The second takeaway: Comey straight up called Trump a liar.
"He did that because he was truly insulted, Comey, that Trump said the FBI is chaos, that it was mismanaged. So, look, I understand the emotion there. But, you know, that was a pretty big thing for an FBI chief to do," O'Reilly said.
Bill O'Reilly joins Glenn every Friday on radio to discuss current headlines. Visit Bill O'Reilly.com to follow Bill, subscribe or purchase his wildly successful "Killing" book series.
Enjoy the complimentary clip, listen to the full segment or read the transcript for details.
GLENN: The one, the only, Mr. Happy Pants himself, Bill O'Reilly joins us, beginning right now.
GLENN: From BillO'Reilly.com. The one, the only, Mr. Bill O'Reilly. Hello, Bill.
BILL: Beck, you back to work, huh? After, what, an eight-week vacation?
GLENN: Wow, I was going to start with something nice, but you go right for the throat.
BILL: Yeah, no. Everybody missed you Beck. People were just stumbling around here in New York City going, where's Beck? Where is he?
GLENN: Right. Right.
So how have you been, Bill? How is your summer going? Hello.
STU: That was it --
GLENN: There he is. Wait. Wait. We lost you for a second. How has your summer been going?
BILL: Everything is good. Just a lot of stuff going on that I have to deal with every day.
GLENN: Like you have to take the dog out to walk. You've been on quite a vacation.
BILL: I'm tweeting. And tweeting makes me nervous because I'm not exactly sure what I'm doing. But this is amazing.
GLENN: Yeah.
BILL: We do live tweets. We're going to do them today in a Sessions hearing. And we did it with the Comey hearing. You heard about that on your vacation.
GLENN: Yes, I did. I watched it. Yeah, I did.
BILL: More than 3 million people were following my tweets.
PAT: Wow.
BILL: And I don't even know what tweets are.
GLENN: Right. Right. Right.
BILL: You know, but I have the machine in my hand, and I'm watching and then I write little comments like, he's lying, or this is ridiculous, or this is a pinhead right here.
BILL: Whatever. 3 million people.
GLENN: Uh-huh.
BILL: And then I understand that one of the Trump sons then was retweeting what I was tweeting, which I don't know what that means either.
GLENN: Right. You have to check the machine for that.
BILL: There was a whole avalanche of things that happened. So that's my life.
GLENN: Well, sounds -- sounds -- sounds good.
BILL: Yeah.
GLENN: So today -- so today you're going to be doing this with Sessions. And what should we expect? Less on the tweets, more on the actual substance.
BILL: You know, Sessions is going to have his rap down, number one. He knows what's coming. So he's got to explain the three meetings that he had as a senator with the Russian people. So he's got -- you got to do that.
GLENN: That's easy.
BILL: So number one, you're looking at a predictable set of Q&A at the top. But then it will get interesting because he has to then say, okay. What do you know about Comey's firing? What did you know about that?
And that's where it gets interesting. Because, you know, you figure Sessions was in on the discussions to sack Comey. You figured that. And how he phrases that, how he puts that, you know, why was he fired, what was the real reason, then you get into interesting territory.
GLENN: What did you pull out of Comey's testimony last week? Give me two highlights that you thought people didn't really understand, or the news media didn't get right.
BILL: Well, the amazing thing is, when he dropped the bomb -- and it certainly was that -- that he is a leaker, that he took notes that he wrote on an FBI computer.
BILL: And gave them to the New York Times. Because, you know, that's what he did. He did it through an intermediary. A friend of his.
BILL: But if you looked at the senators, they didn't even follow up on it. I don't even think they were paying attention. It was like they were somnambulant. And I bolted up. My head bolted up. And I immediately tweeted, "That's huge. That's enormous. He just admitted that he's a leaker." The head of the FBI, going to the New York Times, a paper that hates the president. So there's got to be vitriol there. He didn't go to some neutral place.
GLENN: Yeah, it did show that he wasn't the innocent little Boy Scout.
BILL: No. He's a double dealer.
GLENN: He's a political player. He knows exactly how that works.
BILL: Absolutely. Absolutely.
BILL: And it just destroyed any sense of nobility that he might have had because he was trying -- and every senator -- oh, you're the biggest senator. Paul Revere has nothing on you. You know, all of this crap. All right. And then, oh, by the way, I'm a leaker. I did a leak, that I wanted the president of the United States to look bad and hurt him.
And I went, "Whoa." So that was the biggest thing.
GLENN: What was the second --
BILL: Second thing was he called Trump a liar straight out. And he did that because he was truly insulted, Comey, that Trump said the FBI is chaos, that it was mismanaged. So, look, I understand the emotion there. But, you know, that was a pretty big thing for an FBI chief to do.
GLENN: So, again, you didn't pull what I pulled out. And this is the biggest thing of all, especially with the news that's coming from Bloomberg today. And that is the Russians are coming.
We spent all last week with everybody, talking about Comey and Trump and Hillary Clinton, when we should have been talking about the Russian hack. I don't know if you saw the report that came out today from Bloomberg. It's an exclusive. Thirty-nine states. Now, it didn't affect the election. But as Comey said last week, it will affect 18 or 20 if we don't do something.
They got into 39 states. That's double the amount that has been previously known. And they got into the actual counties -- or, sorry, statewide systems, thinking that it goes statewide up, instead of at the grassroots. That's the only thing that saved us, was they were thinking like Soviets, and not thinking like Americans.
And they -- they hacked in, as many as 7 million voters in Illinois. Could have had their registration wiped clean. That's -- that's significant.
BILL: Absolutely. That's a huge thing. The reason I believe that that didn't get more attention --
GLENN: Sounds sexy.
BILL: And I don't think the Bloomberg thing is going to get a lot of attention either. But you're very smart to point it out, because it is frightening how dependent we are on this technology. And how this technology can be used by criminals, and that's who these Russians are. To, you know, harm a nation.
GLENN: Right.
BILL: But people I think know that. I think they know that espionage was --
GLENN: Yeah, but, Bill, this was different. There was no evidence that they were trying to work for Trump or for Clinton. They were just trying to cause chaos.
BILL: Chaos. Don't you think most Americans know that?
GLENN: I do.
BILL: Don't you think they know?
GLENN: I do. But I think we need to stop all this political nonsense and start focusing on, okay. Wait a minute -- that -- in any -- tell me during Ronald Reagan, that wouldn't have been called an act of war.
BILL: Different world now.
GLENN: It is a different world.
BILL: The primary goal of the opposition party, the Democrats, and the media, the national media, their primary goal is not holding Putin accountable for anything. It's destroying Donald Trump. So anything that doesn't play into that narrative is shunned to the side.
GLENN: So then shouldn't we as, you know, defenders of what we believe is true, shouldn't we then be saying, "Okay. Stop -- watch the other hand. Don't pay attention to this. Pay attention to this, because this is going to actually affect our republic, where the -- where the nonsense of, you know, did Donald Trump -- was he in collusion, that's nonsense. This is important?"
BILL: But here's the problem with a story like that: You don't have any other follow other than what Bloomberg has put out and what we really already know, that Putin and his hackers were trying, as you put -- create as much chaos in our election. Is that an act of war? You could say it's a hostile act. I mean, I don't know whether it rises to the fact that we start to, you know, lob...
GLENN: No, I'm not saying that we do. But I know that in any other time period, that would have been an act of war.
BILL: Yes. I'll cede that point to you, Beck. And it pains me to do it.
GLENN: You seem -- may I say something, getting away from the grind every day, you seem happier than I've possibly ever even suspected you could be.
BILL: Happier.
GLENN: Yeah. You seem almost, dare I say it, less morose.
BILL: I don't have to commute into New York City every day. So that is enormous.
GLENN: Yes.
BILL: The rest of it, I'm not going to comment on. Although, I will tell you that over a 20-year period at Fox News, I worked as hard as a human being could possibly work. And now I don't have to work --
GLENN: No, I saw you. I saw you arrive and I saw you --
BILL: So, you know, those things make you a little more buoyant. Word of the day, "buoyant."
GLENN: Yes. Buoyant. We'll say it with you.
BILL: So another good observation from Beck, after an eight-week vacation. He's refreshed. And ready to go.
(chuckling)
JEFFY: And more buoyant.
When is your vacation up, Bill?
BILL: I never go -- I mean, I have -- somebody has got to keep an eye on this country.
GLENN: Right. Right. So let me go here. It came out last night that Donald Trump may be considering -- may be considering dumping the -- the special counsel Mueller. What do you think of that idea, and what will happen if he does?
BILL: Well, number one, I really don't analyze gossip, and that's what that is. So that would be a bad move if Trump did that.
PAT: Very.
BILL: I don't think he will.
I think there's mischief afoot. Mischief afoot. I think that these kinds of things get out there for a reason.
GLENN: Well, okay. Hang on. Here's how it came out --
BILL: Yeah, I know how it came out.
GLENN: Yeah. The head of NewsMax met at the White House, left, went to PBS. Said it on the air.
BILL: Right.
GLENN: So somebody in the White House was talking to him --
BILL: But Ruddy was clear -- head of NewsMax, Christopher Ruddy. I know him. You know him. He's an honest man. He was clear that nobody told him that. He just surmised it.
BILL: Or he took something -- or this is a deduction of his.
GLENN: Hang on just a second. Wait. Wait. Wait. Just a second. I've been to the White House. I've been in the Oval. You've been in the Oval. That's a condition of even being in the Oval. You can't ever quote the president.
GLENN: You can say I felt that this might have been what he was thinking. Even if he said it to you. That's the way you have to phrase it.
BILL: You know, I just don't -- I just don't analyze gossip and stuff like that.
GLENN: All right. All right.
BILL: To me, if there is an official announcement -- there's something going on because Spicer dealt with it too in a coy way. "Coy," another word of the day. C-O-Y, coy.
Spicer, who is a zombie of the greatest order, you would -- you know, he -- when he starts to play those kinds of games, well, we didn't really say that. But, you know, let's see what happens -- you know that they're trying to create mischief. Which is what the Trump White House does. It plays like a cat and a ball -- plays -- you know, and throws these things out. I don't think that's a good thing for President Trump. I think he probably disagrees with me on that. But they do this, you know, to be provocative. I don't know why. But right now, I don't think Mueller is going anywhere.
GLENN: Okay. We'll be back with Bill O'Reilly. And he's got a book or something for Father's Day that will be great. And it's at BillO'Reilly.com. It's Killing fill in the blank. And your father will love it. Who are you killing this time, Beck?
BILL: You, Beck. You. You. But I'm going to miss you probably more than anyone.
GLENN: Oh, I feel like the end of Wizard of Oz. That is so sweet. Why are you going to miss me? Are you going away?
BILL: No. But I -- in the book, if you --
GLENN: Oh, if I get offed --
GLENN: Okay. All right. At least we know how it ends.
BILL: No, but I would never do that because I'm your biggest defender, and you know it.
GLENN: The killer was Bill O'Reilly.
Welcome to today's Brave New World
New evidence of Ilhan Omar's immigration, tax fraud emerges
Pressley: 'We don't need black faces that don't want to be a black voice'
American Carnage: Author details the Trump campaign's reaction to the Access Hollywood tape
Tim Alberta joined Glenn on the radio program Tuesday to discuss his new book 'American Carnage.' In this clip he gives a detailed account of how the scene unfolded following the release of the infamous Access Hollywood tape and how the Trump campaign dealt with the disaster.
Here's how big tech and progressive Democrats are working together to sway the next election
Have you noticed how the DNC doesn't seem at all concerned about the 2020 election, even though the Democratic candidates are pushing policies that are even too radical for the left? Why would they do that if they're trying to win the hearts and minds of Americans? A website called Spinquark just released a chilling article that reveals exactly how many people who are directly connected to the progressive political machine are now working for big tech to control our conversations online — and they're unquestionably interfering with the 2020 election.
Social media slavery: The government has found a 'constitutional' way to sidestep 1A
Social media companies aren't held to constitutional standards because they're private companies. But they're also protected by the government because they're considered "platforms," not "publishers." Well, the government has figured this out and has seen the power of using social media to circumvent the First Amendment and silence conservative voices.
Brad Thor | Episode 44
Glenn sits down with the author many people call "the master of thrillers," Brad Thor. He is the #1 New York Times best-selling author of nineteen thrillers, including "Spymaster," "The Last Patriot," "Blowback," "Foreign Influence," "The Lions of Lucerne," and his very first book, "Backlash," which was named by Barnes & Noble as one of the best political thrillers ever. Reading his books has been described as being in a time machine one year before an event happens. Glenn talks with Brad about his writing and how closely his novels of international intrigue parallel the real threats facing the world today.


text:
DJI launches the Mini 3 at a cheaper price & weighs only 248 g!
Last Updated on: Dec 12, 2022 by Nagarjun M
Since the release of the lightweight Mini 3 Pro in May, we have been aware that the Mini 3 was soon to follow, but it still seems like the wait has been worthwhile.
Similar in practically every way to the Pro model, the Mini 3 weighs only 248 g (8.75 oz), enabling it to fly unregistered in "many regions of the world." It's a little, incredibly light camera drone got the necessary items for different trips.
Take Cinematic Shots Every Time
The DJI Mini 3 takes photos over an efficient 4K or 30fps in HDR using colors that are real & authentic. The Type 1/1.3 CMOS sensor and a fixed f/1.7 aperture lens can boost low-light performance. The sensor's design boasts pixels "as huge as 2.4 m."
The creator insists, "Greater picture quality, enhanced depth of field, reduced photo sounds in low illumination & robust contrast can be done using this structure. As per the Mini 3's sensor, "4-in1 pixel technology" is available for images with a resolution of up to 48 megapixels.
It implies that the 48MP sensor utilized in the DJI Mini 3 is a 48MP sensor meant to capture additional light by combining four pixels to function as a single pixel for the basic 12MP.
This method is used frequently in cameras and, depending on how the sensor is made, enables a camera to take images with a lower resolution but higher sensitivity or a higher resolution but lower sensitivity.
Within a short span, dual native ISO & chip-based HDR aids in picture-perfect images with great results, even in images using a high-end dynamic range similar to in-depth shadows. When using True Vertical Shooting can turn the camera 90° to produce data for social media sites, this great image quality leads to amazing outcomes.
Fly Longer & Further
According to claims, the Mini 3 can capture stable pictures while hovering steadily in gusts up to 10.7m/s. With the regular battery, DJI promises a maximum flying length of 38 minutes (when traveling at a steady 21.6 kph) and up to 51 minutes with the Intelligent Flight Battery Plus, an additional component that isn't offered everywhere.
The Mini 3 can collect automatically preset QuickShots, just like other current DJI drone models, to create a variety of standard movie shots taken from a helicopter. Dronie, Helix, Rocket, Circle & Boomerang can be executed by Mini 3.
The DJI Mini 3 got the O2 digital transmission device from the firm to send a live 720p or 30fps feed around 10 km distant. The maximum download speed for a QuickTransfer system is 25 MB/s.
The drone has an autonomous takeoff feature and a return-to-home feature. Additional Return To Home (RTH) modes for the DJI Mini 3 include Smart RTH, Low Battery RTH, and Failsafe RTH, which sends the drone home in the event of a controller signal loss.
Even in winds up to 10.7 m/s, hovering stability is achieved by combining GNSS with a downfacing vision system and IR sensors. A subject can be locked on, and the pilot can use pre-programmed flying patterns to capture artistic footage.
Seven variations of the DJI Mini 3 are available for purchase via store.dji.com and authorized retail partners. The Drone solo is offered at a suggested retail price of 469 USD.
Categories Camera


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East of Bethel is thrilled to be part of the spread of God's kingdom around the world. We are active in spreading the word weekly at our East of Bethel Christian Center. We extend welcome to anyone who wishes to come be fed the Word of God.
The purpose of the men's ministry of East of Bethel Christian Center is to encourage men to be the leaders that God created them to be. Through biblical teaching, it is the desire of this ministry to address the various issues that confront today's men marriage, fatherhood, finances, leadership, attitudes, behaviors, thinking process and changing for the better.
The purpose of the women's ministry of East of Bethel Christian Center is to encourage spiritual growth in women through the study of God's word, to cultivate relationships and promote evangelistic outreach. Furthermore, we desire to educate and enlighten young girls and oppressed women – body, mind, and soul.
Prayer cannot be overestimated. Prayer moves the hand of God. Our Church is a body of believers dependent upon prayer, and committed to God's sovereignty. Every Monday, volunteers from our Prayer Warriors team pray for their pastor, a list of church members, any special requests, and missionary prayer needs.
God-honoring worship is something we strive for. Many church members get involved in our music and drama ministry. Some play their instruments. Some are vocalists. Others are involved in the choir. Still, others get involved in drama. These volunteers are an important part of the ministry of our church, using their talents and gifts to glorify God.
Worship dance is our expression of love to God that comes out of pressing into a deeper relationship with Him. Its history goes back to accounts of dancing in the Old Testament. An example is the episode when King David danced before the Ark of the Covenant. Liturgical dancers wear a garment of praise, not a costume. It is symbolic and meaningful in design, colors and fabrics. Our purpose is not to dazzle audiences but to deepen their religious experience, to make dance into a prayer.
The purpose of the finance ministry of East of Bethel Christian Center is to manage the financial aspects of the church – including but not limited to the church's operating fund, investments, and disbursements. Furthermore, this ministry was established to provide financial guidance to the congregants and community through the teaching of applying biblical principles to everyday budgeting and long-term financial planning.


Three of NGA's gymnasts competed at the NCGA Floor and Vault in Mansfield on Sunday 13th May.
Florence and Tiffany began the day, competing the under 8s floor routine and a squat on stretch jump vault. Both girls enjoyed performing to the crowd and their vaults in particular were fantastic!
This was Tiffany's first competition experience and she took part in all aspects of the competition from marching on to warm ups, to watching other members of her group compete, and of course the presentation!
Florence also attended the competition last year and her independence has grown leaps and bounds since - she competed her vaults and went round the competition with her group that was made up of gymnasts from other clubs, too. This was a fantastic step forward for Florence. Both girls had a fantastic morning with lots of smiles and some super selfies with their coach! Florence came second AA in the I'm in Category, and Tiffany came 1st AA.
Caitlin competed in the Primary 2 category in the afternoon session. She competed a fantastic floor routine which included her newly-learnt half spin, and her two vaults. Despite finding the vault hard work, Caitlin showed excellent spirit and persevered - each vault from her first warm up vault to her last competition vault improved. Caitlin also enjoyed socialising with gymnasts from the other clubs, and to top it all off came 1st AA in the 9 year old, "I'm In" Category.


text:
/*
  Include declarations.
*/
#include "magick/studio.h"
#include "magick/blob.h"
#include "magick/blob-private.h"
#include "magick/exception.h"
#include "magick/exception-private.h"
#include "magick/magick.h"
#include "magick/memory_.h"
#include "magick/pixel-accessor.h"
#include "magick/quantum-private.h"
#include "magick/registry.h"
#include "magick/static.h"
#include "magick/string_.h"
#include "magick/module.h"

/*
  Forward declarations.
*/
static MagickBooleanType
  WriteMPRImage(const ImageInfo *,Image *);

/*
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
%                                                                             %
%                                                                             %
%                                                                             %
%   R e a d M P R I m a g e                                                   %
%                                                                             %
%                                                                             %
%                                                                             %
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
%
%  ReadMPRImage() reads a Magick Persistent Registry image as a blob from
%  memory.  It allocates the memory necessary for the new Image structure and
%  returns a pointer to the new image.
%
%  The format of the ReadMPRImage method is:
%
%      Image *ReadMPRImage(const ImageInfo *image_info,
%        ExceptionInfo *exception)
%
%  A description of each parameter follows:
%
%    o image_info: the image info.
%
%    o exception: return any errors or warnings in this structure.
%
*/
static Image *ReadMPRImage(const ImageInfo *image_info,ExceptionInfo *exception)
{
  Image
    *image;

  assert(image_info != (const ImageInfo *) NULL);
  assert(image_info->signature == MagickSignature);
  if (image_info->debug != MagickFalse)
    (void) LogMagickEvent(TraceEvent,GetMagickModule(),"%s",
      image_info->filename);
  assert(exception != (ExceptionInfo *) NULL);
  assert(exception->signature == MagickSignature);
  image=(Image *) GetImageRegistry(ImageRegistryType,image_info->filename,
    exception);
  if (image != (Image *) NULL)
    (void) SyncImageSettings(image_info,image);
  return(image);
}

/*
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
%                                                                             %
%                                                                             %
%                                                                             %
%   R e g i s t e r M P R I m a g e                                           %
%                                                                             %
%                                                                             %
%                                                                             %
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
%
%  RegisterMPRImage() adds attributes for the MPR image format to
%  the list of supported formats.  The attributes include the image format
%  tag, a method to read and/or write the format, whether the format
%  supports the saving of more than one frame to the same file or blob,
%  whether the format supports native in-memory I/O, and a brief
%  description of the format.
%
%  The format of the RegisterMPRImage method is:
%
%      size_t RegisterMPRImage(void)
%
*/
ModuleExport size_t RegisterMPRImage(void)
{
  MagickInfo
    *entry;

  entry=SetMagickInfo("MPR");
  entry->decoder=(DecodeImageHandler *) ReadMPRImage;
  entry->encoder=(EncodeImageHandler *) WriteMPRImage;
  entry->adjoin=MagickFalse;
  entry->format_type=ImplicitFormatType;
  entry->stealth=MagickTrue;
  entry->description=ConstantString("Magick Persistent Registry");
  entry->module=ConstantString("MPR");
  (void) RegisterMagickInfo(entry);
  entry=SetMagickInfo("MPRI");
  entry->decoder=(DecodeImageHandler *) ReadMPRImage;
  entry->encoder=(EncodeImageHandler *) WriteMPRImage;
  entry->adjoin=MagickFalse;
  entry->format_type=ImplicitFormatType;
  entry->stealth=MagickTrue;
  entry->description=ConstantString("Magick Persistent Registry");
  entry->module=ConstantString("MPRI");
  (void) RegisterMagickInfo(entry);
  return(MagickImageCoderSignature);
}

/*
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
%                                                                             %
%                                                                             %
%                                                                             %
%   U n r e g i s t e r M P R I m a g e                                       %
%                                                                             %
%                                                                             %
%                                                                             %
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
%
%  UnregisterMPRImage() removes format registrations made by the
%  MPR module from the list of supported formats.
%
%  The format of the UnregisterMPRImage method is:
%
%      UnregisterMPRImage(void)
%
*/
ModuleExport void UnregisterMPRImage(void)
{
  (void) UnregisterMagickInfo("MPRI");
  (void) UnregisterMagickInfo("MPR");
}

/*
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
%                                                                             %
%                                                                             %
%                                                                             %
%   W r i t e M P R I m a g e                                                 %
%                                                                             %
%                                                                             %
%                                                                             %
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
%
%  WriteMPRImage() writes an image into the Magick Persistent Registry
%  image as a blob from memory.  It allocates the memory necessary for the
%  new Image structure and returns a pointer to the new image.
%
%  The format of the WriteMPRImage method is:
%
%      MagickBooleanType WriteMPRImage(const ImageInfo *image_info,Image *image)
%
%  A description of each parameter follows.
%
%    o image_info: the image info.
%
%    o image:  The image.
%
*/
static MagickBooleanType WriteMPRImage(const ImageInfo *image_info,Image *image)
{
  MagickBooleanType
    status;

  assert(image_info != (const ImageInfo *) NULL);
  assert(image_info->signature == MagickSignature);
  assert(image != (Image *) NULL);
  assert(image->signature == MagickSignature);
  magick_unreferenced(image_info);
  if (image->debug != MagickFalse)
    (void) LogMagickEvent(TraceEvent,GetMagickModule(),"%s",image->filename);
  status=SetImageRegistry(ImageRegistryType,image->filename,image,
    &image->exception);
  return(status);
}

redpajama_set_name: RedPajamaGithub


Tom Davis, editor and publisher of Old Mountain Press, recently released WORDS, a Poetry and Prose Anthology. Brenda Kay Ledford's poem, "Reach of Words," appeared in this collection. Her works have been included in all 15 Old Mountain Press anthologies. Blanche L. Ledford' short story, "Fainting Words," was included in WORDS. Barbara Ledford Wright' essay, "Fruit of Our Words," also appeared in this collection. Only writers previously published in Old Mountain Press were eligible to submit to WORDS, or persons recommended by these contributors. Writers with works in this anthology include: Jim Clark, Celia Miles, Nancy Dillingham, Al Manning, Debra Elramey, C. Pleasants York, Ed Cockrell, Tom Davis, Jo Barbara Taylor, Dr. Lynn Veach Sadler, Mark Harden, and others. Marian Kaplun Shapiro, thrice Senior Poet Laureate of Massachusetts, volunteered to judge the contest for this anthology. Mark E. Harden's poem, "Gun Line," won the Best of the Best Award. Honorable mentions included: Michael Colonnese, David Treadway Manning, and Jerome Norris. For more information about WORDS, go to: www.OldMountainPress.com.


MARTINSBURG, W.Va. - State Sen. Clark Barnes announced Friday that he filed for a second four-year term in the West Virginia State Senate.
According to a news release, Barnes said his decision to run again was to continue the progress that has been made in the past three years.
"Serving the residents of the 15th District has been and will continue to be his first priority," Barnes said in the news release.
Now Senate Minority Whip, Barnes of Randolph County, said he was the first Randolph County Republican to win a seat in the State Senate since 1929.
He defeated Randolph County Democrat Mike Ross, who filed for the seat on Jan. 14, according to West Virginia Secretary of State Betty Ireland's office.
Barnes said he delayed filing for office because he received "pressure" from GOP operatives who considered him as a candidate for the statewide offices of Secretary of State or Governor.
The 15th district is the largest of the State Senate's 17 and includes Randolph, Pocahontas, Pendleton, Hardy, Hampshire, Morgan, and portions of Upshur, Grant and Berkeley counties.
Barnes cited his involvement in issues pertaining to private property rights and victims of sexual predators, and noted his avid support of Second Amendment gun ownership and the rights of homeowners to defend their homes and families without fear of liability from lawsuits.
"It has been a great pleasure to serve the citizens of the 15th Senatorial District," Barnes said.
Can GOP win over Latino hearts?