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MGA2 and SPT23 are functionally and genetically redundant homologs in Saccharomyces cerevisiae. Both genes are implicated in the transcription of a subset of genes, including Ty retrotransposons and Tyinduced mutations. Neither gene is essential for growth, but mga2 spt23 double mutants are inviable. We have isolated a gene-specific activator, SWI5, and the Δ9 fatty acid desaturase of yeast, OLE1, as multicopy suppressors of an mga2Δ spt23 temperature-sensitive mutation (spt23-ts). The level of unsaturated fatty acids decreases 35–40% when the mga2Δ spt23-ts mutant is incubated at 37°. Electron microscopy of these cells reveals a separation of inner and outer nuclear membranes that is sometimes accompanied by vesicle-like projections in the intermembrane space. The products of Ole1p catalysis, oleic acid and palmitoleic acid, suppress mga2Δ spt23-ts and mga2Δ spt23Δ lethality and restore normal nuclear membrane morphology. Furthermore, the level of the OLE1 transcript decreases more than 15-fold in the absence of wild-type Mga2p and Spt23p. Our results suggest that Mga2p/Spt23p control cell viability by stimulating OLE1 transcription.
IN the budding yeast Saccharomyces cerevisiae, insertion of a Ty retrotransposon into the promoter region of a gene often results in alteration of the transcription of that gene (reviewed by Winston 1992). Several key genes required for general transcription have been identified as extragenic suppressors of Ty-induced promoter mutations and are named SPT (Suppressor of Ty) genes. These include genes encoding the TATA-binding protein TBP (SPT15; Eisenmannet al. 1989), histone core proteins H2A and H2B (SPT11 and SPT12; Clark-Adamset al. 1988), and proteins involved in transcription elongation by RNA polymerase II (SPT4, SPT5, and SPT6; Hartzoget al. 1998).
SPT23 was isolated as a multicopy suppressor of Tyinduced promoter mutations (Burkett and Garfinkel 1994). SPT23 shares considerable homology with MGA2, a gene identified as a multicopy suppressor of a snf2-imposed block on STA1 expression in S. cerevisiae var. diastaticus (Zhanget al. 1997). Although null mutations in either of these genes have only modest effects on cell growth, mga2 spt23 double mutants are inviable. Because previous work suggests that Mga2p and Spt23p are transcriptional coactivators with overlapping specificities, we have been interested in identifying genes that are functionally related to or controlled by Mga2p/Spt23p. Therefore, we screened a multicopy plasmid library to identify suppressors of the cellular lethality resulting from the absence of Mga2p/Spt23p. The strongest plasmid-borne suppressor of mga2Δ spt23-ts lethality isolated from the screen is OLE1, which encodes the Δ9 fatty acid desaturase of yeast.
The fatty acid synthesis pathway in S. cerevisiae has been established using biochemical and genetic approaches (Figure 1; Paltaufet al. 1992; Schneiteret al. 1996; Schneiter and Kohlwein 1997). Yeast cells utilize exogenous free fatty acids through activation by at least four acyl-coenzyme A (CoA) synthetases (Faa1p–Faa4p; Johnsonet al. 1994) or synthesize saturated fatty acids de novo to form the major saturated long chain fatty acids (SFAs) palmitoyl-CoA (16:0) and stearoyl-CoA (18:0). Three enzyme systems participate in this process: acetyl-CoA carboxylase (Acc1p), fatty acid synthetase (Fas1p and Fas2p), and elongase (Elo1p). A small portion of the long chain fatty acids can also be elongated to form very long chain fatty acids (C20-C30; Welch and Burlingame 1973).
In animal and fungal cells, monounsaturated fatty acids (UFAs) are synthesized from fatty acid precursors by an aerobic microsomal enzyme system that includes cytochrome b5, NADH-dependent cytochrome b5 reductase, and Δ9 fatty acid desaturase (Dailey and Strittmatter 1978, 1980; Stukeyet al. 1990). The OLE1 gene encodes the sole Δ9 fatty acid desaturase in S. cerevisiae and is essential for viability (Stukey et al. 1989, 1990). Ole1p catalyzes the formation of a double bond between C9 and C10 of palmitoyl-CoA (16:0) and stearoyl-CoA (18:0), forming palmitoleic acid (16:1) and oleic (18:1) acid. Under laboratory conditions, yeast cells synthesize only monounsaturated fatty acids, which comprise about 70% of the total fatty acids in the cell. The correct ratio of saturated to unsaturated fatty acids is important for maintaining the optimal levels of membrane fluidity and curvature, which are essential for a variety of cellular processes (Krulwichet al. 1990; Carratuet al. 1996). Interestingly, an OLE1 temperature-sensitive allele, mdm2, causes aberrant mitochondrial segregation at the nonpermissive temperature and UFAs decrease about 2.5-fold (McConnellet al. 1990; Stewart and Yaffe 1991).
The steady state level of OLE1 mRNA is regulated by transcription and RNA turnover, and both regulatory processes are affected by the presence of fatty acids (Bossie and Martin 1989; Choiet al. 1996; Gonzalez and Martin 1996). Addition of an exogenous UFA represses the transcription of OLE1 and promotes the decay of OLE1 mRNA. cis-acting sequences in the OLE1 5′ noncoding region as well as trans-acting factors are implicated in the transcriptional regulation of OLE1 (Choiet al. 1996). Two fatty acid activation genes, FAA1 and FAA4, the transcriptional activator HAP1, and acyl-CoA-binding protein have been reported to regulate OLE1 transcription. Our current studies suggest that Mga2p and Spt23p stimulate transcription of OLE1. Loss of functional Mga2p/Spt23p results in a lower level of UFAs and subsequent cell death. Plasmid-borne expression of OLE1, or UFAs present in the growth medium, rescues the conditional mga2Δ spt23-ts mutant at the nonpermissive temperature or a mga2Δ spt23Δ double mutant. Electron microscopy shows that the mga2Δ spt23-ts mutant has a morphologically altered nuclear membrane at the nonpermissive temperature, a defect that is also reversed by addition of UFAs. Therefore, our results suggest that OLE1 is an essential target gene affected by Mga2p/Spt23p.
Strains and media: Yeast strains are listed in Table 1. The ole1::LEU2 mutant SZ66 was constructed by one-step gene disruption (Rothstein 1991) using a SacI-HindIII fragment from pSRZ163. Correct gene replacement was verified by Southern blot analysis (S. Zhang, unpublished results). OLE1-URA3 was integrated at the ura3-52 locus of strains DG1555 and DG1667 by linearizing the YIp plasmid pSRZ176 at a unique StuI site in URA3. Strain SZ67 was obtained by tetrad dissection of a diploid strain heterozygous for spt23Δ-hisG and mga2Δ::LEU2 on an unsupplemented YPD plate, followed by replica plating to YPD containing unsaturated fatty acids (UFAs). Standard growth media were used as described by Rose et al. (1990). Media supplemented with UFAs were made by adding equimolar amounts of palmitoleic acid (16:1) and oleic (18:1) acid to a final concentration of 0.5 mm. Tergitol (Fluka Chemical, Buchs, Switzerland) was included at a final concentration of 1% (v/v) to solubilize the UFAs, as described by Stukey et al. (1989). Media supplemented with a saturated fatty acid (SFA) were made by adding palmitic acid to a final concentration of 1.2 mm. Brij 58 (Aldrich Chemical, Milwaukee) was added to a final concentration of 1% (w/v) to solubilize the SFA, as described by Mishina et al. (1980).
Plasmid and DNA manipulations: Plasmids were constructed by standard procedures (Sambrooket al. 1989). Generally, restriction fragments were purified from agarose gels by using glass milk (BIO 101, Inc., Vista, CA). Restriction enzymes [New England Biolabs (NEB), Beverly, MA], T4 DNA ligase (NEB), high-fidelity thermostable DNA polymerase (Boehringer Mannheim, Indianapolis), and the Klenow fragment of DNA polymerase I (NEB) were used according to the suppliers' suggestions. DNA sequencing was performed using a PRISM sequencing kit (Perkin-Elmer, Norwalk, CT). Plasmids were purified from Escherichia coli by a boiling lysis method (Holmes and Quigly 1981) and introduced into yeast cells as described by Gietz et al. (1992). Plasmid pBDG794, which contains the spt23-ts allele, was isolated after mutagenizing plasmid pSRZ47 with hydroxylamine (Zhanget al. 1997). Plasmids pSMS1, pSMS6, pSMS7, pSMS8, pSMS9, and pSMS22 were isolated as suppressors of the lethality conferred by mga2Δ spt23-ts at 37°. Plasmids pSRZ157 (2μ-OLE1), pSRZ161 (CEN-OLE1), and pSRZ176 were constructed by subcloning a 3.1-kb SacI-HindIII fragment containing OLE1 from pSMS7 into pRS426, pRS416, and pRS406 (Sikorski and Heiter 1989), respectively. Plasmid pSRZ159 (2μ-ole1) was constructed by digesting pSRZ157 with Tth111I, filling in the DNA ends with Klenow DNA polymerase, and self-ligation. The ole1::LEU2 gene disruption in pSRZ163 was constructed by a homologous recombination event that replaced OLE1 sequences from codons 14–498 in pSRZ161 with a 2.2-kb polymerase chain reaction (PCR) product containing the LEU2 gene as described by Manivasakam et al. (1995). The relative copy number of the URA3-based low-copy plasmids pRS416 and CEN-OLE1 in strains DG1555 and DG1667 was determined by Southern blot analysis following digestion with HindIII. Total yeast DNA from certain transformants was isolated after cells were shifted to 37° for 6 hr. The resulting filter was hybridized with a 32P-labeled internal NdeI-StuI fragment from URA3. The transformants contained two types of HindIII fragments that hybridize with this probe: cells harboring the pRS416 vector or the CEN-OLE1 plasmid contained a 4.4- or 7.5-kb fragment, respectively, and a chromosomal 2.1-kb fragment from ura3-52. The ratio of the hybridization signals of the plasmid fragments to the signal of the 2.1-kb chromosomal HindIII fragment indicated the relative copy number of the plasmids. The copy number of the integrated pSRZ176 plasmid at the ura3-52 locus in strains DG1555 and DG1667 was determined by Southern blot analysis after EcoRV digestion. The resulting filter was hybridized with a 32P-labeled URA3 probe as described above. The integrants contained two EcoRV fragments that hybridized with this probe: a 4.8-kb fragment spanning an external ura3-52/URA3 chromosomal/plasmid recombination junction and a 6.2-kb fragment spanning an internal URA3/URA3 plasmid/plasmid recombination junction. The ratio of the hybridization signal of the 6.2-kb fragment to that of the 4.8-kb fragment indicated the pSRZ176 copy number at the chromosomal ura3-52 locus. Hybridization signals were quantitated by phosphorimaging using conditions suggested by the manufacturer (Molecular Dynamics, Inc., Sunnyvale, CA) and ImageQuant software (Version 1.1).
Lipid analysis: DG1555 and the DG1667-based strains were grown in either YPD or synthetic complete media lacking uracil (SC-Ura) to early log phase at 23°. The cultures were either allowed to continue growing at 23° or shifted to 37°, and samples were removed 6 and 15 hr after the temperature shift. Total lipids were extracted by a modification of standard methods (Bligh and Dyer 1959; Martinet al. 1981; Kates 1986). Briefly, yeast cells were broken by vortexing in the presence of glass beads in methanol:choloroform:water (2:1:0.8). Total lipids were saponified with sodium hydroxide, then esterified by transmethylation with boron trifluoride (Morrison and Smith 1964). The fatty acid methyl esters were analyzed by gas-liquid chromatography. Commercially available methyl esters of palmitic acid (16:0), stearic acid (18:0), palmitoleic acid (16:1), and oleic acid (18:1) (Sigma, St. Louis) were used as standards to identify and quantitate cellular fatty acids.
Electron microscopy: Strains DG1555 and DG1667 were grown to early log phase in YPD broth at 23°, then either kept at 23° in YPD or shifted to 37° in either YPD or YPD supplemented with UFAs, and grown for an additional 6 hr. The ole1 mutant strain SZ66 was pregrown to early log phase in YPD medium supplemented with UFAs at 30°. The cells were washed with 1 and 0.5% tergitol to remove residual UFAs. The washed cells were incubated in either supplemented or unsupplemented YPD medium at 30° or 37° for 6 or 14.5 hr. Cells collected by vacuum filtration onto a 0.45-μm nitrocellulose filter (Millipore, Bedford, MA) were washed once with water and once with buffer A [40 mm KPO4 (pH 6.5), 0.5 mm MgCl2], then fixed overnight at 4° with 4% formaldehyde and 2% glutaraldehyde in buffer A. After two additional washes with buffer A, the cells were resuspended in a buffer containing 200 mm Tris-HCl (pH 9.0), 20 mm EDTA, and 100 mm β-mercaptoethanol, then incubated for 10 min at room temperature. The fixed cells were washed three times with 0.1 m KPO4 (pH 7.5), digested with Zymolyase 100T (250 ng/ml final concentration; ICN Corp., Aurora, OH), and postfixed with 1% osmium tetroxide by standard techniques (Byers and Goetsch 1991).
RNA analyses: Total yeast RNA was isolated and Northern filter hybridizations were performed as described by Schmitt et al. (1990) and Zhang et al. (1997), respectively. 32P-labeled OLE1, ACC1, and GAL80 hybridization probes were synthesized using the Megaprime DNA Labeling System (Amersham, Buckinghamshire, UK). The template for labeling OLE1 was a 1.3-kb SalI-PacI fragment of pSRZ157. The template for the GAL80 probe was a 1.3-kb genomic fragment amplified by PCR using primers 5′-CCACTCCCGTCATGGAC-3′ and 5′-GGGGGCCAAGCACAGG-3′. The template for the ACC1 probe was a 1.9-kb genomic fragment amplified by PCR using primers 5′-CCGGACAAGGGCTTC-3′ and 5′-GTACCCCTTC CCACAG-3′. Hybridization signals were quantitated by phosphorimage analysis as described above. RNA differential display analysis (Liang and Pardee 1992) was performed using RNAimage (GenHunter Corp., Nashville, TN) under conditions suggested by the supplier. Residual DNA was removed from yeast RNA using MessageClean (GenHunter Corp.).
Isolation of plasmid-borne suppressors of mga2Δ spt23-ts: The synthetic lethality of an mga2 spt23 double mutant suggests that these genes share functions that are essential for cell viability. To identify genes functionally related to or controlled by Mga2p/Spt23p, we screened multicopy plasmid libraries for clones that suppress the lethality of an mga2Δ spt23-ts mutant, DG1667, at the nonpermissive temperature of 37°. Strain DG1667 carries the spt23-ts allele on plasmid pBDG794 and contains chromosomal deletions of MGA2 and SPT23; therefore, we used plasmid segregation to determine whether a given suppressor plasmid is dependent on spt23-ts for its activity. As expected, DG1667 grew well at 23° but not at 37° (Figure 2, vector), compared with the MGA2/SPT23 wild-type strain DG1555 (Wt./vector). Two URA3-based multicopy yeast genomic libraries (Carlson and Botstein 1982; Nehlinet al. 1989) were introduced into DG1667 at 23° to allow all possible transformants to grow. Primary transformant colonies were then replica plated to fresh SC-Ura plates and incubated at 37°. A total of 20 transformants grew at 37° from more than 400,000 screened. Suppressors pSMS1, pSMS6, pSMS7, pSMS8, pSMS9, and pSMS22 were plasmid borne and restored growth at the nonpermissive temperature to various extents (Figure 2; S. Zhang, unpublished results). The suppression by the other 14 isolates probably resulted from chromosomal mutations; we will characterize these suppressors elsewhere.
To identify the suppressor genes, a partial DNA sequence from the genomic inserts of the six plasmidborne suppressors was submitted to the Saccharomyces Genome Data Base (Stanford University). As expected, we reisolated MGA2 twice (pSMS8 and pSMS9; in Figure 2, pSMS8 is shown) and SPT23 once (pSM6). Strain DG1667 containing pSM6, pSM8, or pSMS9 grew well at 37°, compared with the DG1667 (vector) mutant and DG1555 (Wt./vector) wild-type control strains. The suppressor plasmids pSMS7 and pSMS22 contained overlapping inserts, as demonstrated by DNA sequence and restriction enzyme analyses (S. Zhang, unpublished results). The suppression by pSMS7 and pSMS22 was independent of spt23-ts and growth temperature, because the loss of plasmid pBDG794 did not affect the suppressor's activity at 23°,30°,or37°. The spt23-ts-independent suppression by pSMS7 and pSMS22 also showed that these plasmids suppressed lethality in an mga2Δ spt23Δ mutant. Deletion and site-directed mutagenesis of the insert in pSMS7 indicated that OLE1 was responsible for the suppression (Figure 3; S. Zhang, unpublished results), because a subclone containing only the OLE1 open reading frame (ORF) suppressed mga2Δ spt23-ts (2μ-OLE1) and a frameshift mutation in the OLE1 coding sequence no longer suppressed mga2Δ spt23-ts (2μ-ole1). As expected, either 2μ-MGA2 or 2μ-SPT23 suppressed the temperature sensitivity of DG1667. Surprisingly, a low-copy centromere-based plasmid containing OLE1 was also capable of suppression (CEN-OLE1).
Plasmid pSMS1 suppressed mga2Δ spt23-ts weakly (Figure 2) and the suppression was dependent on the spt23-ts plasmid, suggesting a possible interaction between the suppressor and Spt23p. Analysis of its genomic insert by DNA sequencing and mutagenesis indicated that SWI5 conferred the suppressor activity (S. Zhang, unpublished results). SWI5 encodes a transcriptional activator necessary for expression of the HO endonuclease gene and of EGT2, a gene required for mother-daughter cell disjunction (Breeden and Nasmyth 1987; Kavacechet al. 1996). Further analysis of the possible interaction between Spt23p and Swi5p will be presented elsewhere.
Fatty acid content and requirements in the mga2 spt23 mutants: The Δ9 fatty acid desaturase encoded by OLE1 is the only desaturase identified in S. cerevisiae (Stukey et al. 1989, 1990). Under standard laboratory growth conditions in minimal or rich media, yeast produce monounsaturated fatty acids (UFAs), which constitute about 70% of the total fatty acids in the cell. Because an ole1 null mutant requires exogenous UFAs for growth and plasmid-borne OLE1 expression suppresses the mga2Δ spt23-ts mutant, we reasoned that OLE1 restored UFA synthesis at 37°. Therefore, we determined the saturated fatty acid (SFA; 16:0 and 18:0) and UFA (16:1 and 18:1) content in mutant and wild-type cells (Table 2), and determined whether the mga2 spt23 mutants grew when supplemented with an SFA or UFAs (Figure 4). The wild-type (MGA2 SPT23) and mutant (mga2Δ spt23-ts) strains contained about 73% UFAs at 23°, and the wild-type cells contained 77.8% UFAs 15 hr after the shift to 37° (Table 2, total UFAs). However, the percentage of UFAs in the mga2Δ spt23-ts mutant decreased to 53.5% after a 6-hr incubation and to 48.1% after 15 hr at 37°. When UFA levels in the mga2Δ spt23-ts mutant at 23° and 37° were compared, a loss of almost half of the 16:1 UFA palmitoleic acid occurred after 15 hr at 37°. As expected for cells lacking Ole1p activity (mga2Δ spt23-ts at 37°), the levels of 16:0 and 18:0 SFAs increased to over 30 and 16%, respectively, at 37°. The presence of plasmid-borne OLE1 in the mutant cells restored UFA synthesis to wild-type levels (2μ-OLE1).
We then determined whether direct supplementation of UFAs (16:1 and 18:1) to the growth medium suppressed the conditional lethality caused by mga2Δ spt23-ts and the unconditional lethality of the mga2Δ spt23Δ double deletion (Figure 4). Inclusion of the SFA palmitic acid or UFAs in the growth medium was mildly toxic to wild-type cells (Figure 4A), because these cultures reached stationary phase at a lower cell density. However, the supplemented cells maintained their normal shape, budding pattern, doubling time (80 min), and had no other obvious defects (S. Zhang, unpublished results). In contrast, the mga2Δ spt23-ts mutant only grew at the nonpermissive temperature of 37° in medium supplemented with UFAs (Figure 4B, 131 min doubling time). Addition of an SFA did not support growth, indicating that the suppression by UFAs was specific. Furthermore, supplementation with UFAs also suppressed the lethality of the mga2Δ spt23Δ null mutant at the standard growth temperature of 30° (Figure 4C, 120 min doubling time). Taken together, these results suggest that the absence of Mga2p and Spt23p results in a significant decrease in intracellular UFAs and subsequent cell death at 30° and 37°.
MGA2/SPT23 and OLE1 affect nuclear membrane morphology: Certain fatty acid-deficient mutants have nuclear membrane defects. In particular, a mutation in the yeast acetyl-CoA carboxylase gene acc1-7-1 causes severe alterations in nuclear envelope morphology (Schneiteret al. 1996). Therefore, we examined the cellular structure of the mga2Δ spt23-ts mutant (Figure 5) and an ole1 null mutant (Figure 6) by electron microscopy. The mga2Δ spt23-ts mutant showed normal nuclear envelope morphology at 23° (Figure 5D), which was similar to that observed for the wild type at 23° (S. Zhang, unpublished results) or 37° (Figure 5A). The nuclear membrane in the mga2Δ spt23-ts mutant was severely distorted at 37° (Figure 5B). Like acc1-7-1, the mga2Δ spt23-ts mutant showed a separation of the inner and outer nuclear membranes and some cells contained vesicle-like structures in the intermembrane space. Addition of UFAs to the growth medium corrected the nuclear membrane defect in the mga2Δ spt23-ts mutant (Figure 5C).
Because the cell growth and morphological defects present in the spt23 mga2 double mutants are reversed by supplementing the growth medium with UFAs, we determined whether ole1 and mga2Δ spt23-ts mutants had similar phenotypes. We disrupted one copy of OLE1 in an OLE1/OLE1 diploid strain by single-step gene disruption using a restriction fragment containing ole1::LEU2. The correct disruption was verified by genomic Southern blot analysis (S. Zhang, unpublished results). Dissection of the resulting ole1::LEU2/OLE1 diploid on medium supplemented with UFAs resulted in four viable spores in each of five tetrads. As expected, there was a 2:2 segregation for the UFA requirement and all UFA– cells were Leu+. To examine the cellular morphology of the ole1 mutant after starvation for UFAs, strain SZ66 was grown to early log phase in YPD broth supplemented with UFAs and washed extensively to remove residual UFAs, as previously described by Stukey et al. (1989). The ole1 mutant was resuspended in either supplemented or unsupplemented YPD media and incubated for 14.5 hr at 37° to further deplete UFAs from the unsupplemented cells. Cells treated in this way have UFA levels <10% of total cellular fatty acids (Stukeyet al. 1989). Electron microscopy of the ole1 mutant that was either supplemented (Figure 6A) or starved (Figure 6B) for UFAs revealed severe morphological defects when the mutant was depleted of UFAs. Many membrane-like structures separated by unstained material were apparent, but morphologically recognizable subcellular structures, such as mitochondria and nuclei, were not observed in these cells. UFA starvation for 6 hr or incubation at 30° resulted in similar morphological defects (S. Zhang, unpublished results). The severity of the ole1 mutant phenotype suggests that UFA synthesis is not completely blocked in the mga2Δ spt23-ts mutant.
GAL80 and OLE1 transcript levels in the mga2Δ spt23-ts mutant: To identify additional target genes activated by Mga2p/Spt23p, we performed RNA differential display followed by Northern blot analysis with RNA extracted from the wild type and the mga2Δ spt23-ts mutant grown at 37°. Candidate genes having a lower level of transcripts as well as differential display products included CRY1, UBI2, YLR388w, and YMR142c (S. Zhang, unpublished results). However, we were unable to reproducibly obtain the same reduction in candidate gene transcript levels from independent RNA preparations. These results also raised the concern that an Mga2p/Spt23p-independent RNA polymerase II transcript, which would be required as a loading control for further analysis of OLE1 transcription, would be difficult to obtain. Therefore, we identified RNA differential display products that remained constant under all conditions. One of the strongest constant display products was from the GAL80 gene. This result was verified by Northern blot analysis using independent RNA preparations (S. Zhang, unpublished results). Because the level of the GAL80 transcript was not controlled by MGA2 or SPT23, GAL80 was used as an internal loading control to quantitate the relative level of the OLE1 transcript.
We performed Northern blot analysis using total RNA from different strains to determine whether Mga2p/Spt23p are required for OLE1 transcription (Figure 7). Similar levels of the OLE1 transcript were produced in the wild-type strain (Figure 7A, lane 1) and mga2Δ (lane 2) or spt23Δ (lane 3) single mutant strains, compared with the level of the GAL80 transcript. When the mga2Δ spt23-ts mutant was analyzed at 37°, however, very little OLE1 transcript was detected (lane 4). Introduction of a multicopy plasmid carrying OLE1 increased the OLE1 transcript well above (lane 5) the wild-type level. Adding exogenous UFAs did not increase the level of OLE1 transcript in the mutant at 37° (S. Zhang, unpublished results), minimizing the possibility that an alternative pathway mediates suppression of growth by UFAs and expression of OLE1. In addition, the transcript level from ACC1, another gene required for fatty acid biosynthesis (Figure 1), remained unaltered in the mga2Δ spt23-ts mutant (S. Zhang, unpublished results), suggesting that not all fatty acid biosynthetic genes require Mga2p/Spt23p.
To quantitate the level of the OLE1 transcript in the absence of functional Mga2p/Spt23p, we performed Northern blot analysis with RNA extracted from the wild type and the mga2Δ spt23-ts mutant 0, 10, 30, and 60 min after shifting the cultures from 23° to 37° (Figure 7B). Phosphorimage analysis of the resulting filters indicated that the level of the OLE1 transcript remained constant, compared with the GAL80 transcript loading control throughout the time course in wild-type cells (lanes 1–4). The OLE1 transcript level in the mutant (lane 5; 0 min) was about threefold lower than that in the wild type (lane 1; 0 min) at the time of the temperature shift. The lower level of the OLE1 transcript in the mga2Δ spt23-ts mutant at 23° is probably due to a moderate loss of activity of the Spt23-ts protein at the permissive temperature. Shifting the mutant cells to 37° decreased the level of the OLE1 transcript 15-fold after 10 min (lane 6), and 30-fold after 30 (lane 7) or 60 (lane 8) min, compared with the level of the OLE1 transcript in the wild type at equivalent time points.
Suppression by CEN-OLE1: We considered two models that might explain the suppression of mga2Δ spt23-ts when OLE1 is carried on a low-copy centromere plasmid. Mga2p/Spt23p may be required for centromere function, thereby allowing the plasmid copy number to increase significantly when Mga2p/Spt23p are absent. Alternatively, the amount of the OLE1 transcript produced by the mga2Δ spt23-ts mutant may be just below the threshold required for growth, so that even a modest increase in the copy number of OLE1 would allow cell growth. Therefore, we determined the relative copy number of the URA3-based centromere vector pRS416 and CEN-OLE1 compared with the chromosomal ura3-52 locus by Southern blot analysis in both wild-type and mutant cells at 23° and 37° (refer to materials and methods). A 1.5-fold increase in the copy number of pRS416 or CEN-OLE1 was observed in the mga2Δ spt23-ts mutant at 37° (data not shown).
To determine the effect of OLE1 copy number on mga2Δ spt23-ts, we directed the integration of the YIp plasmid pSRZ176, which contains the same OLE1 insert as that in CEN-OLE1 (pSRZ161), to the ura3-52 locus of strain DG1667 by linearizing pSRZ176 with StuI, which cleaves pSRZ176 once in the URA3 gene. Ura+ transformants containing one to four copies of pSRZ176 integrated at the ura3-52 locus (refer to materials and methods) were analyzed for growth by endpoint dilution and for OLE1 transcript level at 37° (Figure 8). Increasing the copy number of OLE1 in the mga2Δ spt23-ts mutant cells improved cell growth (Figure 8A), indicating that the mutant is sensitive to OLE1 gene dosage. Northern blot analysis revealed that the level of OLE1 transcript is also sensitive to the copy number of OLE1 in both the wild-type (Figure 8B, lanes 1 and 2) and the mga2Δ spt23-ts mutant (Figure 8B, lanes 3–7) strains. However, there was a discrepancy in the level of the OLE1 transcript (Figure 8B, lane 8) and the degree of cell growth for the mutant cells carrying CEN-OLE1 (Figure 8A, CEN-OLE1): these cells grew as well as a mutant integrant containing two additional copies of OLE1, but the level of the OLE1 transcript contributed by CEN-OLE1 was lower than expected. It is possible that a combination of small differences in the stability and copy number of the CEN-OLE1 plasmid influence the suppression, because our data clearly show that the OLE1 transcript level in mga2Δ spt23-ts cells is just below the threshold required for growth at the nonpermissive temperature.
Here we report the isolation of SWI5 and OLE1 as plasmid-borne suppressors of mga2 spt23 lethality. Cells deficient in Mga2p/Spt23p have a lower level of UFAs and pronounced nuclear membrane alterations. These phenotypes are not as severe, however, as those observed in an ole1 null mutant. Suppression of mga2 spt23 or ole1 lethality also occurs when the growth medium is supplemented with the enzymatic products of Ole1p, palmitoleic and oleic acids. Finally, the loss of function of both MGA2 and SPT23 results in at least a 15-fold decrease in the level of the OLE1 transcript. Taken together, our results suggest that MGA2 and SPT23 are redundant activators required for full transcription of the essential metabolic gene OLE1.
In the absence of Mga2p/Spt23p, the expression of OLE1 and the resulting synthesis of UFAs are below the threshold required for growth. Although the ratio of SFAs to UFAs is important for maintaining optimal membrane-associated enzyme activities and transport processes (Krulwichet al. 1990; Schneiter and Kohlwein 1997), analyses of mutations affecting fatty acid biosynthesis have revealed complexities in membrane structure and function that are not completely understood. In particular, the mdm2 mutation causes temperature-sensitive growth and failure to transfer mitochondria into the growing bud at the nonpermissive temperature and is an allele of OLE1 (McConnellet al. 1990; Stewart and Yaffe 1991). The mitochondria fragments and aggregates in the mother cell and the UFA level comprises only 30% of total fatty acids. Electron microscopic analysis of the mdm2 mutant, however, does not reveal any morphological alterations other than retention and fragmentation of mitochondria in the mother cells during mitotic growth. In contrast, UFAs are present at a higher level in the mga2Δ spt23-ts mutant than in mdm2, yet the nuclear envelope morphology is altered in the mga2Δ spt23-ts mutant. In addition, when rat liver stearoyl-CoA desaturase replaces Ole1p, the level of UFAs in these cells is slightly less than that observed when Mga2p/Spt23p are absent (42 vs. 48.1%, respectively), but nuclear membrane alterations are not observed (Stukeyet al. 1990).
Another membrane-associated process that responds to a change in fatty acid content is exemplified by a mutation in ACC1, which encodes acetyl-CoA carboxylase (Figure 1). The acc1-7-1 mutant was isolated in a screen for genes involved in mRNA transport to the cytoplasm (Kadowakiet al. 1994). Like mga2Δ spt23-ts, the acc1-7-1 mutation causes nuclear membrane separation and the appearance of vesicle-like structures in the intermembrane space (Schneiteret al. 1996). The acc1-7-1 mutant fatty acid profile shows a remarkable reduction in the level of very long chain fatty acids, such as C26, but not in the levels of other fatty acids. We have not determined the level of very long chain fatty acids in the mga2Δ spt23-ts mutant; therefore, a reduction in the C26 level may also contribute to the nuclear membrane defect observed in our work. However, three results suggest that the mga2 spt23 and acc1-7-1 mutations act differently. First, our results suggest that the transcript levels from several genes decrease in the mga2Δ spt23-ts mutant, instead of being retained in the nucleus. Second, unlike acc1-7-1, both the lethality and the morphological defects in the mga2Δ spt23-ts mutant are reversed by supplementation with UFAs. Third, Mga2p/Spt23p are not required for ACC1 transcription.
Because the lethality and the morphological defects observed in the absence of Mga2p/Spt23p are reversed by supplementation with UFAs, these phenotypes are probably caused by a deficiency in UFA synthesis. Results from our work, as well as the results from others mentioned above, show that correlating UFA levels with morphological alterations and cell viability is not straightforward and suggest that other lesions in the mga2Δ spt23-ts mutant contribute to the morphological alterations in the nuclear membrane. Therefore, identifying other cellular processes influenced by MGA2 and SPT23 will be necessary to completely explain their roles in the cell. An attractive starting point for these studies is to understand the molecular basis of mga2Δ spt23-ts suppression by the gene-specific transcriptional activator Swi5p.
Our results implicate OLE1 as the essential target gene whose expression is dependent on Mga2p/Spt23p. Mga2p/Spt23p probably affect OLE1 transcription, although we cannot exclude the possibility that Mga2p/Spt23p influence OLE1 mRNA stability. Mga2p/Spt23p do not affect general mRNA stability, however, because GAL1 mRNA decayed at the same rate when wild-type or mga2Δ spt23-ts cells were shifted from galactose to a repressing carbon source, glucose, at 23° or 37° (S. Zhang, unpublished results). If MGA2/SPT23 stimulate transcription of OLE1 and Ty1, and probably other genes, how does this happen? No known DNA-binding motif has been found in either gene. We have proposed that MGA2/SPT23 encode related transcriptional coactivators that may act by changing chromatin accessibility (Zhanget al. 1997). These proteins may also interact with sequence-specific DNA binding proteins. Interestingly, the promoter region of the Histoplasma capsulatum OLE1 gene (OLE1-Hc) has been analyzed by deletion, gel mobility shift, and DNase footprinting analyses (Toscoet al. 1997). An AP1 binding site (TGACTAA) 740 bp upstream of the initiator ATG is required for OLE1-Hc transcription and binds nuclear proteins from the yeast and mycelial phases of H. capsulatum. The S. cerevisiae and H. capsulatum OLE1 genes encode virtually identical proteins that are also closely related to higher eukaryotic Δ9 fatty acid desaturases (Stukeyet al. 1990; Anamnartet al. 1997; Gyorfyet al. 1997). Therefore, we scanned the sequence 900 bp upstream of OLE1 (OLE1-Sc) for potential AP1 binding sites. The OLE1-Sc region contains one putative AP1 binding site (TGACTAT) at position –636. The OLE1-Sc AP1 site is contained within the subclones used in our analyses, and deletions encompassing this sequence lower the level of OLE1-Sc transcripts (Choiet al. 1996). Furthermore, there are eight yeast AP-1-like bZIP proteins identified by sequence and mutational analyses, in addition to the well-studied AP-1/bZIP transcriptional activator Gcn4p. Fernandes et al. (1997) have shown that several of the yeast AP-1 factors (designated Yap proteins) are transcriptional activators with distinct biological functions. We are currently investigating whether Mga2p/Spt23p act as coactivators in concert with Yap proteins and the Snf/Swi chromatin remodeling complex to stimulate OLE1 transcription through its AP1 binding site.
Our studies suggest that GAL80 has unique features, because its transcription appears to be independent of Mga2p/Spt23p. GAL80 is, in fact, unusual in that two pathways are used to initiate transcription (Sakuraiet al. 1994). The first is constitutive, stimulated by a specific upstream activating sequence (UAS), independent of the TATA box, and transcription initiates at a specific start site called +1. The second pathway is induced by galactose and repressed by glucose and, under the control of the Gal4 activator protein and a UASGAL binding site, depends on the TATA box and initiates transcription at several sites downstream of +1. Because all the RNA samples used in our analyses are from cells grown in media containing glucose, the GAL80 constitutive transcript is not under Mga2p/Spt23p control. Whether Mga2p/Spt23p-independence is characteristic of all TATA-independent transcription units or involves specific upstream activating sequences remains to be determined.
In summary, our continued investigation of MGA2 and SPT23 illustrates the power of using suppression of Ty-induced mutations to identify components important for gene expression (Winston 1992). We have now linked a fatty acid and Ty gene expression through the identification of OLE1 as a target gene for MGA2/SPT23. This result is consistent with the idea that Ty elements and the yeast genome have coevolved such that the cell can modulate the level of Ty gene expression and hence transposition, through multiple regulatory pathways (Boeke and Sandmeyer 1991; Garfinkel 1992).
We thank K. Nagashima and R. Shen for help with electron microscopy, H. Issaq for fatty acid analysis, and A. Arthur and S. P. Moore for helpful comments. Research is sponsored by the National Cancer Institute, Department of Health and Human Services, under contract with Advanced BioScience Laboratories. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the United States Government. We acknowledge the British Universities North America Club for sponsoring Y.S.
Communicating editor: F. Winston
- Received September 14, 1998.
- Accepted October 19, 1998.
- Copyright © 1999 by the Genetics Society of America


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Darhl Peterson discusses Certificates of Deposit, or CDs, explaining their coverage by the FDIC, varying time deposits, and other characteristics. Buying CDs can be done not only in a bank, but online through brokerages.
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The words you entered did not match the given text. Please try again.

subset: commoncrawl


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https://i1.wp.com/www.newwaysministry.org/wp-content/uploads/2018/03/Youth-Synod-2018-Survey-Facebook-Ad-650px.jpg?fit=400%2C400&ssl=1 400 400 Robert Shine, Managing Editor https://www.newwaysministry.org/wp-content/uploads/2017/09/logo_nwm-1.png Robert Shine, Managing Editor2018-03-21 01:00:412018-06-28 11:20:16Are U.S. Youth's LGBT-Positive Views Being Represented at Pre-Synod Meeting This Week?
Are U.S. Youth's LGBT-Positive Views Being Represented at Pre-Synod Meeting This Week?
This week in Rome, some 300 youth and young adults are meeting in advance of October's Synod on Young People, the Faith, and Vocational Discernment. Included among the attendees are three people from the U.S., but given their backgrounds, will the overwhelmingly LGBT-positive views of U.S. Catholic young people be fairly represented? Read More


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Abh mod/Bird of Prey Disruptor Cannon 2
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|crew move speed:
Abh 0.5.3 RC2
|DPS against shields:
|DPS against hull:
|DPS against armour:
|area damage:
|part power drain:
|area power drain:
|fire chance:
The Bird of Prey Disruptor Cannon 2 was introduced in 0.5.3 RC2. Fashioned after Star Trek Klingon's Bird of Prey weapon. This is the larger variant. There are left and right version.
This part requires power. It can be buffed by the Phaser Chamber. Crew automation can be applied in the future.
As of 0.6.2 its power levels can be set but is limited by the size of its storage. Not all power settings may work due to this limitation.
Building recommendations
- Build like any other peripheral weapon part.
- 0.6.2 Health was raised from a ratio of 1.5 for armored parts to 3.0, inline with vanilla, which was raised from 2.0 to 3.0 to 4.0 in Cosmoteer 0.14.7.
See Also
Retrieved from "https://wiki.cosmoteer.net/index.php?title=Abh_mod/Bird_of_Prey_Disruptor_Cannon_2&oldid=2835"


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My husband loves DIYs just as much as I and without our mutual love, none of our DIYs could be possible mainly because anyone who enjoys doing it themselves knows a creative mind thrives in chaos and believe me, our house is always littered with glitter, power tools, and my bits and bobs. Most of the time those things are put away but it feels like majority of the time I'm tripping over cords and various crafts in various stages of not doneness.
Once and a while I'll be featuring a DIY that's been spear headed by my husband. Most things involving wood and power tools are his brain child, which I love. He's very creative and even has his own Pinterest account (SWOON). He was hiding his secret Pinterest addiction by bookmarking his ideas rather than making an account. Finally I convinced him to just make account.
This is his latest creation. We don't drink so we can't quite call it a "Wine Rack" but that's essentially what it is! Right now it houses a funky old wine bottle that had been collecting dust along with one of his other cool DIYs he's done recently.
He started with a scrap piece of wood he found at the cottage. It was doing nothing but collecting dust, quite literally. He got some rail way spikes from our Brother In-Law that he had salvaged from a rail yard.
He figured out where he wanted the spikes to go.
Drilled holes.
and Hammered in the spikes!
Whenever something involves power tools and wood, I tend to back away in fear I might do some permanent damage to my project. But in this case, it couldn't have been easier!
My husband wants to put a finish of some sort on the wood to protect it — what do you think? How should this wine rack be finished?
This post will be shared across the blogosphere on these lovely blogs!


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PIC32 USB Starter Kit II(DM320003-2)The PIC32 USB Starter Kit II provides the easiest and lowest cost method to experience the USB and CAN functionality of the PIC32 microcontrollers. The board contains an on-board programming/debugger, standard A USB and micro A/B connectors, and an expansion header compatible with the PIC32 Starter Kit and expansion board such as I/O Expansion Board (DM320002) or for prototyping your own. Users can develop CAN applications using I/O expansion board (DM320002). The board contains everything needed to develop USB embedded host/device/OTG applications by combining this board with Microchip's free USB software.
The USB Starter Kit II has the same form factor and expansion connector as other PIC32 Starter Kits.
- Free MPLAB IDE v8.41 or newer
- Free MPLAB C32 C Compiler with no code size limit
- Example project files for HID-class device mode, MSD-class host mode (see note below), CDC-class device mode. Additional projects will be available in the future.
- Integrated programmer - all you need is the included USB Cable
- Program and verify a full 512k image in under 9 seconds
- Integrated debugger - all you need is the included USB cable
- Full C source based debugging in MPLAB IDE
- PIC32 running at 80 MHz with 512K Flash, 128K RAM, 8 ch. DMA + 4 ch. DMA dedicated to USB and CAN
- USB powered board
- USB connectors, user switches, and LEDs
- Expansion connector enables addition of Microchip's PIC32 expansion board or create your own
- Majority of MCU signals are present on the connector [Hirose: FX10A-120P/12-SV1(71)]
Write Code
Program Memory
Execute Code
Тесты и Инструкции
Протестируйте плату по готовым тестам
Печатные платы и ПО
Ускорьте разработку по готовому дизайну


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Choose your room
Property Location A stay at Wei Di Hotel - Zhangjiajie places you in the heart of Zhangjiajie, steps from Tianmen Mountain Cable Car and close to Bailong Elevator. This 4-star hotel is within the vicinity of Great Gate at Tianmen Mountain and Tanimeni Mountain.Rooms Make yourself at home in one of the 180 guestrooms. Complimentary high-speed (wired) Internet access is available to keep you connected.Rec, Spa, Premium Amenities Enjoy the recreation opportunities such as a sauna or make use of other amenities including concierge services.Dining Enjoy a meal at a restaurant, or stay in and take advantage of the hotel's room service (during limited hours).Business, Other Amenities Featured amenities include dry cleaning/laundry services and a safe deposit box at the front desk. This hotel has 3 meeting rooms available for events.
Useful information:Other names for Wei Di Hotel - Zhangjiajie:
Wei Di
Vide Hotel
Zhangjiajie Vide


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So a while back, I had this problem. A lot of the neighbourhood cats had been coming into my backyard, mostly to hide from the packs of savage dogs out there beyond the fences of my little world. I felt kind of bad about that, but, to paraphrase John Howard ;
I will decide which animals come into my yard and by which means they come here. So what I did was catch the cats that were in my yard and put them in cages down the back, near the shed. Then I electrified my fences to stop any more cats coming in.
A few cats tried to cross the electric fence, they may have died, I don't know. I think the feral rottweiler might have got the rest, but it's okay, 'cause it happened next door.
So, here's my question. How long is it humane to keep the cats caged up before throwing them back to the feral rottweilers? It's been a few years now.
I mean I feed them, I'm not a monster. (Okay, I pay a dodgy company to poke food through the bars sometimes, but I don't like seeing the cages. Or talking about them, apparently. )*
The neighbours complained about me keeping them locked up, so I gave a heap of cats to the dodgy kid next door to deal with, only now, the neighbours tell me the dodgy kid's been abusing the cats I gave him.
Some people are never happy.
I hate it when people tell me what to do.
Especially, when I'm being an arsehole.
.*N.B. Just got the bill from the guy's feeding the cats down the back yard. $570,000 per cat seems a bit excessive.
.Update. Some protesters tried to free the cats again but I slipped the cops a twenty and they took them away for trespassing. Get off my lawn, hippy.
Dear reader. If you feel bad for the cats, how much more those trapped in the hell Offshore,(and onshore) Indefinite Detention. These are people, human beings, not illegals, queue jumpers or boat people. Women, children and men, traumatised by the rottweilers all over the world, and we keep them in cages, feed them back to the Rottweilers and console ourselves as we throw them to the neighbours, out of sight and out of mind. Somebody Else's Problem.
If you've supported Australia's treatment of refugees, I hope this touches your heart because if it doesn't, you are beyond redemption.


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Trauma both small or big due to accidents is devastating for everybody. Often the results of trauma damage the mental and physical health of a person completely. This is where medical intervention is needed so that the healing process is done in a specialized way. It is here that physicians in the field of internal medicine, critical care medicine and emergency care step in to help both the patient and his/her family through this challenging phase.
Dr. Roger Olade- helping patients and their families cope with mental and physical trauma
Dr. Roger Olade is an esteemed name in the field of internal medicine, emergency care, and critical care medicine. He has over two decades of invaluable experience in the above fields. He has completed a number of international fellowships across the world and is widely respected in the healthcare industry in Texas. He currently is associated with Genesis Health in Houston. He says that when a person suffers from an injury or trauma, there is a lot of internal pain. One might report nagging back pain and body ache. The physician here needs to treat every patient with care and compassion.
A systematic approach towards healthcare
Dr.Olade says that when it comes to the treatment of a trauma patient, the approach must be extensive and systematic. No two trauma patients are the same. Even though the case might be similar, every patient reacts uniquely. Some patients become victims of life-threatening health disorders and disease. They suffer from immense pain and agony from the incident. He says that in all cases, not only does the patient suffer but the family of the patient faces a tough ordeal as well. Therefore, it is the primary need and onus of the treating physician to be compassionate when communicating with the patient and his/her family.
Customized treatment plans and their importance
Customized treatment plans contain the course of action that doctors and nurses follow when it comes to the treatment of patients recovering from a trauma. Most of the time, these patients are kept in intensive care units. They need to be supported by tubes, machines and other forms of medical equipment for healing and recovery. These treatment plans need to evaluated frequently. There are cases where the health of a patient might suddenly deteriorate, so the customized healthcare plan for the patient should make provisions for such changes.
Dr. Roger Olade says that in the event of any trauma, the patient needs constant attention, care, and monitoring. He says that in the event of trauma, the families of the patient are confused and often find it emotionally difficult to deal with the situation. The physician needs to provide them with rock-solid support so that they can face the ordeal with faith and courage. Dr. Olade says the internal pain and agony for both the patient and family is immense. Doctors must guide them through the healing and recovery process in a personalized way so that they can cope with the challenges of life-threatening disease and disorders better!


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Facebook engineer ports Doom on the MacBook Pro's Touch Bar
But does it run Crysis?
Apple announced a new MacBook Pro back in October 2016 that did away with the function keys on its keyboard in favor or a Touch Bar. The Macbook Pro has started to make its way to customers, with some developers working on new ways to use the Touch Bar with their applications. One such developer is Facebook engineer Adam Bell who decided to port Doom onto the Touch Bar.
Surprisingly, Doom appears to run relatively well on the Touch Bar as we can see the Marine make his way through the first level of the game. As you'd expect from such a thin display, the game is extremely stretched out, which should make playing the entire game quite challenging. This is definitely one of those things
[Via 9to5 Mac]
-Shack Staff posted a new article, Facebook engineer ports Doom on the MacBook Pro's Touch Bar
It has been ported to a printer: https://www.youtube.com/watch?v=NPWi5yJK3zo


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Sascha Cordner joined WABE 90.1 FM as a Morning Edition Producer/Reporter in October 2018. She is now with All Things Considered.
She has more than 10 years of public radio experience, working at NPR member stations: WFSU-FM in Tallahassee and WUFT-FM in Gainesville. She's worked in both radio and TV, serving in various capacities as a reporter, producer and anchor. She's also a graduate of University of Florida with a bachelor's degree in telecommunications.
She is the recipient of 17 awards from the Associated Press, Society of Professional Journalists (SPJ), and Edward R. Murrow. Her award-winning stories include her coverage on the infamous "Dozier School for Boys" and a feature titled "Male Breast Cancer: Lost in the Sea of Pink."
When she's not reporting, Sascha likes catching up on her favorite TV shows, singing and reading.
Follow Sascha Cordner on Twitter: @SaschaCordner.


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The following is a rough translation of the official rules translated from the official web site . If there are any glaring errors, please notify me, and I'll promptly make the corrections as necessary. (Or simply edit this page with your own corrections!)
A View of the CardsEdit
There are three types of cards in the FF-TCG, and the card type can be distinguished by the text box in the upper left-hand corner.Forwards (shown left) comprise your attack force, and are a central part of your deck. They are easily distinguished from the other types by the Power number in the bottom-right hand corner. You can have an unlimited number of Forwards in play.
Backups (shown right) are responsible for providing a steady source of Crystal Points, which are needed to play cards from your hand. You can have a maximum of five Backups in play at any time.
Monsters (not shown) are played in active state and can use abilities on the turn they are played, but they can't attack abilities on the turn they are played and do not provide Crystal Points.
Items (not shown) are equipped to a character to give them new abilities. Only 1 Item of each type can be equipped to a single character.
1. Cost and Attribute: Indicates the amount and type of CP needed to play the card.
2. Card Name: In addition to the name of the card, the bar may also have an icon on the right side.
- Any number of copies of this card may be in play at any time. Usually, only one copy of a card with a given name can be on the field at a time.
- The card has an EX Burst effect (see below).
3. Card Type: The card's type. To the right-hand side is the character's job.
4. Text: The card's effects. Other than the name written on the card itself, this helps identify the card.
5. Card Number: Card rarity and serial number.
6. Power: Only appears on Forward cards. This figure is used to determine how much damage a card deals while attacking, and how much damage it can sustain while defending. If a card receives damage equal to or greater than its Power, it Breaks (is discarded).
There is also help in tight moments, and summons are like a trump card.
1. Cost / Attribute The cost required to summon this monster. You must always pay at least 1 CP of the same attribute as the card.
2. Card Name Similarly to characters, cards with EX Burst have an icon on the right-hand side.
3. Card Type Summon Monsters do not have jobs, so only "Summon Monster" is written here.
4. Text Once you resolve the effect written here on the card, put it in the Break Zone.
5. Card Number Card rarity and serial number.
Summon Monsters are placed on the Field, used only one time, and each have a powerful effect. Summon Monsters, too, cards can be used like Forwards and Backups to interrupt your opponent's actions using the abilities written in the text field (see page 5). Summon Monsters must have a full set of valid targets when played, but not when resolved.
Only 3 of the same card are allowed in a deck. For cards with the same name, any number can be on the Field if they have the three-card icon. If a card shares the same name but does not have the three-card icon, only one of those cards may be played (however, this does not include your opponent's cards).
Red Crystal – FIRE– Many attacks during a battle from Summon Monsters and Characters will use the fire attribute.
Light Blue Crystal – ICE– The Ice attribute excels at keeping your opponent from being able to perform abilities during battle. Green Crystal – WIND -With this attribute, a game will be quick and characters will be light and fast.
Yellow Crystal – EARTH – Characters with this attribute are substantially larger and sturdier with solid defense.
Purple Crystal – LIGHTNING – This attribute will determine victory or defeat by removing characters with a single blow.
Dark Blue Crystal – AQUA – This attribute helps draw more cards, and you'll indirectly gain an advantage.
Clear Crystal – LIGHT
Black Crystal – DARKNESS
– Light cards utilize the power of order and virtue, while Darkness cards utilize the power of chaos and evil. Because of this, only one can be used on your Field.
Special Rare - Rare - Uncommon - Common - Entry
Degrees of Rarity
High <———————————————- Low
The cards have 4 categories of rarity, S, R, U, and C, while E is only contained in the Entry Set.
Using CardsEdit
All cards cost Crystal Points (CP) to put into play. There are two methods of generating CP.The first way is to discard cards from your
hand. Each card you throw away to generate CP generates 2 CP of the same attribute as that card.
The second way is to Dull Backup cards (a mechanic similar to "tapping" in Magic: The Gathering). Each Backup that you Dull for CP generates 1 CP of the same attribute as that backup.
Using CP: After making these preparations and paying these costs, use your cards.
*POINT* When playing a card, the card requires at least 1 CP of the same attribute. However, it's acceptable to use any attribute of CP when playing a Light or Darkness attribute card. Since only Light and Darkness attribute cards are special, be careful because you cannot trash these cards to generate CP. Once you generate the required cost of CP, you cannot use any remaining CP to play another card or ability.
1. Forwards are placed in the upper half of your side of the field. Forwards come out Active on the turn they are played but cannot use Dull abilities or attack on the turn when they are placed on the field. When a Forward is defeated in battle, they "Break", and are sent to the Break Zone.
2. Backups are placed in the lower half of your side of the field. Backups come out Dulled on the turn they are played. Remember that a maximum of 5 Backups may be in play at one time.
3. Damage Zone. Each time your opponent deals Direct Damage to you, take one card from the top of your Deck, and place it face-up here. You lose the game if 7 cards are placed in the Damage Zone.
4. Your Deck. At the start of each turn, you draw cards from here and place them in your hand. If there are no more cards in the Deck when you need to draw a card, you lose the game.
5. Break Zone. Forward cards defeated in battle, cards discarded from the field, as well as cards you discard from your hand, are placed face-up here.
Setting Up A New GameEdit
At the start of the game, you and your opponent perform the following steps:
1. Prepare a deck of exactly 50 cards for yourself. Only 3 copies of any card are allowed to a deck. Cards with identical names but different abilities do not count as copies of the same card. (Example: You may have three copies of Black Belt (1) and three copies of Black Belt (2) in the same deck.) Exception: You may only have up to 3 cards named "Garnet" in your deck.
2. Thoroughly shuffle your deck.
3. Flip a coin to determine who plays first.
4. Draw a hand of 5 cards from the top of your own deck.
Flow of GameplayEdit
Reset Phase --> Draw Phase --> Main Phase 1 --> Attack Phase --> Main Phase 2 --> End Phase
Reset Phase
(Note: This phase is effectively ignored during each player's first turn.)
Change any Forward and Backup cards in the Dulled state (horizontal) to the Active state (vertical).
Draw Phase
Draw 2 cards from the deck and add them to your hand. (Exception: If you are playing the first turn of the game, draw only 1 card.)
Be careful; if you have no more cards in your deck at the start of the Draw Phase, you lose.
Main Phase 1During this phase you may, in any order, play cards to the field and/or use abilities.
- Playing Character Cards: After paying the required CP cost, as detailed above, play a Forward or Backup card (or cards) from your hand to the Field. Backups are played in the Dull state and can't use abilities with the Dull icon in the turn they are played. Forwards are played in the Active state, but cannot attack or use abilities with the Dull icon in the turn they are played. (Exception: Forwards with the "Haste" ability are not hindered in this way.)
- Playing Summon cards: After paying the required CP cost, show a Summon Card to your opponent, resolve that card's effects, and place it in the Break Zone immediately after. Also, your Summons can be used to interrupt your opponent's Summons and abilities.
- Playing Items: After paying the required CP cost, play an Item card from your hand onto a character you control. The character types which can equip an Item card are listed on the card. The character gains all the abilities listed on the Item. A character can only equip 1 Item of each type. (eg: A character can equip 1 [Weapon] and 1 [Armor], but not 2 [Weapons]).
- Using Character abilities: Preceding the description of any Action Ability or Special Ability is its cost. By choosing to pay it, you activate its effects. You can also interrupt your opponent's Abilities and Summon Monsters.
Attack Phase
This is where combat takes place. If you prefer not to attack this turn, skip to the End Phase.
1. Prior to attacking, if you are the first attacker, you can also use abilities and summon monsters to block.
2. To attack with one of your Forward cards, Dull that Forward. Forwards that are already Dulled cannot attack, and Forwards without the "Haste" ability cannot attack on the turn they are played to the field.
3. Your opponent may choose to Block with a defending Forward. They may select any Forward on their side of the field, so long as it is in Active mode.4a. If your opponent Blocks, the attacking and defending Forwards deal damage
to each other. The defending Forward receives damage equal to the attacking Forward's Power, and vice versa. If either Forward suffers damage greater than their Power rating, that Forward Breaks. For any Forwards that do not Break after combat, the damage they took will persist until the end of the turn.
4b. If your opponent does not (or cannot) Block, they take one point of Direct Damage.
5. If you wish to attack again, return to Step 1.
Main Phase 2 * The same things can be done in this phase as in the first Main Phase.
End Phase Subtract damage dealt by Forwards, and if you have more than 5 cards in your hand discard back down to 5 cards.
- Abilities and Summon Monsters can be used during these times.
Forming parties: When selecting Forwards to attack, any number of Forwards can form a party to attack as a single character providing that the Forwards could all otherwise attack and:
- The Forwards are all of the same element OR
- The Forwards all have the P ability symbols
When in a party, the power of the party is equal to the sum of the power of its members.
When in a party, if one of the Forwards cannot be blocked or has First Strike, it is only effective if all the Forwards in the party have that ability.
When a party receives damage, the defending player decides how that damage is distributed.
If there is only one Forward remaining in a party, that Forward is no longer in a party.
There are three categories of abilites that may appear in card descriptions. Using them effectively will bring you closer to victory.Field Abilities have continuous effects on gameplay, e.g. reducing CP costs for playing cards from your hand, boosting your Forwards' Power.
Action Abilities can be activated during any Main Phase or Attack Phase. To use them, you must first pay a cost, represented by various symbols following the ability name. Here is what the symbols mean:
- : Pay 1 CP of matching color. If there are multiple crystal symbols, pay for all of them (for example, means you must pay 1 Fire and 2 Light CP).
- : Dull this card. If the card is already Dulled, or has been played this turn (without Haste), you cannot use this ability.
- : From your hand, discard one card of the same name as the card this ability is printed on. Cards with identical names but different pictures are acceptable to use.
Auto Abilities trigger when the stated conditions are met. For example, "When this card is attacked by a Forward, deal 1000 Damage to that Forward."
Haste: With this ability, it's possible to do things like attack and use Dull abilities on the turn they've been played onto the Field.
Brave: Does not Dull when attacking (however you can only attack once per turn as usual).
Preemptive Strike: When attacking and blocking, your Forwards are able to deal damage before your opponents.
Level UP: When this character breaks a Forward or damages an opponent, you may put this card in the Break Zone to search your deck for a Forward with the same name and place it on the field in the Dull position.
Link: When this card enters the field, you may play one Forward with a cost equal or less than the listed cost at no cost.
Assist: When this card is enters the Field, you can choose to place this card in the break zone to activate the following ability.
War God: You can send one character with the listed job to the break zone to reduce the casting cost of this card by the cost of the card sent to the Break Zone. The casting cost must remain higher than 0.
Overdrive: At casting time, you can increase the casting cost of this character by the overdrive cost to give this character the following ability.
Awakening: By paying the cost, you can search your deck for a card with the same name as this card and put is on the field. This can only be done on your turn. (This will usually force you to put one of the two cards in your break zone).
Blue Magic: Pay the following cost and discard this card from your hand to use the following ability. This can only be done while this card is in your hand.
Remove: Return the Item card to your hand.
Job Change: Pay the following cost to put 1 Backup with a cost equal to or less than the given number from your hand into play.
Ability Notes:
If an ability says something like "Choose a "something".", the choice of target or targets must be made when the ability is used. If a target becomes invalid at resolution time, the ability does nothing to that target only.
If an ability says something like "Choose a Forward. Dull it", you MUST choose a Forward and Dull it.
If an ability says something like "Choose a Forward. You may Dull it", you MUST choose a Forward and activate its on-targetted abilities. You can choose to Dull it or not.
If an ability says something like "You can choose a Forward. Dull it", you can choose to choose a Forward or not. If you choose one, you MUST Dull it.
If an ability says something like "Choose up to 2 Forwards", you can choose 0, 1 or 2 Forwards.
Dulled cards are valid targets for Dulling. Active cards are valid targets for turning Active.
If a card prevents a Forward from receiving damage (ex. Minwu), that card doesn't even receive 0 damage. On damage abilities don't get triggered.
Abilities that don't have "choose" in their text are not targetted and so don't trigger on-targeted abilities or abilities that prevent targetting or abilities that force a choice of target.
Abilities that ask your opponent to choose a target are not targetted by you and so don't trigger on-targetted by opponent abilities, abilities that prevent targetting by opponent or abilities that force a choice of target on opponent.
Characters can be put on the Field even if their on Field auto abilities don't have a full set of valid targets.
If a Forward's Power becomes 0 or less due to an ability, it is immediately put in the Break Zone. This doesn't count as Breaking.
When the phrase "Alternatively if" is used, the following instructions apply instead of the previous ones if the following condition is true.
Damage and EX BurstEdit
If your opponent attacks with a Forward, and you do not Block, you take one point of Direct Damage. When this happens, take the top card of your deck, and place it face-up in the Damage Zone. The moment you accumulate 7 Direct Damage, you lose the game.
NOTE: If the card turned over has an
EX Burst ability, you can use that effect.
Important EX Burst PointEdit
If a card gets turned up with EX Burst, the effect can be used immediately, and no one's summon monsters or abilities can interrupt it. Also, in a situation where the card flipped states "When this card is placed on the Field…", the card does not get placed on the field, but the described effect is activated.


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Property Details
81 ST LAWRENCE Boulevard, London, Ontario
Sales Representative
eXp Realty Of Canada, Inc., Brokerage
Tel: 519-868-7355
$319,900 - Sold
Fantastic prime location in Norton Estates with this meticulously maintained, bright and open, spacious 3 level backsplit. Lovely finished lower family room and extra 4th lower bedroom/den. Thousands spent on upgrades over the years, which include: complete all new electrical receptacles and light fixtures throughout, new shingles on south facing roof, new driveway, new extra wide front door, updated kitchen and bathroom with granite, energy audit '09, the list goes on! This home has hardwood and ceramic, a side patio and gazebo with a fully fenced private back yard. All appliances included! Large picture window overlooks popular Sir Isaac Public School and playground. Great value in a very sought after area. Buyer to verify taxes and room measurements.
- Property Address:81 ST LAWRENCE Boulevard, London, Ontario
- Property Type:Residential
- Acreage:Under 0.5 Acres
- Sewage Type:Municipal
- Water Type:Municipal
- Taxes:$3,401 (2017)
- Structure Type:Single Family
- Structure Style:3 Level (split), Back Split
- Building Age:Estimate (1972)
- Bedrooms:3+1
- Bathrooms:2
- Heating Type:Forced Air-Gas
- Exterior Type:Brick
Sales Representative
eXp Realty Of Canada, Inc., Brokerage
Tel: 519-868-7355


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The construction of a new border fence between Germany and continental Denmark has started, Danish Radio reported.
The fence, which aims to keep German wild boars off Denmark's territory, has been railroaded through by the Scandinavian country's powerful pork lobby. It will be 1.5 metres high and go 50 centimetres into the ground, costing circa DKK 80 million ($12 million). The Danish pork industry will fork out DKK 30 million ($4.5 million) of the cost.
The border wall aims to prevent African swine fever from entering Denmark. Should the deadly disease ever find a way into the Scandinavian country, its pork exports would stop immediately. Danish pig exports account for DKK 11 billion annually ($1.7 billion)
The fence is the Ser Hegn construction company's largest current project.
"It is a great task that we are taking up with great humility and respect. We are very excited to get started," Ser Hegn director Henrik Hallenberg Rasmussen told Danish Radio.
Se den første stolpe til vildsvinehegnet langs den dansk/tyske grænse blive sat op #dkpol #dkbiz #dkgreen #dkeksport pic.twitter.com/yXa9scdzZm— Miljø & Fødevaremin. (@MFVMin) 28 января 2019 г.
The fence will be completed in a matter of months through varied terrain. To achieve the goal in record time, the workers will work 24/7 in 12-hour shifts. The fence will end at Schengen border stations.
Martin Brink, a project manager for the boar fence with the Danish Nature Agency, stressed that the giant construction won't serve as a 100-percent guarantee against the dreaded disease.
"Sure, it will reduce the possibility of wild boars entering, but it cannot rule it out completely," Brink told Danish Radio. "The openings shall be monitored, so that the animals coming through will be shot, Brink suggested.
Previously, the fence came under fire from biologists, who claimed it violates centuries-old animal migration routes, and the EU authorities, who claimed that it sends out a "wrong kind of signal".
Den første stolpe i det kommende vildsvinehegn langs den dansk/tyske grænse er oppe. #dkbiz #dkpol pic.twitter.com/5rOzpCq4Gw— Naturstyrelsen (@naturstyrelsen) 28 января 2019 г.
Land owner Henrik Gotborg Hansen, whose land, which has been in his family's possession since 1875, will be cut in half by the fence, called it "a day of sorrow".
In 2018, France began raising a similar anti-boar fence along the border with Belgium following an outbreak in southern parts of the country. Previously, swine pest has been encountered in Latvia, Lithuania, Poland, the Czech Republic, Romania and Bulgaria.
Om under ti minutter sættes den første stolpe til vildsvinehegnet op #dkbiz #dkeksport #dkgreen #dkpol pic.twitter.com/mqThydDuqi— Miljø & Fødevaremin. (@MFVMin) 28 января 2019 г.
While it doesn't affect humans, swine pest (also known as swine fever or swine flu or ASFV) can have devastating consequences for a nation's economy, by bringing pork exports to a halt.
Around 5,000 pig farms in Denmark produce approximately 28 million pigs annually, which is several times Denmark's human population of 5.6 million.


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In 2014, while fishing during an annual family vacation to Turks & Caicos, founder Jim Lambert hooked a vision. He envisioned wearing a pair of stylish, comfortable trunks and wanted to capture his passion for the Caribbean and all the region has to offer in a brand and lifestyle
Their trunks are accented with a patterned trim waistband adding unique nautical style to every pair. They're made from performance, stretch material for an athleisure feel, perfect for beach activities. The Caribbean influenced the bright colors you see in our products and content
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SEC Proposes Securities Offering and Disclosure Reforms for Business Development Companies and Registered Closed-End Funds: SEC Proposes an Overhaul of the Registration, Offering and Communications Processes and Other Aspects of the Disclosure and Regulatory Framework for Business Development Companies and Registered Closed-End Funds to Provide Parity with Operating CompaniesSullivan & Cromwell LLP - April 1, 2019
On March 20, 2019, the Securities and Exchange Commission (the "SEC") issued a release (the "Release") proposing rule and form amendments to implement certain provisions of the Small Business Credit Availability Act (the "SBCAA") and the Economic Growth, Regulatory Relief, and Consumer Protection Act (the "EGRRCPA"), which direct the SEC to provide parity in securities offering regulation between certain types of closed-end investment funds and operating companies. Consistent with the statutory mandates, the SEC's proposed changes would allow business development companies ("BDCs") and registered closed-end funds ("CEFs" and, collectively with BDCs, "Affected Funds") to use the more streamlined and flexible registration, offering and communications processes that have been available to eligible operating companies since 2005. Although the SBCAA and the EGRRCPA principally direct the SEC to level the playing field for Affected Funds and operating companies regarding securities offering regulation, the SEC has also proposed to revise other aspects of the disclosure and regulatory framework for Affected Funds. These sweeping securities offering and disclosure reforms, if adopted as proposed, would have broad application in the closed-end industry, albeit to varying degrees depending on fund size and type. The reforms do not, however, affect restrictions under the Investment Company Act of 1940 ("Investment Company Act") on the issuance of securities by Affected Funds, including the general prohibition on the issuance of shares of a fund's common stock at a price below current net asset value. The SEC is seeking comment from the public on all aspects of the proposed reforms. Comments on the Release will be due 60 days following its publication in the Federal Register.


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It's beginning to look a lot like Christmas…and the festive cheer is all around!
With the season dedicated to gatherings, you'll be spending many hours of celebrating around the dining table. Put some extra thought into this year's table to make it memorable, cheerful and most importantly, gather-worthy. Follow these tips to a festive setting:
Think outside the box when it comes to your colour scheme – we've combined millennial pink, delft blue and touches of festive sparkle for an eye-catching setting.
When deciding on crockery, keep the menu and type of dishes you'll be serving in mind. Food generally looks and taste best on simple country pottery. Opt for solid colour plates, rather than patterned plates, to highlight the detail and beauty of the food. If you want to add interesting, colourful crockery, do it with side plates, underplates and dish-up bowls.
Bring colour to the table with tablecloths and napkins. Balance out patterns and solids to create an elegant and cheerful setting.
What's a festive table without fresh flowers? When arranging blooms and greenery, keep the height and size of the arrangements in mind to allow easy conversation and eye-contact.
Decorative accessories such as vases, candle holders and ornaments can be used to tie together the festive theme of your table. Remember to always keep it classy and in line with your chosen colour scheme.
For extra dimension and aesthetic appeal, mix and match your chairs. Combine classic chairs with contemporary seating options or use two different chairs at the head of the table for an interesting look.
Make sure your table does not wobble. This might sound pretty basic, but it has happened to the best of us. Opt for a sturdy, solid table and secure the feet of the table with rubber or textured pads.


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SCC Summer Lunches
The Food Service students enrolled in the Restaurant Fundamentals and Buffet Decorating class will be preparing and serving luncheons during the Summer Quarter. By attending and supporting these luncheons, you are providing our students with an experience that will help them meet their education goals.
Due to limited seating in Room E2/E3, a reservation is required to attend these meals. You may make your reservation(s) by contacting Marcia Blender in advance at (402) 437-2446 or email@example.com.
You may pre-pay for the luncheons by sending your payment to Marcia Blender at :
Southeast Community College
Attn: Marcia Blender
8800 "O" Street
Lincoln, NE 68520
Or stop by Room D6 to make your payment. We only accept checks or cash at this time.
If you are having a party of 6 or more attending one of the luncheons, please pay for the meals in advance.
RESTAURANT FUNDAMENTALS LUNCHEONS: The doors to the College Inn Restaurant will open at 11:00 a.m. on the following Thursdays during the Summer Quarter and will stop taking orders at 12:00 noon in Room E2/E3 on the 8800 "O" Street campus in Lincoln. At the luncheon you will have your choice of soup or salad; choice of three entrees; and choice of three desserts plus bread and beverages. Cost is $13.00 per person.
Thursday, August 9
Thursday, August 16
Thursday, August 23
Thursday, August 30
Thursday, September 6
Thursday, September 13
BUFFET AND DECORATING CLASS LUNCHEONS: The doors for the Buffet Luncheons will open at 11:00 a.m. on three Tuesdays during the Summer Quarter and will stop serving at 12:00 noon in Room E2/E3 on the 8800 "O" Street Campus in Lincoln. Cost is $13.00 per person.
Tuesday, August 14 ….. Salads and Dessert Buffet
Tuesday, August 28….. Hors d'oeuvres and Desserts
Tuesday, September 11…… Brunch Buffet
August 9 and August 16
Choice of Soup or Salad:
· Wild Rice and Mushroom Soup
· Green Salad with Duck, Raspberries and Bleu Cheese
Choice of one of the following Main Entrée:
· Hearty Shepherd's Pie
· Bangers and Mash
· Pork Smokehouse Cuban Style Sandwich with Gaufrette Potatoes
Choice of one Dessert:
· Freeform Apple Pie with Honey Ice Cream
· Chocolate Kahlua Soufflé
· Vanilla Bean Crème Brulee
August 23 and August 30
Choice of Soup or Salad:
· Smoked Salmon and Bacon Chowder
· Spinach and Bacon Salad
Choice of one of the following Main Entrée:
Served w/Maple Polenta and Braised Kale
· Ham Steak with Caramelized Apples
· Sausage Stuffed Pork Loin with Robert Sauce
· Stuffed Chicken Leg
Choice of one Dessert:
· Cream Puffs w/Pastry Cream, Chocolate Ganache and Fresh Fruit Garnish
· Lemon Soufflé with Whipped Cream
· Fruit Tart
September 6 and September 13
Choice of Soup or Salad:
· Navy Bean and Ham Soup
· Wilted Spinach Salad w/Bacon Vinaigrette
Choice of one of the following Main Entrée:
Served w/Rice Pilaf and Brussels Sprouts
· Carpet Bagger Steak
· Chorizo Crusted Snapper
· Roast Loin of Pork with Honey Mustard Sauce
Choice of one Dessert:
· Chocolate Pots de Crème
· Dried Fruit Bread Pudding
· Pound Cake w/Vanilla Bean Ice Cream and Berry Compote
Marcia Blender
Division Secretary
Community Services and Resources Division
Ag, Food and Natural Resources Division
Telephone: (402) 437-2446
E-Mail: firstname.lastname@example.com


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HOT ROD Engine Masters is all about innovation. Jon Kaase's vintage-class-winning MEL engine is that and more.
When the Engine Masters Challenge was conceived more than a decade and a half ago, the concept was to encourage talented builders to push the envelope of engine design through an open rulebook. That naturally encourages builders to take the road less traveled when it comes to building big power. This wild, Mercury/Edsel/Lincoln (MEL) build by Jon Kaase and Royce Brechler is a perfect example of that approach.
"I teamed up with Royce Brechler," says Kaase, the man at the helm of the Jon Kaase Racing Engines in Winder, GA. "He ran this engine here last year and was nice enough to let me use it this year. We've done a bunch of modifications to it for this year's competition. It's kind of an odd, old engine called the MEL Series." Kaase explains that these engines were normally reserved for FoMoCo's luxury brands and didn't see use within the Ford marque itself, making the MEL a rather unusual motor simply by virtue of its rarity.
But if the oddball block wasn't enough to catch folks' attention, we'd imagine that some of the parts in the combination will do the trick. "We built this a couple of years ago for a project that we did for a presentation at AETC [Advanced Engineering Technology Conference] in Indianapolis," Kaase says while pointing to the large clear plastic chamber that's installed between the intake manifold and the pair of carburetors. "There's no advantage to this plastic, but because it's a little different and provides a little more area inside, it actually makes a little more power than the original top that would have."
Outfitted with a steel, Lunati crank, this MEL motor now displaces 473 cubic inches and features no shortage of original hardware. "The distributor is 1958, the oil pan is 1958, so we've got a little mixture of good and old," he jokes. "It's a neat project – and it's surprisingly good on the power."
Diamond Pistons played a crucial role in Kaase's build as well. "We made a model of what we wanted and sent it to Diamond," he explains. "Diamond is really good about taking your model and making exactly what you want. When they're all done they get inside and CNC-mill around so there's not a lot of dead weight in it. So that's our choice for just about everything we build."
For more photos of this wild creation, check out the gallery on HOT ROD.com


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Making its first appearance on the runways of Paris Fashion Week, the Neon Fluorescent Spike Runner Shoes have a refreshing design that gathers inspiration from the 90's with a hint of neon. The sneakers have lifted, chunky soles with a mesh upper and fluorescent neon details to instantly make a bold statement. Wear this pair with a mesh top and distressed jeans to amplify the retro influence.
• We recommend purchasing your normal size – this design has a true fit
• Italian sizing
• Vetements X Reebok collection
• Technical mesh
• Lace-up fastening
• Brand logo on tongue and sides
• Round toe
• Fabric lining
• Chunky rubber sole
• Comes with a dust bagProduct Code: 212773812
Led by creative director Demna Gvasalia, design collective Vetements specializes in putting a new spin on style while cleverly commenting on the zeitgeist. Previous designers of the Maison Margiela powerhouse, Vetements fearlessly flips what we know as fashion firmly on its head.
Free for orders above 100 OMR 6 OMR for orders below 100 OMR


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Our Programs
Strong guidance from a close knit recovery community
Checkout all that we have to offer, and don't hesitate to ask us any questions
The mission of 10 Acre Recovery is to impart "real recovery" to the men & women in our care. We are known far and wide as the place that many very difficult cases have been turned around. There simply is no substitute for the caring and close-knit culture of support at 10 Acre Ranch. Our 4 to 1 staff to client ratio allows for close guidance and much clinical interaction. Many of the 10 Acre Ranch staff members have had their lives touched by addiction, so they personally understand and relate to what the men and women in treatment are going through.
In addition to providing an initial detoxification and 30 to 90-day residential treatment, we also have an outpatient program for the man or woman to participate in as he/she moves off site into a sober living environment (SLE), or returns to his home in the Riverside area. All evidence from recent medical studies on the outcome of addiction treatment points to a strong correlation between length of treatment and length of sobriety after treatment. Simply put, the longer a person commits to sobriety while in the program, the longer they'll commit outside of it.
A Social Model Program with Cutting Edge Treatment and 12-Step Principles
Our program is for men & women who are seeking and willing to get help to overcome the disease of addiction. Our Southern California rehab is a social model program, which means therapeutic processes takes place in a group setting. Our meetings and group therapy sessions help men adapt to living with one another and ultimately helps them adjust to living in society outside of addiction treatment.
Building a support system of peers in recovery is essential because men struggling with addiction tend to isolate. To break that pattern, our program features a serene and intimate setting that is staffed around the clock by our credentialed addiction treatment staff. Our staff will keep a close eye on the man who is in treatment, as will his peers, who are also in various stages of their first year of sobriety.
Our staff will facilitate therapeutic processes. These sessions include:
All of these activities prepare the men to address stressors and triggers that they will encounter without turning to drugs or alcohol. We also offer daily trips to the Tournament House, a private racquetball club and gym, for exercise.
On Fridays, there is a softball tournament where 10 Acre Ranch clients and alumni challenge the other local treatment centers in a friendly game. On Saturdays, the clients enjoy an afternoon out at the movies and pizza, always supervised by qualified 10 Acre Ranch Staff.
The Entire Continuum of Care & Support
10 Acre Ranch offers a multitude of addiction treatment programs in order to provide the appropriate level of care to each individual. The program best suited for you or your loved one will depend on the length and nature of your addiction prior to entering treatment. To find out more about the various stages of treatment follow these links:
12-Step Support Community
Our spiritual 12-step based curriculum guides our clients toward opening their minds and their hearts. When the men learn to accept some higher power, they are able to unburden themselves by letting go of control.
A goal of our clinicians is to expose the men to the 12-step program that is most appropriate for their belief system and substance abuse history. The most frequently available 12-step meetings available are Alcoholics Anonymous, but some clients prefer Narcotics Anonymous, or Cocaine Anonymous.
The 12-step community is an invaluable support system, and is widely available in every locale in the United States and beyond. This community will provide invaluable support for the man upon leaving our program.
Get Help Today
If you are interested in learning more about our treatment program or would like information on our treatment services and programs, please contact us at 1-877-228-4679.
Contact Form
This form is completely confidential – a recovery staff member will call you back.


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Thyroid No Surgery 101: Massive Goiter Treated Without Surgery
The patient from the east coast was worried about a thyroid surgery because of weight problem and the loss of her thyroid function and the scar.
Patient above before the RF treatment
When I saw her she had a very large nodular goiter which was visible from across the room. After consultation she was told that ablation with radiofrequency could decrease the size and relieve her symptoms and correct the cosmetic issue.
I informed her that multiple treatment sessions were needed to reduce the size enough to correct her symptoms and cosmetic issues.
The original size was 9.1 x 4 x 6.6 cm or 127 ml volume.
After three sessions her nodules was 4.5 x 3 x 2.8 or 19 ml.
The size reduction was 85% and her symptoms and cosmetic issues were corrected.
Smaller nodules can be ablated with one visit. I am doing Thyroid RFA in my office.
Call me at 310-393-8860 or email to [email protected]


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Optimized levels list for large amounts of levels
Not sure if it's the root cause of #48, but the levels list would display ALL levels in the current directory (even those offscreen) and more importantly, filtered levels every frame. That's fine if there are not many levels, but large amounts of levels will start to cause the performance to worsen. Now levels are only filtered once every time the input changes, and offscreen levels are not displayed. [pre06]
parent dea8e2f0
Showing with 70 additions and 46 deletions
mentioned in issue #48 (closed)Toggle commit list


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I want to start things off by saying - I'M A COMPLETE FAILURE.
Have you heard of puzzle rooms, or escape rooms? They're popping up all over the country, and are basically a giant interactive game that requires people to gather in a themed room with an intricate storyline and 'break out' by completing a series of puzzles and investigations in an allotted amount of time. Groups of families, friends and even co-workers hoping to team-build flock to these closed rooms. You rely solely on your brain and team work. No phones allowed!
It sounded intriguing, so a group of pals and I tried one called Quest Factor - and this is where the FAILURE comes in.
I had no idea what to expect, and was kind of nervous going in. The thought of being trapped in a room wasn't...appealing to me. Why on earth would I willingly lock myself in a room? But I was pleasantly surprised as our group of four walked into a room that looked like an old historic study - not a prison. As soon as the door shut the timer started and we all ran around like chickens with our heads cut off looking for clues.
We had sixty minutes to complete the 'mission', and if (slash when) we got lost along the way we had walkie talkies we used to ask for clues. Despite the clues....we came super close to solving the mission, but ended up failing, mostly because of my husband. But even sad sacks like us could lose, and still have a good time. And all of you judging me out there, it's much harder than you may think.
There's three different Quest Factor locations that all have different escape room themes: Redmond, U-District and Shoreline (we went to the Shoreline location). Here's what each location offers:
- Dracula: You are adventurers who discovered vampire crypt. The front door slammed shut behind you and unfortunately your group got trapped inside... Hurry and save yourself before twilight!
- Bank Heist: You and your team are an infamous but very successful crime syndicate specializing in safe cracking. Today, you'll face your greatest challenge yet.
- Pirates of the Caribbean: During the Caribbean voyage, you got attacked by pirates. They destroyed your ship and took you and your team hostage! Today the pirates arrived at the harbor and dropped the anchor not too far from the shore.
- The Castle: Are you ready to transport inside a medieval castle and retrace the steps of the Knights of the Round Table to find the long lost treasure?
- Da Vinci: Following the centuries-old clues and secrets, your team of historians and amateur cryptologists finally locate the hidden workshop of the one and only Leonardo Da Vinci.
- National Treasure: As a team of historians, you learn that the original document which established the New World is not located at the National Archives Museum and is just a cover up.
- Mad Scientist: Mad Professor Manifesto is furious at the world as his latest invention gets him ridiculed within the science community and fired from the greatest lab in the world.
Tickets are $30 a player and part of the proceeds benefit Early Life Speech and Language that's all about helping kids struggling with speech development. In fact, kids are a huge demographic that come through the puzzle rooms, and learn to work as a team.
"These kids are often successful at complete the puzzle because they work together and don't take themselves seriously," said Quest factor's Jeffrey Lucas. "Adults can over-complicate things."
So...my group is just really mature, and that's why we failed, right?
Well played Quest Factor, well played.


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In a recent post, we mentioned that the recent American Taxpayer Relief Act of 2012 gave the estate planning community a long-awaited dose of permanence regarding tax policy.
After changes to tax law, estate planning face more certainty
How did the estate and gift tax change in the fiscal cliff deal? P.2
In our last post, we informed our readers about the new gift and estate tax rules that came out of the recent fiscal cliff deal. As we noted, the potential change has been a huge issue in the estate planning world for some time, attorneys not knowing what the future would hold and clients not sure what to do with their wealth. The bottom line is that the system that has been in place over the last two years was extended indefinitely.
How did the estate and gift tax change in the fiscal cliff deal? P.1
We want to update our readers on how the fiscal cliff deal will affect gift and estate tax planning in the coming year. This has, as our regular readers know, been a big issue in the estate planning world, and had many people taking last minute precautions over the last several months, so let's take a look at what happened.
Trusts and LLCs in estate planning, P.2
In our last post, we began speaking on how trusts and LLCs can be used for real estate in planning one's estate. The topic is important, as failing to protect one's real estate from lawsuits and probate after one's death can create extra troubles and costs. These can be avoided by prudent use of a trust or LLC.
Trusts and LLCs in estate planning, P.1
Asset protection, like Medicaid planning, is an important aspect of estate planning. In truth, Medicaid planning is a form of asset protection, which includes a number of other considerations like tax minimization, planning for special needs children, and ensuring avoidance of costly will contests.
Estate planning mistakes which increase tax liability, P.2
In our previous post, we began looking at five common ways people's estate plans mistakenly leave money to the IRS. The combination of income, estate, capital gains, and gift taxes can take a substantial sum out of an estate. With some proper planning, though, these mistakes can be avoided.
Estate planning mistakes which increase tax liability, P.1
A recent article out of Vending Times took up the important topic of how to avoid passing your wealth on to the IRS. This is a particularly important issue for those who are wealthy, as they have a greater risk of losing their wealth to various forms of taxation. The topic is worth considering even for less wealthy readers who want to maximize their tax savings.
Estate planning matters women, and men, should know about, P.2
In our previous post, we began looking at a list of estate planning issues which were suggested as important for women to understand. We present them as important for anybody to understand.
Estate planning matters women, and men, should know about, P.1
In a recent post entitled "Women and estate planning," we looked at an article written by Deborah Jacobs of Forbes online. The article was about the need for women to become more involved in the estate planning process, not help in the work of planning for the family's future, but also for their own sake. The fact is that women are more likely to see the effects of poor planning than men. And even when the planning is thorough, it isn't always in women's favor.
Estate tax can be a moving target for estate planners
A recent Wall Street Journal article which goes into the uncertain state of estate taxation among the states highlights that estate planning can often be one of the more challenging aspects of estate planning.


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We cover everything from group days out; parties and events, to corporate and private bookings.
Corporate bookings
If you're looking for a great way to get you, your colleagues or clients out of the office and exploring Bristol by bicycle, Cycle the City can help.
We've designed a number of ways which are ideal for team building, a staff outing or even showcasing your city to visiting clients.
We can tailor a tour to stop at a restaurant, involve an activity or focus on a topic which suits your group.
Private parties
Got a large group? Looking for something different to do in Bristol? Choose any of our tours, or packages and we'll make it private for your group and work around what you want.
Perfect for:
Stag groups
Hen dos
Birthdays and celebrations


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Junior Suite
junior suite
Our precious Junior Suite is decorated with fined fabrics and exclusive furnishings, it has a beautiful bathroom with an hydromassage shower and a private balcony with Etna view.
With one double bed, this room can accommodate up to a maximum of two people; on request, it's possible to add an additional bed or a cot.
In room climate control
flat screen TV with
international channels and radio
Complimentary Wi-Fi
international TV channel
Safety box
Hair dryer
Slippers and bathrobes
Design toiletries
How to reach us from: Aci Bonaccorsi, Aci Catena, Acireale, Aci Sant'Antonio, Adrano, Agira, Caltagirone, Bronte, Belpasso, Enna, Biancavilla, Catania, Siracusa, Paternò, Giarre, Messina, Ragusa, Palermo, Misterbianco, Zafferana Etnea, If you're interested to Rooms & Suite.