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We have used a novel method to activate the DNA damage S-phase checkpoint response in Saccharomyces cerevisiae to slow lagging-strand DNA replication by exposing cells expressing a drug-sensitive DNA polymerase δ (L612M-DNA pol δ) to the inhibitory drug phosphonoacetic acid (PAA). PAA-treated pol3-L612M cells arrest as large-budded cells with a single nucleus in the bud neck. This arrest requires all of the components of the S-phase DNA damage checkpoint: Mec1, Rad9, the DNA damage clamp Ddc1-Rad17-Mec3, and the Rad24-dependent clamp loader, but does not depend on Mrc1, which acts as the signaling adapter for the replication checkpoint. In addition to the above components, a fully functional mismatch repair system, including Exo1, is required to activate the S-phase damage checkpoint and for cells to survive drug exposure. We propose that mismatch repair activity produces persisting single-stranded DNA gaps in PAA-treated pol3-L612M cells that are required to increase DNA damage above the threshold needed for checkpoint activation. Our studies have important implications for understanding how cells avoid inappropriate checkpoint activation because of normal discontinuities in lagging-strand replication and identify a role for mismatch repair in checkpoint activation that is needed to maintain genome integrity.
EUKARYOTIC cells have the ability to detect DNA damage and stalled replication forks, and, if problems are found, checkpoints are triggered to control cell cycle progression, induce DNA repair enzymes, stabilize replication forks, remodel chromatin, modulate gene expression, and many other activities. Coordinated action of these processes prevents cell death while preserving genome integrity; however, if surveillance and repair activities are not successful, the resulting genomic instability produces cell death or is mutagenic and even potentially carcinogenic in human cells (Hartwell and Kastan 1994; Myung et al. 2001; Nyberg et al. 2002; Brown et al. 2003). Given the multitude of genotoxic lesions that can be produced by endogenous and exogenous agents and the formation of aberrant DNA structures that can occur during normal DNA replication (Tourrière and Pasero 2007), it is not surprising that S-phase checkpoint activation and responses are complex.
Briefly, checkpoint pathways are protein kinase cascades that begin with sensing DNA damage in the context of single-stranded DNA (ssDNA) that is coated with ssDNA binding protein (RPA) (Figure 1). The PCNA-like Ddc1-Rad17-Mec3 clamp complex (the Saccharomyces cerevisiae 9-1-1 DNA damage clamp) is loaded near DNA damage by the Rad24-clamp loader complex. Mec1/Ddc2 (yeast ATR/ATRIP) is loaded onto ssDNA regions at gaps and the resected ssDNA ends produced at double-strand breaks (DSBs) (Zou and Elledge 2003). Mec1 phosphorylates Ddc2, and colocalization with the DNA damage clamp allows phosphorylation of Ddc1. Mec1 also phosphorylates Rad9, which is a signaling adapter that brings Mec1 and Rad53 together and facilitates phosphorylation of Rad53, the yeast ortholog of the effector kinase Chk2 (Sweeney et al. 2005). Rad9 also facilitates the phosphorylation of Chk1. Mrc1 (yeast claspin) appears to fulfill a similar role as Rad9 in Rad53 phosphorylation, but in response to stalled replication forks (Alcasabus et al. 2001). Thus, the DNA damage checkpoint is mediated by Rad9 and the replication checkpoint by Mrc1.
Rad53 phosphorylation (Figure 1) is a key step in the signal transduction cascade (Tercero and Diffley 2001; Nyberg et al. 2002). Once activated, Rad53 phosphorylates protein targets including Dun1, which in turn phosphorylates proteins that lead to the up-regulation of deoxynucleoside triphosphate (dNTP) synthesis and the transcription of DNA repair genes (Huang et al. 1998; Bashkirov et al. 2000; Zhao and Rothstein 2002; Ahnesorg and Jackson 2007). Rad53 also acts either alone or in combination with Chk1 to arrest cells at G2/M, which prevents the premature segregation of damaged or broken chromosomes (reviewed in Hsieh and Yamane 2008; Branzei and Foiani 2009; Schleker et al. 2009).
Traditionally, the favored experimental approach to invoke the S-phase DNA damage checkpoint response is to treat cells with a high dose of a DNA-damaging agent. While studies using a variety of chemicals and radiation treatments have provided a wealth of information on checkpoint pathways, controversy remains due, in part, to the different types of genotoxic agents used, varying dosages, and the wide range of lesions generated from even a single type of treatment. One controversy concerns the role of mismatch repair (MMR) in checkpoint activation. In the "signaling" model, MMR complexes are proposed to bind to DNA damage and form a protein scaffold that recruits sensor and effector checkpoint kinases, which facilitates phosphorylation and checkpoint activation (Brown et al. 2003; Wang and Qin 2003; Adamson et al. 2005;Yoshioka et al. 2006). For the "processing" model, MMR DNA degradation activity is proposed to generate persisting ssDNA gaps that produce DSBs when encountered by advancing replication forks (Mojas et al. 2007). These proposed roles of MMR in checkpoint activation, however, are paradoxical since checkpoint activation is a mechanism generally thought to help cells survive DNA damage, but the absence of MMR activity often renders mammalian cells less sensitive to killing by several DNA-damaging agents (reviewed by Jiricny 2006; Hsieh and Yamane 2008). The MMR status of cancer cells is important for therapeutic treatments because the absence of MMR can confer resistance (reviewed by Karran 2001; Hsieh and Yamane 2008).
We report here a new approach to probe the role of MMR in the activation of the S-phase DNA damage checkpoint in S. cerevisiae by using a drug-sensitive DNA polymerase (pol) δ (Li et al. 2005). The yeast L612M-DNA pol δ, which was modeled on the bacteriophage T4 L412M-DNA pol (Reha-Krantz et al. 1993; Reha-Krantz and Nonay 1994), has a Leu-to-Met substitution in the conserved motif A sequence in the polymerase active center. Both the phage T4 and yeast mutant DNA pols are sensitive to the antiviral drug phosphonoacetic acid (PAA) (Reha-Krantz et al. 1993; P. Burgers, personal communication). Mounting evidence suggests that there is a division of labor at DNA replication forks with DNA pol ε performing leading-strand replication (Pursell et al. 2007) and DNA pol δ serving as the lagging-strand DNA pol (Nick McElhinny et al. 2008); thus, PAA is expected to inhibit lagging-strand replication in cells expressing the L612M-DNA pol δ (pol3-L612M cells). All other DNA pol δ functions in DNA repair and recombination will also be compromised in the presence of PAA.
PAA-treated pol3-L612M cells arrest with the classic dumbbell morphology—a large mother cell and an attached daughter bud of similar size with a single nucleus located in the bud neck (Figure 2A) as observed for yeast treated with DNA-damaging agents and for temperature-sensitive DNA pol δ (cdc2) cells at the nonpermissive temperature (Weinert and Hartwell 1993). A fully functional MMR system is required to maintain viability of pol3-L612M cells exposed to PAA (Li et al. 2005). pol3-L612M cells lacking any component of MMR except for Msh3, but including Exo1, are killed by very low concentrations of PAA (≤0.2 mg/ml). Although a large increase in mutations may be suspected of causing cell death, error catastrophe is unlikely in the case of EXO1 deficiency because only a modest increase in mutations is observed (Li et al. 2005).
Here we tested the hypothesis that the severe PAA sensitivity of MMR-defective pol3-L612M cells is because MMR is needed to assist activation of an S-phase checkpoint.
Materials and Methods
Media, strains, and the synthetic genetic array screen
Cells were grown in either glucose or galactose standard synthetic media supplemented with amino acids; the type of sugar used did not affect PAA sensitivity. PAA gradients were made as described previously (Li et al. 2005). We find that spotting a constant number of cells (50–100) across a gradient is more useful for determining chemical sensitivity than assays in which serial dilutions of cells are spotted on a chemical-containing plate because high cell concentrations mask sensitivity.
All strains are described in Table 1. Gene inactivation was done by replacement of an endogenous wild-type gene with the kanMX cassette in POL3/pol3-L612M heterozygous diploids. Haploids containing the desired mutations were identified by tetrad dissection or by random spore analysis. All gene replacements were verified by PCR and the DNA pol δ gene was sequenced to confirm the presence of the pol3-L612M allele.
The pol3-L612M query strain Y6295 was constructed for the synthetic genetic array (SGA) analysis by transforming the Y5563 starter strain (MATα can1Δ::MFA1pr-HIS3 lyp1Δ ura3Δ0 leu2Δ0 his3Δ1 met5Δ0) with the URA3 YIP-5 pol3-L612M plasmid (Li et al. 2005). The transformant had a truncated pol3 gene and a full-length pol3-L612M gene as confirmed by DNA sequencing. The linked URA3 marker was retained to select for pol3-L612M double mutants. The SGA screen was performed in triplicate as described (Tong et al. 2001, 2004). The final step was to test the double-mutant array strains on plates with and without PAA. The PAA sensitivity of all double mutants was confirmed (Supporting Information, Figure S1, Figure S2, and Figure S3).
DNA labeling, cell morphology, cell viability
Because fungi do not have a nucleoside salvage pathway, yeast were engineered to express the human nucleoside transporter and herpes virus thymidine kinase from the GAL1-10 promotor (Vernis et al. 2003), which allows [3H]thymidine to be taken up by yeast cells and phosphorylated to [3H]dTMP. [3H]dTMP is then further phosphorylated by cellular enzymes to [3H]dTTP, which can be used in DNA replication. A total of 20 μCi/ml [3H]thymidine was added to the labeling cultures at the start of the experiment. To measure [3H]dTMP incorporation into DNA, 0.2-ml samples of labeled cells were mixed with 0.2 ml of yeast carrier cells (109 cells/ml) plus 0.7 ml 20% trichloroacetic acid (TCA). The cells were pelleted and the supernatant was removed by aspiration. The cells were then suspended in 0.5 ml 0.1 M NaOH, and then 0.5 ml of 15% TCA was added to reprecipitate acid-insoluble molecules including DNA. Suspensions were filtered through GF/C filters (Whatman) to capture DNA, and the dried filters were counted in scintillation fluor.
To estimate the amount of DNA replication in PAA-arrested pol3-L612M cells, we first determined the amount of label in a single cell. pol3-L612M cells were evenly labeled with [3H]dTMP in the absence of PAA. The amount of label per dumbbell was then determined for fully arrested pol3-L612M cells (4 hr exposure to 1 mg/ml PAA). Since nearly all of the arrested cells are large-budded cells, the amount of label per arrested cell was divided by the amount of label expected for two genomes; this value was ∼70%.
In most experiments, early exponential-phase cells were synchronized by adding α-factor to a final concentration of 5 μg/ml and incubating for 2.5 hr. The cells were then pelleted by centrifugation, resuspended in fresh medium, pelleted again, and then resuspended.
Cell morphology was monitored by microscopic examination of cells throughout the experiment. Cell viability was determined by comparing the cell titer from counting cells in a hemocytometer to the number of cells that can form visible colonies in 3 days.
Western blots
Standard procedures were followed. Cells were synchronized with α-factor and then exposed to PAA for various times. Protein extracts were prepared by the TCA method. Briefly, 2 × 108 cells were washed first with water and then 20% TCA. The cell pellets were suspended in 100 μl 20% TCA and stored at −20°. Extracts were prepared by mixing an equal volume of glass beads with the TCA-treated cells and then by vigorous vortexing until the majority of the cells were broken as determined by microscopic examination. The broken cells were transferred to a fresh tube, and the glass beads were washed twice in 100 μl of 5% TCA. Protein was pelleted by centrifugation and resuspended in 100 μl Laemli buffer and neutralized by adding 50 μl of 2 M Tris. The sample was then boiled for 3 min and centrifuged. The supernatant, which is the protein sample, was then transferred to a fresh tube. Protein concentration was determined by SDS gel analysis. Rad53 phosphorylation was revealed by goat polyclonal antibodies to yeast Rad53 (Santacruz catalog no. sc6749) and rabbit anti-goat secondary antibodies conjugated with horseradish peroxidase (HRP) (Sigma catalog no. A5420). H2A phosphorylation was revealed by rabbit polyclonal antibodies to yeast phosphorylated H2A (phosphorylated Ser129) (Abcam catalog no. ab15083) and goat anti-rabbit IgG HRP (Santacruz catalog no. sc2004).
Gross chromosomal rearrangement rates
Gross chromosomal rearrangement (GCR) rates were determined as described (Motegi and Myung 2007). Deletion of the left arm of chromosome V, which contains the endogenous CAN1 gene and the URA3 gene inserted into the nearby HXT13 gene, was detected by plating cells on plates containing both canavanine and 5-FOA. Parallel cultures in synthetic dextrose medium were grown either with or without 1 mg/ml PAA starting with ∼0.7 × 105 cells or with a single 3-day colony. The pH of the PAA-containing medium was adjusted to 4.5 with NaOH. PAA-containing cultures were grown for 20 hr with shaking and then pelleted and resuspended in 10 ml YPD and grown overnight. Cultures without PAA were grown overnight. Cells were concentrated 10-fold and titered to determine the total number of cells and the number of cells that could grow on plates containing both canavanine and 5-FOA. Under these conditions, we could not detect any CanR 5-FOA-resistant mutants in cultures of wild-type or pol3-L612M cells not exposed to PAA or in cultures of wild-type cells exposed to PAA, but we assume that the GCR rate is the low reported rate of ∼0.3 × 10−9 as previously reported (Motegi and Myung 2007). As a control, we determined the GCR rate for xrs2 cells and observed a rate of ∼190 × 10−9, which is similar to the reported rate (Chen and Kolodner 1999) (Table 2). Rates were determined as described (Motegi and Myung 2007); the median rate is reported.
PAA-treated pol3-L612M cells arrest growth and DNA replication
PAA-treated pol3-L612M cells arrested as large-budded (swollen) cells with the mother and a similarly sized daughter sharing an undivided nucleus (Figure 2A). To examine the progress of DNA replication following addition of PAA, cells were engineered to incorporate [3H]dTMP into newly replicated DNA as described in Materials and Methods. In the absence of PAA, the doubling time of pol3-L612M cells in galactose medium was ∼2.5 hr (Figure 2B, solid circles); DNA replication, as measured by [3H]dTMP incorporation, paralleled cell growth (Figure 2C, solid circles). In contrast, cell division ceased after 1 hr in the presence of 1 mg/ml PAA (Figure 2B, open circles), but DNA replication continued for ∼3 hr (Figure 2C, open circles). About 70% of the nuclear genome of PAA-arrested pol3-L612M cells was replicated as determined by comparing the amount of [3H]dTMP-labeled DNA per cell for cells not exposed to PAA to the amount of label per cell in cells exposed to PAA (see Materials and Methods). A similar value was observed by Conrad and Newlon (1983).
In contrast to pol3-L612M cells, wild-type (POL3) cells did not arrest in the presence of PAA, but progressed through the cell cycle at a slower doubling rate of ∼3.5 hr compared to ∼2.5 hr in the absence of PAA (data not shown). Thus, PAA slows growth of wild-type cells, but triggers cell cycle arrest in pol3-L612M cells.
PAA-triggered cell cycle arrest in pol3-L612M cells depends on the DNA damage S-phase checkpoint
Both the replication and DNA damage S-phase checkpoints arrest cell cycle progression (Figure 1). The two checkpoints share several components in common, but a distinguishing feature is the dependence of the DNA damage checkpoint on Rad9 and its independence on Mrc1, which is required to mediate the replication checkpoint at stalled replication forks. To determine which checkpoint components are required for the PAA-triggered arrest of pol3-L612M cells and to uncover other DNA pol δ interactions that affect PAA sensitivity, we carried out a SGA in which the pol3-L612M strain was crossed to ∼4700 deletion mutants (Tong et al. 2001, 2004). The double mutants were placed into two groups on the basis of PAA sensitivity.
For one group, PAA increased the slow growth phenotype as demonstrated by mrc1 pol3-L612M cells. PAA sensitivity was measured by spotting cells on a plate with a gradient of PAA from 0 to 1 mg/ml. Increased PAA sensitivity was observed for the double-mutant mrc1 pol3-L612M compared to the single-mutant strains at 3 days (Figure 3A), but was less apparent after 6 days incubation (Figure 3B). Deletion of TOF1, CTF18, CTF8, or CTF4 in the pol3-L612M background enhanced the slow growth or synthetically sick phenotype to the same extent as observed for deletion of MRC1 (Figure S1).
For the second group, cell death was observed at very low PAA concentrations as demonstrated for rad24 pol3-L612M cells (Figure 3A). This is the killing or synthetic lethal phenotype because growth was observed only at the lowest PAA concentration after 6 days (Figure 3B). In addition to RAD24, this group includes the DNA damage clamp genes DDC1, RAD17, and MEC3 as well as RAD9 and MEC1 (Figure S2 and Figure S3). The synthetically sick and lethal phenotypes were observed only in the presence of the pol3-L612M allele (Figure 3, A and B; Figure S1; Figure S2; Figure S3). The stronger dependence of PAA-treated pol3-L612M cells on RAD9 function compared to MRC1 indicates that survival is dependent on the DNA damage checkpoint.
Two methods were used to confirm the presence or absence of S-phase checkpoint activation. If an S-phase checkpoint is not activated, DNA replication is expected to continue unabated in the presence of PAA as observed for the human ataxia telangiectasia mutant or related (ATM/ATR) cells in which replication continues in the presence of DNA damage (Painter and Young 1980). rad24 pol3-L612M cells failed to arrest cell growth in the presence of PAA (Figure 3C, open triangles) or to curb DNA replication, as measured by [3H]dTMP incorporation (Figure 3D) in contrast to pol3-L612M cells in which DNA replication plateaued at a low level after 3 hr (Figure 2C). Cell viability declined sharply; <10% of rad24 pol3-L612M cells were viable after 5 hr exposure to PAA (Figure 3E). rad24 pol3-L612M cells also did not produce the high numbers of large-budded cells that were observed for arrested pol3-L612M cells in the presence of PAA. The continued cell cycle progression and DNA replication observed for PAA-treated rad24 pol3-L612M cells suggests that the severe PAA sensitivity of these cells is caused by failure to activate an S-phase checkpoint.
In contrast, mrc1 pol3-L612M cells (open squares) arrested growth as large-budded cells in response to PAA as observed for pol3-L612M cells (open circles) (Figure 3F). This experiment was done with asynchronous cells, but the doubling times for pol3-L612M (solid circles) and mrc1 pol-L612M (solid squares) cells in the absence of PAA were ∼2.5–2.7 hr (Figure 3F) as observed with synchronous cells (Figure 2B). The doubling time of mrc1 pol3-L612M cells as measured by incorporation of [3H]dTMP was ∼2.7 hr (Figure 3G, solid squares). Cell viability declined gradually to ∼37% after 32 hr of continuous exposure to PAA (Figure 3H). The slow decline in the viability of mrc1 pol3-L612M cells is in contrast to the sharp decline observed for rad24 pol3-L612M cells exposed to PAA (Figure 3E). Thus, the slow growth/synthetically sick phenotype observed for mrc1 pol3-L612M cells is due to a combination of apparent checkpoint activation and to a loss of viability upon prolonged exposure to PAA.
A second test for checkpoint activation was to measure Rad53 phosphorylation in PAA-treated pol3-L612M cells. Rad53 phosphorylation is a key indicator of checkpoint activation (Figure 1). Rad53 phosphorylation was detected in pol3-L612M cells after 1 hr treatment of synchronized cells with 1 mg/ml PAA in glucose medium in which the doubling time is ∼90 min; phosphorylation increased further after 2 and 3 hr exposure (Figure 4, top). Rad53 phosphorylation was also observed for mrc1 pol3-L612M cells (Figure 4, bottom), which is consistent with the PAA-induced cell cycle arrest observed for these cells and for the cessation of DNA replication (Figure 3, F and G). Little Rad53 phosphorylation, however, was detected for rad24 pol3-L612M cells (Figure 4, bottom), which is consistent with the absence of cell cycle arrest observed for these cells (Figure 3, C and D). Rad53 phosphorylation was also not detected for wild-type cells exposed to PAA (Figure 4, middle). Note that these later experiments were done with asynchronized cells, but swollen dumbbell-shaped cells quickly accumulated for PAA-treated pol3-L612M and mrc1 pol3-L612M cells as expected for checkpoint activation, but they did not accumulate for PAA-treated wild-type or rad24 pol3-L612M cells, and cell growth did not arrest.
pol3-L612M cells exposed to PAA require DSB repair
We next examined the possibility that DSBs were produced in pol3-L612M cells exposed to PAA. Deletion of the recombinase (RAD51) or RAD52 genes, which are required for homologous recombination, increases the PAA sensitivity of pol3-L612M cells (Li et al. 2005). More severe PAA sensitivity was observed by deletion of genes that encode components of the yeast MRX complex (Mre11-Rad50-Xrs2) (Figure 5), but less than that detected for deletion of genes required for the DNA damage checkpoint such as RAD24 (Figure 3, A and B). The PAA sensitivity of pol3-L612M cells in the absence of MRX components may reflect the role of these proteins in checkpoint signaling as well as in DSB repair since the DNA damage checkpoint can be initiated but not sustained in the absence of the MRX complex (D'Amours and Jackson 2001). PAA-treated xrs2 pol3-L612M cells arrested initially as swollen dumbbell-shaped cells as observed for pol3-L612M cells, but viability was just 42% by 5 hr and many abnormal multi-budded cells started to accumulate that were not observed for pol3-L612M cells. Viability declined to 25% by 10 hr and was just 8% by 24 hr (data not shown).
We also tested the possibility that DSBs produced by exposure of pol3-L612M cells to PAA increased GCRs. An isogenic set of strains (Table 1) was constructed with a GCR reporter (Chen and Kolodner 1999; Myung et al. 2001; Motegi and Myung 2007). GCRs are detected in the reporter by deletion of the nonessential end of the left arm of chromosome V, which contains the endogenous CAN1 gene and the nearby URA3 gene that was inserted into the nonessential HXT13 gene. The major class of GCRs observed by the simultaneous loss of the CAN1 and URA3 genes is terminal deletions of the end of chromosome V that are healed by de novo telomere addition (Motegi and Myung 2007). A 20-fold increase in the GCR rate was observed for pol3-L612M cells exposed to PAA (Table 2), which suggests that PAA treatment induces DSBs and that repair is not completely error free.
Role of mismatch repair in protecting pol3-L612M cells exposed to PAA
We reported previously that deletion of MSH2, MLH1, PMS1, MSH6, or EXO1 (but not MSH3) markedly increases the PAA sensitivity of pol3-L612M cells (Li et al. 2005). The dependence of pol3-L612M cells on MMR was confirmed here (Figure 6A). To avoid complications produced by the high genetic instability of completely MMR-deficient pol3-L612M cells, msh6 pol3-L612M and exo1 pol3-L612M cells were examined. These cells are nearly as PAA sensitive as completely MMR-deficient pol3-L612M cells, but are 80–100% viable in the absence of PAA, and only a small increase in mutations was observed for the exo1 pol3-L612M strain (Li et al. 2005). Cell growth and DNA replication were monitored as described above; PAA-treated msh6 pol3-L612M and exo1 pol3-L612M cells did not arrest cell growth or curb DNA replication as observed for checkpoint-deficient rad24 pol3-L612M cells (Figure 3, C and D). Cell viability declined for PAA-treated msh6 pol3-L612M cells (Figure 6B) at a slower rate than observed for rad24 pol3-L612M cells (Figure 3E), but <5% cells were viable after 26 hr exposure, which accounts for the observed PAA-induced killing/synthetic lethality phenotype. A similar pattern of PAA sensitivity was observed for exo1 pol3-L612M cells (Li et al. 2005).
Little if any Rad53 phosphorylation was observed for synchronized msh6 pol3-L612M cells over a 3 hr exposure to 1 mg/ml PAA (Figure 6C, top) under the same conditions in which extensive Rad53 phosphorylation was observed in pol3-L612M cells after just 1 hr (Figure 4). For synchronized exo1 pol3-L612M cells, no increase in Rad53 phosphorylation was visible after 1 hr, but by 3 hr a small amount of Rad53 phosphorylation was detected at ∼20% the amount observed for PAA-treated pol3-L612M cells (Figure 6C, lower panels); however, there was much less of the most highly phosphorylated Rad53, which has the slowest rate of migration. The low amount of Rad53 phoshorylation for PAA-treated exo1 pol3-L612M cells may reflect the ability of other exonucleases to substitute for Exo1 in MMR; other possibilities are suggested in the Discussion. Despite the small amount of Rad53 phosphorylation observed, PAA-treated exo1 pol3-L612M did not arrest cell growth as observed for pol3-L612M cells.
H2A phosphorylation is produced in response to PAA for MMR-proficient pol3-L612M cells but not for msh6 pol3-L612M or exo1 pol3-L612M cells
We attempted to detect DSBs in PAA-treated pol3-L612M cells by using a fluorescent protein–Rad52 construct to detect DSB repair foci (Lisby et al. 2003). If DSBs are responsible for activation of the DNA damage S-phase checkpoint, then Rad52 foci are expected to be visible in most of the PAA-treated, MMR-proficient pol3-L612M cells. Although MRX deficiency strongly increases the PAA sensitivity of pol3-L612M cells (Figure 5), Rad52 foci were observed in only ∼20% of PAA-treated pol3-L612M cells. Thus, we tried a more sensitive test to detect DNA damage: antibodies to phosphorylated histone H2A, which is a well-characterized response to DNA damage (Figure 1). H2A phosphorylation was observed for α-factor-synchronized pol3-L612M cells after just 1 hr exposure to 1 mg/ml PAA in glucose medium, which did not increase after 2 and 3 hr exposure (Figure 7). In contrast, little H2A phosphorylation was observed for msh6 pol3-L612M or exo1 pol3-L612M cells even after 3 hr exposure to PAA (Figure 7). The appearance of phosphorylated Rad53 (Figure 4) and H2A (Figure 7) in pol3-L612M cells just 1 hr after exposure to PAA indicates that DNA damage and checkpoint activation occur in the first S phase.
Can HU rescue MMR-deficient pol3-L612M cells from PAA?
If the role of MMR in activating the S-phase DNA damage checkpoint in PAA-treated pol3-L612M cells is to increase the level of checkpoint-inducing DNA damage, then another method for creating damage is expected to protect msh6 pol3-L612M and exo1 pol3-L612M cells from PAA. DNA damage treatments sufficient to activate the S-phase damage response on their own could not be used because of the essential roles of DNA pol δ in recombination and repair, which are compromised in PAA-treated pol3-L612M cells. For example, we have observed that PAA increases the UV sensitivity of pol3-L612M cells, which indicates a role for DNA pol δ in the gap-filling step of nucleotide excision repair that cannot be replaced by other DNA polymerases (L. Reha-Krantz and Md. Siddique, unpublished data). Thus, we tested if a low concentration of HU (10 mM) could substitute for MMR. Partial rescue of the PAA sensitivity of msh6 pol3-L612M and exo1 pol3-L612M cells was observed on a 1-mg/ml PAA gradient plate; colony formation was observed in the presence of 10 mM HU (Figure 8, bottom) at higher PAA concentrations than observed in the absence (Figure 8, top). HU may be acting in several ways. For example, HU could increase the length of ssDNA gaps in the lagging strand and/or uncouple leading- and lagging-strand replication; however, in all scenarios, it is likely that ssDNA is increased.
DUN1 is required for pol3-L612M cells to survive PAA
If the S-phase DNA damage checkpoint is required for pol3-L612M cells to survive PAA, then downstream targets of Rad53 phosphorylation such as Dun1 are also expected to be required for viability. As expected, dun1 pol3-L612M cells are as PAA sensitive as rad24 pol3-L612M cells in which Rad53 is not activated (Li 2004). Thus, one or more Dun1-regulated activities is required for pol3-L612M cells to survive PAA. We have verified by the SGA screen that one Dun1 phosphorylation target—Rad55, which functions in DSB repair, is needed for pol3-L612M cells to survive PAA. Deletion of SML1, however, did not affect the PAA sensitivity of pol3-L612M cells (Figure S3). Sml1, an inhibitor of ribonucleotide reductase, is phosphorylated by activated Dun1, which relieves inhibition of ribonucleotide reductase and restarts dNTP synthesis (Chabes et al. 2003). If increased dNTP synthesis alone were sufficient to rescue PAA sensitivity, then deletion of SML1 would be expected to decrease the PAA sensitivity of pol3-L612M cells. As this was not observed, other Dun1 and damage checkpoint targets, likely in addition to inactivation of Sml1, are required for pol3-L612M cells to survive PAA.
POL32 is required for pol3-L612M cells to survive PAA
Deletion of the POL32 gene, which encodes the DNA pol δ PCNA-binding protein (Johansson et al. 2004), produced severe PAA sensitivity; no growth was detected even at the lowest PAA concentrations tested (Figure S2). Although POL32 is not essential in S. cerevisiae, a slow growth phenotype is observed in its absence and POL32 is essential in other eukaryotes. The strong dependence of pol3-L612M cells on Pol32 in the presence of PAA suggests that PCNA tethering of DNA pol δ at replication forks and/or at other sites of DNA pol δ action is required for viability. A role in maintaining the stability of stalled replication forks is another possibility because deletion of POL32 also increases HU sensitivity (Parsons et al. 2004).
PAA treatment of pol3-L612M cells activates the DNA damage S-phase checkpoint. Survival requires all components of the DNA damage checkpoint, including Mec1 (the yeast ATR Ser/Thr kinase), the signaling adapter Rad9, and the PCNA-like DNA damage clamp (Ddc1-Rad17-Mec3) and damage clamp loader (Rad24) complex (Figure 3; Figure S2 and Figure S3). pol3-L612M cells deficient in any of these proteins are highly sensitive to PAA and, as shown for rad24 pol3-L612M cells, fail to activate the DNA damage checkpoint as demonstrated by the failure of cells to arrest cell cycle progression, to curb DNA replication and to phosphorylate Rad53 and by subsequent loss of cell viability (Figures 2–4⇑⇑). pol3-L612M cells have less dependence on Mrc1, the checkpoint adapter for stalled replication forks (Figure 3).
An important finding is that while DNA damage as detected by phosphorylated H2A is observed for PAA-treated pol3-L612M cells after 1 hr, only a little phosphorylated H2A is observed for PAA-treated msh6 pol3-L612M or exo1 pol3-L612M cells after 3 hr (Figure 7). Note that phosphorylated H2A is a well-known marker for DSBs, but there is a possibility that phosphorylated H2A is also produced in response to ssDNA. Detection of phosphorylated H2A is correlated with Rad53 phosphorylation, which is readily detected for PAA-treated pol3-L612M cells after 1 hr (Figure 4), but not for PAA-treated msh6 pol3-L612M cells even after 3 hr (Figure 6C, top) and only a small amount is observed for exo1 pol3-L612M cells (Figure 6C, bottom left).
The dependency of PAA-treated pol3-L612M cells on Exo1 for survival suggests that ssDNA gaps produced by MMR are required for checkpoint activation; however, Exo1 is reported to play several roles, including a structural role in stabilizing MMR complexes that is independent of its enzymatic role (Amin et al. 2001). Outside of MMR, Exo1 has many functions, including processing and stabilizing stalled replication forks, controlling DNA damage checkpoint activation, and in recombination (Tran et al. 2004; Cotta-Ramusino et al. 2005; Segurado and Diffley 2008). Furthermore, Exo1 is reported to function in checkpoint activation as part of the nucleotide excision repair pathway (Giannattasio et al. 2010). Note that DNA replication by DNA pol δ is also involved in these processes. Thus, the severe PAA sensitivity of PAA-treated exo1 pol3-L612M cells may indicate the need of Exo1 function and/or the combined need of Exo1 and DNA pol δ function in multiple pathways. The low level of Rad53 phosphorylation detected after 3 hr exposure of exo1 pol3-L612M cells to PAA (Figure 6C) may be caused by DNA damage arising because of the absence of Exo1 in several different processes, but with regard to the PAA sensitivity of pol3-L612M cells, Exo1 function is epistatic to Msh2 (Li et al. 2005), and, thus, Msh2 and Exo1 are working in the same pathway.
We propose that MMR creates ssDNA gaps that persist in PAA-treated pol3-L612M cells (Figure 9). DNA pol δ is responsible for resynthesis of MMR-created ssDNA gaps (Kadyrov et al. 2007), but since PAA inhibits the L612M-DNA pol δ, gap filling will be slowed. This proposal is supported by the observation that a low dose of HU, which is expected to provide an additional source of ssDNA, can partially substitute for MMR activity (Figure 8). These data together suggest that MMR is required to increase ssDNA/DSBs above a threshold that is needed to activate the S-phase DNA damage checkpoint.
A threshold amount of ssDNA is required to activate S-phase checkpoints to prevent damage-like DNA structures present during normal DNA replication from accidentally triggering checkpoint activation (Shimada et al. 2002). This reasoning certainly applies to lagging-strand replication, in which individual Okazaki fragments are primed, elongated by DNA pol δ, and eventually joined together in reactions that also require DNA pol δ (Jin et al. 2004); these steps will be compromised in PAA-treated pol3-L612M cells. We propose that lagging-strand replication is not monitored for ssDNA gaps or nicks that exist during the maturation of Okazaki fragments because these strand discontinuities are a normal aspect of lagging-strand DNA replication; however, there are likely more gaps and nicks in the lagging strand of PAA-treated pol3-L612M cells than wild-type cells, and, more importantly, the gaps/nicks are predicted to heal slowly. Experiments presented here suggest that lagging-strand discontinuities fall under the radar of checkpoint surveillance, while larger gaps produced by MMR activity and DSBs are detected. S-phase checkpoint activation for PAA-treated pol3-L612M cells is lifesaving because cell cycle arrest provides more time and additional resources (dNTPs, DSB repair enzymes) for the drug-sensitive L612M-DNA pol δ to fully complete lagging-strand replication and to repair DSBs before the cells enter mitosis.
We examined the possibility that hard-to-replicate DNA sequences are likely to be even slower to replicate in PAA-treated pol3-L612M cells. Previous studies suggest that unreplicated regions like the ribosomal DNA (rDNA) locus are not detected by S-phase checkpoint sensors and, as a consequence, do not prevent premature cell cycle progression into mitosis (Torres-Rosell et al. 2007). Pulsed-field gel electrophoresis analysis of chromosomes from PAA-treated pol3-L612M cells, however, did not reveal problems with replication of rDNA (data not shown). Thus, the most reasonable causes of cell death in PAA-treated pol3-L612M cells in the absence of checkpoint activation are unrepaired DSBs and persisting strand discontinuities in the lagging strand that produce more DSBs when encountered by advancing replication forks in the next cycle of DNA replication.
Even in the presence of checkpoint activation, DSBs are a problem for pol3-L612M cells as demonstrated by the increased PAA sensitivity observed when the genes required for DSB repair—RAD51, RAD52, XRS2, or MRE11—are deleted (Figure 5). The slow growth/synthetic sickness phenotype that was observed for pol3-L612M cells deleted for MRC1, TOF1, CTF4, CTF18, or CTF8 (Figure 3; Figure S1) is also likely related to the function of these genes in the sister-chromatid cohesion that is required for recombination repair of DSBs (Xu et al. 2004). Furthermore, since DNA pol δ is required for the replication phase of homologous recombination (Maloisel et al. 2008), PAA inhibition of the L612M-DNA pol δ is expected to adversely affect recombination if reactions initiated are slow to be completed. This could explain the 20-fold increase in the GCR rate detected for PAA-treated pol3-L612M cells (Table 2).
In conclusion, we have provided additional evidence from studies of PAA-treated pol3-L612M cells for the proposal that a threshold level of DNA damage is required to activate the S-phase DNA damage checkpoint in yeast. Lagging-strand replication problems produced by drug inhibition of the L612M-DNA pol δ do not appear to provide sufficient DNA damage to trigger checkpoint activation even though continued cell cycle progression results in cell death. MMR activity, however, increases DNA damage within 1 hr of PAA exposure, as demonstrated by phosphorylated H2A (Figure 7). In a related observation, Exo1 independent of MMR is observed in noncycling cells to enlarge ssDNA gaps produced by the removal of UV lesions, and these gaps are reported to drive Mec1 kinase activation (Giannattasio et al. 2010). Exo1-enlarged gaps in these experiments were detected as a "cloud" or smear of chromosomes that migrated only a short distance from the loading well during pulsed-field gel electrophoresis. We did not observe a chromosome cloud in PAA-treated pol3-L612M cells, which may indicate a lower level or shorter ssDNA gaps in our experiments compared to UV-treated cells in the experiments by Giannattasio et al. (2010). Thus, Exo1 degradation as part of MMR in our studies or at sites of UV lesions appears to provide the means to engage the checkpoint response under conditions where checkpoint activation may not occur.
The absence of MMR and the checkpoint response in PAA-treated pol3-L612M cells has serious consequences for the few cells that survive. An unusually high mutation rate for production of PAA-resistant cells is observed for survivors (Figure 6A), which appears to diminish in the presence of HU (Figure 8). PAA resistance is due to the acquisition of a second mutation that encodes an amino acid substitution that suppresses the PAA sensitivity conferred by the L612M substitution (L. Li, A. Oladarin, L. Reha-Krantz, unpublished data). Furthermore, we observed many examples of unusual yeast cell morphology in PAA-treated MMR-deficient pol3-L612M cells (Figure 10). We cannot rule out the possibility that MMR is signaling checkpoint sensor kinases as proposed for mammalian cells, but because ssDNA is required for sensor kinases to bind DNA (Zou and Elledge 2003), we propose that MMR is needed primarily to produce ssDNA gaps. To explain the apparent interaction between MMR proteins and sensor kinases in mammalian cells, we suggest that MMR complexes remain associated with the ssDNA gaps (Figure 9). This is a reasonable proposal because some DNA repair proteins have been observed to remain associated with partially repaired DNA to reduce the chance of DNA repair intermediates becoming genotoxic lesions (Mol et al. 2000).
We gratefully thank J. Cobb for providing instruction in detecting phosphorylated Rad53 and for the suggestion to measure phosphorylated H2A; G. Brown for advice on detecting Rad52 foci and other helpful comments; H. Kremer for constructing the mre11::kanMX strain; and A. Radziwon for technical assistance. Research was supported by the Canadian Institutes of Health Research (grant 14300 to L.R.-K. and grant 41567 to C.B.). L.R.-K. acknowledges support as a Scientist of the Alberta Heritage Foundation for Medical Research.
Communicating editor: J. A. Nickoloff
- Received July 21, 2011.
- Accepted September 6, 2011.
- Copyright © 2011 by the Genetics Society of America


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Village Life
Village life photos
Collection of photos of village life as from the archive
Tennis Club
Tennis Club history – Upper Broughton Youth and Social Club
Auntie Annie
a well known Upper Broughton character
Samuel Wright
Samuel Wright is one of the latest people we are researching. New information has come to hand recently as we have been contacted by Charles Wright a relative of Samuel's and also Father Edward Martin who has Samuel's prayer book in his collection. We have also discovered two large slabs behind the Church which we believe lead to a vault where Samuel is buried.

subset: commoncrawl


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How to Variety Uncomplicated Smart Property? This specific dilemma might be found in this intellects of people who wish to uncover more about a smart house. The actual question could be asked olh regular men and women all about the entire world regarding property or home in addition to that's been plunged therein.
To be a design and style household in which is the nearly all green, minimalist house must have special features, which include:
Baca Selengkapnya di URL: http://www.rumahminimalisin.com/62/bentuk-rumah-minimalis/


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Play through the 36-mission singleplayer marketing campaign, experience the life-like terrain, and wield the unimaginable powers of the Gods. The primary game of the Command & Conquer series was released in 1995 and was one of the first games in the RTS genre.
The administrators are additionally more than happy to listen to player feedback, allowing you to affect the route that the sport takes sooner or later. Not solely does the sport provide intense combat and an interesting main storyline it additionally presents varied ethical decisions, though unlike different games all ethical choices fall heavily in a grey space.
You see, zombies could be fooled, however gamers can't. The PlayStation three version additionally permits gamers to file their gameplay and upload it which allows you to share your favorite recreation facets with others. The unique Crackdown features a large sandbox surroundings that players are free to discover after deciding on one of many brokers accessible.
The game is on the market throughout many genres and is a superb recreation for these followers who enjoy the flip-based combat model of Pokémon. The Elder Scrolls video games and Fallout sequence are my favorites. Now I know video games to play in the meantime. Just Trigger is an open-world action journey game the place players play as Agent Rico Rodriguez.
Nevertheless, remember that many players were launched to the Elder Scrolls series with Skyrim and easily don't contemplate the previous video games in the collection after they take pleasure in it a lot. In Rise of Nations, players can select from 18 completely different civilizations and guide them through eight different stages of world historical past.
The game presents 5 completely different game areas, four races to play and three different class bushes (Warrior, Rogue and Mage) with each offering a variety of different skills. I can't communicate extremely sufficient of the game and how much I loved my time with it. It's most likely the closest sport I've found to the Fable sequence.


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Finished a drawing of Art Gish, peace activist who died this week in a farming accident (Read the stoty at:http://www.athensnews.com/ohio/article-31680-prominent-local-activist-dies-in-farming-accident.html). I read Art's book The New Left and Christian Radicalism back in the late 70s, when I was looking for anything that was Christian and radical. I met Art at a regional peace gathering I helped organize in Columbus, Ohio around 2004. We were at a table with a Christian Zionist who had a totally different perspective on Israel/Palestine than Art and the rest of us at the table! And yet, Art treated him with respect, even though he has been on the ground in Israel/Palestine with Christian Peacemaker Teams and knows well the real situation there and not just some idealogy.
Art became recognized internationally through an Associated Press photo of him confronting an Israeli tank ready to destroy a Palestinian marketplace (See picture above and read the story at :http://mideastchristians.virtualactivism.net/articles/amongapples.htm)
May his wife, Peggy, find comfort in her loss and the world weep upon the loss of one of her prophets.
Video of Art and Peggy Gish, Old Radicals:
Old Radicals from matthew leahy on Vimeo.


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Upper Bearwallow Falls
This is a long slide in two main parts. Only the lower part is visible from the overlook constructed by the park. It's a pretty nice falls, and the view from the overlook isn't bad – however, the cluster of dead hemlocks in the foreground detracts from it some.
Driving Directions
From downtown Brevard, it is just over 18 miles on US 64 West to the intersection with NC Hwy 281 South – turn left. From Highlands, it's just over 20 miles on US 64 East, thru Cashiers, to NC 281 South – turn right. Go 0.9 miles South on NC 281 from US 64 to the park entrance road on the left. It's less than a mile to a left turn for the loop road and picnic shelters – turn there. The picnic shelters are just ahead on the right, down the hill – park at the second picnic shelter (or the first one for a slightly longer hike).
Trail Directions
The trail is just over 1/2 mile round-trip and moderate, with a smooth surface and a moderate climb. Start just across the parking area from the picnic shelter. It descends some rock steps, crosses the road, and then goes downhill through 5 switchbacks in a nice forest to the falls.
– by northcarolinawaterfalls.info


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Event detail
ITS Seminar: Future of the Interstate Highway System
Seminar | September 8 | 4-5 p.m. | 290 Hearst Memorial Mining Building
Steve Heminger, Metropolitan Transportation Commission
Institute of Transportation Studies
The Interstate Highway System (IHS) is a key component of the US transportation system. While it makes up only 1.2 percent of roadway line-miles of the country's public road system, it handles nearly 25 percent of the total vehicle miles traveled (VMT) annually and almost 40 percent of the nation's total truck traffic. The IHS of today, with a network little changed since its inception, serves more traffic than the entire U.S. road network served when the IHS was authorized in 1956. However, what was once a premier system that stood as a symbol and enabler of American growth and economic vigor is showing its age.
The Future Interstate Study is being done pursuant to Section 6021 of the Fixing America's Surface Transportation Act of 2015, which calls for the Transportation Research Board to conduct a study on the actions needed to upgrade and restore the Dwight D. Eisenhower National System of Interstate and Defense Highways to its role as a premier system that meets the growing and shifting demands of the 21st century. This talk will present updates from the study.
Alumni, Faculty, General Public, Staff, Students - Graduate, Students - Prospective, Students - Undergraduate
Alumni, Faculty, General Public, Staff, Students - Graduate, Students - Prospective, Students - Undergraduate
Cookies and beverages
email@example.com, 510-642-3585


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For this week's blog, I thought I would take you on a tour of my home town, and a typical night spent looking for new music in bars and dives.
The Tabu is dank. Small and dank, and over in the corner is a sign saying The Hellfire Club, where men fatter and older than me congregate in small circles, wearing rubber and scour the room for the one attractive person. You can almost taste the semen. On one side, there's a gentleman's club (which means the door staff wear suits) and on the other, an old-fashioned peep show where you can buy a couple of hundred tickets for a few dollars. A few rocker types litter the tables, occasionally crawling up the stairs to smoke on Brunswick Street alongside the homeless and pimps; there's Tom from Rocking Horse Records (Brisbane's oldest independent record store) and Jo from Brisbane's own Breeders tribute band, Butcher Birds, neither feeling as psychedelic as the night before.
The Tabu feels like home, which in this case is a succession of Midlands venues, starting with Edwards No 8 in Birmingham, and culminating with snatched bursts of sleep during drum checks. There's no one here – well, very few – to catch pick-up garage band the Young Liberals featuring local rock hero Ben Salter race through a turbulent, thrilling and frankly un-together set of smart-Alec, raw-as, deconstructionist rock songs such as the Art Brut-esque brace We're in a Band and My Neighbour's Band; only matched for poignancy by the Shop Assistants/Velvet Underground duet Let's Go Swimming, during which Butcher Bird Jo provides Mo Tucker vocals to Salter's deeply evocative rasp, honed by years of busking and smoking. A disconcertingly sober Englishman clambers onstage to provide caustic spoken word, but mercifully the florid interruption is brief.
It's not long before I make a sharp exit, taking a deserted St Paul's Terrace all the way up to Musgrave Road, in Red Hill, where I arrive at Lofly Hangar. Whoa. All of a sudden, I feel like I'm in Portland, Oregon (a compliment) or a neat venue like Bottom of the Hill in San Francisco. There's no bar, no formal set-up – just a disused tyre factory with a bunch of (self-styled) indie kids standing round drinking from bottles, a room with comfy, vagrant armchairs and a makeshift stage. On stage are a bunch of cute indie kids pretending to be A Silver Mt Zion or Godspeed You Black Emperor! or someone that it's quite likely no one's seen play up here, so they won't notice the overt similarities. And hence this band – who may or may not be called Mt Augustus, it's hard to tell – are pretending they've been into gospel and off-mic singing their entire lives (ie the last three months), and use brass and a female cheerleading section dressed in dungarees to help whip the refined indie kids in their refined indie clothes up into something not resembling a ferment. It's all very polite and everything – the sort of music you wouldn't be worried about taking home to meet your parents.
But the music is OK, and suitable for this warm spring evening. I'm stood by one of the doors where Phil Elverum from touring Anacortes' Washington band Mount Eerie. Phil's set – solo, with his feet perched up on a monitor like a kindly visiting uncle laying bare the wonderment of the soul – is as gentle, poetic and vaguely off-key as desired, even if I do only watch five or seven songs before wandering off, buoyed by the Lofly Hangar's oasis of vagrant sofas in this city of wealth, to brave the breathalysers along Waterworks Road.
One visit and the Hangar is already close to being my favourite Brisbane hangout, just behind the city-funded Powerhouse in New Farm, and Audiopollen, operating out of a West End loading bay. Here, Elverum is selling his wares to a succession of star-struck kids; a beautifully hand-produced photo book here, a recording of the mountain gods in his hometown there ... and now I really feel like I'm in Portland.


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Professional & Social Networking for Published & Aspiring Writers of the Northwest
Founded as a society of professional writers in 1921, Seattle Free Lances has grown its membership in recent years to include publishing industry professionals and writers actively working toward publication. It is our goal to support the continued success of our published members while encouraging the aspirations of those writers whose ultimate aim is to become published.
Things Every Writer Should Think About:
Acknowledge yourself as a writer and be proud that you are one.
Realize that you cant be writer, editor and critic all at the same time.
Learn how to hear and when to accept feedback.
Identify the kind of writing you write.
Keep a journal of your ideas.
Recognize the time of day or week that is the best time for you to write.
Respect your writing time. Write daily if at all possible.
Use your writers voice to speak to readers with all the joys, sorrows and celebrations that real people experience.
Understand that writing creates shape and meaning that real life may not have.
Read, read, read current and classical works.
Learn to read genres you dont currently read and step back to discover why it works, not why it doesnt work.
Dissect your story if it goes off track. Examine each chapter or section and make it as powerful and focused as possible.
Become knowledgeable about the publishing business.
Build a network of publishing contacts and maintain them.
Remember that discipline and focus makes the difference between wannabe writers and published writers.
Know that every writers path to publishing has its own story just as every writer works differently.
Read rejection letters more than once and give yourself time between readings.
Celebrate the steps you take to discovering your writing life, as this process is known as success.
About Jennifer
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Jap Tsukihime runs on NScripter, but that doesn't support English very well. That's why we use ONScripter-En. It's the same thing, only better for English. So yeah, Tsukihime doesn't have its "own" exe file. It never had.Zogundar wrote:Hmm, I see.. I think. The game doesn't have its own actual .exe file? Or is it a function of the translation.. stuff?
Detail enough if you take the time to read it, I guess. But basically:Zogundar wrote:How much detail does the Readme go into about the music replacement? (Also I can't make a decision if it's an either/or thing, which one do I want??)
1) Rip the music to .ogg (.mp3 will also work, if you're going to upgrade to the latest ONScripter-En)
2) Rename the files to Track01.ogg through Track10.ogg (or .mp3)
3) Create a directory called "CD" in your game directory
4) Move the ogg files to that CD directory.
I just found this on google, in contains our instructions, and a nice visual guide: http://myanimelist.net/forum/?topicid=4907
As for either/or, they are all the same tunes/songs as the original game, only with different arrangements/instruments. I prefer EVER AFTER over the Tsukibako version, because it is less monotonous and more atmospheric. My recommendation: Use tracks 01 through 09 from EVER AFTER. Use track 10 from the original game. (EVER AFTER does not have this track, but it's only played once so the original is good enough.)


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one last promise emo jon calvert
Pan-fried Corona Beans & Kale Recipe - 101 Cookbooks
Feb 28, 2010 . The other day I found myself in the basement of San Francisco's Anthropologie store. I'd fallen for the rose & tuberose solid Frazer Parfum on .
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recipe with corona beans Location
Giant Chipotle White Beans Recipe - 101 Cookbooks
Jan 7, 2009 . I love his original recipe, but the last time through I decided to add a few of . 1 pound of large, dried white beans (corona, giant limas, gigantes, .
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recipe with corona beans Building
Gigante Beans Recipe : Anne Burrell : Food Network
Food Network invites you to try this Gigante Beans recipe from Anne Burrell. . 1 pound gigante or corona beans; 1 large onions, skin removed, root end left on, .
White Beans with Pancetta and Sage Recipe : Tyler Florence : Food ...
Please sign in to save this recipe to your Recipe Box! . 1/2 pound large dried white beans, such as corona beans; 3 bay leaves; 3 garlic cloves · All-purpose .
Pan Fried Corona Beans and Kale Recipe
from 101 cookbooks.com. We used Giant Lima Beans instead of Corona Beans.
Sausage and Corona Beans with Sage and Arugula
Sep 22, 2011 . Sausage and Corona Beans with Sage and Arugula I found a lot of unusual dried beans at the Whole Foods Market cooking section, a new .
Sidewalks and Streetscapes
Corona Bean Salad with Grilled Baby Octopus : CIAProchef.com
2 lb/907 g Corona Bean Salad with Basil (recipe follows); 20 lemon wedges; 2 1/ 2 oz/71 g flat-leaf parsley, leaves only; 1 oz/28 g fennel fronds; 5 oz/142 g frisée, .
Recipe: Giant Crusty and Creamy White Beans with Greens :: Heidi ...
Plan ahead for this recipe, as you need to soak the uncooked beans overnight. . Giant corona beans, cellini beans, or white cannellini beans are the best .
Corona Bean Salad with Basil and Rosemary
1 pound giant Corona beans, soaked overnight; 1/2 cup olive oil, or to taste; 1 sweet onion, chopped; 2 cloves garlic, chopped; 2 cups chopped fresh tomato (or .
Extra Thoughts
Grilled Octopus With Stewed Corona Beans
Grilled Octopus With Stewed Corona Beans. Beans 2 cups dried corona beans, soaked overnight (can substitute cannelloni) 1 stalk celery, cut into 3 pieces .
Cookstr - Stewed Beans
A great recipe for Stewed Beans by Mario Batali from Spain. . 1 pound dried Corona beans (or other large white beans), soaked overnight in water to cover .
Florentine Corona Beans - Foster & Dobbs : Authentic Foods ...
Florentine Corona Beans. Posted by Luan in Recipes. January 10, 2012. Ah, corona beans! Huge, plump, ivory-colored beans with a rich flavor and a tender, .
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Meg Wolff: Plant-Based Meals: Fit-for-a-King Corona Beans!
Sep 27, 2011 . Cancer Diet , Vegetarian Recipes , Corona Beans , Meg Wolff . According to the Italia Gourmet website, "Corona beans are a gourmet and .
Tuscan Kale, White Bean, and Ciabatta Soup Recipe | MyRecipes.com
Save Recipe; Add to Menu; Add to Shopping List. 1 cup dried white beans such as Corona* or cannellini; 2 to 2 1/2 qts. reduced-sodium chicken broth; 10 garlic .
Dried Beans - Cooking Light
Corona beans. Qualities: Slightly mealy texture and meaty taste. Best in recipes that call for long, slow cooking. Serving suggestion: Combine with tuna, onions, .
Sous-Vide Octopus | Zen Can Cook
Nov 2, 2011 . Grilled Sous-Vide Octopus w/ Corona Beans, Chorizo . at home I'm going to post some easy recipes for the kitchen Indiana Jones out there.
I think this hotel will be a hit!
Goaoaa Beans WITH Srtcv Roasreo PRAWN5
Combine beans and water in large saucepan and bring to a gentle boil over medium high heat. . I eap reset-and roalring liquid t recipe ranked Corona lira n. -.
blissfulb - bliss blog - blissful eats with tina jeffers: Corona beans ...
Mar 15, 2012 . This is a very easy recipe to alter and adapt, if you can't find corona beans, cannellini or navy would work equally well. Add the tomatoes or .
Corona Beans Lazio Italy from ChefShop.com
Dried corona beans are already about half again as large the typical white . Or try them in this delicious recipe with fennel, olives and a lemon-mint vinaigrette.
What I'm Eating Now » A Gigante Bowl of Comfort
Jan 21, 2010 . Recipe: Tamara's Whole Foods Gigante Bean Salad Knockoff . Italian Corona Beans work just as well-as a matter of fact, hard to tell the .
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Pan-fried Corona Beans & Kale A few notes related to the recipe - be sure to wash the kale well, so you don't end up with grit in your beans. I use dried beans .
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Search by keyword, recipe or item # Search . Flageolets with Fennel, Herbs and Olives · Fried Corona Beans with Garlic & Sage. Rated 4 out of 5. (1) .
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1 pound medium to large dried white beans (I love to make this recipe with giant Corona beans or Rancho Gordo white cannellini) 3 tablespoons salt for the .
Isn't the mural mentioned along Peace?
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Florentine Corona Beans. Posted by Luan in Recipes. January 10, 2012. Ah, corona beans! Huge, plump, ivory-colored beans with a rich flavor and a tender, .
Tumis Kacang & Kubis (Pan-fried Corona Beans & Kale) Dual ...
I used giant corona beans, but you could use runner cannellini, or something . http://www.101cookbooks.com/archives/panfried-corona-beans-kale-recipe.html .
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Nicola DeLaurentiis is driven to tirelessly invent, improve, innovate and refine his products. A Recipe with Corona Beans - from the folks at The Taste of Oregon .
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bean recipes: 101 Cookbooks. . This is a version made with Christmas Lima beans, but you can substitute cannellini or giant corona beans. Dried Fava Soup .
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Chorizo and Gigante Bean Cassoulet: Recipe: bonappetit.com
Recipe by The Bon Appétit Test Kitchen . 3 cups dried gigante, corona, or large lima beans; 4 tablespoons extra-virgin olive oil, divided, plus more for drizzling .
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Healthy Recipe Group Watch the master golf tournament online coverage CBS Tv · Healthy Recipe Group Cilantro, Lemon and Corona Bean Salad (g-free, .
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Dinner: Veggie
Repin Like. Corona beans with braised kale. This was very tasty! I calculated 7 WW points per serving, if you reduce the oil to just 2 Tbs. Recipe serves 4. 1 repin .
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Jan 23, 2012 . Pan-fried Corona Beans & Kale A few notes related to the recipe - be sure to wash the kale well, so you don't end up with grit in your beans.
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Jan 9, 2011 . A can of cannellini (white) beans can be substituted for the heirloom beans. "Pan- Fried Corona Beans & Kale Recipe" is another delightfully .
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Buy corona beans wholesale (sweet white runner beans). . Full line of dried heirloom beans available. . Product Info; Storage; Recipes & Tips; Reviews .
10 places deserving of love | The Columbus Dispatch
Sep 30, 2011 . View an archive of all recipes made by Food Editor Robin Davis in the . in his pork-and-beans entree — huge white corona beans topped with .


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Art Music Photography
#cassettes #posters and prints
Cassette Tape Cyanotypes
New-York based artist Christian Marclay is a visual artist and composer who explores the intersection of sound recording and photography.
As a performer and sound artist Christian Marclay has been experimenting, composing and performing with phonograph records and turntables since 1979 to create his unique "theater of found sound." A dadaist DJ and filmmaker, his installations and video/film collages display provocative musical and visual landscapes. He was born in California, raised in Switzerland and now works between New York and London.
These striking blue cyanotypes are made by resting cassette tapes on photosensitive paper, like a photogram, and making multiple exposures with a light source. (via ex-chamber)


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When organizations need new sources of innovation, efficiency, and cost-control, PWT's flexible staffing provides the project management and technology-specific skills that meet your needs and reduce associated recruiting, training, and overhead costs. PWT identifies, qualifies, and deploys professional IT staff for your organization by screening, skill-testing, providing background checks, and monitoring performance using established processes.
- PC and server network management
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Policy formation
This content was COPIED from BrainMass.com - View the original, and get the already-completed solution here!
Policy Formation
i) Choose a topic on which to write the policy. This topic may address a needed change in technology, staffing, billing, diagnosis, etc.
ii) construct a policy using the 7 steps of policy formation. Address the following:
(1) Define the problem.
(2) Assemble the evidence of the problem or need for a policy.
(3) Construct alternative solutions to the problem.
(4) Select the criteria for choosing the correct solution from the alternatives.
(5) Predict the outcomes of the policy changes.
(6) Confront the trade-offs of not having a policy or addressing the problem.
(7) Decide on a recommendation.
iii) explain your policy's intended use and evaluate the effect that your policy may have on patient care in America.© BrainMass Inc. brainmass.com October 10, 2019, 1:17 am ad1c9bdddf
Solution Preview
Hello student!
The policy I have chosen is staffing volunteers. At my current organization volunteers are used for a wide variety of services. They are an integral part of the organization. I have set a foundation for you to expand the policy with your ideas and feelings.
1. The problem is volunteers do not have a set list of job responsibilities. Volunteers are treated like employees but do not have guidelines to follow. Having guidelines will help resolve some of the issues with what volunteers should be doing while on duty. They are normally told what to do by the manager but nothing is on paper.
2. The need for the policy came into action when a volunteer was found to be doing something that was not asked of them. The volunteer was ...
Solution Summary
The solution is comprised of information on how to formulate a policy to staff volunteers at an organization. The policy formation can be used in many different situations.


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We not only provide a desired IT solution to our clients rather we crave to provide value added services to them which makes them give more than hundred per cent satisfaction.
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We just do not provide a solution, we provide solution with quality and that makes our value system stronger and stable. Delivering the quality remains our top priority and we never ever compromise with quality parameters.
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We create a strong platform of relation with the clients based on
Once you create a relation with us, it's our responsibility to make it productive and sustainable!
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We make you believe in our strengths and our attitude. A strong belief can only help us serve you for long. We do believe in our capabilities, skills and strengths which ultimately help us serve you better.
It is only because our continuous assessment of our work we are able to grow steady and strong. Assessment requires for healthy relationship with the clients and our middle management makes it sure that the strategy developed by the top management is diligently executed by our top class developers.


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Throggs Neck, NY - The USMMA baseball team overcame a 9-2 deficit and scored nine runs in the finall inning en route to a 19-9 victory over Maritime (NY) Thursday night. Senior Warren Hartmann (Wyckoff, NJ) posted three hits in the game and with a two-single in the eighth inning became the Academy all-time career hits leader with 178. The seniors in the middle of the Mariners batting order, including Hartmann, Danny Collins (Sleepy Hollow, NY), and Jake Martin (San Diego, CA), combined to go 9-of-13 at the plate with 10 RBI and nine runs scored.
Merchant Marine (9-5-1) tallied two runs in the top of the first inning with the opening run coming on a Matthew Thomson (Memphis, TN) and Hartmann double steal with a throwing error allowing the run. USMMA stole eight bases in the game without being caught, Collins led the way with three steals for the night and is a perfect 11-for-11 on steal attempts this season. Nathan Slusarczyk (Woodbury, NJ) earned the win on the mound, tossing 4.2 innings of relief with five strikeouts and two earned runs.
Maritime (NY) (4-10) plated all nine of its runs in the third inning to grab a 9-2 advantage. Pat Kirk and Joe Longo ended the rally with back-to-back two-RBI hits as the Privateers also scored with the help of a USMMA balk as well as an error. USMMA would begin its comeback in the top of the fifth with four runs with RBI rips from Hartmann, Collins, and Martin.
The middle of the order would strike again in the top of the fifth as this time the combination of Hartmann, Cameron Stadele (Alpharetta, GA), and Martin drove in runs. Martin plated the tying and go-ahead runs by hammering a 1-2 pitch up the middle to bring home both Collins and Stadele. The flood gates opened in the eighth as Merchant Marine had its biggest inning of the season. Richard Kernan (Blythewood, SC) and Andrew MacKinnon (Buffalo, NY) each helped to blow the game open with two RBI hits, pushing the score to 14-9. Hartmann, Collins, and Tyler Alger (Fairfax, VA) also added RBI down the stretch with Hartmann's record-breaking single driving in MacKinnon and Thomson.
Hartmann is the reigning Landmark Conference Player of the Year and is hitting .517 this season and has a .396 career batting average. His career stats are available HERE. The USMMA record book, which will continue to be attacked this season is available HERE (last updated before start of 2014 season).
The Mariners will return to action this weekend for a three-game Landmark Conference series against Scranton.


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The home secretary has said authorities must be "ruthless in purging" police wrongdoings following claims the Met wanted to smear the family of murdered teenager Stephen Lawrence.
Speaking to the House of Commons, Theresa May confirmed she has asked the Independent Police Complaints Commission (IPCC) to supervise the allegations against the Metropolitan Police by former undercover officer Peter Francis.
Francis told the Guardian that the police wanted him to find "dirt" on the family of Lawrence, shortly after he was murdered in a racist attack in 1993.
Following the claims, May said Operation Herne - originally set up by Met police and overseen by Derbyshire Chief Constable Mick Creedon - will now investigate the allegations.
May told the Commons she wished to show her support for the "unspeakable tragedy" the Lawrence family suffered in the wake of their son's murder.
She said: "Their pain was compounded by the many years in which justice was not done and these allegations still coming 20 years after Stephen's murder only add to their suffering".
After confirming the allegations will be looked at by the IPCC, May added: "I am determined that we shall have zero tolerance of police corruption and wrongdoing".
Neville lawrence, the father of the mudered teenager, said that he finds the announcement "completely unsatisfactory".
He added: "I am convinced that nothing short of a judge-led public inquiry will suffice and I have no confidence that the measures announced today will get to the bottom of this matter".
The allegations surround Francis's claims he was asked to target people launching a campaign against the Scotland Yard, who were angry at their failure to bring Lawrence's murderers to justice. He also claims he was asked to target Duwayne Brooks, one of Lawrence's friends who witnessed the murder.
Posing as an anti-racism activist in the mid-1990s, Francis claimed he was under "huge and constant pressure" to find information on the family, which would ultimately weaken the campaign against the police.
Prime Minister David Cameron previously described the allegations as "dreadful" and said it is important that the truth is revealed "as rapidly as possible".
He added: "One can only think of the Lawrence family who suffered so much from the loss of their son and now suffer again, hearing that potentially the police that were meant to be helping them were actually undermining them".
Lord Condon, the Met police commissioner at the time of Lawrence's murder, said he never authorised any smear campaign.
He said: "I can say categorically that at no time during my time as commissioner did I authorise or condone or was aware of the sort of smear operation that has been described in the Guardian.
"I don't know if it's true or not, but I can certainly say that from a senior level, as far as I'm concerned, there was no such operation".
It's a disgrace
Mayor of London Boris Johnson said he has met with the Met Police mommissioner Sir Bernard Hogan-Howe to discuss the allegations.
Johnson added: "I cannot think of anything more despicable than the police attempting to smear Stephen Lawrence's family.
"If there was a campaign to discredit those seeking justice, we need to know what the orders were and who gave them. We need to know whether there was any criminal activity, and we shouldn't rule out the possibility of criminal proceedings.
"It is critical that the investigation led by the Chief Constable of Derbyshire and the IPCC gets to the bottom of these allegations as quickly as possible".
Howe said he is "shocked" by the claims, adding: "If these allegations are true, it's a disgrace, and the Metropolitan Police Service will apologise".
Scotland Yard has so far refused to confirm or deny the claims by Francis.
The force added: "The Met must balance the genuine public interest in these matters with its duty to protect officers and former officers who have been deployed undercover, often in difficult and dangerous circumstances.
"We are therefore not prepared to confirm or deny the identity of individuals alleged in the media to have been working undercover, nor confirm nor deny the deployment of individuals on specific operations.
"At some point it will fall upon this generation of police leaders to account for the activities of our predecessors, but for the moment we must focus on getting to the truth."


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News Ticker
- NPAs under PM Modi's Mudra scheme jumped 126% in FY19
- shows RTI
- RTI query reveals banking frauds of ₹ 2.05 Trillion reported in the last 11 years
- 509 per cent rise in cases under child labour law: Study
- The Central Information Commission has allowed disclosure of file notings on the mercy petition of a rape and murder convict, rejecting the government's contention that the records cannot be disclosed as these are privileged documents under Article 74(2) of the Constitution.
- Electoral bonds worth over ₹5,800 crore were bought by donors to fund political parties between March 1, 2018 and May 10, 2019, a Right to Information reply has said.
- Don't pay 500/- for answer sheet now- Supreme Court says if Answer sheet is asked under RTI, RTI Fees will be governed
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Showing results for tags 'information definition'.
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u/s 2(f) only information "relating to" can be provided and not "held by"
njit posted a question in Ask for RTI SupportKind Attention of all RTI pros, 1. FAA has rejected my appeal by citing the reason that only information "related to" private body can be provided and information "held by" private body cannot be provided. 2. This decision is dated and has been sent well past the 45 days deadline. If this is upheld by CIC then it seems it will be another death knell to RTI act 2005. Please give your valuable views, opinions, suggestions. Thanks and Regards.


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The No-scrub Way To Clean Your Stove Burners
Stove burners are expected to have some stains and greases from everyday use. It does not matter how careful a cook you are there will always be splatters that form on the burners and grates.
You likely have tried soaking them in hot soapy water in the sink and scrubbing them. This method can be successful in cleaning but not always. It is time-consuming and somewhat frustrating getting the desired result.
There is an easier way that requires a lot less of your energy and time.
Ammonia is the secret ingredient. Ammonia is a chemical compound consisting of nitrogen and hydrogen that forms a very strong smelling liquid or gas. Likely, your grandmother had ammonia in her home but these days we tend to stay away from this toxic substance. Ammonia is part of many common window cleaners in a diluted state.
Ammonia is a superb cleaner and when used correctly is the solution to cleaning those disgusting burners.
You do not want to breath this in, so hold your breath when doing this.
It is the fumes from the ammonia that will cut the residue, not the scrubbing process.
Just place the stove burner into a ziploc bag. Place onto a baking sheet. Pour 1/4 cup of ammonia into the bag and seal it. Place the cookie sheet outside someplace so there is no chance of breathing the fumes from the ammonia. You can even do the entire process outside if you like. Leave overnight. The fumes will dissolve all the grease and hardened oil from the burner. Remove the burner and just wipe clean with a sponge.
And that is it! The results will amaze you. Give this a try, save time, frustration and energy scrubbing. Do be very careful with the ammonia and NEVER mix ammonia with bleach. The two combined are very toxic. Store your ammonia, well out of reach of children.
For photos and details, please visit ' The V Spot' website below.
Learn MORE / Get RECIPE at the V spot
To help with slow website load, we have put all photos for this article here: View photo gallery.


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Meeting & Event Planners - Multiple Disruption - (CIYMP901)
Overview: Participants are the meetings & event management team of their trade association's three-day annual conference. The meeting has an unfriendly demonstration, looming severe weather threatening all outside activities, loss of the keynote speaker, conference attendees complaining of a stomach illness and the news media begin to swarm. How would a team handle such a disaster, especially with your Senior Meetings Director injured and a drunken board member arrested? See how your meetings management staff will respond.
Exercise Focus: Emergency Response / Crisis Communications / Media Relations
Delivery: This Conduct It Yourself Tabletop Exercise is available for immediate download.


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In the velvety blackness of Lunar Samhain our coven, Sisterhood of the Sacred Veil, celebrated the Dark Moon with our annual overnight gathering. The synchronicities, both by timing and by theme, were perfect for me.
I'm currently digging down into my own bedrock in a class in which we're exploring working side-by-side and hand-in-hand with our Shadows. The information that is pouring forth from my inner essence as a result is almost too much to become conscious of all at once, and I'm feeling spectacularly connected to the mistiest, most profoundly obscure and mysterious parts of myself.
As I engage with these experiences during the Dark Time of the Wheel, the work feels so right, important, needed, and necessary. My relationship with the Dark is ever-changing, expanding, intensifying, becoming more and more powerful as the seasons wind on. I don't recall any particular time I was ever "afraid" of the Dark per se, but I do know there were many, many years where my relationship with it was not strong and was certainly not intentionally cultivated.
One of the things I remember really bothering me as a young person becoming rooted into the Pagan community was the "love and light only" energies I constantly seemed to bump up against. It appeared that a large expanse of people believed that "love and light" was the only answer, the only possibility, the only way to create a "positive" life, and everything not of it was disparaged, even vilified in some cases.
This didn't fit into my worldview—I knew that everything wasn't glitter and gems, pale beauty, and visible perfection. Difficult things happen. Paths get snarled. Houses burn down. Evil is elected into roles of great power. Loved Ones die. Dreams perish. And I had already realized that the Dark was just as natural, right, and organic as the Light…and just as essential for growth and change, perhaps even more so.
I recognized that this full attention placed upon only one part of the spectrum of life—Light—didn't speak to me; it felt unbalanced, unrealistic and, at times, delusional. This model wasn't my reality. Further, I was feeling an intense from the (then unnamed and unknown) Dark Goddess to answer Her call. I could find no satisfaction in love and light as the totality of my existence.
I was also sensing that part of my fledgling Priestess mission was to deepen my own connection with the shrouded, swirling Dark, and then to midwife these energies forth in ways more tempting and delightful to others. I felt called to express, in words and in actions, that there is nothing to fear in the Dark, that the Dark is just as crucial and fundamental and good and holy as the Light. I wanted to share how the Dark is fierce and raw yet also tender and soothing, that the Light isn't the only place that holds awe-inspiring comfort—and that this is a big patriarchal lie.
Without the Dark, seeds won't gestate, be held, or grow. Without the Dark, there is no Light. The Light and the Dark are One—not polar opposites, not either-or. The Dark and the Light are each individual sides of the very same coin. The Dark and Light exist together in harmony, as One, pushing and pulling, holding us close while also helping us to expand. The Dark is healing, kind, and loving.
The Dark is where we sleep, regenerate, refresh, rejuvenate. The Dark is the place of birth, death, and rebirth—both literal and metaphorical. Before we are born we reside in the dark of the womb, the dark of the void, the dark of the cauldron of transformation—we were formed in the darkness, the blackness, and we will return to this same beautiful inky stillness at the end of this lifetime. The Dark is the place of both endings and beginnings, of compost and breakdown, of sprouting and growth. The Dark is the liminal zone, the boundary, the threshold.
Our own personal darkness brings alchemy between seeming polarities. When we know our own darkness and ask our Shadows to transform into our Beloveds, we then become real, pure, unveiled. Our Shadows cannot hurt us—in fact, I'm learning that my Shadow sincerely wants only absolute excellence and joy for me and does her best to protect and provide safety for me. When we greet our own Shadows by our own free will at the Altar of Courage, we greet our whole Sweet Selves.
Accepting our own darkness—which includes reveling fully in the dark parts of ourselves and owning our sacred Shadows—is what makes us whole; it is what comprises the wholeness, the holiness, of our identities. Our authenticity, our very sovereignty, is encompassed and held by the Dark. This is full point of our power: coming to terms with our darkness, our Shadow, that which isn't always pretty or nice or "light", to master—to know—Self. Darkness can be the source of Light, of life, and all parts of us are right and valid—all parts of us necessary and valuable.
As Witches, Pagans, Magicians, etc. we need to cultivate the ability to walk in all of the worlds, between all of the worlds. To banish the Dark, to banish the Shadow, is to banish our whole Selves. We are infinite, multifaceted, and perfect as is. And the Dark is our mirror; the Dark is our holy ally.
For now, I invite all of us to contemplate our answers to these questions:
Do I have the courage to step into this sacred place? What seeds am I planting in the Dark? What am I blessing in the Dark? What about the Dark can I bless? How can I best integrate my Light with my Dark? Where do they meet? What is dying that needs to be released to the Dark? What needs to be renewed in the Dark? What cycle has ended or is ending? Who am I? What is my mission in this lifetime?
I look forward to exploring the Dark further with you, Dear Ones, in future blog posts, online, and together in ritual.
With Love,
Lady Jesamyn
Art: 1) Priestess Sabrina Moon; All the rest) Yours Truly


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Facts About Trust Deeds
Credit products and services remain to provide a facilitating grip to the people developing credit problems. While individuals desire revenue web hosting expenses when in household emergencies and additionally stuff like that, currently taking credit ratings provided by home loan organizations can be a strong option. That is the whites of the coin. It an indication in order to all people of which having a payday loan is one thing, repaying is actually another. On the other hand, consumer credit rating organizations supply you with some help to make sure you these people in the event that the debt will get also amazing to remain reimbursed, which often is the situation today. It's not to make a lousy picture of most of these lending institutions to be the cause about making people today within the orchestra pit about indebtedness.The unhappy truth in the present day will be we now have simply just many instances wherever people can do not deal because of their repayments stemming from high-interest charges as well as stuff. Go to the below mentioned site, if you are hunting for additional information concerning trust deed scotland glasgow.
But still, it in its final stages that can put the blame concerning another person else. Anytime this will happen, an optional might be to seek out credit debt advice. Different consumer debt software providers present such a plan to people who want to work out their very own money owed the moment and also for all. Endure, a lot of people lately possess propensity so that you can stall mainly taking care of debts. These people maintain putting rid of it for another person time till the time period arrives going without shoes has grown and also is unmanageable. It can be empirical anytime bills beginning pressure anyone, seek personal debt advice. Thus, what do you think may be the basis associated with unsecured debt suggestion?Might it be vital to purchase one when you can actually compromise a person's requirements on your own and you just just have inspiration together with the sincerity to take action?
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Peer to Peer Lending also known as Social Lending is emerging as a popular way to lend and borrow money. There are two companies at the top of the social lending space. Pertuity Direct and Lending Club both offer the opportunity to lend and borrow money.
Lending Club is a social lending network that brings together investors and creditworthy borrowers to offer value beyond traditional banks. Borrowers with good credit can get personal loans from $1,000 to $25,000 at interest rates that are often significantly better than rates from conventional sources.For lenders, money invested goes immediately to Lending Club's approved borrower members. Most lender members spread their investment across tens or hundreds of qualified borrowers.
Social lending is the new way to borrow. We call it Mutually Responsible Banking – it's people working with people, where everyone comes out ahead. It cuts out all of the high fees and rates that the big banks charge, which means you can make smarter financial decisions. Pertuity Direct provides savers and borrowers with a new, efficient and secure way to meet their financial goals.
Typically reserved for people with good credit scores of 660 and higher to reduce risk. It may not be available to everyone but one thing is for sure everyone is talking about Peer to Peer Lending. From the Wall Street Journal and Yahoo Finance to National Public Radio the topic is buzzing.


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For some traveling, it can be to know the world, for others to identify themselves, to reconnect with the family, to look for lost treasures, to get lost in the jungle or to de-stress from a hard year of work.
Whatever the meaning for each person, what is certain is that traveling is one of the most important things for most people. It is also inherent in traveling the fact of having to manage all the preparations of the trip: look for means of transport, accommodation, make the route of roads, a selection of the most critical places in the city, where to eat and even what outfit we will wear. However, it is almost impossible that we do not forget anything. There are so many preparations! And each one more decisive.
When the journey you are going to make is destined for a foreign country, the preparations increase and have to be much more careful, since some of them are vital for you to travel to certain countries or so that once there you can enjoy without worrying about what you forgot so far The issue of documentation that should accompany you is, for example, much more cumbersome when traveling outside the border of your country. That's why we give you a series of travel tips that will help you make your trip perfect.
Check the validity of your documentation: Check the expiration date of your ID if you are traveling to any country in the European Union or your passport if you travel outside Europe. At the time you are going to board, and you realize (instead the customs workers will notify you) that your passport or identity document is expired, the shipment will be canceled entirely and there will be nothing to do. Nothing will be of any use to the airport staff; it is an irremovable rule. That is why you should be cautious and check this before undertaking anything else.
Photocopy the documentation: first, you will have to keep your documentation well. But, just in case, it is better that you carry a copy (in addition to the original) or several distributed in the different bags of luggage that you sell. Also, thanks to the technological development you will be able to scan your documentation and take it with you in your smartphone or your email account. This way your documents will be in the cloud, and you will not have problems to prove yourself in cases of theft or loss.
Vaccination: we recommend that you inform yourself beforehand about the health policies of the country to which you are traveling as, in some cases, you will not be able to do so if you do not have the necessary vaccinations. In other cases, it is not mandatory, but it is recommended. If you are a person suffering from different allergies (to insects, pollens or plants), it is recommended that if you are going to travel to the Central American jungle, where there are vast amounts of tropical mosquitoes, take the appropriate measures regarding vaccination.
Money: keep your money safely in small compartments of your belt, clothes or backpack. Also, try to distribute it among your handbags (those that you will have control at all times) because if you suffer the theft of the wallet or a bag, you will have money in another.
Travel insurance: get travel insurance when you go abroad. The insurance will cover both medical expenses and legal expenses or theft. There are many comparators where you can get insurance at an excellent price. Detect your needs and choose the best coverage with which to cover them. Remember that in the case of traveling through Europe, if you have a medical problem, you can use the European Health Card.


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"The Supreme Court on Wednesday refused the federal government's request to salvage a six-year-old law seeking to ban minors' access to sexually explicit material on the World Wide Web. Acting on that law for the third time, the Justices simply declined to review a Third Circuit Court decision last July striking down the Child Online Protection Act of 1998. The Justices' action came without comment and with no noted dissents in Mukasey v. American Civil Liberties Union, et al. (08-565)."
Wednesday, January 21, 2009
Child Online Protection Act put to rest by Supreme Court
In a case that's been watched by the digital rights community for may years, the Child Online Protection Act of 1998 was finally killed off for good in the latest US Supreme Court decision on the Act today. Scotusblog says:
Posted by Ray Corrigan at 8:49 PM
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Some 50 people have been injured when a morning commuter train crashed into the buffers in a station in Barcelona.
Some 48 people were treated after the accident and 18 taken to hospital, five of them in serious condition, Catalan regional emergency services said.
Footage from the train crash at a station in Barcelona in which dozens of people were injured pic.twitter.com/tfBKmu23Y1— RTÉ News (@rtenews) July 28, 2017
The accident happened at Francia station on the northern side of the city.
Television images showed many passengers being tended to on the ground in the station.
The train driver was among those seriously injured.
The train had left the southern coastal town of Sant Vicenc de Calders and arrived at Francia station at 7.15am local time.